Poster session abstracts - CyberLeninka

Poster Session Abstracts 211

Poster Session Abstracts

CFTR

1*

A TP-DEPENDENT PROTEOLYSIS OF CFrR-NBDIIN VITRO. L. Millen, E. Strickland, G. DeMartino, and P. Thomas. Department of Physiology, UT Southwestern Med. Ctr., Dallas, TX.

A TP-dependent degradation of CFI'R is catalyzed by the ubiquitin­proteasome pathway. Recent studies (Ward, eta/., [1995) Cell83:121; Jensen, et al., [ 1995] Cell 83: 129) have shown that immature fonns of the Cystic Fibrosis Transmembrane Conductance Regulator (CFI'R) retained in the ER are polyubiquitinated and rapidly degraded. The complex pathway of events leading to degradation includes: substrate recognition, ubiquitination (marking the incompletely folded protein for degradation), and translocation of the tagged substrate through PA 700 into the core 20S proteasome. To understand the mechanisms by which the t.F508 mutant is preferentially recognized by this proteolytic machinery, we have developed an in vitro system for ATP-dependent degradation of the arnino-tenninal nucleotide binding domain (CFI'R­NBD I). The system employs rabbit reticulocyte lysates as the source of proteasome and ubiquitin-conjugating enzymes. The lysate supports the fonnation of a ladder of higher molecular weight NBD I species which are more prominent in the presence of ubiquitin aldehyde, a potent inhibitor of ubiquitin isopeptidase activity. Degradation of NOB 1 under these conditions is A TP-dependent and the initial rate of proteolysis of the unfolded domain is at least an order of magnitude faster than that of the native domain, indicating it is preferentially recognized. Notably, depletion of the molecular chaperone Hsc70 from the lysate retards the initial rate of degradation of the unfolded but not the folded domain. Thus, the results are consistent with a model in which Hsc70 is not only central to the folding of CFI'R but to the identification of incompletely folded confonners by the proteolytic machinery. (Supported by NIDDK and the Welch Foundation).

2* RECONSTITUTION OF THE CFTR DEGRADATION IN THE ENDOPLASMIC RETICULUM D. Ferguson, F. Zhang, N. Kanner and G L Lukacs. Hospital for Sick Childeren and University of Toronto, Toronto, Canada

The Incompletely folded wild-type (wt) and mutant (~S08) CFTR are degraded In a pre-Golgl compartment, most likely In the endoplasmic reticulum (ER). Recently, It has been demonstrated that certain misfolded proteins are translocated in a retrograde manner from the ER to the cytosol In order to be degraded by the cytosolic proteasomes. lbis translocation process requires metabolic energy and Is Inhibited by ATP depletion. Interestingly, the degradation of AFSOSCFTR and the Incompletely folded wtCFTR was accelerated following the depletion of cellular A TP (Lukacs et al., EMBO J. (1994) 13, 6076). In an attempt to investigate this discrepancy and to delineate the molecular requirementa of CFTR degradation we have reconstituted the process In permeablllzed cells and in Isolated mlcrosomes. The half-Jives of the wt and the mutant CFTR were determined following metabolic pulse-labeling of stably transfected cells with lmmunoprecipiation and quantitative phosphorimage analysis. Prior to the chase period, the plasma membrane was permeablllzed by hypotonic swelling and scraping or with digitonin. The half-lives of both the AFS08CFI'R and the Immature CFTR were shorter In permeabillzed (S min) than In Intact cells (3S-40 min). Although >95% of the cytosollc lactate-dehydrogenase activity was depleted upon permeabillzatlon, ruling out possible effects of tracer amounts of cytosol and A TP, the experiments were repeated on mlcrosomes, Isolated from metabolically labeled cells. Neither cytosol nor A TP was required for the proteolysis of unfolded CFTRs, since the turnover of the immature wt and AS08CFTR was accelerated In microsomes (T112 2-3 min,) compared to that observed In permeablllzed cells. The degradation can not be attributed to the deterioration of microsomes since folded CFTRs were resistant to proteolysis. Furthermore, the discordant protease sensitivity of unfolded and folded CFTR was

preserved following the removal of peripherlal membrane proteins or the depletion of luminal proteins from Isolated mlcrosomes. Meanwhile, these perturbations diminished substantially the activity of proteosomes associated with the microsomes, as determined by the cleavage of fluorogenic peptides. Collectively, these rcsulta suggest that Incompletely folded CFTRs, can also be degraded by a proteolytic mechanism, which appears to be distinct from proteasomes and to be strongly associated with or Integral in the phospholipid membrane of the ER. Supported by MRC and CCFF Canada.

3*

ATP Dependent CFTR Degradation in a Cell-Free System. X. Xiong.~ Departments of Molecular and Cellular Engineering and Medicine, Institute for Human Gene Therapy University of Pennsylvania.

While recent studies have indicated that CFI'R degradation in vivo is mediated through the ubiquitin-proteasome pathway, the mechanism by which newly synthesized CFTR is recognized and targeted for degradation remains unknown. To better understanding Ibis process, we have reconstituted CFTR synthesis and degradation in vitro using a rabbit reticulate lysate (RRL) expression system. Under standard translation conditions, synthesis of full length CFI'R was completed in 90 minutes and reached a maximum at approximately 2 hours. Newly synthesized protein was core glycosylated with high mannose carbohydrates as indicated by Endoglycosidase H digestion and was fully integrated into microsomal membranes as determined by extraction of chains in 0.1 M sodium carbonate (pH 11.5). Immunoprecipitation revealed tight association of nascent CFI'R with HSP73. Microsomal membranes containing radiolabeled protein were pelleted through a sucrose cushion to remove cytosolic components, resuspended and incubated in either sucrose buffer or fresh reticulocyte lysate. In the absence of cytosol, newly synthesized CFTR was highly stable in 3 mM OTT but was rapidly converted to a high MW complex (>300 kD) under oxidizing conditions in an ATP independent manner. This high MW complex could not be resolved even after prolonged incubation in 1.0 M OTT and 4% SDS. When microsomes were incubated in ATP-depleted RRL, CFI'R chains were stable. However in the presence of ATP and RRL, full length CFTR chains were converted to a series of high MW intennediates which were degraded over a four hour period. Degradation of high MW CFTR complexes in RRL was blocked by hemin, a known inhibitor of the 26 S proteasome, but was unaffected by addition of MG 132 or ALLN. These studies thus define a cell free system for de novo synthesis, glycosylation and membrane integration, of CFTR which reconstitutes key features of HSP73 association and cytosoi/ATP-dependent degradation. Future studies will be aimed at dissecting the role of cytosolic components involved in the degradation process. (supported by the CFF and NIH DK51818)

4 Redundant Translocation Mechanisms Direct Topology of CFTR N-terminus Transmembrane Segments. Y. Lu, X. Xiong, A. Bragin, K. Kaimi, W Skacb Departments of Molecular and Cellular Engineering and Medicine, Institute for Human Gene Therapy University of Pennsylvania.

Recent studies have demonstrated the existence of redundant topogenic infonnation encoded within the first two transmembrane segments (TMI and TM2) of human P-glycoprotein (MDRI) (J. Bioi. Chern. 268:23552). It has been unclear however, whether this information reflected two truly independent mechanisms of transmembrane assembly or rather, different aspects of a single

212 1997 Cystic Fibrosis Conference

mechanism. Here we show that like MDR I, TM I and TM2 from CFfR each function as independent, internal signal sequences to target the nascent chain to the endoplasmic reticulum, initiate translocation, and span the membrane with complimentary specificity. However, while TMI directed translocation of C­terminus flanking residues, this signal sequence activity was significantly impaired by the presence of endogenous charged residues, E92 and K95, located within the TMI hydrophobic core region. Consistent with this observation, TMI signal sequence activity was neither required nor sufficient for directing CFfR N­terminus topology. In contrast, TM2 signal sequence activity was both efficient and specific in translocating N-terminus flanking sequences, and as a result, TM2 was able to direct CFfR N-terminus topology independent ofTMI through a novel, ribosome-dependent posnranslational mechanism. Thus both TM I and TM2 participate in CFfR biogenesis by directing two distinct and redundant pathways of polytopic protein transmembrane assembly. During this process TM2 provides a back-up mechanism to direct topology of chains which fail to target and/or translocate via TM I. This unique arrangement of topogenic information thus provides a general mechanism for ensuring proper orientation of TM segments which, due to primary structural features, are unable to efficiently achieve correct transmembrane topology through conventional, cotranslational pathways. (Supported by CFF and NIH (DK 51818))

s* ROLE OF C-TERMINAL CYTOPLASMIC DOMAIN IN THE BIOSYNTHETIC PROCESSING OF CFI'R. M.A. Loo. L. Cui, Y.-X. Hou, X.-B. Chang, and J.R. Riordan. Mayo Clinic, Scottsdale, AZ, USA.

As nascent CFfR chains are synthesized in the endoplasmic reticulum (ER), they are scrutinized by members of the intertwined pathways of maturation and degradation, including multiple molecular chaperones and proteolytic systems. In attempts to determine which features of the nascent CFfR molecule make it more susceptible to degradation than maturation, we found that sequences in the C-terminal extension beyond NBF2 had a major influence. Truncation to remove this entire domain of -90 residues resulted in even more rapid degradation and reduced the proportion that could mature and form functional channels at the cell surface. This effect was entirely reversible by proteasome inhibitors such as lactacystin, indicating that this truncated molecule was an excellent proteasomal substrate. It was less susceptible, however, to degradation by the alternate ATP-independent proteolytic pathway, which acts on full length nascent CFfR. Truncations at more C-terminal locations had quite different effects, making the molecule a poorer substrate for the proteasome while remaining sensitive to the alternate pathway. Manipulations of these sequences have similar effects on the rates of degradation of nascent Ml508 CFfR. We postulate that sequences within the C-terminal extension participate in intra- and inter- molecular interactions that influence the biosynthetic lability of the molecule and the pathway of proteolysis to which it is directed. (Supported by NIDDK of NIH).

6* RESTORATION OF oAMP-aTIMULATED CFTA CHLORIDE CHANNEL ACTIVITY IN AFIOI CELLI BY DEOXYIPEAGUALIN. S.L. Fq, S.P. O'Connor, S.G. Nadler', D.W. L .... D.M. J.ttereorf, J.M. KapiM, A.E. Smith, C. JIMg and S H Cbtoq. Genzyme Corpogtion, One Mouruln A*, F111mlngham, MA 01701, 'Brilt(j.Myers Squibb, Suttle, WA, ITulta Unlvll'llly, ao.ton, MA.

Deletion ol the codon encoding phenylelllnine 508 (AF508) Ia the moat common nUation In cyatJc IIHoala (CF) and IMUita In e ao-cded trafllcldng deled. M1Dnt AF508-CFTA protein ,..Ina lunctionalectivlty but the ne-.c protein Ia recognized u ebnormel and In conuqu811Ce, Ia l'lllelned In the endoplumic l'llllclalm (ER) and aubaequently degrllded. h Ia thla ln8blily to trllllic normelly to the llpiclll membrane that Ia the baala for the diaeaH. h '- bHn propoMd thllt thla l'lllention In the ER Ia medlatad, at INII In pert,

by the molecular chaperones Hsp70 and calnexin. This observetion raisea the poaaibility of therapeutic intervention in CF by agents capable of interfering with the normal functioning of these chaperones. We heve investigated the ability ol deoxyspergualin (OSG), an immunosuppressant known to compete effectively for binding with Hsp 70 and Hsp90, to promote trafficking of AF508-CFTR to the cen membrane. We first analyzed DSG· treated recombinant C127 cells stably transfected with 4F508-CFTR (C127· AF508), for evidence of mature band C-CFTR which would ba indicativa of pnx:essing of the mutant protein in the Golgi. Biochemical analysis of lysates from these cells showed no discernible evidence of the mature band C form of CFTR suggesting that very little W any AF508-CFTR had exited the ER. To ascertain whether a sman amount of &F508-CFTR, below the sensitivity of detection w~h the biochemical assay, may have traversed to the plasma membrane, the more sens~ive SPQ fluorescence helide assay was utilized to test for the presence of functional CFTR chloride (CI") chennel activity at the ceU surface. Approximately 17% of C127·AF508 cells thet had been cultured lor 3 days w~h 10 Jlg/rnl DSG exhibited measurable cAMP-stimulated CFTR Ct channel activity. This effect was specWic for DSG and wea not replicated w~h the structurally related analog spermidine. The number of responding cells wealo- then that observed when the C127-AF508 cells wera treated with sodium butyrate (90%) or grown at a raducad temperatura (90%), two other intervention• shown previously to effect the presence of AF508-CFTR at tha eel surface. Similarly, treating JMEICF15 cells (airway epithelial ceU line derived from a AF508 CF patient) and IBE-1 cells (intrahepatic cell line derived from a AF508/G542X CF patient) with 50 Jlg/rnl DSG for 3 days also resuhed in the appearance of cAMP-mediated Ct channel ectivity in up to 20% of the treated ceUs. Bacauae meny studiea heve indicated that these cells lack cAMP-dependent c1· channel activity other than CFTR, the observed response after DSG treatment was most likely due to AF508-CFTR at the cell surfece. Together, these resuha suggest that agents capable ol aharing the normal functioning of molecular chaperones may provide yet anolher means of restoring CFTR Ct channel activity.

7* IDENTIFICATION OF PHOSPHORYLATION SITES IN CFTA FROM CALu-3 CELLS BY MASS SPECTROMETRY. D. C. A. Neville, W. Ankbeiner, A. S. Verl<man and R. R. Townsend. Cystic FibrOSis Research Center, U.C.S.F., San Francisco, CA, U.S.A.

CFTR c1· channel activity Is regulated by phosphorylation of specific Ser residues In the R domain by protein kinase A. Analysis of phosphorylated residues In CFTR has usually been done following metabolic labelling of cells using ["P], trypsin digestion of lmmunoprecipated CFTR, and 20 peptide mapping. The advantages of mass spectrometry are that neither exchange of an exlsiting phosphate with ("P) nor protein purification are required. Plasma membranes from unstimulated and stimulated ( 1 0 11M forskoHn for 15 min) Caiu-3 cells (an adenocarcinoma pulmonary epithelial cell line) that constitutively express CFTR (-1 prnoV1o" cells) were isolated following ceH lysis and high speed centrifugation. Membrane proteins were separated by SDS-PAGE, and peptldes from the CFTR­containing region were Isolated following In situ get digestion with trypsin. The peptides were purified by micro-scale reverse-phase chromatography and analyzed using nano-electrospray ionlzation­quadropole ion trap mass spectrometry (nanoESI-QITIMS). NanoESI-QITIMS haa the ability to trap and accumulate lone so that masses can be observed using femtornole amounts of peptide. Peptides containing phosphorylated Ser rssldues are Initially characterized by the loss of H,PO, from the parent rnolectJar lona. Molecular Ions with mass-to-dlarge (m/z) ratios of Interest are then selected and fragmented to determine peptide sequence and site of phosphorylation. Mass spectrometric analysis was performed on an aliquot of CFTR equivalent to that Isolated from one mllion cells (-10 fmol, 1. 7 ng CFTR). An unanticipated finding was the Identification of phosphorylated sites (Ser eeo, 737 and 753) In unstimulated Calu-3 cells. Phosphorylation of Ser 680 and 737 have been reported In full. length CFTR following forakolln stimulation. Phosphorylation of Ser753 has only been reported In a 10S-A mutant CFTR moleaJie and In n vitro phosphorylated full-length CFTR Isolated from Sf9 cells. This Is the first ldentiflcetlon of phosphorylated CFTR residues In unstimulated mammalian cells and suggests that certain residues may have a slow turnover rate in CFTR. Supported by NIH-NCRR and CFF.

8 BINDING OF NUCLEOTIDES TO NBD1 STRONGLY INHIBITS A-DOMAIN PHOSPHORYLATION BY PROTEIN KINASE A. D.C.A. Neville, A.A. Townsend, C.R. Rozanas and A.S. Verkman. Cystic Fibrosis Research Center, U.C.S.F., San Francisco, CA, USA.

To study interactions between the contiguous NBD1 and R domains of CFTR, NBD1-R (amino acids 404-830, in fusion with His. tag) was expressed as a single protein in E. coli. NBD1·R (10-25 mg/liter culture) was purified from inclusion bodies in 8 M urea by Ni-affinity chromatography, and renatured by rapid dilution at pH 5. In vitro phosphorylation by protein kinase A increased the apparent size of NBD1-R from -52 kDa to -56 kDa by SDS-PAGE. The fluorescent ATP analog TNP·ATP bound to renatured NBD1·R with high affinity (K,"" < 1 j.tM) with a stoichiometry of -1 TNP·ATP site per NBD1·R molecule; TNP-ATP binding was reversed by ATP with K, -2 mM. TNP-ATP binding to NBD1·R was not affected by R domain phosphorylation. Secondary structure analysis by circular dichroism gave 19% a-helix, 43% ~-sheet and tum, and 36% 'other" structure. To study the influence of nucleotide binding to NBD1 on R domain phosphorylation, NBD1·R was In vitro phosphorylated with the catalytic subunit of protein kinase A and [y"P]A TP in the presence of AMP-PCP, AMP-PNP or TNP·ATP. NBD1·R phosphorylation was quantified by autoradiography of SDS-PAGE gels. Phosphorylation of NBD1·R was reduced> 75% by AMP·PNP or AMP-PCP (0.25 mM) and> 50% by TNP-ATP (0.25!1M) in a dose-dependent manner. Two types of control experiments indicated that the inhibition of NBD1·R phosphorylation was related to NBD1-R domain-domain interactions, rather than to nucleotide Interactions with protein kinase A or the R domain: Phosphorylation of the protein kinase A substrate Kemptide (assayed by HPLC) was not affected by the nucleotide analogs, nor was phosphorylation of a GST·R domain fusion protein (assayed by [y"P ]ATP incorporation) processed identically to the NBD1·R protein. The Inhibition of A domain phosphorylation by nucleotide binding to the NBD1 domain indicates significant domain-domain interactions and suggests a novel mechanism for regulation of CFTR phosphorylation. (Supported by CFF).

9* BASAL PHOSPHORYLATION OF SER 768 KEEPS WT CFTR CHANNELS CLOSED IN UNSTIMULATED XENOPUS OOCYfES. K W Cbau1, S.S.

Smi!h3, L. Csanadyl, A. C. Naim2, D. C. DawiOD3, D. C. Gadsby!, Laboratories of I Cardiac/Membrane Physiology, and of 2Molecular and Cellular Neuroscience, The Rockefeller Uoivenity, New York, NY, and 3Depl. of Physiology, University of Michigan Medical School, Ann Arbor, Ml, USA.

Serine 768 in the R domain of CFTR ia a good protein kinase A (PKA) IUbatrate and ia rapidly phosphorylated by PKA in vitro. Xerwpua oocytea expre88inl Ser-Ala mutant CFTR (S768A) show enhanced sensitivity to activation by IBMX in the presence of forakolin, suggestina that phosphorylation of Ser768 exerts an inhibitory influence on CFTR channel activation. When oocyte• expreaaine wild type (WT) or S768A CfTR were exposed to a Na + • and ca2 +-free solution, we consbiCIIdy observed spontaneOUS. tranoient activation of CfTR ct· conductance. This "basal" CfTR ct· conductance reached, within 3-!1 min, a peak amplitude of up to 7!1" (WT), or 90"(S768A), of the maximal CfTR conductance subsequently activated in the I&JDe

oocyte by I mM IBMX plus SO 11M forskolin. After reaching the peak, the basal CFTR conductance spont.meOUSiy declined within !1-7 min for WT (0.6 na cRNA), but penilted for>40 min for S768A (0.6 na cRNA). This baeal Cl" conductance reflected activity of WT or S768A CfTR channels because It wu not seen in uninjected or H20-injected oocytes, il waa raveraibly diminished by adding 10 mM

SCN" to the bath, and II wu prevented by pre-injection o{ the PKA antagonist RpcAMPS (SO I'M). Pre-injection or 0.!1 11M microcyatin markedly slowed decay of !he basal WT CFTR ct· conductance, Indicating that !his deactivation required dephosphorylation by phosphatase 1 or 2A. Activation of the basal CFTR conductance wu prevented by injection of 2mM BAPT A, auggesting that it depended on intracellular Ca2 +release. But, regardlen of the precise mechanism of the bual activation of the CFTR channels, the llrikin& difference in dtactivtJliOII between WT and S768A mutants muot be attributable to the Ser to Ala point mutation. Because the mutant is expected to differ from WT only under conditio111 in which Ser 768 is phosphorylated, these reeulta both oupport an inhibitory role for pbospboserine 768 and ouggeat that Ser 768 ia phoaphorylated in vivo, in

Poster Session Abstracts 213

unstimulated oocytel. Apparently, basal phosphorylation of Ser 768 is sufficient to keep WT CFTR channels closed at a time when S768A channels are open. The single-channel basis for these phenomena is being analyzed. Supported by NIH OK 51767 (DCG & ACN) and OK 45880 (DCD).

10 DEPHOSPHORYLATION OF CFTR BY PROTEIN PHOSPHATASES 2AAND 2C. K W Chan1, J. Qin1, B. T. Chait2,

D. C. Gadsby', A. C. Nairn•, Laboratories of 'Cardiac/Membrane Physiology, ofl~Mau Spectrometry and Gaseous Ion Chemistry, and of "Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY, USA.

In stimulated cells, CFTR Cl· channels become phosphorylated at multiple R domain serines by protein kinase A (PKA). The channels require phosphorylation before they can be opened by ATP, or other hydrolyzable nucleoside triphosphate&, acting at CFTR's nucleotide binding domains. Our single-channel studies have indicated that incremental phosphorylation of CFTR by PKA is associated with increases in channel open probability (Po), reflecting longer average open times. These distinct gating modes display differential sensitivity to specific inhibitors of cellular protein phosphatases (PPases), suggesting that they are controlled by particular phospho-serines acting alone or in combination. We have suggested that a serine(s) essential for opening a channel is(are) dephosphorylated by PP2A, while dephosphorylation of serine(s) associated with the higher Po states of more fully phosphorylated CFTR is/are mediated by PP2C. Previously, we have reported the use of matrix-assisted laser desorption/ionization-ion trap mass spectrometry (MALDIJITMS) to resolve the sequence and kinetics of phosphorylation of the R domain of CFTR by PKA. We have now performed similar studies of the dephosphorylation of the R domain (phosphorylated to high levels by PKA) by the catalytic subunits of PP2A (rabbit muscle) and PP2C (recombinant bovine PP2Cp). The results suggest that PP2A and PP2C display overlapping, but distinct, kinetic specificities for individual phosphoserines in the R domain. In addition, complete dephosphorylation of the R domain appears to require both PP2A and PP2C. These findings support the suggested role in the regulation ofCFTR function of an incremental, differential phosphorylation of R domain serines by PKA, and of their selective dephosphorylation by PP2A and PP2C. Supported by NIH, DK51767 (DCG & ACN), HL49907 (DCG) and RR00862 <BTC).

11* MEMBRANE TARGETING OF cGMP-DEPENDENT PROTEIN KINASE IS REQUIRED FOR CFTR-Ct CHANNEL ACTIVATION. A. B. Vaandrager', A. Smolenski2, S.M. Lohmann•, and H R de Jonge'. 'Department of Biochemistry, Erasmus University Rotterdam, The Netherlands. and 'Institute of Cinical Biochemistry, Medical University Clnic, WOrzburg, Germany.

A recently cloned iaoform of cGMP-dependent protein kinase (cGK), designated type II, was lmpncated as the mediator of cGMP­provoked Intestinal Ct secretion, based on Ita localzation In the apical membrane of enterocytes and on Ita capacity to activate CFTR-Ct channels (1). In contrast, the soluble type I cGK was unable to activate CFTR In intact cells, atthough both cGK I and cGK II could phosphorylate CFTR In vitro. To Investigate the molecular basis for the cGK II isotype-speclficlty of CFTR channel gating, we c<Hxpressed cGK II or cGK I mutants displaying different membrane binding properties by means of adenoviral vactors in a CFTR-transfected Intestinal cell Ina (IEC-CF7) and examined the ability of cGMP to phosphorylate and activate the CFTR-Ct channel. Mutation of the cGK II N-terminal myristoylation sHe (Giy2 -+ Ala, I.a., G2A) reduced cGK II binding to the membrane and severely Impaired the activation of CFTR. Conversely, expression of a chimeric protein consisting of the N-terminal anchoring domain of cGK II fused to the N-terminus of cGK IP resutted in association of the cGK I chimer with the membrana and activation of the CFTR Ct channel. The potency order of cGK constructs for increasing CFTR-mediated '"'!· efflux from IEC-CF7 cells In response to cGMP (cGK II ,. membrane-bound cGK I chimer • non-myristoylated cGK II (G2A) ,. cGK I p) correlated with the extent of 02P incorporation into CFTR determined in parallel measurements. These results strongly support the concept that targetting of cGK to the membrane is a major determinant of CFTR-Ct channel activation in intact cells. (1) A.B. Vaandrager eta/. (1997) J. Bioi. Cham. 272: 4195-4200.

214 1997 Cystic Fibrosis Conference

12* 14* ACTIVATION OF CFI'R CHLORIDE CHANNELS BY TYROSINE PHOSPHORYLATION. x.......na, F. Seibert, X.-8. Chang*, J. R. Riordan*, J. W. Hanrahan. Dept. Physiol McGill Univ. Montreal QC and •S.C. Jolmson Res. Center, Mayo Clinic Scottsdale, Scottsdale, AZ.

CFI'R Is a phosphorylation regulated chloride channel which is defective In cystic fibrosis. Protein kinase A (PKA) is the primary activator of the CFIR cr channel, however, constitutive phosphorylation by protein kinase C is also required and plays a pennissive role In PKA activation (Jia et al, J. Biol Cbem. 272:4978, 1997). Although tyrosine phosphorylation was not observed in early studies of CFI'R, an increase in CFI'R mediated current was observed recently by FISCher and Machen (Biophys. J. 71:3073, 1996) when excised patches were exposed to the !Esine kinase Sn: in the presence of PKA We have used patch clamp, P04 labeling and immunoblotting to study CFIR regulation by tyrosine kinases and phosphatases (PTPase), and to assess whether CFI'R is itself a substrate for tyrosine phosphorylation. When patches excised from CFI'R transfected BHK cells were exposed to the tyrosine kinase p6()"""' (30 u/ml) and A TP (1 mM) in the presence ofPKA Inhibitor, CFI'R channels were activated to a level which was comparable to that seen during PKA stimulation. A TP alone caused partialiClivation of CFIR when patches were excised from cells that had been transfected with v-Sn: eDNA, and channel activity was further enhanced by the PTPase Inhibitor dephostatin ( 10 IJM). Expresaion of v-Sn: elevated tyrosine phosphorylation of CFI'R in vivo according to Western blotting with an antlphosphotyrosine antibody. In vitro phosphorylation was also observed after incubation of immunoprecipitated CFI'R with p60"""' in the presence of nP-ATP. Sn: and PKA responses were compared using CFTR channels bearing mutations at 1!5 monobasic and dibasic PKA sites. PKA activation was attenuated by about 8!5 'II In this mutant whereas Sn: activation was not decreased. These resulta suggest that direct tyrosine phosphorylation can be a potent, PICA-independent mechanism for activating CFTR chloride channels. Supported by CLA, CCFF, MRC. and NIH (NIDDK).

13* CFTR Cr Clwlnelo Phyoicllly end Funcllonllly lnter..:t with Synt1xin 1A.

~· M.W. Quick, B • .Jovov, A. Touuon, J. Pev1Mr, M.K. Blnnen, O.J.

hnoo lnd K.L Kirk. Oepertment of Phyliology lnd Blophyolco. Gregory

Fleming Jornoo Cyme Fibrooio Re111rch Center, Univeroity of Allbomo 11

Blrminghern. Birmlnghern, Allblme, 35294-00011.

At pr~~~nt- know llttll lbout the kind1 of moleculel thlt reguiltl

CFTR function It the eplciiiUifecn of epithllill colo. We hiYI oburved thlt

colonic epltheliel clllo IT .. lnd HT29·CL 1 9AI expron two eyntiXin looformo

C1 A lnd 31 lnd 1 eyntain binding protein n·S.C 1 . Such mollculeo hiVe been

impHCited in the docking lnd lulion of oyn1ptic vllicleo It the nervi termin81,

lfthough - -uy the cirect r.gulltion of Cllcium chennell by oynuxln 1 A

In br8ln hll been reported. Synuxin 1 A ond oyntuln 3 IICh loclllze ot or ne• the epicll pole of colonic epithllt81 Cillo. Syntlldn 1 A but not eyntiXin 3 blndl

to CFTR with 8Ubmlcromoltlr efflnlty Itt vltto. SyntiXIn 1 A inhibited CFTR·

medloted Cr currento when lhlll moleculn were co-exproaed In Xenopus

oocytn, whlrlll oyntllcin 3 hll no ouch effect. Thi1 negltivo r•l•tion of

CFTR by eyntiXin 1 A could be riVIrlld by trlltmlnt with Botulinum d. which

cle1V11 eyn11x1n lA off-· MlcrolnjiCtion of the cytOIOAc domlin of

eyntuln 1 A heel no effiCI on CFTR currentl In oocytll but did rncue CFTR

from Inhibition by full length eyntlldn 1 A. Thutl, lyntlllin 1 A mUll be

membr~n~-enchored to regulltl CFTR function. N·S.C 1 Inhibited the binding

of CFTR to· eyntiXin 1 A In v1rto lnd 1110 riYirlld the neg11iv1 regullltlon of

CFTR current~ by oynt1xln In 1 A obHrved in oocvtn. Converlely, on n·S.C 1

point mut1n1 thlt dol8 not bind eyn1IXIn 1 A did ncrc racue CFTR from inhibition

by rnembrMI -.cl oyntiJdn 1 A. Our obelrvlltlono lndlc1t1 thlt: II

evn1IXIn 1 ...-em cr chlnnlla 1n llklltion to N-type cr • chenneio 1nc1

ill n-S.C1 lllo regul- CFTR function by conlrOIIIng theiVIillbllity of eyntlxln

1 lor CI'TR.

ANALYSIS OF THE REGULATION OF MURJNE CFfR. K.A. Robinson, S.J. Delaney•, D.P. Lunn•, S.A. Thomson*, B.J. WairJwriabt1

and D.N. Sheppard. Depar1mcnt of Medicine, University of Edinburgh. Edinburgh, UK and •Centn: for Molecular and Cellular BioiO&Y, University of Queensland, Brisbane, Australia.

When expressed in CHO cells, wild-type murine CITR fonns a regulated cr with a signific:aotly reduced open probability (P .) compared with that of human CITR (Robinson eta/. (1997) J. Physiol. 499P, 59P). To UDdenland why murine CFTR has a reduced P., we investigated the regulation of murine CITR cr channels in excised insidc-ilUt membrane patches. For comparison, we studied wild-type human CITR expressed in Cl27 ceiiJ. ATP (0.3 mM) and PKA (75 nM) were continuously present in the intracellular solution. In excised inside-out membrane patches, human CFTR CJ" channels are stimulated by drugs that: I) alter gating behaviour (e.g., fluoride; F"), 2) inhibit membrane-associated pbosphltues (e.g., (-) bromotetramisole), and 3) inhibit tyrosine kinases (e.g., genistein). F" (10 mM) significantly increased the P. of both human (144 ± 17%, P<0.01; n: 4), and murine CFTR (JS6 ± 12 o/o, P<O.OJ; n = S). (-) Bromotetnunisole (I mM) increased the P, of human CITR from 0.41 ± 0.07 to O.S4 ± 0.07 (P<O.OJ, n = 4), but decreased the P. of murine CFTR from 0.10 ± 0.02 to 0.07 ± 0.02 (P<O.OS, n = 4). In contrast to previous results, genistein (30 j1M) did not alter the P. of 1aunan CFTR (n : 4 ); it wu also without effect on the P. of murine CFTR (n: 4). However, genistein (100 j1M) decreased the P. of human (36 ± 7 %, P<O.OI; n = 4) and murine CFTR (6S ± 7 'Yo, P<O.OJ; n • 6).

Genistein (I 00 j1M) also decreased the singlo-dlannel current amplitude of both human and murine CITR (human; 82 ± 4 %; n = 4, murine; 76 z S %; n = S). These results suggest that the(-) bromotctramisole sensitive phosphatase that inactivates human CFTR may not regulate murine CFTR. They also suggest that both human and murine CFTR are stimulated by F" and inhibited by high concentrations of genistein. Supported by the Cystic Fibrosis Trust, BBSRC and NH and MRC of Australia.

15*

POSSmLE CONTRIBtmON OF THE PEPI'IDB BACKBONE TO CFI'R ANION SELECTIVITY Paul Yoldc!L Sbu·XIan Zbena llld Jolla W. Hannban. Dept. l'bylloloay. MeOW Untvaalty, Moa~Ra~. Quebec. Clll8dl.

We baw ~ tbe etrec:t ot mutatln& tbrecnlne 338 In tbe slxtb lnlllsmcmlnlle bellx ol CFIR on cbannel ICicctlvlty. Mulallooa at tbil pooldon lnlt.r!ered wttb protein proceu~n1; !be IIDOUDt ot lllltUre protein produced followlna IWIIe lnlllllectlon In BHK cella - WT > T338V > T338S > 1'3381 > T338N > T338A > T338F. Patdl dlmp I'OIXJI'din& tran tarae. 1ns1c1e out mcmlnne patdlel maled JllllalliCIOPk: PICA- llld ATP-depeadent a· cumoll fer Ill mutanll except T338F; It II Dot cJe.lltbil rdlecta lou ol cblallel ftmcdlla In Ibis mutlllt or Ia related to 111 mllproceuJaa. Atthoup wr .cFrR exbtblta a ampletely llaelr curreat-witaae relalloalblp Ia eiCIIed plldlea wttb ~ a· c:a~CZ~~tndonl, ICIIIe mutaaulbOMd llnlll& (1'338N) or mild (1'3381, T338V) Inward rec:tllk:atloa UDder tbele coadltlonl. 111e aeloctlvtry ot wr 111c1 mutant cblaaell - ~ by repladaa cr 1a lbe bllb IDiuUon wttb ~ 1111oa1. Selectivity aequeace~ for dlftereot cbanael wriiDll were:

W,: SCN >NOs> Br > Cl > I> ao, >formate> F > PF, T338A: SCN> I O!:NO,> Br> CJO,> Cl > PFo> formale > P T338S: SCN >NO,> I> Br >a> 00,> formale > Pl'o> P T338N: SCN>NOs> Br>l•a > PFo>aO,>formale>P T331V: NOs> SCN > Br • a> 1 > ao, > PFo> rorma~e > P

Note tbat uo ~ wrilllll bad !be - ~ All YINaiiiCiecled llnlii&IY qaiall ~ aailllla auc:b u F llld lbrm8le, IU&&eatla& tbat permeability II CODtrolled by a 1)'0tlllplc or wall: llcld -&tb lite. ~. !be Nlallw permabillty ol cli&reDt J)'Oinlplc •111111, Ia plr1lcullr PF., 00. llld SCN, were IIJUI&ly dclcled by muwtona at 1'338. All I)'OirCJplc IDiona tested ab..-1 lbe pcrmablllty IICqlmCII T338A > T338S > wr. IUJPIIID& a llzo.depcadeat etrec:t ol tbele mlllllionl. Howewr, tbe permabilltlel ot T338N llld T338V wae more dlftlcult to latapet. Purtbamore, tbe cban&CII Ia permabillty oblened did Dot - to CIIJIIUie llnlii&IY w1tb 1be ..-e otlbe llllluo ICid R paup at pooldon 338. 1bla, CODpled wltb lbe fact tbat tbll R poup doel Dot line tbe IQ1ICOUI pore olCPTR (Cbeua&.t Abbu. 1996, Blophyl. J. 70:2688-269S) IIUI&CIII tbat lbCie lllUI8lloal may aller CPTR aelecllvity by cbla&la&lbe orielllllioa d tbe peptide blctboae, wttb lbe lllllde Dlll'Oiea poulbly ICtla& u a biadln& lite fer 1)'111nlplc llliolll. Suppor11d by tiN C4NidJtM CF FOIUIIIDtlotl, MRC tllld NIH (NIDDIC).

16* INTERACTION OF CHANNEL BLOCKERS WITH R347l).CFJ'R

Paul Linsdel! and JClbn W. Haorabao. Department d Pbysiology, McGill University, Montreal, Qutbcc, Canada.

A number d large anionic compouoda are able to enter lhe CF1R channel pore from lbe cytoplasmiC end aDd block cr CXlllduCIIOD. We have previously sbowo lbat 1 positively charged arginine residue located dose to lhe cytoplasmic end oldie lllxlh transmembrane helix at CFIR, R347, 0011ttibutea bolb to a a· and SCN" binding sile aDd to lhe site at wbicb lhe slilbcoes DNDS aDd D!DS block lbc cbanne! (Tabcbaraol et al., 1993, Na1ur1 366:19-82; Linsdell &: Hanrahan, 1996, J.PhysloL 496:697-@3). We have Investigated block d maatlS(X)pic wild type (WI) and R347D-CFI"R currents in large, Inside out patches excised 6'ool BHK cells by lhe inttacellular blockers glibcoclamlde, DPC aDd benzoate. In cacb case, lbc apparent block« affinity at OmV, Kd(O), aDd apparent block~ valency, z6, were eslimaled. Glibcoclamide caused 1 Slrollgly Wlltage dependent block c:L wr -CFTR (Kd(OJ 67±9J!M, z6 0.37±0.03). Glibeoclamide blocked R347D wilh 1 slightly lower affinity (Kd(O) 128±191LM) and, lnttrestingly, 1 mucb sba!lower Wlltage dependence (z6 0.13±0.01). As 1 consequence d cbls change In Wlltage dependence, glibcoclamlde &bowed· 1 lllgnificantly bigb~ affinily for wr lbao R347D at negative membrane potenlials, but 1 similar affmity at positive potenliaJJ. Reducing lbe extracellular or inlrliCC!!Uiar CJ" CODCCDtratioo increased tbe affinity ol &libeoclamlde block d bolb Wf and R347D, but did Dot eliminate lbC difference in block« Wlltagc dependence. Similar changes In Wlltage dependence were IICCD for DPC (Kd(O) 46l±Slj.IM, r6 0.27±0.05 for wr, Kd(OJ 6l2±66JiM, r6 0.12±0.03 for R347D) and benza~te (Kd(O) 2!l±3mM, z6 0.37±0.05 for wr, Kd(OJ 21±3mM, z6 0.14±0.03 for R347D). In fact, for benza~te block, lhe diff=t Wlltage dependencies meant lbat Ibis block« bad 1 high~ affinity for wr at negative potentials, but 1 bigber affmity for R347D It positive potenliaJJ. Our resulll do not support 1 stroog role for R347 In forming lbe biDding site for lhese block«s, u It appears to for DNDS and DIDS. ~. lbe llxed charge at position 347 appears to inOuence lhe Wlltage dependence d block. This may be due to 1 direct coottibulioo to lhe electric lleld wilhiD lhe pore, or to altered iDteraaloos belwecn block« and cr looL A direct role appean more likely, since R347D IIi!! sbows reduced block« Wlltage dependence when extracellular aod lnlniCe!lular cr coacentrationsarc reduced. Supponed by the Canadlall CF FoiUidatlo11, MRC twJ NIH (N/DDK).

17* SINGLE CHANNEL PROPERTIES OF DISEASE· CAUSING MUTATIONS WITHIN THE R DOMAIN OF CFTR. E A Pasyk, X.K. Morin, P. Zeman, K. Galley, L-1. Huan and C.E. Bear, Hospital for Sick Children, Toronto, Ontano, Canada

CFTR functions as a plasma membrane cAMP-activated Cl· channel. Cyclic AMP causes activation of CFTR Cl" channel largely through phosphorylation of serine residues. within th~ putativ~ R do~a1!1· Interestingly,_ mo~t of the R domam disease causmg ~utauons reside tn a region which IS d1sUnct from the phosphorylation Sites, a regiOn referred to as RD1 (Dulhanty & Riordan, 1994). Our previous study showed that some of the R domain mutants, A604-634 and L619S, known 10 cause severe disease and L610S, for which disease severity has not been yet defined, failed to exhibit cAMP-activated macroscopic CFTR Cl" current when expressed in Xenopus oocytes. On the other hand, the 1618T mutant, which causes moderate disease and the L633P mutant, for which the clinical phenotype has not been yet defi_ned, h_ad intermediate whole-cell currents. The 06140 mutant, causmg m1ld disease, exhibited normal macroscopic cAMP-activated Cl" curr~nt (Morin eta/., 1997; Society of Gen. Physiol. Abstract). Defective macroscopic current of these mutants could be due to lower expression at the plasma membrane and/or lower intrinsic CFTR Cl· channel activities. The objective of this study was to examine single channel properties of those RDl mutants that exhibit panial activily at the whole­cell leveiiO determine if these variants possess altered channelgaung or permeation properties. Multi-level and single channel activities were recorded in the cell-attached patches of oocytes expressing wild type CFTR (n=l6), D614G (n=6), 1618T (n=IZ) and L633P (n=6) when oocytes where stimulated with an activation cocktail containing 10 I!M forskolin, I mM IBMX and 20 l.lM CPT -cAMP. Single channel analysis revealed that conductances of channel activities ofD614G, 1618T and L633P mutants were unchanged compared wilh the wild type CfTR (7±0.9 pS, n=3). Probability of openmg (Po) of single channel wild type CFTR was O.S, whereas the PQ. values of 06140 and 1618T mutants were reduced to 0.26 and 0.30, respectively. Probability of opening of L633P was close 10 that of wt CFTR (O.S). The reduced PQ of 06140 and 1618T mutants were likely due 10 prolonged channel closed time. Whereas the mean closed time for wild type CFTR was 45 ms, the mean closed time for 06140 and 1618T mutants were 600 ms and 480 ms, respectively. Since some of these CFTR variants exhibit altered channel gating at the single channel level, it may be hypothesized that the RD 1 region of R dom111n interacts with other domains of CFTR protein involved in channel gating, possibly its nearest neighbour, NBD 1. Supported by Canadian Cysnc Fibrosis Foundation.

Poster Session Abstracts 21 5

18* Effect d CO\'aleat Modific1tloa ol the Nucleotide Blndla& Domalnl ol CFTR. J F CQUen and M.J. Welsh, Howard Hughel Medical Institute and the Departments of lntemal Medicine and Physiology and Bicphylict, Uolvenity of Iowa College of Medicine, Iowa City, Iowa 52242

The development of 1 model of CFTR piing by A TP II complicated experimentally by the presence of two Interdependent nucleotide binding domains (NBD I and NBD2). To undel'lland beDer nucleotide regulation of CFTR channel activity and to probe tbe llnlcturO and fllnctjon of the NBDs, we Introduced cysteine residues by lite-<lirected mutagenesla Into the NBDs of the CFTR· CSJlA mutant, 1 mutant which behaves like wild-type CFrR, but is unalfected by 100 11M cytosolic N-cthyl malcimide (NEM). A cysteine residue wu Introduced Into tbe Walker A motif of either NBDl (GSTGAGK -+ GSTGCGK, A462C, termed •NBDI-Cys") or NBDl (GRTGSGK -+ GRTQCGK, Sl248C, termed "NBDl-Cys"). We traolicntly expressed lhese mutants In HcLa cells and examined their respo11111 to 100 11M cytosolic NEM In excised, lnsici«lut membrane macropatchcL NEM Irreversibly Inhibited channel activity In both mutanlL The rate of modification at bolh NBDs - u determined by the lit of 1

lingle decay-exponential - wu slowed by increasing the MgA TP concentration. ThiJ lllggCib lhat MgA TP "proleccs" the introduced cysteines from NEM· modification. The rate ofmodif~e&lion ofNBDl-Cys wu much faster lhan lhat of NBD 1-Cys even at high MgA TP concentralions. NBD 1-Cys activity wu completely Inhibited by NEM-modilicalion in the presence MgA TP. however tbe NBD2-Cy1 channel retained significant activity when modified in tbe presence of MgATP. Magnesium pyrophosphate (MgPPi) protected NBDl-Cys, but only the in presence of MgA TP. MgADP also protected the NBDl-Cys from modification. Gi~n the interdependency between tbe NBDs, caution II warranted in lnterprelin& lhese daiL The results 111gges1 that both NBDI Interact with MgA TP and conttibute to channel piing. The results also 111ggest that NBD2 intcracll wilb bolb MgADP and MgPPi, and lhat tbe MgPPi interaction is dependent upon Interaction of MgATP at 1 llCCOIId lite, perhaps NBDI. The differences in reactivity between tbe two NBDa may be revealing an asymmetty in the piing cycle or SIJUciWal differences between tbe two NBDL

19*

TilE SECOND NUCLEOTIDE BINDING FOLD OF CFTR FUNCTIONS AS AN ACTIVE ATPue, GTPue AND ADENYLATE KINASE. c.....Randak'. P. Nethu, E.A. Auerswald2

, C. Eckerskom3, 1. Assfalg-Machleidl', W. Machleidt'. 'Depanment of Pedialrics, 'Department of Clinical Chemislry and Clinical Biochemislry, 'Mu-Planck-lnstilut fiir Biochemic, Maninsried, 'Department of Physiological Chemistry, Ludwig-Muimilians-Univenitit Miinchen, Gennany.

The second nucleotide bindin& fold (NBF-2) of CFI'R seems to be involved in both channel opening and closing u well u in inhibition of CFTR channeloclivity by cytoplasmic ADP. In order to ochieve 1 betttr understanding of the individual regulatory eveniS that arc required for channel octivalion and inllibition and of the effecll of CF causing mutations the enzymatic properties of isolated CFI'R domai111 have to be sludied and related to known electrophysiotogical data. We have recently demonstrated that a recombinant CFI'R-NBF-2 polypeptide is able to bind A TP, ADP and AMP with high affinity (Randak et al., FEBS Len. 363, 189-194) and that there ex isiS a common binding site for A TP and GTP (Randak et at., FEBS Lett. 398, 97. 100). For the studies reported here NBF-2 was expreased in fusion with the maltose­binding protein (MBP) and purified from the soluble E. coli proteilll to homogeneity u indicated by SDS·PAGE, HPLC, N-tenninal omino ocid sequencina and mass spectroscopy combined with N-tenninal sequencing of protein fragments gained by Lys-C digestion, that cover 70% of tbe NBF-2 sequence. In conlrast to MBP alone or 1 fusion protein of MBP and the a-subunit of ~-galactosidase, the recombinant NBF-2 wu shown to catalyse tbe reaction A TP -t ADP + P1 by three different assays, monitoring A TP turnover, fonnation of ADP and release of P1 IJ(. 86 f'M, rate constant 0.37 min·'). The reaction product ADP inhibits this A TPase octivity even a1 concentralions one to two magnitudes lower !han the A TP concenlration present in the assay. In a similar manner the hydrolysis of GTP to GDP and P, wu dcmonslrlled IJ(. 40 11M. rate constant 0.29 min·'). In the presence of AMP the A TPase reaction was quickly superseded by the fonnalion of two ADP from ATP and AMP (adenylate kinase reoction). Bolh ATPase and adenylate kinase activilies were inhibited by AMP­PNP. As typical for adenylate kinases a dislinct AMP binding site could be verified for CFI'R·NBF-2 by the inability of TNP-ATP and AMP to compete for binding. All three enzymatic activilies of NBF-2, ATPase, GTPase and adenylate kinase activity, were readily inhibited by the symmettic double-substrate-mimicking inhibitor Ap,A. that needs a three- and a monophosphate position for binding and did not inhibit a mere ATPase like the (Na•,tc:•)A TPase. It il tempting to speculate that the inhibitory effect of ADP on CFTR channel sating, mediated by NBF-2 (Anderson & Welsh. Science 257, 1701-1704), is the consequence of interference with octivatins ATP hydrolysis. The strong inhibition or CFTR c1· currents in the presence of cytoplasmic AMP (Quinton & Reddy, Nature 360, 79-81) could be the result or high ADP formation at NBF-2 that consecutively inhibits ATP hydrolysis (Supported by the Deutsche Fonchungsgemeinschafl Ra 68213-1).

216 1997 Cystic Fibrosis Conference

20* A STRUCTURAL STUDY OF THE NBFI DOMAIN IN CFTR: IMPLICATIONS FOR CF. I P Anoca:uul, J. Boucher!, M. Schneider!, F. Bontemsl, J. Barthe3, S. Blanquet2, G. U:noir3, J.Y. Lallcmandl, V. Stovenl. t· RMN dea proU!inea DCSO, Ecole Polytoobnlquo, 9tl28 Palaitoau, Franoo 2· BlOC, Ecoto Potytoc:bnlquo, 91128 Patalooou, Pronoo. 3· P6diotrio &4n4ralo, HOpilll Neokor-Enfanll Malad .. , 149 rue de stvr .. , 7~743 Paris ood<tx IS.

In most cases, cystic fibrosis Is caused by mutation~ In the NBFI domain or CFTR. in particular AFS08. This deletion leads to a folding defect in NBFI, and disables CFTR to complete its post-translational processing. Therefore, detennlnation or the three dimenslonnal (30) suucture of NBFI is a key step in understanding this folding defect. We have built a new model for NBFI (I) by homology modeling with the known 30 structure of the nucleotide binding domain of bovine F1 • ATPase<2>. Unlike previous models<3.4), this model is consistent with the phenotype associated to AFS08, and other functional characteristics of NBFI. Moreover, It leads to reconsider the initially defined limits of NBFI within the complete primary sequence or CFTR. According to Ibis model, NBFI should extend approximally 70 residues into the region previously atulbuted to the regulatory R domain<S>. This new definition enables to explain the phenot)'pe associated to mutations situated into the formerly defined R domain<6>. Furthennore, we have produced this revisited NBFI domain by heterologous expression in E.coli. We have obtained a pure, stable recombinant domain, with high yield. The domain has been characterized by recoanition of an anti-body (MATGI061 Transa~ne), and by ATP binding experiments. NMR structural and functional studies are currently perfonned on this domain. According to primary results, these studies provide an unexpected and new function for CFTR which could lead to a new therapy. (I) J.P. Annereau et al, C.R. Act~d. Sci. Paris, 320, 113-121 (1997). (2) Abrahams et aJ Nature, 370,621-628 (1994). (3) Hyde et aJ. Nature, 346, 362·36S (1990). (4) Mimura et aJ. Proc. Nat/. Acad. Sci. USA., 88, 84-88 (1991). (S) Riordan et al, Science. 245, 1066-1072 (1989). (6)J.P. Annereau et al, FEBS letters, in press, (1997). Supported by the A.F .L.M.

21 EFFECT OF R·DOMAIN PHOSPHORYLATION SITE MUTATIONS ON CHLORIDE CURRENT IN FRT EPITHELIA. HA Bmpr, 0 Baldursson, AP Thompson, MJ Welsh, HHMI, DepL lnL Mecl., Univ. of Iowa, Iowa City, lA 52242.

PhOiphorylation of the R domain reauJates the activity of CFI'R. To understand better the reauJalion of a· channelletivity, we mutated Individual ICI'ines In CFI'R and expressed the varianllin Filcber rat thyroir!:nthelia. We measured transepithelial a· CWTent after~! a al cAMP stimulation (10 mM fonkolln) 111d com current by the mutants: currents were S737 A > S79 A f!: WI' ~A f!: S-Quad-A> S813A. (S-Quad-A contains Set to Ala mutations at S660, S737, S79S,IIld S813.) Mutation of the serines also altered rates or current activation and Inactivation. We found that the activlllion rate following addition of 10 J.LM rorstonn was S737A >WI' CFI'R >S-Quad-A. In addition all of the single serine mutants and S-Quad-A had faster Inactivation than WI' CFI'R, eYeD wbcll absolute current was area=. Mutations also altered the a· curmlt response to increasing concenlrations of roralr:oUn; the sensitivity to rontoUn was S737 A> WI' CFJ'R > S­Quad-A. Pinally, we found that COIISINCts containing any three serine mutations had current similar to that or S-Quad· A. suqestlng that phospborylation or no one serine was able to restore Wild-type levels of currenL However, the S660,795,813A construct, which retains Scr737, had a faster lnacdvation rate than S-Quad-A. It is interesting that these results are different from thoae observed In studies or single channels In excbed patches or membrane. sugestlnf that other IICliviliea In the Intact cell may lnf1uenc:e CFI'R f'unclion. The data IUgelt that four R domain ICrinea contribute to channel reaulation but that they have dift'erent effects on absolute current and IIClivalion and inactivation n1e1. Scr737 was put1cu1ar1y lntereslina because it lnfluencecllelllidvity to rontofln llld actfvalion rates differently from the other mutants. 'Thole effec:IS may explain the increased current obselwd with the S737A mutanL

22 DIFFERENTIAL MODULATION OF GAP JUNCTIONAL COUPLING BY CAMP IN CFPAC-1 AND PLJ-CFTR HUMAN PANCREATIC DUCT CELLS. M Chanson, I. Duperrut, J. Coclet-Ninln, P. Meda' and S. Suter. Dept. of Pediatrics and 'Morphology, University Hospital and Medical School, Geneva, Switzerland. .

Gap junctional communication (GJC) and chloride secretion have been shown to be concurrently regulated by cAMP in a human intestinal cell line expressing the cystic fibrosis transmembrane conductance regulator (CFTR). To assess whether GJC and CFTR-dependent chloride secretion are related, we have now compared connexln (Cx) expression and GJC In a human pancreatic duct cell line (CFPAC-1) homozygous for the deltaF508 mutation, and in a clone of CFPAC-1 cells (PW-CFTR) Infected with a retrovirus containing wild-type CFTR eDNA. 62% of PW-CFTR calls ellcltad large chloride conductances (2.95 :1: 0.9 nS, n=34) In response to 500 11M cpt-cAMP and dB-cAMP whereas modest changes (0.35 ± 0.3 nS) were detected in only 2 out of the 13 CFPAC-1 cells studied. Amplification products corresponding to mRNAa of CFTR, Cx45 and Cx43 but not of Cx32, Cx37 and Cx40 were detected by RT-PCR in both CFPAC-1 and PW.CFTR cells. lmmunolabeiing showed that Cx45 was expressed al cell-cell contacts whereas Cx43 was not detected. Consistent with Cx45 properties, no Intercellular diffusion of Lucifer Yellow or neuroblotin was observed In both CFPAC-1 and PW-CFTR cells. In addHion, gap junctional currents with steep dependence on transjunctlonal voltage could be detected In both cell types using the dual patch-clamp approach on cell pairs. Interestingly, large electrical coupling was detected In CFPAC·1 cell pairs, which showed junctional conductances (OJ) averaging 2330 ± 381 pS (medlan•1820 pS, n•27). In contrast, PW-CFTR cell pairs exhibited lower OJ values (610 ± 258 pS, medlan-o pS, n•14). AHhough the distribution of gj values was not affected In CFPAC-1 cella by adding Sp-cAMP to the electrode solution (median-1382 pS, n-8), the non-hydrolyzable analog of cAMP Increased (P<0.02) Oj of PW-CFTR cell pairs (median-2333 pS, n•10). Single­channel analysis of gap junctional currents In CFPAC-1 and PW-CFTR cella studied under control condHions and in the presence of Sp-cAMP showed one population of gap junction channels with sinilar unitary conductance of 30·35 pS. These resu•s suggeatthat expression of wild­type CFTR affects Cx45-mediated GJC by regulating tha cAMP­dependent changes In gj. The defective regulation of gap junction channels may add to that of other membrane channel& to contribute to the pathogenesis of cystic fibrosis.

23 ISOLATED NBD-1 LOCALIZES TO TilE CEIL MEMBRANE OF COS. 7 CELLS AND INCREASES BASAL HALIDE PERMEABILITY BY A GLIBENa.AMIDE-SENSmVE PATHWAY. I P Ct•nqE, Z. Bebok, J.S. Hq, EJ. Soncher. Deplrtmela of Pedlatrlca, CeU BioloaY, and Medlclae, Univenlty of Alabama at Birm1Jiibam, BlrtniJiibam, AL 35294 USA.

We tlllled wbeb1r NBD-1 (M 4~588) of CFTR II capable of tarptin& die eubryollc cell membrane after n111iea1 over apreaaloo In COS-7 cella. NBD-1 antiaen wu detecled at die c:eU IUrface by dl&ltal IXlllfial lmmuno lllia'c.copy q I polyclonll nbbit IJIII•NJID..l antibody. 1be preaenc:e of die AP5al mullllon In NBD-1 did not llplftcalldy alser die membrane locallzadon paaem. We...- ballde penneablllty etrect. of llolaled NBD-11n cos-7, 2CfSMEo., and Cl27 cella by SPQ analysis. NBD-1 increued ballde permeabillty of die CQS-7 cella COIIIplred with cella upreaina an unrelated poleln of a1mllar molecular wei&ht (adenovlrua ftber prolein, "21 kDa). Care wu talren In tbele eltperimenll to - tbat anion permeability ell'ec:ll wen speclllc tor !be expreuion of CFrR NBDI. Other proeeiniiiUCh u LacZ, T -7 polymeraae, and muldple vTF7-3 (vacclnla vector-derived) polypepddea expread u COIIIl'dJ did not Influence die penneablllty In any of die c:eU typea llludled by SPQ. By SDS-P AGE and/or Wellenl blot analysil, exprealon Ieveli of many of tbele proteinl wu at or above die level of NBD-1. In addldon, incteued halide peraabillty wulellliiM to die llllfonyl urea recepllll' blocker &libenclunlde {7100 ,.M), wblcb bu beelllbown previoully to bind to die opeo-1111e wild-type CFTR Ct ~- Toplber, tbe1e ftnlliDp Indicate tbat !be NB0-1 can direct ill own lll'pdaa to die eubryodc ceU membrane, and Influence ballde penneablllty In 1 aUbeDcl•mJc!Helllldve manner. 1be nucleotide blndln& doma1nl (NBDI) wltbln die traf8c A TPue family lban slpliftcant IIOqlleiiCe bomolo&Y ("30") and Mvenl, lncluclin& Hill' and MalK have been lbown to tarpt die prolwyodc ceU membrane and 111111118 1 tnmmembnne conft&undon. Our re.dtllndicale tbat 1bla ame property of NBD1 a11o ...,0. to eubryodc c:elll and to CFTR. Becaule there II no _.. to~ dial- remainder~- ftdl..lqdl CFTR tDOiecule ICII to conatraln NBD-11n IUCh a way u to pnveacalllrOIIIIendency to biDd and/or eneer die p1uma membrane, • cilia ..--s here IUppOfllaiDplllopc model in wblcb NBD·lipllll die plum& membrane. (Supported by - CPP and NIH. J.P. Clancy II a Leroy P. Maabewl Award reclpleat.)

24 A Functional CFTR-NBF1 Is Necessary for ROMK2-CFTR Interactions Carmel M. McNicholas', Malcolm w. Nason, Jr.•, William B. Guggino', Erik M. Schwiebert', Steven C. Hebert', Gemard Giebisch1 & Marie E. Egan•. Depts. of Cellular & Molecular. Physiology' & Pediatrics", Yale University School of Medicine, New Haven, CT; Dept. of Physiology & Biophysics', UAB, Birmingham, Al, Dept of Medicine4

, Vanderbitt University School of Medicine, Nashvme, TN, Dept. of Physiologf, The Johns Hopkins University School of Medicine, Baltimore, MD.

ROMK2, an alternative spliced lsoform of the ROMK gene, Is a renally derived ATP-sensitive W channel (!,.."'). Unlike native 1 .. ,.. channels that ara inhibited by sulfonytureas such as glibendamide, ROMK2 channels are relatively insensitive to these compounds. In excised inside-out patches we racenUy demonstrated that coexpression of CFTR with ROMK2 restores glibenclamlde sens~ivity to the W channel (PNAS, 93: 8083-8088, 1996). In order to examine which regions of CFTR are Important to this interaction, wild type(WT) CFTR or mutant CFTR constructs were coexpressed w~ ROMK2 In Xenopus oocytes and the glibenclamide sensitivity of ROMK2 was assessed. In two-microelectrode voltage-damp studiea, when WT -CFTR was expressed with ROMK2 52% of currents were inhibned by glibenclamide (n=13). This Is significanUy different from the 15% decrease observed when oocytes expressing ROMK2 alone were exposed to glibenclamide (p<0.001, n=8). These data are consistent with our previous patch clamp findings. To examine If the first nucleotide binding fold (NBF1) was Involved in this lntereclion, ROMK2 was expressed w~ either the CFTR mutant G551D or A455E. In oocytes expressing G551D-CFTR and ROMK2 there was only a 14% decrease In current after glibenclamide treatment (n=6). likewise, glibenclamide lnhlbtted only 25% of current In oocytes coexpresslng A455E­CFTR and ROMK2 (n• 10). These data suggest that alterations In NBF1 abolish the CFTR-ROMK2 Interaction that leads to ilicreased sulfonylurea sensHivity. Furthermore, when ROMK2 was expressed w~ K370X-CFTR, a construct truncated prior to NBF1, glibencalmide had no effect on the Ba·­sensitlve K• currents (n=12). In contrast, when a truncated CFTR construct that contains an Intact NBF1 (K593X), was expressed w~ ROMK2, 39% of current was lnhlbHed by glibenclamide (p_s 0.001, n=8). Taken together these data suggest that the Interaction between CFTR and ROMK2 may require a funclional CFTR-NBF1. Moreover, the iicreased sensitivity of ROMK2 to glibenclamida can occur when a truncated form of CFTR Is expressed provided NBF1 Is present. These data aupport our previous findings which demonstrate an interaction between CFTR end ROMK2. (Supported by NIH &CFF)

25 SUR1 Can Substitute for CFTR In the CFTR-ROMK2 Interaction. Malcolm W. Nason, Jr.', Tong-VI Yao', Pamela Thomas•, & Marie E. Egan'. Dept. of Pediatrics', Yale University School of Medicine, New Haven, CT; Dept. of Pediatrics, University of Michigan School of Medicine, Ann ArbOr, MI.

CFTR, a member of the ABC transporter auperfamlly, functions as a channel regulator by Interacting with a number of ion channels, Including amiloride-sensitive epithelial aodlum channels (ENaC), outwardly rectifying chloride channels (ORCC), and renally derived ATP-sensitlve potassium channels (ROMK). We have previously demonstrated that CFTR can regulate ROMK2 channel activity by altering the glibenclamide sensitivity of this K' channel. Other membera of the ABC superfamily Including SUR1 (the beta call sulfonylurea receptor) also function as channel regulators. Moreover, cartain ABC transportera can aubstitute for dysfunctional family members, I.e., mdr3 can restore the transport ability of ste6-def1Cient yeast. In order to Investigate whether enother ABC transporter could substitute for CFTR in the CFTRIROMK2 Interaction, we coexpressed SUR1 with ROMK2 In Xenopus oocy1es. Using the two mlcroelectrode voltage-damp technique, we confirmed that the expression of ROMK2 was associated with the appearance of ea•-aensillva K• currenta that are Insensitive to glibenclamide (n=8). When CFTR was coexpreased with ROMK2 aimilar Ba·-sensitlve K• currents were seen, however these currents were significantly Inhibited by glibenclamide (50%)(n•12). In addition, when SUR1 was coexpressed with ROMK2 38% of the K• current was gllbenclamlde sensitive (n=5, p<0.005). Thus, expression of either SUR1 or CFTR with ROMK2 enhances the glibenclamide sensitivHy of the K• channel. These data suggest that SUR1 can act as a ROMK2 channel regulator and substitute for CFTR. Furthermore, H suggests that there are common motifs In ABC transporters that allow for these lnteractlona. We are presently investigating which domains and residues are essential for thil coupling. (Supported by NIH &CFF)

Poster Session Abstracts 217

26

EFFECT OF COMMON MUTATIONS ON INTRAMOLECULAR INTERACTIONS IN CFTR. Scon A Kjni, Eric J. Sorscher. Department of Physiology and Biophysics, Department of Medicine, Umversity of Alabama at Birmingham, Birmingham, AL, USA.

Understanding intramolecular interactions within CFJ'R may illuminate fimcti~nal and biogenic processes that are disrupted in disease states, Assoctattons between the f.rst and second halves of the CFfR protein have been demonstrated. Coexpression of an N-terminal truncated CFI'R (M837X, ending after the R domain) and a C-terminal CFfR (A837) produce dramatically enhanced halide permeability well beyond what is present when either construct is exfressed alone as assayed by SPQ florescence. Halide movement out o the cells coexpressing both halves of CFI'R is highly conslitutive; however we have shown that the addition of forskolin moderately enhances halide efflux implying that some CFfR-Iike regulation ts maintained by the association of the half molecules. Physical interaction of the two halves was determined by coimmunoprecipitation with antibodies specific to the first nucleotide binding domain {NBDI) or the last 4 amino acids of the C-terminus. lm~unoprecipitates of lysates from cells coexpressing both halves using anu-NBDI antibody specific for M837X were highly enriched in A836~ implying physical binding between the N-terminal and C-terminal halves. Another smaller truncation {R553X) missing the R-domain and the last 33 amino acids of NBDI was also seen to coimmunoprecipitate with A836. The R553X did not, however, compliment A836 functiOnally. In contrast to other studies(Ostedgaard, Biochemistry, 1997), we founa that w1ld type or AF508 NBDI {a.a. 433-596) coimmunoprecipitated with A836; implying that interactions of the two halves may involve NBDI. Two common mutations occurring in NBDI were also examined, AF508 and G551D. The AF508 mutation in M837X eliminated the enhanced halide_penneability seen with wild type M837X. However, the AF508 M837X retained the ability to coimmunoprecipitate with A836. Surprisingly and in contrast to the AF508 mutation, the G551 D mutation in the N-tenninal prolein did not disrupt either the functional or physical events seen with the wild type M837X. The functional defect conveyed by the G551D mulation may be allevialed by removal of a constraining domain or interaction that exists in the full-length G55 lD CFfR. Our findings demonstrate the usefulness of studying separately exl'ressed domains of CFfR to detennine the effect of disease causing mutaltons on functional complimenlation and physical interactions. (Supported by CFF)

27 COMPARISON OF CFrR REGULATION BY ENDOGENOUS AND EXOGENOUS PROTEIN PHOSPHATASES ...1....l.!li. M.D. Pato2, P. S. Seibert1, X.-B. Chana•. J. R. Rltl"dan• and J.W. Hanrahan1

, 10ept Pbyslol., McGiU

Univ., Mootrl!al, QC, Canada; 2Dept. Blocbem., Univ. Sask, SK., Canada; 'Rea. lnst., Hosp. for Slclt CbUdren, Unlv. Torooto, Torooto, ON, Canada; "s.c. Jobosoo Medical Rescardl Caner, Ma)'O Clinic Scottsdale, AZ. U.S.A.

The Cyslic Abrosls Transmembrane CooductaDce Regulator {CFm) dllorlde dianne! Ia tightly regulalcd by procein ldoasea and phospbatuea. crrR cbanoela dial are ICIIVI!ed m-cell usually beoame. deactivated In excised membrane patcbea due to a rollusl, mcmbrano-assodated phosphatase IIClivlty. We bave atudled the spoolalleoua rundown of crrR cr cbannels In Calu-3 llld transfected BHK cells, and bave canpared II wltb the deactlvatlm Induced by expooure to exogenous protein pbosphatasea. RundowD was obsened In mae than 75% of patcbea l'rom Calu-3 cella but only In about 28% of patcbcs li"om BHK cella. Thus there Ia variation In membrano­assodated phosphatase ICIIvlty between diiTereot ceU types, and also between palcbea excised l'rom tbc same cell culture. Rundown was Insensitive to calycullo A and lnbibltcr 2. and was revened In the presence of low PKA ICilvlty by F, 1 non-specific protein phosphatase lnbibltcr. The relative potencies and ldoetlca of deactiVIIion were canpared by adding candidate pbospbatascs to those excised patcbcs that did not rundown spontaneoosly. Both PP2A and PP2C caused deactlvatlm, but the time counc of deactlVIIioo and elfecu on cbannel gating were markedly diiTereoL PP2C was the most potent deactivata wben expressed per mg of phosphatase and caused deactivation with the II8IIIC time coune u spoolalleou& rundown obsened In other palcbes following exclslm. PP2B 111d PPI bad DO

effect on dianne! activity wben tested at canparable phosphatase cmcentratlon. Slmllsr results were obtained wben pbospbatascs were tested In the presence of a low level of PKA. We cmclude that deactlvatlm by exogenous PP2C c:losely resembles spootaneoua rundown obsened In palcbea excised l'rom epithelial md BHK cella. Sllflporltd by Outodiall CF FoiUidatiolt aNI NIH (NIDDK).

218 1997 Cystic Fibrosis Conference

28 GENISTEIN INHmiTS INACTIVATION AND DEPHOSPHORYLATION OF THE CFI'R. J F Murphy. S. Raman and W. W. Reenstra. Dept. of Pediatrics, duPont Hospital for Children, Thomas Jefferson Univ., Wilmington, DE, 19803

It has been proposed that genistein (gen), a soy derived isoflavone, increases CFTR activity by inhibiting channel dephosphorylation. To test this hypothesis we examined the effects of gen on CFTR inactivation and dephosphorylation following the removal of agonist. CFTR activity was assayed in Xenopus oocytes by two electrode voltage clamping. Oocytes were held at the resting potential, and pulsed to -60 mV for 0.5 sat 12 s intervals. Data were collected over the last 20 ms of each pulse. With I 0 J.1M forskolin (fsk) the current increased by 5.18±1.44~LA (n=7); stimulation with 10 J.1M fsk plus 50 J.1M gen increased the current by 6.67±1.41 11A (n=8). Maximal currents were stable for up to I hr. Currents were shown to be due to the CFTR because no currents were seen in mock injected oocytes and currents reversed at -20 m V. The rate of current inactivation following the removal of stimulant was also measured. Removal of both fsk and gen led to a decrease in current that could be fit with a rate constant of0.17±0.04 min'1 (n=3). In contrast when fsk was removed in the continued presence of gen, currents were stable for up to I hr (n-4).

In vivo dephosphorylation of the CFTR was assayed in NIH-3T3 cells that stably expressed CFTR. Time courses for in vivo dephosphorylation were determined. Cells were bathed in 3zPi for 2 hr prior to stimulation with I J.1M fsk plus SO J.1M gen. After 2 min fsk, or fsk plus gen, was removed and cells were lysed at times between 0 and 8 min after agonist removal. CFTR was immunoprecipitated, resolved by SDS-PAGE, and quantified by scintillation counting. In the presence of gen the rate of in vivo dephosphorylation of the CFTR was reduced by -50%. These results support our hypothesis that gen inhibits dephosphorylation of the CFTR. (Supported by the Nemours Foundation and CFF.)

29 HeterozY&ote advantaae In CF: decreased susceptibility to typhoid fever due to use of CFI'R by Salmora•lltl t:yplli, but not S. t:yphimurlum, to tnnslocate from the aastrolntestlnal (GI) lumen.~. M. Grout, T. Zaidi. Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School, Boston, MA.

Decreased fluid secretion during diarrhea bas been suggested to pro­vide a survival advantage in CFI'R ~~rozygote& .. ~ow~ver, the actual effect of CFI'R on aastrointeatinallununal composition 1s unresolved, and heterozygote CF mice do not sbow any difference from homozy,ous, wild-type CPTR mice in clectropic chloride secretion in gut epitheba. Recent studies identified CFI'R-mediated ingestion of Puudomonlls uruginosa as a critical host response for clearing this pathogen from the respiratory tract. Therefore we cletermitled if other pathogens used CFI'R as a cellular receptor for epithelial ceU uptake. Using both C 127 murine cells transfected with eDNA for AFSOS and wild-type CFTR, and isogenic airway epithelial ceU lines derived from a -CF patient homozy­gous for the AFS08 mutant allele of CFI'R (CFrl cells) and a corrected version of this line expressing wild-type CFI'R (CFrl-LCFSN cells), we found that S. typhl, but not S. typhlmurlum, had an 8-10 fold increase in epithelial ceU uptake in ceUs expressing wild-type CFI'R. Epithelial cell ingestion of S. t:yphl, but not S. t:yphlmurlum, by CFrl-LCFSN, Hep2 and T84 colonic tumor cells was inhibited by a monoclonal antibody (Mab) and synthetic peptides corresoonding to the first ptedicted extracel­lular domain of Cf'IR- Control Mabs to intracellular domain& of CFTR and a scrambled version of the flf'St-domain peptide had no effect. Trans­location of S. t:yphl across the Gllumen in transgenic adult, heterozygous AFSOS CFI'R mice (+I- mice) was reduced 86~ in comparison to homo­zygous wild-type CFI'R mice (mean Jog to cfu translocated/gm 4.889 ± .257 [SE] in +1- mice va. 5.755 ± .251 [SE] in +I+ mice, P <.001, ANOVA). S. typhl translocation was virtually eliminated in mice bomo­zysou• for the mutant AFSOB CFTR. (mean log to cfu translocated/gm 0.109 ± .109 [SE] -/-mice). A 10-mer peptide correspondi~:~ amino acids 106-115 in the flrstpredk:tedextraceUulardomain of inhibit-ed S. t:yphl ingestion by T84 ceUs as weU as bacterial translocation from the GI lumen in Balblc mice. CFI'R could also be seen by immunoelec­tron microscopy to bind to S. t:yphl during entry into epithelial cells. Use

of CfTR as the epithelial-cell receptor for translocation of S. typhi from the GI lumen could be the basis for the high-level maintenance of mutant CfTR alleles by promoting enhanced resistance to S. typhi infection and the occurrence of typhoid fever in heterozygote individuals.

30 EXTRACELLULAR LABELING OF RECOMBINANT CFI'R. D. Gruis, C. Korte, ~- Dalton Cardio. Research Center and Dept. Vet. Biomed. Sci., Univ. Missouri, Columbia, MO 65211 USA.

Using the membrane-impenneant reagent NHS-biotin as a probe to extracellularly label proteins, it was demonstrated that individually expressed (in HiS insect cells) NBFl and NBF2 domains are surface­accessible. In further support of this conclusion, it was also shown that NBFl was capable of interaction with the plasma membrane (Gruis and Price, Biochemistry, (1997), in press). Based on these labeling results of the individual domain&, it is reasonable to hypothesize that these domains, in the context of the full-length protein, are also capable of being surface-accessible. To address this question, intact Hi-S insect cells expressing full-length, epitope-tagged ("flagged'') CFTR were surface-labeled with NHS-biotin. The flagged CFTR from the extracellularly labeled cells was solubilized, immunoaftinity purified and analyzed via protein blots probed with streptavidin-horseradish peroxidase (HRP). The results indicate that CFTR was indeed biotinylated, suggesting that at least some region(s) of the protein was extracellularly labeled by NHS-biotin. The immunoaftinity purified material was further purified via preparative SDS-PAGE and digested with hydroxylamine. The hydroxylamine digest was analyzed via protein blots probed with streptavidin-HRP. Hydroxylamine is predicted to digest CFTR into 4 major fragments at Asn-Gly bonds. Two of these fragments have a predicted molecular weight of 33kD and correspond to residues 423-722 (NBFI-R) and residues 1185-1481 (NBF2-C-term). These contain none of the predicted transmembrane domains. The other two fragments of predicted molecular weights 49kD and 52kD correspond to the N­term-TM6 and TM7-TM12, respectively. Preliminary analysis showed a biotin-labeled band of approximately 35kD which suggests that a portion of the presumed cytosolic domains of the CFTR (the NBFI-R and/or the NBF2-C-term fragment) is indeed accessible to the cell surface. Supported by the CF Foundation.

31 CONSEQUENCES OF CO-EXPRESSION OF NBF OR R DOMAINS WITH FULL-LENGTH .6FS08 CFI'R. D. Gruis, C. Korte, L. Clarke, ~. Dalton Cardio. Research Center and Dept. Vet. Biomed. Sci., Univ. Missouri, Columbia, MO 6S211 USA.

The major consequence of the deletion of phenylalanine 508 of the CFTR is a deleterious effect on the processing and targeting of the protein. The mutant protein is not fully glycosylated and little or none of the AFS08 CFTR reaches the plasma membrane. Considerable effort has been invested in the search for compounds or conditions that can circumvent this processing defect. To date, conditions such as low temperature or compounds such u glycerol or dimethylsulfoxide have proven useful at achieving some degree of processing correction. To further pursue the goal of AFS08 correction, individual domains of the CFTR (NBFl, NBF1AF508 orR) were co-expressed in Cl27 cells that had previously been stably transfected with the full-length MS08 CFTR protein. This approach was designed to teat the hypothesis that the retention/degradation machinery responsible for retaining or degrading the AFS08 CFTR recoanizes specific regions of the mutant protein. Co-expression of the appropriate domain containing this specific region atona with the full-length MS08 CFTR may interfere with this recognition and facilitate correction of the processing defect. The eDNA encoding the NBFl, NBF1AFS08 or R domain was subcloned into the expression vector pZeoSV. Cl27 cells stably expressing Msog CFTR were electroporated with the individual

constructs and transfectants selected in 200uglml zeocin. Individual colonies were cloned and expanded into stable cell lines. Correction of the 6FS08 CFTR processing defect was determined via inununoprecipitation of the mutant protein followed by in vitro phosphorylation and SDS-P AGE. Correction is accompanied by an increase in the molecular mass of the protein due to complete glycosylation. Preliminary data indicate that these constructs, especially NBFI, may indeed partially correct the M508 CFTR processing defect. Supported by the CF Foundation.

32 A NOVEL, MORE EFFICIENT METHOD FOR THE PURIFICATION AND FUNCTIONAL RECONSTITUTION OF CFTR PROTEIN. M Ramjeesinr:h, C. Li, E. Garami, M. Hewryk, L.-1. Huan, Y. Wang, K.A. Galley, C.E. Bear. Hospilal for Sick Children, Toronto, CAN

Our published sttategy for purification of CFTR from Sf9 cells was critical for our subsequent demonsttation of the channel and A TPase activities ofCFTR (Bear, C.E. etal., 1992) and (Li, C.etal., 1996). This procedure required extraction of CFTR from Sf9 cell1ysates with sodium dodecyl sulfate followed by a two step purification by h~xyapatite and gel filtration chromatography. We estimated that the )'leld of purified CFrR (>95%) using this method was approximately O.S mg/109 Sf9 cells and approximately 10-20% of this protein was functional following reconstitution into model phospholipid membranes. The purpose of the present study was to assess the efficacy of a novel, rapid purification strategy for CFTR which combines the use of the nonionic detergent, sodium perfluorooct~n~a!C (NaPFO) wit~ metal affinity chromatography. F1rst, a polyhistJdme tag (xlO Hu) was attached to the 3' end of the human CFTR ORF. Then recombinant baculovirus containing the engineered CFTR ORF was transfected into Sf9 cells. Whereas CFTR is usually intractable from cell membranes using nonionic detergents, NaPFO (8%, in low ionic strength buffer) is capable of solubilizing 80% of CFTR-His, comparable to that fraction solubilized by SDS (2%). NaPFO solubilized CFTR-His bound with moderate affinity to nickel nitrilotriacetic acid columns and could be selectively eluted with buffer pHs6.8. We estimate that this rapid new procedure increased yield of purified protein (>95% pure) by 140%. More importantly, the overall activity of the protein following reconstitution into phospholipid liposomes is enhanced. ATP (1 mM MgA TP)-dependent electrogenic uptake of 36Cl by proteoliposomes containing phosphorylated CFTR purified using the new method was approximately 2.9±0.3 (nmOI/Ilg/hr) compared with 1.5±0.3

· (nmolll!g/hr) estimated using the conventional method. Likewise, maximal A TPase activity of CFTR purified using the new method was increased to 4.2±0.6 from 3.0±0.2 (nmoVI!g/hr). Single channel studies in planar lipid bilaycrs conf1nned that the Increase in overall activity of purified CFfR reflects a higher percentage of functional molecules rather than a change in function of individual molecules. ThLse studies were supported by CCFF and NIH

33 PURIFICATION OF CLC-2 FROM Sf9 INSECT CELLS. M Ramjccsin&h, E. Garami, L.-1. Huan., Y. Wang, K.A. Galley, H. Xiong, C. Li, C.E. Bear., Hosp. for Sick Children, Toronto, CAN.

C1C·2 is a member of the CIC family of putative chloride channels which is expressed ubitquitously throughout several tissue types, including epithelial cells. It has been hypo~csi~ ~at <?C-2 may be able to functionally compensate for CFTR m ep1thebal ussucs .. However, to date, there is little direct evidence that CIC-2 conb1butes to epithelial cell fluid transport. Although expression of ClC-2 in Xenopus oocytes (Thiemann etal., 1992) and in Sf9 cells (Xion~ et.al., unpublished observations) leads to the appearance of swelhn~­activated chloride currents, dcfmitive evidence supporting the func:uon of CIC-2 as a chloride channel awaits its purification and reconstitution in phospholipid bilayers. The purpose of the present study was to determine the efficacy of CIC-2 purification by metal affinity chromatography for subsequent reconstitution studies. First. a polyhistidine tag (X6His) was attached to the 5' end of the ClC-2 ORF. The polyhistidine tag did not interfere with CIC-2 function, . assessed as hyperpolarization-activated chloride conductance followmg expression in Xenopus oocytes. Then, recombinant baculovirus containing the engineered ClC-2 ORF was transfected into Sf9 ~lis. Expression of ClC-2-His protein was con finned by immunoblotung using a polyclonal antibody generated against the amino terminus of

Poster Session Abstracts 219

ClC-2 (aa's 34-71). CIC-2 protein was poorly solubilized by CHAPS, cholate, dodecyl maltoside, Triton X-100 and SB 12. On the other hand, the relatively novel nonionic detergents, ammonium (APFO) and sodium pcrfluorooctanoate (NaPFO) were as effective as sodium dodecyl sulphate in the complete solubilization of ClC-2-His. NaPFO solubilized ClC-2-His was effectively purified to high degree of homogeneity (>85%) by nickel chromatography as assessed by silver stained SDS PAGE. The yield of purified ClC-2-His protein was high; 1mgllo' Sf9 cells. NaPFO detergent was substituted with phospholipids (PE:PS:PC: ergosterol, 5:2:1:1 by wt) by dialysis for the purpose of assessing the chloride channel function of purified, reconstituted ClC-2-His protein. These studies will permit direct assessment of the structural basis for chloride conductance associated with ClC-2 expression and evaluation of its putative role in epithelial cell chloride secretion. Studies supported by operming grants awarded to C.E.B.by MRC (CanDda), CCFF and NIH.

34 ATP Release In Heterologoua Flbroblull and In Epithelial Monolayen Ia Enhaneed Markedly by the ABC Transporten, CITR and •tlr .Erik..M. ~ and Tamas Jilling. Dept of Physiology and Biophysia and G~ry Fleming James CF Research Center, U•iv. of Alabama at Birmingham, Birmingham. AL. 35294 The role of ABC transporter~ in facilitating cellular A TP effiux is

controversial. A TP biolumineacence release usaya were perfonned on cells grown to eonfluence in 35 mm c:ollagen-c:oated dishes ("bulk" A TP releue) and/or on epithelial monolayera grown in air-fluid interface cultwe for 8-10 days ("vectorial" A TP release) to detennine whether cells expresaing wild-type CFTR or mdr had an enhanced capability to release A TP. All cells were handled in a similar manner (washed 2X in PBS and atudied immediately in real time within the sealed chamber of the luminometer in a ICrum-free medium (OptiMEM-1) eontaining 2 mglmlluciferase-luciferin (L:L) detection reagent). Appropriate controls were done on every prcpar11tion. Nonnal bronchoal epithelial cell monolayen (16HBEt4o") expreasing the mature band C fonn of CFTR were compared to CF (41'508 homozygous) bronchial epithelial cell monolaycrs (CFBE4fo·) lacking a signal for CFTR protein. Apical-directed A TP release, measured by inclusion of the L:L detection reagent only in the apical medium, had a magnitude of 8.007±0.897 arbitmy light unita in 16HBEI4o" (n-26) versus 2.401±0.427 in CFBE4lo· (n•l9). Basolateral­directed A TP release (L:L reagent in the basolateral mediwn only) was not different between 16HBEI4o· (1.209±0.303, n•l4) and CFBE4fo· (0.917±0.311, n•IO) monolayer~. Parental CFPAC-1 epithelial monolaym (41'508 homozygous) were compared to a wild-type CFTR-c:omplemented CFPAC clone, CFPAC-PU-WT-20 either ~elected or not selected with G418 (I mglml). Apical-directed ATP release values were 1.862±0.338 (CFPAC-1; n•l5), 5.486±0.481 (clone WT-20, no G418; n•l5) and 12.86±1.440 (clone WT-20, G418; n•IS). Basolaler~l ATP release values were not different among the CFPAC series (n•5 each). Similar differential results were obtained in bulk A TP release assays comparing heterologous fibroblasts expressing wild-type CFTR (124.4±14.92; n-49; expressed primarily band C fonn ofCFTR) to mock fibroblasts (44.31±3.978; n"""9) or fibroblasts expressing 41'508-CFTR (63.03±9.310; n-49; expressed only band B fonn of CFTR)(generous gift of Michael Welsh). Higher basal A TP release was also measured from fibroblasts expressing mdr (89.07±7.598; n•77) when compared to parental NIH-3D cells (15.34±1.542; n•87)(generous gift of Suresb Ambudkar). Taken together, these results mggest that CFTR and mdr enhance apical A TP release from epithelia and bulk A TP release from heterologous fibroblasts under unstimulated conditions; the cellular and molecular mechanisms that underlie this enhanced basal A TP release are under investigation. (Supported by CFF)

35 CITR and Mtlr Potentiate Markedly ATP Release Induced by Hypotonic Cell Swelling: Role of ABC Transporten In Cell Volume Regulation .J;.till; M Schwiebcrt. Dept. of Physiology a11d Biophysia a11d Gregory Flemi•g James CF Research Center, Univenity of Alabama at Birmi~~gham, Birml•gham. AL. JJ]94

Stimulation of A TP c:onduction or release in CFTR-expressing cells with cyclic AMP agonists has been met with significanl variability. Because of small, transient and variable A TP release signals after eyclic AMP agonist addition and because purified CFTR in planar lipid bilayera fai!J to c:onduct A TP, other c:onditions or alimuli were c:onsidered as being more important for CFTR- and mdr-associated A TP c:onduction or release. A TP bioluminescence release assays were performed measuring either '"bulk" A TP release from fibroblasts grown 10 eonfluence on 35 mm c:ollagen-c:oated cultwe dishes or "vectorial" A TP release into the apical or basolateral mediwn by epithelial monolayers grown on eollagen-c:oated filter aupports for 8-10 days in air-fluid interfac:e culture. Nonnal bronchial epithelial monolayera (16HBEI4o") responded 10 increasing

220 1997 Cystic Fibrosis Conference

dilutions of the medium osmolality (hypotonic cell swelling) with profound increases in A TP release into the apical medium (APM basal, 5.30±0.92 arbicrary light units; 2S% dilution, 18.52±4.86; 33% dilution, 34.21±10.92; and 41% dilution, 51.46±17.46; n•8) and significant albeit smaller increases in ATP release into the basolateral medium (BLM basal, 0.81±0.38; 25% dilution, 3.34±0.45; 33% dilution, 4.56±0.69; 41% dilution, 5.41±0.90; n•3). Responses from 16HBEt4o· monolayen were 6-7 fold greater in apical ATP release and 3-5 fold greater in basolateral A TP relesae than those observed from CF (AF508/AF508) bronchial epithelial monolayers (CFBE4lo')(APM basal, 1.48±0.23; 25% dilution, 3.47±0.37; 33% dilution, 5.37±0.68; 41% dilution, 8.39±1.01; n•8XBLM basal, 0.35±0.07; 25% dilution, 0.79±0.28; 33% dilution, 1.21±0.37; 41% dilution, 1.52±0.36; n•3Xaenerous gift of cells from Dieter Gruenert). Similar heightened responses to swelling (4-5 fold) were found in bullc A TP release assay• comparing fibroblasts expreosing wild-type CFTR and mdr when compared to mock, parental or AF508-CFTR-expreosing counterparts. Taken together, these results SUBSe&t that CFTR and mdr regulate positively a aeparate swellins-senaitive A TP release pathway that is expreosed ubiquitously and that is present, but far less active, in CF epithelia expressing the &FS08-CFTR mutation. Because CFTR {Valverde et at. PNAS 92:9038, 1995} and mdr {Valverde et at. NaJure 3SS:830, 1992} have been implicated in cell volume regulation and because conductive A TP relesae is essential for autocrine control of cell volume (Wang eta!. PNAS 93:12020, 1996}, ABC transporten may play an essential role in aenain& and/or transducin& osmotic insults into reaulatory volwne decrease (RVD) reoponseo. [Supported by CFF}

36

CFI'R: Probln1 the Pore with Penneant Ions. S S Smjtht, E.D. Steinle*, V. Pecoraro*, M.E. Meyerhoff*, and D.C. Dawsont. tDcpt of Physiology and *Dept of Chemistry, University of Michigan, Ann Arbor, MI 48109.

CFTR functions as a cAMP-activated, Cl-selcctive channel, but the structure of the anion selective pore is unknown. Pcrrncant anions arc uniquely valuable as probes of the ~x.~re because they must, of necessity, enter, traverse and interact wtth the structural clements that form the conduction pathway. Previous studies (Tabcharani ct al, Nature 366:79-82, 1993: Mansoura ct al, Ped Pulm Sl0:79, 1994) suggest that the permeation of SCN may shed light on pore structure because this ion binds tightly in the pore. The behavior of SCN suggested that the ion enters the pore readily (P sot" q > I) but sticks in the pore (Ssa/~ < 1 ). To determine if this behavtor ts characteristic of polyatomic anions we investigated the permeation of a panel of pscudohalide anions that vary widely in their size and shape. We estimated permeability ratios by measuring shifts in reversal potential in Xenopus oocytes expressing wtCFTR and assayed for the binding of anions in the pore by determining the blockade produced by the addition of SmM of the anion to the perfusate. Monovalent and divalent pscudohalides were investigated, including OCN, SCN, ScCN, N(CN)z, Au(CN)3 C(CN),, Ni(CN)4, Pb(CN)4 and Pt(CN)4•

The apparent permeability ratios ranged from around unity to about 8 • Blockade by SmM addition ranged from 2% to SO.. and was voltage­dependent for the larger divalent species, like Pt(CN)4• We used an anton selective electrode (PranitiS ct al, Crit Rev Anal. Chern 23(3): 163-186, 1992) to estimate the relative hydration energies of monatomic and polyatomic anions. Permeability ratios were highly ~latcd with the case of dehydrating the anion, as expected i( the energy barrier to the entry of anions into the channel is strongly influenced by the energy required to dehydrate the anion. The efficacy of block was also ~Jated with the case of dehydration, suggesting that large, highly polarizable anions may interact favorsbly with amino acid residues that form the lining of the pore. Because of the wide variation in their size and shape, polyatomic pscudohalide ions should be uniquely valuable as probes of the CFTR pore. (Supported by grants from the NIH and Michigan 0.1 Peptide Center).

37 CFI'Il·ASSOCIATBD CHLORIDE AND ATP CONDUCTANCES: DISTINCT POIUUI REGULATED BY COMMON GATES. M Sysjll 1. K. PolbU. Deplnmeat of Physiolo&Y. Univenity of Pennaylvlllia, Philadelpbla. PA. USA.

1be cystiC fibrosiall'lllllllelllbnae CCIIIductaneo ~epalalor (CFill) is a chloride channel that is J1lllllalld by pholpborylallon of the R domain and A TP hydrolysis at two aucleotlde biDdina domains (NBDI). It ia not ctw whether CFrR itself conducta ATP, or wbelber CFrR mipl be closely MaOCiated with a eeplrille A TP COIIIIuctance. To chlrlclerize A TP c:hannela IIIOCiated with CFTR, we lllllyzed ct· 11111 A TP sinaJe c:hannel-amcnts in excised inside-out membnae paldlel from MOCK cella transiently expreuins CFill. With 100

mM Na,A TP in the pipette and 140 mM NMDG-Cl in the bath, A TP channels were associated with CFTR Cl' channels in two thirds of patches that included CFTR (n=99). CFTR Ct· channels and CFTR-associated A TP channels hd slope conductances of7.4 pS (belween 30 mV and 90 mV) and 5.2 pS (berween -30 mV and -90 mV), respectively. ATP channels were also observed in -10% of patches that did no! include CFTR ct· channel activilies (n=74), bur the conducllnCCS and kinetics were distincl from !hose associated wilh CFTR. CFTR-associated A TP channels exhibited slow gating kinelics thai depend<d on the presence of PKA and cytosolic A TP. similar to CFTR Cl' channels. Diphenylamine-2-carboxylic acid (DPC) caused a fliclcery block of CFTR Ct· currents, bur was withoul effect on A TP currents. 4,4 • -diisothiocyanoslilbene-2.2'-disulphonic acid (DIDS) in the pipette was withoul effect on CFTR ct· currents, but completely blocked CFTR-associaled A TP channels. A hydrolysis­resistanl ATP analogue (adenosine 5'-0-(3·thiotriphosphate); ATPyS) alone failed to open PICA-phosphorylated CFTR Cl" channels or A TP channels. whereas A TPyS or 5' -adenylylimidodiphosphate (AMP-PNP) enabled CFTR Cl' channels or A TP channels opened by A TP 10 remain open, by increasing !he open bunt duration of both conductances. Mutation of the Wallcer A motif lysine in NBD2 (Kl2SOA) locked both CFTR-IISOCiated Cl" and ATP channels inro prolonsed open stales. A deletion mutant (CFTRAR-S660A). which lacks much of the R domain and replaces Ser-660 with alanine, eliminaled CFTR­associated A TP channel activities. However, a mutant containina eight serine-to. aspartate substirutions in the R domain generated CFTR-IISOCialed A TP channels as well as CFTR ct· channels !hal opened wirhout PKA phosphorylation. These results sunesl thai CFTR-associated A TP channels may be regulated by phosphorylation of the R domain and A TP hydrolysis at NBD, althou&h the permeation pathway for A TP is distinct from the ct· channel in CFTR. (Supported by CFF)

38 REGULATION OF CFTR CHLORIDE CHANNEL BY CHARGE OF THE CONSENSUS PKA PHOSPHORYLATION SITES IN THE R DOMAIN. Junxja Xje Mitchell L. Drumm, Jianjie Ma, and Pamela B. Davis. Department of Physiology and Biophysics, and Pediatrics, Case Western Reserve University, Cleveland, OH 44106.

Phosphorylation by cAMP-dependent protein kinase (PKA) at multiple sites in the R domain is required for opening of the CFTR chloride channel. Six serine residues have been identified as the major in vivo PKA phosphorylation sites in human CFTR (S660, S700, S737, S768, S795 and S813). These serine residues reside in the consensus motif: RRJKNS. To examine the role of R domain charge interaction in CFTR channel function, we constructed two CFTR mutants, R-P6N CFTR, in which the six positively charged amino acids at the -2 positions were changed to negatively charged amino acids (R658D, K698E, R73SD, R766D, K793E & RBI IE) and IU6-P6N CFTR, in which the corresponding serine was also mutated to alanine. lllese mutations introduced two changes in CFTR: first, a net change in charge of -12 in the R-domain; second, the consensus sequence around the major sites was altered, probably reducing PKA phosphorylation and its regulation. When exprelled in 293 HEK cells, processing of R-P6N and R46-P6N is similar to the wild type CFTR. as mature glycosylated CFTR proteins were detected using Western blot. SPQ assay showed that both R-P6N and R46-P6N CFTR expressing cells have greater basal chloride efflux rates (~.19 ± 0.01 relative fluorecence unit changelminule (Rfuc.lminute)) than that of either untransfected or WT CFTR expressing cells (~.040 ± 0.005 Rfuc./minute). Forskolin stimulation further increased chloride efflux rate for R-P6N but not for R46-P6N CFTR expressing cells, suggesting possible phosphorylation of serine residues in R-P6N CFTR with increase in channel activity. In planar lipid bilayer, without PKA but with ATP (2 mM), both R-P6N and R46-P6N CFTR fonncd open chloride channels. Similar to WT CFTR, both mutants exhibited three conductance states: -3 pS (L), -5 pS (M) and 8 pS (H) in 200 mM KCI. The H state ofR-P6N has average open probability (PJ of0.047 ± 0.009 (-PKA, 2 mM ATP). Addition ofPKA increased the open probability of the H state to 0.087 ± 0.0 I 0, still significantly less than wild type CFTR ( P. • 0.318 ± 0.022). We conclude that electrostatic interactions arc likely to be involved in the gating of the CFTR channel, but the confonnation of the R domain is essential for maintaining optimum channel function. (Supported by Cystic Fibrosis Foundation, grant P30 DK276S I and ROt HUDK49003).

39 CONSTITUTIVE PKA AND PKC-DEPENDENT PHOSPHORVLA TION OF CFTR REVEALED BY 2-D PEPTIDE MAPPING. K A Yurllo-Mauro and W. W. Reenstra Dept. of Pediatrics. duPont Hospital for Children, Thomas Jefferson Univ .• Wilminaton. DE. 19803.

We have previously shown thai in stably transfected NIH-3T3 cells both

forskolin (FSK) and phorbol ester (PMA) increase in vivo CFTR phosphorylation and channel activity (Ped. Pul. 135:225). In order to identuy PKA and PKC­dependent in vivo phosphorylation sites. imunoprecipitated CFTR was subjected to tryptic digestion and 2-D peptide mapping. Peptidcs were identified by comparison with published maps. In all cases agonist dependent phosphorylation was tenninated after 2 ntin. a time shown previously to give maximal stimulation of channel activity. Stimulation with I 11M FSK increased CFTR phosphorylation on peptidcs containing S737, S768, and S795. Stimulation with 200 nM PMA increased CFTR phosphorylation on peptides containing 5768, S795. and 3 other unidentified peptidcs. (Since specific sites were not detennined, phosphorylation could occur on other serines within each of the identified peptides.) In the absence of agonist a low level of basal phosphorylation was seen in all6 peptides. To detennine if this phosphorylation was due to a constitutive PKA or PKC activity, basal phosphorylation was assayed in the presence of the PKA antagonist, H89 and the PKC antagonist, staurosporine. These antagonists were present during "Pi labeling and stimulation. In cells treated with 10 11M H89, basal phosphorylation ofpeptides containing 5737, S768, and 5795 was decreased, but phosphorylation of the 3 unidentified peptidcs was not inhibited. H89 also decreased FSK-dependent phosphorylation of peptides containing S737, S768, and S79S. In cells treated with 1 .,M staurosporine, basal phosphorylation of all 6 peptides was reduced. Staurosporine decreased PMA-dependent phosphorylation of peptides containing S768. S795, and the 3 unidentified peptides. Staurosporine also decreased FSK· dependenl phosphorylation of peptidcs containing S737 and S768, but not S79S. These results suggest that under basal conditions specific sites on the CFTR are phosphorylated by PKA and PKC. The observation of basal PKC phosphorylation of the CFTR is consistent with a recent proposal that PKC-dependent phosphorylation is required for PKA-dependent activation of the CFTR (JBC 272:4978). Taken together these studies suggest that kinase-dependent regulation of the CFTR may he more complex than previously thought. (Supported by the CFF and the Nemours Foundation).

40 MOLECULAR CLONING OF HAMSTER PROTEIN PHOSPHATASE 2Ca. I....Z!nl. S. X. Zheng, and 1. W. Hanrahan. Department of Physiology, McGill University, Montreal, Quebc!c, Canada.

Activity of the cystic fibrosis transmembrane conductance regulator (CFTR) runs down In excised membrane patches due to a membrane­associated protein phosphatase activity. Patch clamp studies using CHO and airway cells In the presence of different exogenous protein phosphatases and phosphatase inhibitors suggest that protein phosphatase 2C (PP2C) is responsible for this rundown (1. Luo et a!., submitted). Since PP2C is usually considered a soluble emyme, we have studied the distribution of PP2C activity In different cell tractions. Membrane-associated PP2C activities were assayed In BQ1, BHK. T84 and Calu-3 cells using ,2P labeled casein as substrate. The activities were 0.117, 0.196, 0.21, and 0.113 mU/mg, respectively, about one third of the PP2C activities measured in the cytosollc fractions. To enable further studies of dephosphorylation in CFI'R­transfected hamster cells (CHO, BHK), we have cloned a eDNA encoding hamster PP2Ca. Three overlapping cDNAs encoding hamster PP2Ca, including one which is full length, were produced by RT-PCR and 3' end RACE-PCR, using hamster cell first strand DNA as the template. PCR products were cloned into pCR2.1 vector for sequencing. The hamster PP2Ca coding sequence is 88 % Identical to that of human, 92 % to mouse, 88 % to rat, and 93 % to rabbit PP2Ca. At the amino acid level, sequence Identities are 99 % to human. 98 % to mouse, 99 % to rat, and 99 % to rabbit PP2Ca. These results Indicated that a signifiCant fraction of PP2C activity is membrane-associated, and that PP2Ca is highly conserved among mammalian species. Supported by NIH (NIDDK), Montreal Chest Institute and MRC

41 Characterisation of R-domain CFTR mutation• Anne yankeerberohen', Lin Wet', Martine Jaspers', Bernd Nilius•, Jean­Jacques Cassiman' and Harry Cuppens' Center for Human Genetics', Department of Physiology', KULeuven, Leuven, Belgium

Poster Session Abstracts 221

The R-domain plays a crucial role In the regulation of the CFTR chloride channel since phosphorylalion of this domain Is necessary to open the channel. Despite this, missense mutations located In the Rodomaln and found in CF patients have not been studied yet We therefore characterised all the R-domaln mutations that have been reported to the CF Genetic Analysis Consortium. The effect of these amino acid changes on the maturation of the corresponding mutant proteins, heterologously expressed in COS cells, was analysed. According to the maturation pattens obtained for the different mutant proteins, the Rodomain can be subdivided into two subdomains. Mutations localed In the first domain (1601F, L610S, A613T, 1618T, L619S, H620P, G628R and L633P) gave rise to mutant CFTR proteins that matured up to the core glycosylated form and were subsequently degraded without reaching the mature form. Mutations In the second domain (H620Q, G622D, 0648V, T665S, F693L, R766M, R792G, A800G, 1807M, E822K and E826K) did not alter the maturation pathway; the mature 190 kDa form was detected. Interestingly, a new model for the first nucleotide binding fold of CFTR has recently been proposed by Annerau et al. (FEBS Letters; in press). According to this model, tha first nucleotide binding fold extends to amino acid 650 and thus contains more than 60 amino acids located in exon 13 which Is thought to encode completly for the R-domain. It is in this first part of axon 13 that all the mutations that cause a maturation defect are located. Mutations that had no effect on maturation were further analysed at the electrophysiological level in Xenopus oocytes. Compared to wildtype CFTR, T665S, R792G, E822K and E826K CFTR produced, when activated with cAMP, significant lower membrane currents while H620Q and ASOOG gave rise to chloride channels with significant higher membrane currents. The membrane currents of the other studied mutant CFTR proteins were not significant different The significance of these data is currently further analysed at the single channel level. These studies should provide a better insight into the effect of mutations, located In the Rodomain, on the maturation and the electrophysiological characteristics of the corresponding mutant CFTR proteins and thereby Increase our knowledge of the structure and function of this domain.

42

MECHANISMS OF THE POTENTIATION BY ADENOSINE OF ATP INDUCED CALCIUM RELEASE IN TRACHEAL SMOOTH MUSCLE CELLS. M C Michoud, F. Tao, A.A Pradhan, and J.G. Martin. Meakins-Christie Laboratories and Montreal Chest Institute Research Center, McGill University, Montreal, Quebec, CanadL

We have previously retorted that extracellular ATP produces an increase in intracellular Ca ([Ca~1) mediated by P:ru-purinoceptors in rat tracheal smooth muscle cells. We also have shown that adenosine (ADO) alone has no effect on [Ca~1 but potentiates the response to ATP. The aims of this work were 1) to determine the mechanisms of the increased Cal+ peak response to ATP in presence of ADO and 2) to detennine if this effect of ADO was specific for ATP. All measurements were made on cultured rat tracheal smooth muscle cells (lst-Jrd passage). (Ca~·]1 was measured using Fura-2 and dual excitation wavelength microfluorometry. Total inositol phosphates were measured by ion exchange chromatography and the resulting fractions quantified by liquid scintillation counting. Peak [Ca~; was 600±76 nM ( mean±SE, n=20) for ATP (10~ alone and 954:!:211 nM following ADO (lO"'M) + ATP (10~ stimulation (pS0.05). Increases in inositol phosphates were (m % of baseline): 218±30 for ATP (10~ (nz6) vs 190::1:19 (n=6) for ADO(lO''M)+ATPqo~. After nifedipine (10''M) (a Cal+ channel blocker), peak [Ca 11 was 572±92 nM (nsl8) for ATP (lO~M) and 614±80 nM (n•l8) for ADO (10"'M)+ ATP(lO~M)(p- ns). Following incubation with AACOCF, (10-'M}, a blocker of phospholipase A,. the ADO enhancement of ATP induced release ofCa2+ was abolished and peak [Ca21; release by ATP (10~ was significantly reduced to 281±40 nM (p<O.OS). Finally, peak [Ca~1 release produced by S-liT was also enhanced in presence of ADO (559±48 nM for 5liT(lO"'M) and 854±126 nM for AD0(10'5M)±SliT(10"'M) pSO.OS). We conclude that the potentiation by ADO of the ATP induced Ca2+ release is due to Ca2+ influx from extracellular fluid through a Ca2+ channel modulated by arachidonic acid. Supported by the 1.T. Costello Memorial Research Fund and MRCCanadL

222 1997 Cystic Fibrosis Conference

43 THE NATURAL STRESS PROTECTANT TREHALOSE ALTERS INTRACELLULAR LOCALISATION OF MUTANT CITR PROTEIN. • 1Amaral MD. 1Penque D, 01Farinha C, •1 Gomes A, 1Demolombe S, 1Escandc D & 1Lavinha J. "Dep Quimica e Bioquimica, Fac Ciencias Univ Lisboa, cp• Grande. 1700 Lisboa. 1Dep Gcnetica Humana, lnst Nac Saude Dr. Ricardo Jorge, Av Pde Cruz. 1699 Lisboa Codex. PORTUGAL. 1H6pital G & R Lamncc, Nantes, FRANCE

The disaccharide trehalose has been descnbed to play a critical role in the survival of micro-organisms by protecting them against structural and functional damage caused by stress agents such as high temperature, oxidative stress, dehydration, ethanol, etc. We have tested here the effect of trehalose on both the CFPAC-1 cell line, which expresses the mutant ~F508-CFTR, and on a HeLa cells stably transfected with CFTR eDNA with the A561E mutation, which also leads to mislocalization of CFTR. We have exposed these cell lines to I 0% glycerol, 1-1 00/o trehalose or to heat shock, HS ( 42.5"C), and to HS in the presence of each of those two chemicals. We have observed that the level of the HSP70 mRNA, an indicator of the cells 'stress state', is reduced under HS in the presence of trehalose in CFPAC cells by comparison with non-treated cells also under HS. Moreover, by invnunocytochemistry with anti-CFTR antibody, trehalose also alters intracellular localisation of mutant (M508 and A561E) CFTR protein. Indeed, some protein can be observed in the cell membrane. SPQ halide-efflux assays are being carried out in the same cells treated with trehalose. The lower induction of HSP70 mRNA under HS in the presence of trehalose suggests that this compound may remove the stimuli for HSP70 induction, i.e., misfolded proteins by stabilising them in the native conformation under HS. Similarly, for CFTR, we suggest that trehalose prevents mutant protein from being recognised as 'abnormally folded' by HSP70 or other members of the 'cellular quality control machinery', escaping from its abnormal localisation. We thank Dr. W Ou1111ino for 169 anti-CFTR antibody and JNICT for partial fundins.

44 CELL DIFFERENTIATION AND AF508 CFfR PROCESSING. ZL ~C. I. Venglarik, z. P6ncz61, T. Jilling, K. Kirk and E. J. Sorscher. Greaory Flemina James Cystic Fibrosis Research Center, Department of Medicine, and Physiology and Biophysics University of Alabama at Birminsham, Birmmsham, AI. USA.

The 4FS08 mutation in CFTR results in the retention of the CFTR protein in the ER and rapid degradation by the proteosome. We reported earlier that in LLC-PK1 cells exprcssins 4FS08 CFTR, srowth .as a polarized monolayer on permeable supports, or treatment of cells w1th 2'11 DMSO for 4-S days resulted in cAMP ~tivated Cl· secretion. ~is was detected in cell monolayers by an Usstng chamber assay, or m isolated cells using the patch clamp technique. lmmunoprecipitation of 4FS08 CFI'R from these cells indicated a fully glycosylatcd form of the protein after DMSO treatment. To investisate the mechanism by which DMSO overcomes the ~FS08 maturational defect, we designed experiment& based on the well described role of DMSO as a differentiatins qent capable of dramatically altering cell surface protein expression in epithelial cells. In order to investigate whether DMSO treatment resulted in a more differentiated phenotype in LLC-PK1 cells, we studied lisht junction formation with immunocytochemistry using an antibody to zonula occludens (Z0-1). LLC-PK1 cells grown on glass coverslips developed more orsanized tisht junctions after DMSO treatment than untreated controls, as observed w1th confocal microscopy. 4FS08 CFTR in this settins localized only to the perinuclear, ER compartment in non DMSO treated cells, but could be detected at the plasma membrane in DMSO treated cells with a distribution very similar 10 wild type CFTR exprcssins cells. Cell surface localization of 4FS08 CFTR required close cellular contact, while in single cells it could be detected throuahout the cytoplasm without extensive cell surface stainins. It his also been reported that treatment of epithelial cells with 1-2'11 DMSO for 4-7 days results in lower Hsp70 expression. In our model system, investisatton of Hsp70 levels by western blotting could not confirm 1 decrease in Hsp70 levels after DMSO treatment, but immunocytochemistry showed redistribution of the cytosolic Hsp70, lncludins 1 stronger sipal around the nucleus and less peripheral llainina. Previous studies of the 41'508 CFTR processing defect have often used non polarized epithelial cells. Our results raise the possibility that induction of polarity in LLC-PK1 cells might increase a transition of 4FS08 CFTR from the ER to the TON. If the 4FS08 CFTR quality control mechanism becomes "lesky" in differentiated cells compared

with the undifferentiated state. the findings could have important implications concerning the in vivo processing of ~508 CFfR, which occurs in well-organized cells within tissues characterized by tight junctions and polarity. (Supported by CFF and NIH).

45 IDENTIFICATION OF NEW AF508 REVERTANT MUTATIONS USING A STE6/CFTR CHIMERA SYSTEM IN YEAST A. DeCanalbo, L.Gaaaberorr aDd J. Teem. Departmeat of Bloloa:r. Florida State Ualnnlt)', FL, USA.

We IIICd a STE6/CFI'R bF508 cbimeno ia yeast to ~Cr=J for oew bF5al revcr1allts wbidl occur within the CFI'R portion of NBDI. The CFTRAF508 NBDI region of the transporter (F494.LS58) was muta&euizcd is...mJ:2 by aror-prooe PCR metbods. and lbeu inlroduc:edby llaD.Iformatioo into a STE61CFfR t.F508 chimera in yeast. Uoina a yeast matina ..... y. we idenlilicd yeast colooics that ovm:ame thc ddcclive matina pheootypc lllllciatcd with the t.F5al mutation. The STE6/CFTR t.F508 plasmid DNA was lbeu J<COVeRd from the yeast revertaDI colonies, 1111 aua1)'1Jcd by DNA oequeac:iJI&. Previously we idcutilicd tbrec t.F508 re­mutatiom (RIDQ, R553M. and R5S5K) in the carboxy terminal region of the CFTR NBDI aexmcnt within the c:bimcra. Ia our presco1 mutant saecn, we apia isolated revcr1allts R553Q and RSSSK and also idcutilicd oew t.F5al re­mutatiom (1539f. 1539K. 1539M. andGSSOE). Most of the revatant mutalioos­isolated multiple times in indcpendent mutant saeens, IU118CStinl that we bave beJun to oatunlle the loa~a for t.F508 revertant mutationa. latcrestingly. one of the revertaat

mutatiooa ocx:un in the c:onaervcd LSOGQ motif of NBDI (GS50E), and the otba tbrec occur al 1539 in the NBD I rep on directly preccdina the LSOOQ motif. Ia Cllb to detcrmioe wbetbcr the revertant mutations could reverse the proceasiaa ddea <:1 CFTRAF508. we introdtwed each revatant mutation into a CFTR4F508 eDNA 1111 cxprcsoecl tbc mutant CFTR alleles in HeU. cells. CFTR protein -immllllll(RCipitatcd. pbosphorylalcd and aualyzcd on SDS polyaaylllllide sel• to 8Siell CFTR proccssiaJ. The revertant mutations 1539f and OSSOE partially lala'cd the proc:csaina of CFI'RAF508 as evidenced by the productioo of the fully glycosylatcd form of the CFTR protein (band C). We arc c:urready elUIDiiaiq the effect of revatant mutatio01 1539f and OSSOE in rcstoriq cAMP-dcpendcul clllcxide cbaaoc:l activity to CFI'RAF508. Our preliminary remits indicare that both t.F5al revatant mutations 1539T and OSSOE aiplificandy inaease the functional activity <:1 CFTRAF5al. Thus. the bF508 revatant mutatiom idcutilicd in yeast partially ...,_ the proc:csaina ddcct in CFTRAF508 mel lad to incrcsscd cbloride chamel activity. Tbia 1U118C51S that the STE6/CFI'R bF508 c:bimenoeffcclively models the effect of thc bF5al mutation on CFTR NBDI. Further. the revertant mutatioaa dd'IIIC a subdamain of NBDI ClltaldinjJ: from 1539-RSS.S wbidl may iDtcract witb the polypeptide region c:onlainina the t.F5al mutation duriq NBDI foldina. RevertaDI mutations within thio aubdomaia may facilitare foldiq ol the mutant NBDI rcmlting in incrcsscdCFTRAF508 proc:csaina and function. Suppol1cd by the NIH.

46 DO P200-CARRIER VESICLES MEDIATE THE TRAFFICKING OF CFTR? P. DoMelly, 0. Mayorga-Wark, S.G. Schultz, Yl.f. ~. Department of Integrative Biology, Pharmacology and Physiology, Univ. of Texas Medical School, Houston, TX, USA.

The most common mutation in Cystic Fibrosis, ~F508, is a trafficking mutant in which CFTR maturation is arrested in the endoplasmic reticulum and prevented from beina transported to the plasma membrane. We have analyzed subcellular fractions in an effort to identifY intermediates in the trafficking of CFTR to the plasma membrane. Previous studies in our laboratory identified a subcellular membrane fraction <Mrs) that appeared to be involved in trafficking of a large conductance anion channel to the apical plasma membrane of CJ· transporting epithelial cells. Immunolocalization of a specific protein marker, p200, in thick frozen sections of the trachea identified small vesicular structures in the cytosol enriched towards the apical domain of the epithelial cells (Am J. Physiol. 263 (Cell Physiol.):C888-C895, 1992). In the present study, M,. from bovine tracheal epithelia and bovine renal cortex were probed with antibodies to the C-terminus of CFTR. The antibodies identified CFTR in a highly purified preparation of M,.. Comparison of CFTR in the airway apical membrane to CFTR found in M,5 showed the protein to be in the mature c- form , or fully glycosylated CFTR. Analysis of Golgi membrane fractions also demonstrated the presence of both CFTR and p200 in the same fraction suggesting that M,. is derived from the Golgi apparatus. Single channel analysis of M,. reconstituted into planer phospholipid bilayers confirmed

the presence a functional CFTR-like, 7.6 pS, cAMP-activated anion channel that could be distinguished from the previously characterized large conductance, SITS-sensitive anion channel. 1be presence of CFTR and other apical membrane constituents in Mrs vesicles, combined with their unique cellular distribution, suggests these vesicles may play a central role in the trafficking and targeting of CFTR to the apical plasma membrane of epithelial cells. (Supported by National Institutes of Health Grants DK-38518 and DK-37260)

47

CELLULAR TRAFFICKING OF CFTR IS DIFFERENTIALLY AFFECTED BY PHOSPHOLIPID COMPOSITION IN L-CELLS TRANSFECTED WITH EITHER WILD TYPE OR AF508 CFTR. S. BarNoy, 0. Eidelman and H. B. Pollard Department of Anatomy and Cell Biology, USUHS, Bethesda, MO.

The nucleotide binding fold domain (NBF-1) of CFTR is the locus of the AF508 mutation responsible for most cases of cystic fibrosis. The recombinant 20 kOa rNBF-1 has been shown, by ourselves and others, to Interact intimately with artificial and natural membranes. More recently, we have shown that the AF mutation changea the lipid specificity for interactions of rNBF-1 with membranes. For example, while both wt-rNBF-1 and AF­rNBF-1 bind phosphatidylserine IPS), the mutant species also interacts efficiently with phosphatidylcholina (PC). This broadened specificity for the .1.F508 mutant was evident in functional assays such as Induction of membrane permeabilization and of vesicle aggregation. This broadened specificity also appeared in structural assays such as tryptophan fluorescence, accessibility to aqueous quenchers and CO. We therefore hypothesized that this broadened specificity might have Implications for the trafficking In vivo of mutant CFTR through the ER. Wa used L-cells transfected with wild type or .1.F508 CFTR in which the lipid composition was chemically manipulated to change PC into negatively charged derivatives. We found that the amount of mature CFTR in these cells was affected by the change in lipid composition. Furthermore. the effect of lipid composition change was different between wild type and AF CFTR. Whereas the amount of mature wild type CFTR was not affected or slightly reduced by the lipid composition change, the amount of mature .1.F508-CFTR was dramatically Increased by replacement of neutral PC with acidic analogs. We conclude from these data that phospholipid interactions affect the stability end trafficking of CFTR through the ER and that these Interactions are especially important in the case of the AF508 mutation.

48 PHARMACOLOGICAL EFFECTS OF THE YEAST ISOPEPTIDASE YUHIP ON THE TRAFFICKING OF AF508 CFTR IN A CF TRACHEAL EPITiiELIUM CELL LINE. 1L. Kempster, 1F.M. Munkonsc, 2R.Cohen, 'D.C. Grucnert, 4S. Cheng, 1J.E. Browning, 'K.V. Lukacs. 1D.M. Ocddcs, E.W.F.W. Alton. 1Ion Transport Unit, National Heart and Lung Institute, London, UK. 'Department of Biochcmistzy, University of Iowa, Iowa City, Iowa, USA, 'Cardiovascular Research Institute, San Francisco, CA, USA, 4Gcnzymc Corporation, Framingham, MA, USA

The mechanisms of AF508 CFTR mistrafficldng and localisation to the ER membrane are not fully understood. PreviOUJ work has suggested that two principal events arc involved. Prolonged association with the molecular chaperones Hst70 and calnexin, and a highly increased degree of degradation by the ubiquitin-proleasOme pathway (>99% compared to -75% in wild-type). The aim of this investigation was to explore the contribution to mistrafficking of AFS08 CFIR by increased degradation and any corrective outcome of inhibiting this process. The S.ctnvlsltu 26 KDa isopcptidasc Yuhlp has been shown to specifically cleave unfolded polypeptide llubstratcs from ubiquitin polymers at the substrate-lysine 48 isopcptide bond, RSUiting in a de­ubiquitinatcd product. Since polyubiquitination is an early event in the degradation of misfolded polypeptides, the effects of ubiquitin cleavage by Yuhlp on degradation and trafficking of AFS08 CFIR were investigated. Stably transformed AF/AF human tracheal epithelium cells (tCFTE29o-) were metabolically loaded with 10 mM SPQ for 12 hours, loaded with and without 3 ~ Yuhlp by llypotonic shock in the presence of cquimolar SPQ to minimise

Poster Session Abstracts 223

leakage and then incubated for 2 hours in the presence of cquimolar SPQ. Halide emux assay by fluorescence microscopy was then carried out with mock­Yuhlp loaded CFTE cells and CfTE cells corrected by transfcction with wild­type CFTR eDNA c:omplexed with lipid 1167 as negative and positive controls respectively. Results demonstrated enhanced halide efflux in CfTE cells loaded with the isopcptidasc (1.95 mMscc'1) as compared to negative control (0.01 mMsec:'1), and cffiux observed for the positive controls (0. 7 :1: 0.22 mMscc'1).

These preliminary data indicate that Yuhlp may inhibit AF508 CFTR degradation and allow it to subsequently enter molecular chaperone-mediated folding, traffick to the apical cell membrane and correct the halide efflux abnonnality in a stably transformed CF cell line.

49

CFI'R BIOSYNTHETIC PROCESSING AND TRAFFICKING IN THE YEAST Saccharomyce11 cerevu111e. G. L. Kiser, A. W. Kloser, and 1. R. Riordan. Mayo Clinic Arizona, Scottsdale, AZ. USA

Many cystic fibrosis disease-associated mutations cause a defect in the biosynthetic processing and trafficking of the protein, not in the function of the cystic fibrosis transmembrane conductance regulator (CFI'R) per se. These protein biosynthetic pathways have been extensively studied in the yeast Saccharomyces cerevlsiae. Yeast mutants, defective at various steps of the secretory. ubiquitination and degradation pathways, have been used to dissect the mechanisms of biosynthctic processing and intracellular transpon of several proteins. 1be related mammalian membrane proteins. P-glycoprotein and multi­drug resistance protein (MRP), when heterologously expressed in this organism, tnrget to functionally relevant sites. lberefore, to exploit the yeast system to study CFI'R, we have employed an expression system in which the CFI'R gene is driven by the promotor of a structurally-related yeast multidrug-rcsistance transponer, Pdr5p, in a Pdrlp transcriptional regulator-dependent manner (E. Balzi et al., 1. Bioi. Chem. 269: 2206, 1994: R. Egner eta/., Mol. Cell. Bioi. 55: 5879, I 995). Studies in rnanunalian cells have shown that the immature CFrR protein is ubiquitinated, possibly as a prerequisite to degradation, and degraded in pan, by the proteasome (1.1. Jensen et al., Cell 83: 129, 1995; C.L. Ward et al., Cell 83:121, 1995). We have observed that CFI'R degradation in yeast is at least partially sensitive to the proteasome inhibitor lactacystin P-lactone, confmning the suitability of yeast as a model system for studies of the biosynthesis of CFfR. We arc currently evaluating the impact of mutations in ubiquitinating enzymes and proteasome subunits on the turnover of CFfR. To define specific components of the CFfR biosynthetic pathway, we arc also investigating the degradation kinetics in other yeast secretory pathway mutants. We found that the absence of the vacuolar protcases Pep4p and Prb I p did not significantly impact the decay kinetics of wild-type CFfR, consistent with our observation that green-fluorescent-protein-tagged CFfR was not seen by fluorescence microscopy in the vacuolar compartment of the yeast cell. (Supponed by NIDDK of NIH and CFF).

so CFTR TRAFFICKING IN POLARIZED EPITHELIAL CELLS USING GFP. B, Moyer , J. Lofting, E. Schwiebert, W. Guggino, G. Langford and B. Stanton. Dartmouth College, Dept. Physiology and Biology, Hanover, NH, Univ. Alabama­Birmingham, Dept. Physiology and Biophysics, Birmingham, AL and Johns Hopkins Univ. Sch. Med., Dept. Physiology, Baltimore, MD, USA.

We examined the trafficking of CFTR in polarized epithelial cells using a green fluorescent protein (GFP)-CFTR expression vector. GFP linked to the N-terminus of CFTR did not alter CFTR function when expressed in IB3-1 cells. In MOCK cells stably transfected with GFP-CFTR, Western blot analyses using anti-GFP and anti-CFTR C-terminus antibodies yielded three bands between 200-230 kDa, consistent with the predicted molecular weights of unglycosylated, core glycosylated, and mature glycosylated GFP-CFTR fusion proteins. Short-circuit current experiments showed that cAMP-activated Cl secretion was three to four times greater In GFP-CFTR stables than In parental, untransfected MOCK cells. Quantitative laser scanning confocal fluorescence microscopy demonstrated that GFP-

224 1997 Cystic Fibrosis Conference

CFTR localized to the apical plasma membrane region under steady-state conditions and that cAMP did not stimulate a detectable translocation of GFP-CFTR to the apical cell membrane. Disruption of the microtubular cytoskeleton with colchicine did not affect cAMP-activated short-circuit currents or localization of GFP-CFTR. Similar results were obtained in CALU-3 cells, a polarized human airway epithelial cell line, and miMCD-1<2 cells, a polarized kidney epithelial cell line from mouse Inner medullary collecting duct, which express endogenous CFTR. Conclusion: cAMP does not stimulate the trafficking of CFTR from an intracellular pool to the apical plasma membrane in polarized MOCK, CALU-3 or m1MCD­K2 cells. (Supported by NIH DK-45881 and DK-51067).

51 P574H and AF!OB ronn "stable B" at 26 'C. Lxnd&..S. Ostcdggrd Bernhardt Zeiher, and Michael J. Welsh, University of Iowa College of Medicine, Howard Hughes Medical Institute, Iowa City, Iowa 52242.

Many CF-associated mutations disrupt the biosynthesis of CfTR. To examine the mechanisms responsible for misprocessing, we studied two CPIR variants, MS08 and PS74H. Previous studies have shown that CPIR forms a core Jlycosylated protein, band B, in the ER which is then funher alycosylated in the Golgi 10 form band C. We found that, like MS08, PS74H made more band C when the temperature was reduced from 37 ·c 10 26 ·c. To learn which procesaing *P. was altered by temperature, we examined the production of 'stable B", a transient precursor of band C. Although neither PS74H nor MS08 made detectable amounts of this intermediate at 37 •c, both formed stable B at 26 ·c. Although both mutants were temperature sensitive, PS74H made more stable B and more band C at the lower temperature than MS08. However, unlike MS08 or wild-type CPIR, at 37 ·c. PS74H accumulated as inclusions that colocalized with the cytosolic chaperone, Hsp70. When the temperature was reduced 10 26 ·c. the mclusions disappeared. Coimmunoprecipitation of PS74H band B with anti­Hsp70 antibodies indicated that PS74H is physically associated with Hsp70 when grown at 37 ·c. Anti-Hsp70 antibodies immunoprecipitated more band B of PS74H than wild-type. Hsp70 also maintained an association with PS74H for lon11er than with wild­type. We asked if formation of stable 8 was coinctdent with release of CfTR from Hsp70. Wild-type stable B was not coimmunoprecipitated with Hsp70 antibodies and thus not associated with Hsp70. These data suggest that release of protein from Hsp70 coupled with the ability 10 aenerate a stable B form are temperature sensitive steps that are defective in MS08 and PS74H.

52 DELETION CONSTRUCI'S ENCOMPASSING THE SECOND HALF OF CFTR CAN FORM FUNCTIONAL CHLORIDE CHANNELS IN X~""JIIU 0«:y1a. S Devjdu. E.M.Sc:hwiebert and W.B.Guuino. Department of PhysloiOI)', Johns Hopkins University, School of Medicine, Bakimore, MD, USA.

The Cystic Flbrolil Transmembrane Conductance Reaulator(CFTR) consists or two 1r1111membrane domains(TMD I, TMD2), two nucleotide-binding domains (NBD I,NBD2) 111d a unique repalatory (R) domain. Studies by Sheppard et ai.(Cell 76,1091-1098) 111d Ougino et al. (Am. J. Physiol. 270, FIOJ8-FI048), have shown 1bst the tint half of CFTR comprising TMD I, NBD I and the R domain c:an fonn functional chloride channels with cbaractcrilla ~aC~~~bling wild type CFTR. Two conslnlcts (RT2N2CFTR and R'T2N2CFTR) which includes the uc:omplete or incomplete" R domain (enc:ompowlna 11111ino Kids 513 to the C terminus or encompowinglllllino Kids 708 to the C terminus), TMD2 and the NBD2 when expreued in Xenopus 0ocytes fonned constitutive chloride cbannell(l-470.527:1:84.51 nA at 60mV) which were further llimulaled by cAMP 11onists (1-676.036±17 nA at V-60mV, n-9XWTCFTR: 1•534.675±39.7 nA at 60mV, n•l). Wiler injected OCIC)'Ies exhibited little or no cumnt ICiivity in the absence (1-90.06±6.11 nA, n-9 at 60m V) or pmenc:e or cAMP ~&onists (1-92.24:1:7.44 nA, n-9 at 60mV). The selec:tlvlty of this channel at the wu Cl'•l' in comparison to wild type CFTR wblc:h wu Cl'>l', An lldditlonal COIISiniCI c:onsiltina of the tlrst nucleotide blndlna domain, the R domain, TMD2 and NBD2 (NIRT2N2 CFTR) alro behaved almiJ.r RT2N2CFTR but the bueline c:onJtituity wu leu. CFTR curm1t1 were chariCieriDicl u chloride curm1t1 by a positive shift of reversal potential ill a chloride he ringer solution. Funber, the CUI'ftllll wu not inhibited by DIDS, but wu inhibited by DPC. An additionaiCOIISinlel (T2N2CFTR) c:omprialna nucleotide• 2523 to the C terminus to include TMD2 and NBD2 had no baseline activity (1•16.47:1:6.62 nA, n•l at 60 mV) and wu lltimullted to a 1111111 extent by

cAMP agonists (1~175.38±17.46 nA, n=B at 60 mV). Ali constructs contained the first 159 nucieotides or CFTR to include the first Kozak methionine for translation initiation. Further, a construct encompassing MS, M6 and the second half of CFTR (A259CFTR) exhibited wild type chloride channel and selectivity characteristics. These results show that the second half of CFTR may be an important contributor to its chloride channel function. In support of the data, McCarty et ai.(Neuron, Voll3, 623-634) have shown a high affinity binding site for DPC on TMI2 as well as TM6. The data is also consistent with previous observations by Anderson et al. (Science%53, 202-205,1991) who showed that residues on M6 confer selectivity to CFTR. A hypothetical model based on the above findings will be presented.

53

Antisense-mediated correction of aberrant splicing in a stable cell line espressina a CFTR mini-gene harboring the CF-eausing mutation, 3849+10kb C>T. K J. Friedman, Y. Wang, L.M. Silverman, M.R. Knowles, R. Kole. Curriculum in Genetics and Departments of Pathology, Medicine and Pharmacology, Univ. of North Carolina, Chapel Hill, NC, USA.

3849+10kb C>T is a splicing mutation carried by a large proportion of cystic fibrosis (CF) patients that exhibit atypically mild clinical disease and normal sweat chloride values. The mutation creates a novel splice donor site deep in intron 19 of the CFTR gene which activates a cryptic acceptor site, thereby promoting the insertion of 84 nucleotides of intron 19 between exons 19 & 20 in the mature mRNA. We modified the L TR-driven CFTR eDNA expression vector LCFSN by inserting 453 bp mini-introns at the exon 19- exon 20 junction that include either the wild-type or mutant allele atfthe 3849+10kb locus, as well as all known consensus splice elements. These modified vectors were subsequently stably transfected into mouse 3 T3 fibroblasts and their expression evaluated by RT-PCR. The stable cell line transfected with the wild-type construct expressed an mRNA that is appropriately processed at the exon 19 - exon 20 junction. The cell line transfected with the construct harboring the 3849+10kb C>T mutation expressed products of aberrant splicing and minimal levels of wild-type mRNA. 2'-0-methyl-phosphorothioate antisense oligoribonucleotides complementary to either of the aberrant splice sites in the mutant construct promoted the formation of wild-type mRNA product in a dose-responsive and sequence-dependent manner. This is an application of antisense oligonucleotides that provides a novel form of gene therapy for a specific class of mutations in CF. This approach is also applicable to other genetic disorders such as 13-thalassemia. This work was supported in part by a grant from the Cystic Fibrosis Foundation.

54

CF MUTATIONS AND TESTICULAR CFTR GENE EXPRESSION IN OBSTRUCTIVE AZOOSPERMIA S Larrihal, Ll. Bassasl, J. Gimenez'. M.D. Ramosl, V. Nunes!, X. Estivilll, T. CasaJsl. 'Medical and Molecular Genetics Center­IRO, Hospital Duran i Reynals. lAndrology Department, I.U.N.A., Fundaci6 Puigvert. Barcelona, Spain

The involvement of the CFTR gene in obstructive azoospermia (OA) has largely been demonstrated (1-4). In an extensive study we analyzed 64 CBA VD patients, 11 CUA VD and 7 patients with renal agenesis and CBAVD (3), CUAVD (3), CUAVS (1). In the CBAVD group we found the genotypes: CF/ST (34.4%), CF/­(23.4%), STI- (10.9%) and CF/CF (14.1%); II patients (17.2%) showed no mutations in the CFTR gene. The variant ST was observed in SO% of the patients, 3 of them with the genotypes 4FS08/SSOP, 4FS08127Sl+3A>G and RSS3XIF1074L, indicaung that these missense mutations and the splice site mutation are associated with a mild phenotype. The CUA VD patients showed the CFIST genotype in 4 samples (36.4%), being the other 7 negative for CFTR mutations. In the 7 patients with OA and renal agenesis we found that 3 (42.8%) showed involvement of the CFTR gene (2 patients ST/-; 1 patient CF/-), suggesting that this group is molecularly heterogeneous, in disagreement with a previous report (S) that suggested that patients with renal malformations were another clinical entity not due to mutations in CFTR. On the other

hand, the expression of CfTR in testis has been demonstrated (6). By means of visualization of fluorescently labelled RT-PCR products using the Applied Biosystem 672 Genescan software, we have determined the relationship between the normal and alternative CFfR transcripts in testicular biopsies from 11 CBA VD patients and controls. Preliminary results show that 90% of CBA VD patients with the 5T allele have a higher proportion of transcripts lacking ex on 9, together with a reduced spermatogenesis in testis.

References: 1- N Engl1 Med (1995) 332:1475-1480. 2- Am 1 Hum Genet (1995) 56:1359-1366. 3- Am 1 Hum Genet (199S) 57:958-960. 4- Hum Reprod (199S) 10:1728-1735. 5- Lancet (1994) 344:1473-1474.6- Am 1 Pathol (1994) 144:906-914 Acknowledgments: This work is supported by grant from FlSS (9612005) and the lCS. S. Larriba is a fellow of Roche SA and the Associaci6 Catalana de Fibrosi Qu!stica.

55 BIOCHEMICAL STUDIES DEMONSTRATE THAT THE NBFI+R COMBINED DOMAINS OF CITR INTERACT WITH THE NBF2, H...L..ldl and P. L. Pedersen, Department of Biological Chemistry, School of Medicine, Johns Hopkins University, Baltimore, MD, USA.

It has been shown that the R domain and the two nucleotide binding domains of CITR arc required for optimal chloride channel activity. It has been proposed also that both phosphorylation and dephosphorylation of the R domain arc involved in the regulation of the channel. However, few biochemical experiments have been performed to explore and fully understand at a molecular level the interrelationships among the NBF I, NBF2, and R domains that regulate clwmel function. Here, we report biochemical studies that demonstrate that the combined NBFI+R domains and the NBF2 domain interact. The NBFI+R (F400-T848, 51.3 kDa) and the NBF2 (WI204-KJ389, 21 kDa) were overcxprcssed in E. colt. The expressed proteins comprise approximately 50% of the total cell protein. These two proteins were purified to near homogeneity under denatured conditions. The purified proteins were then refolded via dialysis at 0 - 4°C. The folded NBF2 binds the trinitrophenyl nucleotides: TNP-ATP, TNP-ADP, TNP-AMP with I<.. values respectively of 3.1 j.IM, 9.3 11M, and 11.2 J.LM. ATP can replace the analogs with a K1 of about 8 mM. The folded NBFI+R binds the trinitrophenyl nucleotides: TNP-A TP, TNP-ADP with I<.. values respectively ofO.I8j.LM and 1.0 11M. The NBFI+R protein is phosphorylated by PKA at pH 6.5 (5S J.LM ATP) resulting in a significant mobility shift on SDS-PAGE. The presence of ADP, AMP, TNP-ATP, TNP-ADP, TNP· AMP have no major effect on the level of phosphorylation and, to date, conditions have not been found to reverse the phosphorylation using alkaline phosphatase. The folded NBF2 domain was labeled with CPM (a fluorescence probe). The addition of unlabeled NBFI+R to 11 pM ofCPM­labeled NBF2 causes a wavelength shift as monitored by fluorescence, consistent with a direct interaction between the two proteins. The results also indicated that the NBFI+R interacts with the NBF2 with a stoichiometry near 1. Furthennore, the mixture ofNBFI+R and NBF2 migrates as a single band on native gels. More experiments are being conducted to obtain independent evidence for the (NBFI+R)- [NBFl) interaction. [Supported by grants from the Cystic Fibrosis Foundation and NIH (NIDDK))

56 EFFECT OF PHORBOL ISrER. ON CFTR mRNA LEVELS IN CLONAL CHO CELL LINES THAT CONTAIN A FUNCTIONAL FULL-LENGTH HUMAN CFTR GENE. Peter J. Mogavzel. Jr.'.l and Melissa A Rosenfeld'. 'National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892 and 2The Johns Hopkins Medical Institutions Baltimore, Maryland, 21287.

Phorbol esters (PE) were proposed as exogenous agents which could potentially modifY cystic fibrosis transmembrane transmembrane conductance regulator gene (CFTR) expression based on the presence of the AP-t sites in the CFTR promoter sequence. The potential effect of PE has been studied primarily in malignant human cell lines (e.g. T84 and HT-29 cells). Their high level CFTR expression allows changes in mRNA levels to be easily monitored and quantified. Prior studies using T84 and HT-29 cells demonstrated that 10-IOOnM PE down-regulated CFTR expression. Performing these studies in other non-malignant human cells lines it difficult because non-transduced human cell lines generally have low level CFTR expression and are thus difficult to quantitatively assess. The present study of PE effect on

Poster Session Abstracts 225

CFTR expression utilizes two recently derived Chinese hamster ovary (CHO) cell lines which express CFTR mRNA at levels detectable by Northern analysis and functional CFTR protein as a consequence of intesration of a single copy of the 230 kb human CFTR gene including the promoter and up to 37S kb flanking sequence (CSIIF2 has 33S kb upstream and 40 kb downstream, CS21CS has 45 kb upstream and 30 kb downstream (Hum. Mol. Genet. 1997, 6, 59-68). These two cell lines were cultured in the presence of I, 10 or IOOnM phorbol 12-myristate 13-acetate (PMA) for 18 hours. CFTR expression was monitored by Northern blot hybridization using a CFTR eDNA probe and mRNA levels were quantified by phosphoimager. As expected, CFTR mRNA levels decreased significantly in T84 cells while P-actio levels increased with IOOnM PMA treatment. In contrast, no change in CFTR mRNA levels were observed in either of the two CHO cell lines and endogenous P-actin mRNA levels were unaffected by PMA treatment at all concentrations. These data suggest that the PE-mediated regulation of human CFTR contained within these CHO cell clones is different from that of T84 cells.

This work was supported in part by the Cystic Fibrosis Foundation.

57 NOVEL A561E CFTR MurATION FOUND IN PORTUGUESE PATIENTS

ASSOCIATED WITH MISLOCALIZATION DISTINCT FROM AF508. 1~ •1Amarai MD, •1Farinha C, •tGomes A ••Domingos P, •1Garcia S, 1Pacheco P, 1Duarte A, •aarreto C, *Demolombe S, *Escande D & 'Lavinha J. 1Dep Genetica Humana, lnst Nac Salide Dr. Ricardo Jorge, Av. Pde Cruz, 1699 Lisboa Codex •Dep Quimica e Bioqulmica, Fac Ciencias Univ Lisboa, Cp• Grande, 1700 Lisboa.; "Serv Pediatria, HSM, Av Prof Egas Moniz, 1700 Lisboa PORTUGAL. *Hopital G & R Latnnec, Nantes, FRANCE

AS61E is a novel CF-associated mutation in the first nucleotide binding domain of CFTR which accounts for -3% of CF chromosomes in the Portuguese population. All patients found to date are compound heterozygotes M508/A561E with a relatively mild disease both pulmonary and pancreatic. This mutation associates with haplotype A, IVSS(CA) repeat 16 and 1VS8 (TGuT1). To investigate the basic cellular defect underlying A561E mutation, we have introduced this mutation into the CFTR eDNA by site-directed mutagenesis and have stably transfected HeLa cells with recombinant pcDNAJ expression plasmid carrying this mutant eDNA Cells were transfected in parallel with wild type (wt) or MSOS-CFTR eDNA The properties of the A561E CFTR protein were studied by immunoprecipitation and immunocytochemistry. In cells transfected with the AS61E mutant plasmid, immunoblotting analysis with anti-CITR (M3A7) antibody revealed the presence of band C, the mature form of CfTR containing carbohydrate structures consistent with processing in the Golgi apparatus, in similarity to cells transfected with wt-CFTR eDNA, but differently from MSDS-CFTR eDNA transfected cells. By immunocytochemistry with anti-CFTR polyclonal antibody, we could observe an intracellular accumulation of A561E-CFrR protein distinct from MSOS. By SPQ halide-efflux assay we could not detect cAMP­stimulated halide efflux. Altogether, these results suggest that A561E major effect must be protein mislocalization post-endoplasmic reticulum. We thank Dr. J Riordan for M3A 7 antibody, Dr. W Guggino for 169 anti-CFTR antibody. This work was partially financed by JNicr.

58 NON-APICAL CFfR EXPRESSION IN ADULT HUMAN VAS DEFERENS EPITHELIUM.~ B. Vander Schucren, G. De Geest, J.-J. Cusiman. Center for Human Genetics, KULcuven, Leu-. BELGIUM.

The majority of the males with CF are infertile due to the absence of the VII deferens. Also CBA VD (congenital bilateral absence of the VII clcfetenS) patients have an increased frequency of mutations in the CFTR JCIIC and CFTR transcripts in the VII deferens have been clescribcd. In order to understand the potential role of CFTR mutations in the absence of the human VII deferens more information will be nec:essaJ)' on the localisation of the CFTR protein in that epithelium. In this context. normal human VII deferens biopsies were obtained from live different patients, who presented to our university hospital for vasectomy for the purpose of birth control. The ultrsstructural features of the epithelium ftom three clilfercnt patients were dcterminecl. The Jocaliution

i' l t I

226 1997 Cystic Fibrosis Conference

of CFTR wu llllalysed with indirect immunolluon:sccnce on formol-medwlol fixed cryoscctions fiom four dill'erent individuals with IICVel'll antibodies directed apinlt dill'crc:nt extra· and intnc:cllulll' domains of CFTR. The epithelium COIIIiltcd of a continuous layer of basal cells and talllhin columnll' CCIII, which were pccudootntilied. As dcocribcd -'icr, three different columrw cell typCI could be observed, lhc principal cells, lhc mitochondrion· rich ccll1 and lhc pencil cells. The localisation ofCFTR wu indcpclldcnt oflhc cell type. With lhc antibody dircc:tcd apinst lhc cxtnc:cllulll' domain, only lhc lateral membrane of lhc columnar epilhclial c:clls wu stained while lhc apical membrane wu U111e11Ctive. The cell membrane of lhc basal cells wu also stained. A c:omparablc result, with lhc antibodies raised apinst dill'cn:nt intnc:cllulll' domains, could be obtlincd. A cyt010lic panular ractivity wu seen below and above lhc nudcus. The cytoplasm of lhc basal cells wu also stained. Only lhc epilhclium wu clearly marked while lhc surroundin1 muscle cells and fibroblasts did not ICIICI. The staininl wu completely blocked in lhc presence ora oynthctic: peptide. The 1pcc:illcity or lhc antibodies wu confumcd on nasal epilhclium, where lhc antibodies ohowcd esiCIItially apical reaction. Cryoocctions of human vu dcfcn:no, treated with lhc inclircct inununopcroxiduc method with lhc I8IIIC antibodies, pvc lhc I8IIIC results. The establishment of cell polarisation •vas dctcrmincd with lhc antibody E· cadhcrin, sivinl rcac;tivity of lhc lateral membrane of lhc columnar epilhclial c:clls and lhc cell membrane oflhc basal cells. MDR·I was not expressed since lhc reaction wu ncptive usin& dift"cn:nt antibodies. It iJ ciCII' lhat CFTR iJ not expressed lllhc apical membrane in human adult vas dcfcn:no epilhciium. The ftlnctionalsipifu:ance of this aberrant expression rcmainl to be dctcrmincd. Thcoc inveotipti0111 have been 1uppol1cd by 'VIaams Instituut voor de bcvordcrin& van hcl Wctcnschappclijk·Tcc:hnolo&iJch Ondcnock in cJe lndustrie (IWT)'.

59 POSTNATAL EXPRESSION OF CFTR IN THE VAS DEFERENS EPITHELIUM OF NORMAL MICE IS DEPENDENT ON CELLULAR DIFFERENTIATION. I Boynacn1 M. Manin2, B. Van dcr Schucn:n', G. De 0-'. J .• J. Cassiman'. • 'Center for Human Genetics, KULcuvcn, Lcuvcn, BELGIUM, 'Physiologic Comparee ct Endocrinolo1ic, Univcrsill! Blaise Puca~ Les Ctzcaux, Aubierc Ccdcx, FRANCE.

The 1\mction of the CFTR protein in the vas deferens and the basis by which mutations in the CFTR acne cause the absence of the vu deferens in human is unknown. The abscn<:e of the vu clefcrens could originate either during embryonic clevelopment or in the postna11J phaH. In orcJer to understand which hypothesis c:an be followed more information is necessary on the development of the vu clefcrens and the Jocalisltion of the CFTR protein. For this purpose, vu deferens tissue scc:tions from normal CO-l mice durin& acveral postnatal lifetimes (,, 7, 8, 10, 20 and 40 days after birth) were prepared. The ultrastructunl features were llll8lysed by means of electron microscopy. The localisltion of CFTR usinl immunohistochemistry wu examined on cryOicc:lions with an antibody directed l&linst the rodent C·terminll domain of CFTR. The cJe&rcc of cell polarisltion of lhc columnll' epithelial cells was dcterminlld with an anh'body apinsl mouse £.cadherin. Until day I 0, no distinction could be mlldc between bull and columnll' epithelial cells. Dividing cells were not localised to a spcc:ltlc rcslon. Tbc epithelial cells were COIIIIeCied only 11 lhc apical stele, when a junctional complex wu formed, wbile a consiclerable Intercellular space bclween the lstcral membrana was present. The cytoplasm still had upects of an Immature epithelium. At lhc epical membrane the llereocllia were - developed. The besement membrane divided the epilhclill cello fi'om the m.-dlymal cells. Difl'cn:ntiltion of mesenchymal cells into flbroblutl and miiiCie cello wu complctcd 11 day 10. From day 20, bUll and columnar c:ells could be dlstlngulshlld. In the columftll' c:el11, which were closely packed, the smooth endoplumic: reticulum wu orpniscd in llnlerprint·like - or in parallel, fldcneciiiCCIIIcs. The stereocilillllhc apical membrane were cleveloped. The localisation of CFill and E-cadherin was analysed on CI)'Oicc:lioal fi'om 10. and 40-clay-old mice. Tbc expreaalon of the E-cadherln Jll'l*in in vas clet'erenl epithelium of I ().day-old mice was absent, wblle the expreuion wu mainly situated in the lalenl membrane near lhc luminal side in the epithelium of 40-dly-old mice. CFTR was absent in the epithelium of I 0-day-old mice. In the epllhclium of 40-day-old mice however the pluma membrane It the apical aide wu clearly stained. The morphol01ical analysis shows thai -Y clevelopmenlll ~ of lhc vu clefercns oc:cur after birth. The parallellam of the cxpreuion of the CFTR protein and the difl'erentiltion of mOIIIC vu deferens epithelium is striking. Thcoc lnvesliplions have boa supported by 'VIums Instituut voor cJe bcvorclerin& van bet Wetenschappelijk· TcchnoiO&isch Onderzoek in cJe lnclustrle (IWT)'.

60 UP-REGULATION OF CFTR. EXPRESSION BY D...-18 IN T84 CELLS. ••Ca1Fenta, E.G.A, •Ooaz6lez Ouerrico, A.M., l»ivetta 0 H and • Santa-Coloma, T .A. "IDatituto de Investigaciones Bioqulmicas­Fundac:ion Campomar and •centro Nacional de Genetic:a Medica, Buenos Aires. Argentina.

The regulation of the cftr gene is poorly understood. So far, only down­modulators such as TPA and interferon-y have been reported. In this work, we show that interleukin-IB (IL-l B) can up-regulated the stady· state levels of the CFTR mRNA in T84 human carcinoma cells. Treatment of T84 cells with IL-l B (0.25 nglml) for 4 h resulted in a clear (two/three fold) increase in mRNA CFTR expression. However, IL-JB at concentrations over 0.5 nglml produce inhibition of CFTR expression. Basal levels of expression of CFTR were inhibited by TPA (I 00 ng/ml), H7 (100 11M), GF109203X (I 11M), cicloheximide (10 J!g/ml), methoxy­verapamil (20 11M), apigenin (12.5 11M) and genistein (100 11M). On the other hand, basal levels were stimulated by II..· I B ( 0. 25 nglml), calcium ionophore A23187 (2 J!M} and genistein (250 JIM}. The stimulation by IL-JB (0.25 nglml) was inhibited by cicloheximide (10 Jig/ml), H7 (100 ~tM), GFJ09203X (I J!M) and partially by staurosporin (SO nM). These results suggest that regulation of CFTR expression involves a very complex mechanism, in which interleukin 1-B is acting through several different parallel signal transduction pathways, including PKC, PTK and MAP kinases and also suggest an important role of calcium in modulation of the cftr gene expression.

61 CFTR EXPRESSION AND FUNCTION IN HUMAN VASCULAR ENDOTHELIUM. Lisa M. Schwiebert, Albert Tousson, Erik M. Schwiebert, and Marcio vaz Sanches*. Department of Physiology and Biophysics, University of Alabama at Binningham, Binningham, AL, USA and "Department of Physiology, Johns Hopkins University, Baltimore, MD, USA.

The cystic fibrosis tranamembrane regulator (CFTR) is a JBOkD glycosylated protein that functions as a cAMP-regulated chloride channel. CFTR is expressed on a variety of cell types including epithelial cells, lymphocytes, and cardiac myocytes. However, the expression of CFTR on human vascular endothelium has yet to be dctennined. Expression on human vascular endothelium may have implic:ations for endothelial cell biology and inflammation in general. CFTR was detected on human vascular endothelial cells isolated from human umbilical vein (HUVEC) as demonstrated through RT-PCR, RNase protection (RPA), and immunohistochemical analyses. RT· PCR analysis revealed CFTR transcripts in HUVEC that differ in length (by approximately SObp) from CFTR expressed in the airway epithelial cell line 16HBE14o· (n'*4). RPA demonstrated that CFTR is expressed at a level approximately 5-fold Jess in HUVEC as compared with CFTR expression in the intestinal epithelial cell line T84 (n=2). Immunohistochemical staining with a monoclonal antibody directed against the R-domain revealed perinuclear and cytoplasmic staining of CFTR in HUVEC (n=2). Moreover, CFTR expressed on HUVEC appears to function as a cAMP· and cGMP· regulated chloride channel as measured via chloride effiux analysis (n=2). A cocktail of 8-Br-cAMP (2SOuM), forskolin (2.5uM), and CPT -cAMP (250uM) or 8-Br-cGMP (250uM) alone triggered an increase in chloride effiux 2-fold greater than controls; the cAMP· initiated response was inhibited in the presence of diphenylamine carboxylic acid (O.SmM). These data suggest that CFTR is expressed and functioning in human vascular endothelial cells.

62

EXPRESSION OF ENGINEERED CFTR. AND ENAC IN STABLE CELL LINES. RA Kandasamy , Boucher, RC and Stutts, MJ. Depanment of Medicine and Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel HilL NC 27599-7248 USA

Investigation of coordinate regulation of apical membrane chloride and sodium permeability requires expression of CFTR. and three ENaC subunits. Construction of stable cell lines expressing a drug resistant marker and a second gene of interest has been problematic. We have constructed three bicistronic vecton that express a drug selection

marker (neomycin, hygromycin or puromycin) and a gene of interest under the control of a single L TR promoter. The poly!inker site proceeding the L TR promotor is linked to the drug resistance selector by a polio virus internal ribosome entry site. Each construct was tested individually using the GFP reporter gene. We found 100% of drug resistant colonies of 3T3 fibroblasts, COS cells, MDCK D cells and 16HBEI4o- cells to express GFP. Expression was stable up to at least S passages. We coinfected MDCK cells with bicistronic vectors containing 13-ENaC + hygro and y-ENaC + puro. All drug resistant colonies expressed 13-ENaC and y-ENaC by Western blot analysis. Three clones were expanded and transfected with a bicistronic vector containing a-ENaC + neor. Triply drug resistant colonies were obtained, cultured on permeable supports and assayed for ion transport function. Each clone formed cultured epithelial with high resistances and amiloride sensitive current that ranged from 1 S to 25 1!Amp/cm2

• Wild type MDCK D cells and cells stably transfected with 13 + y ENaC or a + 13 ENaC variably expressed less than 1 ~amp/cm2

amiloride sensitive I... Simultaneous coinfection of MDCK or 3T3 cells with these bicustronic vectors and selection with neomycin, hygromycin and puromycin has also resulted in cell lines stably expressing ENaC function. With these approaches we will test the effects of mutagenesis on coordinate regulation of CFTR and ENaC. (Supported by HL42384, HL34322).

63 MOUSE BETA-DEFENSIN • 1 JS A SALT-SENSITIVE ANTI·

MICROBIAL PEPTIDE IN THE MURINE AIRWAYS • .I1JIIL.B., M. J.

Goldman, &: J. M. Wilson, Institute for Human Gene Therapy, University of

Pennsylvania, &: Wistar Institute, Philadelphia, PA. USA.

Human beta-defensin I (hBD·I) may play a role in the pathogenesis of cystic

Poster Session Abstracts 227

variant iii intron 8 (ST). while patients with CBAVD have RII7H associated with an efficient splice variant (7l). This observation indicates that the difference in pbenotype between the two patient groups, in particular the ab6ence oC lung disease in CBAVD patients, may be 1 consequence oC higher levels oC full-length CFrR bearing Rll7H. The goal oC the present study was to determine the Cl" conduction properties of airway epithelial cells stably expressing different levels oC R117H· CFTR. Thirty-two 0418-resistant cell lines were aeated in 1 CF bronchial epithelial cell line (IB3-1) by transfection with vector oontaining the nt:t:~1 gene and CFfR bearing Rll7H drivm by the RSV promoter. Four cell lines were selected; all expressed RII7H-CFrR as detected by RT -PCR. Three of the cell lines (RH2, RHIO and RHS) bad different levels of CFfR transcript detectable by Northern blotting, while the otber cell line, RH3, did not have CFTR transcript detectable by this method. None of the cell Jines bad CFrR protein expressed at a level detectable by immunoprecipitation. The level of CFrR expressioo detcctod in the RH3 cell line is similar to the range oC expression detcctod 111 vivo. Since CFTR functions as a cr channel and as a regulator ol the outwardly ftlctifyina chloride channel, these cell lines were anai)'2Cd f<r the presence of CFTR-generated and ORCC-generated Cr currents upon cAMP-stimulation by whole-cell patch-clamp. The RH3 cell line bad cAMP-stimulated current that was outwardly ftlctilied (mean current at +100 mV • 898.28 pA and at -IOOmV• -77S.7 pA; n-4, Pdl.OS) and partially inhibited by DIDS (mean current at +100 mV before DIDS • 971 pA and after DIDS • SI6.S6, n•2). The otber 3 cell lines also generated outwardly rectified, DIDS-sensitive current upon cAMP-stimulation. These results indicate that CFrR bearing the Rll7H mutation retains its ability to regulate the ORCC, evm at very low levels oC expression. Based on these observations, we speculate that severe reduction oCthe level of Rli7H-CFTR. protein, as found when Rli7H is associated with the inefficient ST splice varian~ leads to 1 decrease in total a· conduc:tioo via botb CFfR and the ORCC In airway epithelial cells. Inaeased levels of RII7H-CFI'R, as observlld when RII7H is associated with the efficient 7T splice varian~ causes an ina-ease In the 00111bined cr conduc:tioo of CFrR and ORCC to a level sufficient to escape lung disease.

fibrosia lung disease. It would be very useful to have an animal model to 65 investigate the significance of antimicrobial peptides in the host defense of the

airways. It ia the aim of this atudy to identify mouse beta-defensin(s) present in

epithelial cells and study their antimicrobial activity and tissue distribution.

Using reverse transcriptase PCR. a defensin-like sequence, designated mouse

beta-defensin I (mBD-1), wu cloned from mouse kidney poly(A/ RNA. This

sequence share& many of the hallmark features of other known epithelial beta

defensins includina the ordered array of aix cysteine residues. To demonstrate

antibacterial function, mBD-1 wu aubcloned into an expression vector and

transfected into SW 13 cells. Cell lysates were prepared which showed

antimicrobial activity against gram-positive and -negative bacteria in both

diffusion and liquid broth assays. Antimicrobial activity wu reversibly lost in

high salt concentrations. Nonhern blots detected mBD-1 mRNA in adult mouse

kidney and liver. mBD-1 wu detected in mouse lung tissue by RT-PCR. In situ

hybridization demonstrate• mBD-1 mRNA diffusely throughout the superficial

epithelial cells of the large proximal airways with less expression in the small

distal airways and no expression in the alveolar cells. The results of the present

atudy demonstrate that a beta-defensin homologous to hBD-1 is present in the

respiratory system and other mucousal tlssuea in mice.

64 CFfR bearing the R 1178 mutation retains the ability to regulate the outwardly recllrylng chloride channel Independent ol exp.--Jon level. S(eJlbanie B fulmer-Smcn!Ck. John E. Mickle, and Garry R. Cutting. Department oC Pediatrics and Medical Genetics. Johns Hopkins University Scbool ci Medicine, Baltimore, MD 21287.

One mutation in CFTR. RII7H, is associated with two phenotypes: CF and congenital bilateral aboence of the vas deferens (CBA VD). Prior studies have established that patients with CF have RI17H associated with an inefficient splice

Truncation of the CFTR carboxy-terminus affects ORCC regulation. L...Mi.!;k& M. Macek Jr.', S. Fulmer-Smentck, M. M. Egan, E. Scbwiebert', W. Guggino1

, G. R. Cutting. Ctr Cor Med Genetics, 1

/ Dept. of Physiology, Johns Hopkins Univ Scb oC Med, Baltimore, MD; 'I Uoiv oC Alabama Scb ol Med, Birtningbarn, AL U.S.A; 21 Uoiv Hosp Motol, Prague S , Czccb Republic.

CFfR is I cAMP-activated a· channel that also regulates the outwardly rectifying chloride channel (ORCC). The goal alibis study wu to determine the effect of disease-associated CFfR carboxy terminus truncation on botb functions of CFTR. Previously, we reported a nonsense mutation, S 14SSX. in two individuals with isOlated elevatod sweat a· concentrations. This mutation is predicted to eliminate the last 26 IUDino acids Crom the carboxy terminus. Transient over­expression of SI4SSX-CFTR. in HEK 293 cells produces a 17S KDa protein that can be immunoprecipitated with an R-domaio monoclonal antibody (Genzyme) but not with a C-tcrminus monoclonal antibody (Genzyme) as expected for 1 truncation mutation. Thus. Sl4SSX-CFTR. is stable and ol similar size to the C-band oC wild· type CFrR indicating that SI4SSX did not cause a defect in synthesis. To determine the functioos oC CFrR bearing SI4SSX, whole cell patcb clamp analysis was perfcnned on CPT -cAMP stimulated IB3-I cells transiently over-expressing either the SI4SSX-CFTR. or wild-type CFTR. To assist identification oC transfected cells, a green Ouorescent protein (GFP) reporter plasmid was included at a ratio oC I 0: I (CFTR:GFP). Whole cell recordings fran cells co-transfected with GFP were indistinguishable from cells transfected with wild-type CFrR or S 1455X-CFTR. alone. Cells transfected with the wild-type CFrR consistently exhibited an outwardly rectified chloride current that bad two 00111ponents: an outwardly rectified, DIDS-sensitive 00111ponent attributed to the ORCC and a linear 00111ponent generated by CFTR. For example, DIDS application (SOO jlM) caused 1

34% reduction in whole cell chloride current (n • 13, I' • 0.0009). Interestingly, cells transfected with Sl4SSX-CFTR. fell into two groups. In 9 of 23 whole cell recordings, the I-V relatioosbip did not change after the application oC DIDS (I' • O.S36). Conversely, 14 cells responded to DIDS (I' • 3xl0 .. ) and exhibited 130% decrease in whole cell chloride current, similar to that observlld from wild-type CFfR transfected cells. Glybcnclamide (SO j1M) inbibitod linear current generated by CFrR in wild-type transfected cells and botb subgroups of SI45SX-CFTR. transfected cells. These data indicate that truncation of the C-terminus oC CFfR does Dot alter its a· channel function but does interfere with its regulation o( the ORCC. Thus, defective regulation of the ORCC by CFfR appears to produce a pbenotype that overlaps with CF. Although the mechanism oC ORCC regulation is unclear, the carboxy terminus ci CFrR is bigbly conserved among species, fr<m shark to man, and not is present in other ABC proteins suggesting it bas 1 critical functional role unique to CFTR.

228 1997 Cystic Fibrosis Conference

66 EVIDENCE OF SYNERGISM BY CYCLIC AMP· AND CALCIUM· MEDIATED PATHWAYS ON AMYLASE SECRETION IN ISOLATED PANCREATIC ACINI FROM CF MICE. SbanBSuO Tapg•, Saui Bcbany, Gcraldlne Ken~ and Pcrer R. Durie. Rcacarc:b lnstiiUtc, The Hospital for Sid< Children and Dcpar1mcnt of Pedialrica, Univ. of TPnliiiP, Omario, Canada.

We recently demoutrated that pancreatic anzrme acthltleo of oxon 10 knockout CF mice aro 111niftcantly lower than •l•·matcbed controll fed a oolld or liquid diet (lp, at al. Pedlatr Ru 441:242·24!1, I !1!16), Howner, It lo unclear whether enzyme oocretor)' reapouo to oecrelaiOJDH II alto red In tile CF moue. In the present otudy, we evaluated a variety of oocreta1o1ueo (cyclic AMP· and calcium· madialln1) that are lnvoiYOd In exocytooil and In cltlorldo channel activation. Experlmtnll were performed In ilolated pancreatic acini from llqald·fed CF mice (5-6 waekt of •1•) and •••·matched controll fed a oolld or llqald diet. Amylaoe reloaoe wao expreooed ao porcenta1o of acinar amylaoo content to ollmlnato tho effect of reduced onxyme contenL Acinar amylase content (U/mc protein) wu lower In CF mlco than In tile control• fed botil dlell (P<O.OS). BrcAMP and for1kolln alone Induced lllaller amylue Hcrotlon In the control acini than carbachol (P<0.05). Combinations of carbachol with BrcAMP, and of BrcAMP with forokolln produced additive offecll on enzyme 1ecretlon In tho control acini. These ro1alll oupport tbo concept that enzyme 1ocretlon In tho pancreatic acinar cello Ia mediated by multiple Intracellular pathway• wblcll act In parallel and probably connr1o at a diltal otep In tbe oecretory proceu. Cyclic AMP-mediated amyla1o 1ocretlon In the l1olated pancreatic acini from the CF mica wu not •taU.tlcally different from that obtelned In the a1e· and dlet·matclled control anlmall (n•7-8). Followlq 1thnulallon with combination of BrcAMP and carbachol, there wu a trend for amyluo HCrollon to be enhanced In CF pancreatic acini compared to the llqald·fod controll (NS), However, CF pancreatic acini lbowod • •Ianlftcandy araater amylaH roopouo to the combination of carbacbol and cAMP than the 1um of the Individual rupoue1 In 1eparate experlmenll (P<0.05). Tllil difference In amylue reapoDIO ,... not oblened In acini from either 1olld·fed or llquld·fed controll. In 1ummery, doll 1tudy ougelll that CFTR l1 not o11antlal for enzyme oocrellon 11 evidenced by no reduction In cAMP-mediated am)'lala 10crotlon In CF mice; however, It II rocoanizad that cAMP may act via other Intracellular pethway1. Furtilermoro, CF pancreatic acini axblblted a oyneralltlc oecretory r01poue followln1 BrcAMP· and carbachol-mediated 1tlmulatlon. Tile precilo meclaanilml for tbll obHrntlon remain to be elucidated. Supported by CCFF. •CCFF landed po~tdoctoral follow.

67 AcriON OF GENISTEIN ON CFI'R a· CHANNELS IN PLANAR LIPID BILAYERS. A.A. Aleksandroy. Y.·X. Hou, L.A. Aleksandrov, X.-8. Chang, and J.R. Riordan. Mayo Clinic, Scottsdale, AZ, USA.

From a pharmacological point of view, CFTR is distinguished primarily by the paucity of drugs that have either potent or selective actioN on it. Genistein is one of the few compounds that has been found to reproducibly activ.ate CFTR. Although proposed to do so indirectly by altenng the phosphorylation state of CFTR, recent evidence has suggested that the compound may interact directly with CFTR! as it d~ with several other nucleotide binding proteliUI that 1t modulates. We have examined its mfluence on individual CFTR channels in planar lipid bilayers. To avoid confusion between effects on CFTR phosJ?horylation and other modes of action, we employed a constitutively active form of the molecule, in which serine residues at eight dibasic, PKA coiUieiUius phosphorylation sites ~ere converted to f.utamic acid (SSE): Without added PI<A, Its P 0 inaeased w1th A TP concentration according to a function well fit bv a Hill equation with a slope of approximately 1 and a I<m "' 40 jiM. Phosphorylation by PKA further increased the P0 of SSE to a value similar to wild-type CFTR but the apparent affinity for A TP was not altered. At room temperature, 70 11M genistein increased the P 0 and length of opening of either unphosphorylated or phosphorylatea SSE channelS approximately 3-fold. To test whether the ct~l may act at the nucleotide binding folds (NBFs) of mutatiOIUI in those domaiiUI were introduced into SSE. For example, with 8SE/K1250Q, whi~ .exhibited a prolonged o~ state, no response to gerustetn could be detected, suggesting but not proving that

NBF2 may be one site of action. These experiments support an action of genistein independent of CFTR phosphorylation and mediated by alteration of the state of an NBF. (Supported by NIDDK of Nlli).

68 Actlvadon of CFI'R By Pharmacological Modulators. L Al­~akklllhk and T.-C. Hwang. Dalton Cardiovascular Research

r. esearch Park. University or Missouri-Columbia. Columbia. Missouri. 65211.

Utilizing the cell-attached patch-clamp technique we have examined !it• effects of several pharmacological reagents upon CFI'R activation m NIH3T3 cells stably transfected with wild-type (WT)-CFI'R or AF508-CFI'R. The drugs investigated were: deltamcthrin (a phosphatase 28 inhibitor), cytochalasin D (an actin filament disrupter), 8-cyclopentyl-1,3-dipropylxanthine (CPX, a selective A1-adenosinc· rec~pto.r antagon!st), ~ilrinon~ .<a class I~I Jilhosphodicsterase) and gcmstcm (a tryosmc kmase mhtbttor). Apphcallon of 10 jiM forskolin (a saturating. concentration. yeilding maximal cAMP-stimulation), to cells expresst!lg WT-CFI'R mc~s channel activity. The effect of the phannacologtcal agents menlloned above. was mvestigated in the pre~e!lce of 10 11M forskolin. Deltamethrin (I ).IM) exerted no addtllonal effect. Cytochalasin D (5 J,IM), CPX (25 jiM}, milrinonc (100 J,IM) and genistein (20 J,IM) increased the open probability by 1.27 ± 0.18 (n=4), 1.33 ± 0.27 (n=9), 1.43 ± 0.27 (n=S) and 2.95 ± 0.29 (n=IO) fold r:cspectively. In the presence of 100 nM forskolin, a greater potenllallon of forskolin-dependent CFI'R activity -observed. Cytochalasin D, CPX and genistein increased the open probability by 2.26 ± 0.48 (n=4), and 2.79 ± 0.98 (n=4) and 11.23 ± 4.~7. (n=7) fold respectively. Preliminary data with deltamcthrin and mtlnnone suggest they follow the same trend as CPX and cytochalasin D. After CFI'R channel activation induced by forskolin flus each drug. we monitored the deactivation phase of CFI'R channe current upon removin1 ~onkolin in ~e continuous presence of individual agents. Deltamcthrin, cytochalasm D, milrinonc and genistein all failed to prevent complete channel deactivation. However, CPX alone sustained channel activity. These data suggest that different mechanisms are involved in modulation of CFI'R by these drugs. In cells expressing AF~8-C::FI'R cultured at ~7"C, we observed that followmg the apphcatton. of 10 11M forskohn, cytochalasin D (S J,IM), CPX (25 J,IM), deltamethnn (I jiM) and genistein (20 11M), increased the open probability by 2.S6 ± 0.54 (n=4), 1.30 ± 0.33 (n=3), 1.64 ± 0.31 (n=3) and 21.11 ± 4.27 (n=4) fold respectivel)l. Preliminary data with I 00 11M milrinone suggest it has little or no effect. We conclude from our da~ that. in ~ur cells. of the agents tested, genistein is the most potent m acuvatton of CFI'R, in both WT- and AF508·transfected NIH3T3 cells, and thus presents itself as a potential pharmacological agent in CF therapy. (This work supported by the NIH CF Foundation). '

69 CPX, OTIIER ALKYL-XANTIIINES AND ADENOSINE DIFFERENTIALLY BIND TO TilE WILD TYPE AND ~508 MUTANT FIRST NUCLEOTIDE BINDING FOLD (NBF-1) DOMAINS OF CFI'R. B.E.Cohen1

, G.Lee1, K.A. Jacobson2, Y-C, Kim2, Z.Huang', EJ.Soncher' and H.B. Pollard1

•4• Lab. Cell Bioi. Genetics, NIDDK,

NIH, Bethesda, MD 20892, ~b.of Bioorganic Chemistry, NIDDK, NIH, Bethesda,MD 20892, 1Depts.of Physiology and Medicine, U.A.B., Birmingham,AL., Dept.of Anatomy and Cell Biology, Uniformed Services University School of Medicine, Bethesda, MD. 20814

CPX (1,3-dipropyl-8-cyc!opentylxanthine) and other alkyl·xanthincs stimulate Cl' efflux from cells bearing the homozygous ~F508 mutation common to most cases of cystic fibrosis. We have hypothesized that the CFTR molecule itself might be the site for CPX action, perhaps in the region of the first nucleotide binding fold (NBF-1) domain. Therefore, to test this hypothesis direcdy we have used a rapid membnne fdtration assay to measure the kinetics of association and dissociation of eHJ-CPX to both recombinant wt-NBF-1 and recombinant NBF-1 bearing the ~508-mutation. We report that rHJ-CPX binds with higher affinity to the ~508-NBF-1 domain of CFI'R CK.t=l.O nM) than to wt-NBF-1 of CFTR CK.t=t7.0 nM). These K.. values were corroborated by equilibrium-binding experiments, which gave very similar values. We

have also measured the relative displacement of various xanthines and adenosine from both wt-NBF-1 and 6F508-NBF-t. For wt-NBF-1 the rank order of affinity was 1,3-diallyl-8-cyclopentylxanthine> 1,3-diallyl-8-cyclohexyl-xanthine(DA.'I{) > CPX> caffeine>adenosine >> IBMX> 2-thioCPX. For 6F508-NBF-1 the rank order of aftinity was DAX>CPX>caffeine>1,3-diallyl-8-cyclopentylxanthine>adenosine >>IBMX >2-thioCPX. These relative potencies show close parallels with previously observed relative potencies of these drugs on CF cells, and thus lend strong support to the hypothesis that the mechanism of action of CPX and related xanthines on CF cells may involve direct interaction with the CFTR molecule itself.

70 IBUPROFEN INHIBITS CFTR-MEDIATED cr SECRETION. D.C. Devor and B.D. Schultz. Dept of Cell Biology and Physiology, School of Medicine, Univ. of Pittsburgh. Pittsburgh, PA.

High-dose ibuprofen bas been proposed as 1 pharmacological therapy in cystic fibrosis (CF) based on its ability to slow the annual rate of change in forced expiratory volume in one second (FEV 1) in patients with mild lung disease (K.onstan ct aL, N. Engl. J. Med. 332: 848, 1995). We determined the acute effect of ibuprofen and salicylic acid exposure on cAMP-mediated Cr secretory responses (lsc). In T84 cells, ibuprofen inlubited the forskolin­dependent lac in 1 concentration-dependent manner. The data were fitted by 1

Micbaelis-Menten relation with an apparent K. of 142±32 11M (IFS) and 1 predicted maximal inlubition of 68'Yo. Similarly, salicylic acid inlubited lac with an apparent K. of 646±32 j!M {1F5) with a predicted maximal inhibition of 74%. We dctennined whether ibuprofen would inhibit also the forskolin-mediated lac in primary cultures of mouse trachea epithelia {MTE) and human bronchial epithelia {HBE). Following inlubition of Na• abs01ption with amiloride, forskolin increased lac 29±4 jlA/cm2 in the MTEs and this response was inhibited an average of 59±4% by SOO 11M ibuprofen {rFil). Similarly, in the HBEs forskolin increased Ioc by 16±4 jlA/cm2

,

subsequent to amiloride, and this was inhibited 39±6% by SOO 11M ibuprofen {1F8). These compounds also inlubited Cr currents in CFTR-expressing ~ oocytes, suggesting that ibuprofen and salicylic acid inhibit CFTR. Therefore, we determined the effect of ibuprofen {300 liM) on CFTR in excised. insicJc.out patches from L-cells expressing CFTR. In 3 patches, ibuprofen reduced CFTR cr current by 60±16% and this was explained by an apparent reduction in aingle channel amplitude from 1.07±0.04 pA to 0.59±0.04 pA. Similar results were obtained with salicylic acid {3 mM); CFTR cr current was reduced by 50±8% {n=4) with an apparent reduction in single channel amplitude from 1.08±0.03 pA to 0.48±0.06 pA. Based on these results, we conclude that the nonsteroidal antiinflammatory drugs (NSA!Ds) ibuprofen and salicylic acid inhibit cAMP-mediated Cr secretion in colonic and airway epithelia via 1 direct inhibition of CFTR cr channels. This may reduce their efficacy in conjunction with other therapeutic strategies designed to increase CFTR expression and/or function in secretory epithelia. {Suppl by CFF 8Jllllt5 DEVOR 96 PO and SCHUL 96 PO).

71 ASPIRIN INHIBITS CfTR EXPRESSION IN T-84 CELLS. M. Legros, F. Brouillard, D. Tondclier and A Edelman. INSERM U. 467. Facultt! de Ml!declne Necker, 156 rue de Vaugirard, 75730 Paris cedex IS, FRANCE.

Inflammation takes a large pan In Cystic Fibrosis symptomatology, and both steroldtan and non-steroidian antiinflammatory drugs are included In CF therapies. Since Inflammatory c:ytokines such as Interferon y (IFNy) and Tumor Necrosis Factor a (TNFa) have been shown to decrease genic and functional CfTR expression In T-84 cells, the question was asked whether the non-steroidian antiinflammatory drugs (NSAIDs), and especially aspirin, might antagonize the IFNy- and TNFa-induced CFfR inhibition.

Addition of aspirin (from J0-4 up to 2. to-3 mol/1) to the culture medium of the T-84 cells not only failed to prevent the decrease In CfTR expression Induced by IFNg, but also produced the same effect. Indeed, Northern blot analysis showed that 10" 3 mol/1 of aspirin strongly decreased (by about 80%) the cell coment of CfTR mRNA after a 20 hour Incubation. The immunoblot analysis using anti-R domain CfTR antibodies showed that the amount of 170kD protein was decreased after 48 hours of treatment with the same concemration of aspirin. Functional analysis using SPQ and 36a· assays Indicated that aspirin also Inhibited the cAMP

Poster Session Abstracts 229

fluxes. The decrease In CFI'R mRNA content could be seen after a 4 hour Incubation with aspirin and was additive with that Induced by IFNg. As with the latter, it also appeared to be due to enhanced mRNA degradation.

Other NSAIDs such as salicylic acid, Ibuprofen or sulindac reproduced the inhibitory effect of aspirin, as did high concentrations of indomethacin (5. JO-S mol/1). However. the Inhibition of a cyclooxygenase activity and the suppression of endogenous prostaglandin symhesis do not seem to be directly responsible for the NSAIDs-induced decrease in CFTR expression, since exogenous PGE2 also decreased the cell comem in CFTR mRNA and was unable to prevem the effect of the NSAIDs.

The modulation of CFfR expression thus has to be Included in "the nuclear effects • of aspirin. the molecular mechanisms of which are not clearly defined. This data constitutes new evidence of the adaptative propcnics of CFfR expression in epithelial cells.

72 ACTIVATION OF AF508-CFTR BY GENISTEIN, MILRINONE, JBMX, CPX, AND NS004. W W Reenstrn and S. Raman Dept. of Pediatrics, duPont Hospital for Children, Thomas Jefferson Univ., Wilmington DE, 19803

Genistein (gen), a soy derived isonavone, potentiates cAMP-dependent activation of both wild t)pe (wt) and AF508-CFTR by a mechanism that involves inhibition or channel dephosphorylation. In this study the effects of gen on wt• and 6FS08-CFTR activity were compared 10 those or milrinone (mil), isobutyl-methyl xanthine (IBMX), 8-cyclopentyl-dipropylxanthine (CPX), and the substituted benzimidazolone NS004. Channel activity was assayed by I -efflux in stably transfected NIH3D cells expressing either WI (NIH-CFTR) or AFSOS (NIHAFS08). The basal rate or I -efflux from Nlli-CFTR cells wu 0.06±0.0 I min'1• In the presence of I ~ rorskolin (fsk), SO~ gen. and both agonists rates of0.80±0.04, 0.41±0.02, and 0.79±0.03 min·' were obtained. Like gen. mil, !BMX. CPX. and NS004 did not potentiate the fsk-dependent wt-CFTR activity. For NIHAFS08 cells rates of l~mux (in min"') are shown below.

+fsk +gen +fsk +gen control 0.10±0.01 0.13±0.01 0.12±0.01 0.33±0.01 mil 0.13±0.01 O.IS±O.Ol 0.11±0.01 0.31±0.01 IBMX 0.16±0.01 0.26±0.01 0.22±0.01 0.34±0.01 CPX 0.12±0.01 0.19±0.01 0.12±0.01 0.33±0.01 NS004 0.11±0.01 0.33±0.03 0.17±0.02 0.34±0.03

These results demonstrate that gen. mil, !BMX, CPX, and NS004 potentiate the fsk-dependent activity of AFS08-CFTR. Since mil. IBMX, CPX. and NS004 don't increase AFS08-CFTR activity in the presence of gen plus fsk, and only IBMX (known to increase cAMP) potentiates gen'(fependent activity, gen. mil, JBMX, CPX and NS004 are likely 10 act on the CFTR by a common mechanism.

While SO ~ gen prevented cell growth, 10 ~ gen increased doubling times by <IO'Vo. Alter 3 days with 10 ~gen. basal rates ofl-efflux in NIH-AFSOS cells were unchanged, but the fsk'(fepenclent rate (0.28±0.02 min"1

) was significantly greater than that for untreated cells (0.13±0.02 min"1) or the rate when 1 ~ fsk and 10 ~ gen were added IOgether (0.20±0.01 min"1

). Titis suggests pretreatment with gen doesn't increase the activity of AFS08-CFTR, but potentiates cAMP­dependent activation. If AF508-CFTR can be targeted 10 the plasma membrane gen. or compounds like gen. may be of therapeutic benefit in the treaunent CF. (Supponed by Nemours Foundation and CFF.)

73

EFFECT OF NON-SULPHONYLUREA HYPOGLYCAEMIC AGENTS ON CFTR Cl" CHANNELS. D N, Shepoard and K.A. Robinson. Molecular Medicine Centre, University of Edinburgh, Edinburgh, UK.

The benzoic acid derivatives meglitinde and repaglinide are non-sulphonylurea hypoglycaemic agents that inhibit ATP-sensitive K• channels (K.,., channels). The chemical structure of meglitinide represents the non-sutphonylurea moiety of glibenclamide: repaglinide is a new hypoglycaemic agent that inhibits Km channels in pancreatic Ji-cetls with potency similar 10 that of glibenciamicle. To understand better how sulphonylureas inhibit CFTR, we investigated the effect of meglitinide and repaglinide on CFTR cr channels in excised inside-out membrane patches from Cl27 cells expressing wild-type CFTR. The pipette (external) solution contained 10 mM cr and the bath (internal) solution contained 147 mM cr, PKA (75 nM), and MgATP (0.26 mM) II 37" C; voltage was ·50 mV. Megtitinide (SO. 1000 .,M) caused a concentration'(fependent decrease in CFTR cr current with half maximal inhibition at 288 :t 17 11M (n • 6) and a Hill coefficient of 1.0 :t 0.1 (n • 6); inhibition was readily reversible. To understand how meglitinide inhibited CFTR. we studied single channels. Meglitinide caused a small decrease in current amplitude, but a dramatic change in gating behaviour that was characterised by a large decrease in mean burst duration and a small decrease in mean closed~time between bursts. These results suggest that meglitinide is probably an open-channel

230 1997 Cystic Fibrosis Conference

blocker of CFfR. In contrast 10 glibenclamide, repaglinide (I 0 - 200 jlM) failed to inhibit CFfR cr curren!S. However, it did alter the single-channel activity of CFfR. First, repaglinide caused a small decrease in current amplitude ( 18 :t: I% at 200 jlM; n = 2). Second, repaglinide decreased both mean burst duration and mean closed­time between bursts. However, open probability (P.) did not change (control, P0 = 0.38 :t: 0.03; 200 jlM, P. = 0.39 :t: 0.06; n = 2). These resui!S suggest that the effect of repaglinide on CFfR differs from ilS effect on KATP channels. The data also suggest that the sulphonylurca moiety is not essential for the interaction of hypoglycaemic agenlS with CFfR. Supported by the BBSRC and CF Trusl.

74 FUROSEMIDE AND BUMETANIDE BLOCK CFI'R CI­CHANNELS. Charles J. Vepglarik, Department of Physiology and Biophysics and Gregory Fleming James CF Research Center, University of Alabama at Birmingham, Birmingham AL, USA.

Blockers are needed to uniquely identify CFTR, and to further our understanding of CFTR structure, gating, and regulation. Blocker development may also lead to new classes of drugs will that benefit some CF patients by opening CFTR. My previous studies showed that the diuretic, metolazone, blocks CFTR to a subconductance level with kinetics that reveals two distinct open states. Thus the initial goal of the present studies was to investigate the structure-function relation of the metolazone-CFTR interaction by testing the loop diuretics, furosemide and bumetanide, as possible CFTR blockers. Patch clamp studies were performed using human wt-CFTR expressed in mouse L cells. Addition of furosemide or bumetanide to the solutions bathing the cytosolic surface of excised inside-out patches caused a new population of blocked events to appear in the current recordings. Unlike metolazone, the blocked events evoked by the loop diuretics were indistinguishable from the closed current level. The concentration-response relationships for inhibition of channel open probability were consistent with Michaelis-Menten inhibition functions having K.'s of 40 and S 11M for furosemide and bumetanide respectively (-60 mV). Unexpectedly, these Kt's are similar to those reported for inlubition of transepithelial a- secretion. Since it is possible that the loop diuretics are excluded from the cytosol by the plasma membrane, the CFTR channel activity was then monitored in cell-attached patches as a bioassay for the presence of cytosolic blocker. Addition of 100 11M furosemide or bumetanide to the extracellular bath caused CFTR blockade in cell-attached patches. Thus, in addition to identifying a potent new blocker of CFTR (bumetanide), my data suggests that inhibition of transeplthelial a­secretion by loop diuretics can be mediated, at least in part, by blockade of the apical membrane ct· conductance as originally hypothesized by Candia et al. (Am. J. PhysloL 240: P25-29, 1981), many years prior to the identification of CFTR. These data also raise an Important question regarding the specificity of the loop diuretics for the Na'{IC{CJ· cotransporter. (Supported by the CF Foundation).

75*

Fluid and Electrolyte Transport

NASAL PD MEASUREMENTS SUGGEST TRANSCELLULAR ABSORmON OF SODIUM AND CHLORIDE. Hoat flscher Beate Dlek, llld JOIIIdwl Widdicombe. Children's Hospital Oakland Relearcblllltltute, Oakland CA 94609, USA

Owlq to tile obtlervadon lbat tile NaCl content of the airway surface liquid Is elevated In CF, tile llrway epithelium has been pro­poled to lblorb bodl Na• and CJ- by transceUular routes. To te1t this hypothesi~ wo performed nuaJ potenUII difference (PD) meaaure­meallln bealllly ldult vo111111eer1. We JDeiiUied c:1wJaes lD PD aeuer­lted by amllodde llld by removll of a- rrom buffers. Soludona were latlow-preheatecl to -34"C. 1be laiUII buellne PD with N&Cl buffer Wll-14.9±1.2 mV (a-IS, Inside nepdve). Pertualon with amllortde (50 pM) depolarized PD to -5.8:t;1.2 mV, removll ofCJ· hyperpolar­Ized PD to -15:t;2.1 m V, and perfusion of Isoproterenol (I 0 jlM, an

activator of cAMP/PKA pathway) In CJ·-free buffer resulted In a further hyperpolarization to -21.8±3.5 mV. These maneuvers were used to discriminate PDs due to a Na• conductance from PDs due to a CI- conductance. 1be amplitudes of amlloride-sensiti ve PDs correlat­ed tightly with PDs generated by removal of CJ- under both unstimu­lated (M'Da-rr.e=-1.3±0.4 M'D..;to• p<(J.05) and Isoproterenol-stimu­lated conditions (M'D;..=-0.6±0.2 .aPD _;to• p<ll.05) such that noses with high amlloride-sensltive PDs expressed also high Cl--free PDs. lbls suggested a parallel regulation of both conductances. which Is a pre-requisite for a NaCI-absorptive epithelium that moves both Ions transcellularly. When Isoproterenol was added during perfusion with oormal NaCI buffer, nasal PD depolarized (by 1.3±0.2 mV, p<0.05) consistent with the opening of an apical membrane Cl- conductance and gradient-driven a- absorption aaoss the apical membrane. There­

fore the normal nasal epithelium likely shows transcellular movement of CJ- under both control and cAMP-stimulated conditions.

Thanks to M.M. Reddy for helpful discussion. Supported by CFF and CFRI.

76

CFfR's FAST GATE IS CAUSED BY AN INTRACELLULAR NON-DIFFUSIBLE FACTOR. Horst Fischer Children's Hospital Oakland Research Institute, Oakland CA 94609, USA.

CFIR shows slow and fast gating, which are described by -I Hz and -100 Hz Lorentzlans In the frequency domain. The fast gate (I.e., the 100Hz Loreatzlan) Is strongly voltage-dependent and Is lost after excision In patch clamp experiments. We have previously shown that a tyrosine kinase Increases the activity of the fast gate, and It also has been suggested lhar a blockei-Uke, small-molecular anionic factor may be Involved. We tested whether the fast gate Is sensitive to dialy­sis of the cell to verify the diffusibility of this factor. I obtained an open cell-attached conllguratlon by penneablllzlng the cell in the cell­attached mode with a-toxin, which forms pores with a cut-off at -4 kD (successful permeablllzatlon was vedfted by stimulation of CFIR with free cAMP and A1P-sensltivlty). Open cell-attached recordings were made on Calu-3 and 3T3-wtCFIR cells. Current noise spectra of CFJ'R-actlvtty were recorded with standard cell-attached and the open cell-attached patch clamp configuration. CFTR's fast gate Including its voltage-dependence were totally unaffected by permeablllzlng the cell. Corner frequencies and Intensities of the fast gate were: cell-attached. 119:t;ll Hz, 47.9:t;16'll>; and open cell-attached, 126±18 Hz, 42.8±3.5'll> (pooled -65 to -15 mV). At positive potentials the fast gate was reduced close to background noise levels In both recording modes. In addition, cuiTCnt-voltage relations of multi-channel record­Ings rectified similarly both In the cell-attached and the open cell­attached mode (but not In the excised mode). Since In the open cell­attached mode small molecules and Ions can be expected to diffuse freely Into and out of the cell, these dala suggest lhar CFIR's fast gate Is caused by either a diffusible factor lhar Is bigger than the pore creat­ed by a-toxin, a bound non-diffusible factor, or presents Intrinsic properties of CFIR.

Thanks to Terry Macben for support. Supported by CFF and CFRI.

77* Coordinated Reauladon or Amlloride Sensitive Na+ and CFTR CJ· Conductancea by PKA Phosphorylation in the Native Sweat DueL M.M. Reddy and P.M. Quinton. Department of Pediatrics, Univ. of California, San Dieao. CA- 92103-0831.

Amiloride senaitivo Na+ channels are cruclll for epithelial Na+ absorption. It was shown in ex vivo model systems that amlloride sensitive Na+ channel& are reaulated by PKA phosphorylation and ~rotein ((Smailov eul. 1994; Stutta, eul; 1995). It was

that CFI'R may exen inhibitory control on amiloride sensitive Na+ channels. However, little Ia known about the mechanism or regulating these Na+ channela In native epithelial tissues. We used

basolaterally a-toxin penneabilized sweat ducts as our model system. We followed changes in Na+ conductance (CJN8) as amiloride (10"5 M) sensitive apical membrane conductance and Na+ diffusion potential (generated by 140 mM cytoplasmic KGlu I 150 mM luminal NaCI). CFI"R Ga was monitored by measuring the apical conductance and CJ· diffusion potentials in the presence of luminal amiloride. As previously reported, we found that in the absence a- in the medium, application of 10"~ M cAMP (in the presence of 5 mM ATP) had little effect on GNa (2.8 ± 2.1 mSJcm2, n =12 with cAMP n 2.3 ± 1.2 mS/cm2, n=9, without cAMP; Mean± S.E, n= number of ducts from a minimum of three human subjects). In contrast, when Cl- was present in the luminal perfusate, cAMP significantly increased amiloride sensitive DNa (7.0 ± 2.5 mS/cm2 with cAMP n 1.6 ± 1.2 mS/cm2 without cAMP). Similarly, as reported earlier (Reddy and Quinton, 1992), cAMP also significantly increased CFI"R Go simultaneous with the activation of GNa· We conclude that the amiloride sensitive GNa is regulated by PKA phosphorylation. Such regulation is directly and I or indirectly dependent on the presence of CI- in the medium. The amiloride sensitive GNa and CFrR Ga are simultaneously upregulated by the same intracellular messenger. The absorption of Na+ and a- are controlled together in the same but not in the apposite direction as previously suggested. We thank Mr. K. Taylor for expert technical support. Supported by grants from CFRI and the CF foundation.

78* NITRIC OXIDE-DEPENDENT REGULATION OF AMILORIDE­SENSITIVE SODIUM ABSORPTION IN MURINE AIRWAY EPITHELIAL CELLS. T 1. Kelley and M.L. Drumm. Department of Pediatrics, Case Western Reserve University, Cleveland, OH.

In addition to showing increased chloride secretion in mouse nasal epithelia in response to both class ill phosphodieste~ inhibitors (~~­inhibited PDEs) and to the guanylate cyclase agorust C-type natnurel!C peptide (CNP), we have demonstrated that trans-epithelia! sodium absorption in the nasal passages of wild type and C~ null nuce can be down-regulated by increasing cGMP production w1th CNP. CNP depolarizes lumen negative nasal potential difference (PD) 1.8 ± 0.6 (n=6) mY in wild type mice and 6.8 ± 0.3 (n=4) mY in null mice .. Since airway production of CNP is localiz.ed to vascular endothelial cells and macrophages, another source of cGMP p~~tion may be respo~ible ~or the tonic regulation of sodium transport m auway ep1theha. N1tnc oXIde (NO)-dependent activation of soluble guanylate cyclases may be a candidate for such a role. Other reports have shown that exhaled NO levels from CF patients are reduced compared to non-disease controls and patients with other inflammatol)' airway diseases such as asthma. We report that mice homozygous for either ~ null or AF508 C~ muta~o~ have significantly reduced NO producbon (measured as rutratelrutnte concentrations in BAL fluid) in response to lipopolysaccharide (LPS) from P.seudomofiiU aeruginosa or to the nitric oxide synthase substrate L­arginine when compared to levels produced by non-CF mic~. ~onsistent with these findings, in vitro measurements of NOS acbvtty ID soluble protein preparations from whole trachea excised from either null or AF508 homozygous mice showed significant decreases in specific activity compared to wt/null or wt/t\f508 mice •. respectively. Our. data al~o indicate that a chronic loss of NO produebon could lead to an mcrease m arniloride-sensitive sodium absorption through cGMP-dependent mechanisms. Exposure to the inducible nitric oxide synthase (iNOS) inhibitor S-methylthiourea (SMT) resulted in an increase of basal nasal PD from -8.1 ± 0.7 mY to -15.1 :1: 1.2 mV (n=4) in normal mice. Inhibition of NO-dependent guanylate cyclase activity by the compound ODQ resulted in a hyperpolarization of lumen negative PO by 4:9 ; 1.8 mY(n=5). Similar results were obtained by the inhibiton of protem kin~­G activity with the inhibitor Rp-8-pCPT -cGMPS. Nasal PO changes m response to each of these inhibitors were arniloride-sensitive. These data suggest that a chronic loss of NO production by epithelial iN OS may be in part responsible for the hyperabsorption of sodium characteristic of CF airways. (ThiJ work aupponed by a JTIIll from NIDDK and CFF).

79*

APICAL L-ARGININE ELICITS SIIORT-CIRCUIT CURRENT INCREASE IN POLARIZED MONOLA YERS OF NORMAL AND CF BRONCIIIAL EPITIIELIAL CELLS. L.J.V Galietll1. L. Musante•, S. Lantorol, A. Gazzolol, L. Romano·!, G A. Rossi2• 0. Zegarra­Moran•. •Lab. di Genetica Molecolare, 2Divisione di Pneumologia, 3Centro fibroli Cistica. 1st. Giannina Gaslini. Genova. Italy.

Poster Session Abstracts 231

Ussing chamber experiments, performed on polarized monolayers of human bronchial epithelial cells, showed that apical but not basolateral application of L-arginine (10-1000 11M) elicited a sustained and dose-dependent short-circuit current (11c) increase. The dose-response plot in non-CF epithelia gave a maximal current Imax•3.2±0.3 11A/cm2 and a half effective concentration ECso=81.0±8.3 11M (nc8). Similar values were found for CF cell monolayers, Imax=2.9±0.811Aicm2 and ECso=l09.3±1S.6 11M (n-3). We asked whether the lsc: increase could be due to the activation of ion transport by arginine-dependent nitric oxide (NO) production. Indeed, recent data indicate that the NO-synthase might be present in airway epithelia (Guo et al., Proc.Natl.Acad.Sci USA 92:7809-7813, 1995). Nevertheless, the application of a NO-synthase inhibitor such as Nil-nitro L-arginine methyl ester (L-NAME, 500 11M) did not prevent the L-arginine effect. Moreover, the NO donor S-nitroso-N­acetylpenicillamine (SNAP, 100 11M) was able to produce only a slight and transient increase oflsc:. not accounting for the large current increase elicited by L-arginine. Apical L-lysine, which is not a NO­synthase substrate, activates Isc with lmax=3.9±0.5 11Afcm2 and ECso=75.0±7.3 11M (n"'4). The neutral amino acid proline was also effective, although at higher concentrations (lmax•3.0±0.2 11Aicm2 and ECso=971.3±183.4 11M, n=J). The L-arginine-dependent Ioc seems to be Na•-dependent since the substitution of NMDG+ for Na+ strongly reduced lmu· In conclusion, our data suggest the presence of an electrogenic amino acid transporter in the apical membrane of normal and CF bronchial epithelial cells. This transporter could be involved in the maintainance of an optimal concentration of amino acids in the mucociliary fluid.

This work was supported by Cy.ft ic Fibrosis foundation.

so* CFTR MEDIATES DUODENAL BICARBONATE SECRETION BY TWO DIFFERENT MECHANISMS. M.C. Har1ine and L L. Clarke, Dalton Cardiovascular Research Center and Dept. of Veterinary · Biomedical Sciences, University of Missouri-Columbia, MO. USA.

We have previously reported that CFTR is required for cAMP­stimulated electrogenic bicarbonate secretion across the murine duodenum. In the present study, we investigated the role that CFTR plays in the mechanisms of cAMP-stimulated bicarbonate transport by using pH stat methodology on murine duodenum mounted in Ussing chambers. With physiologic Ringer in the serosal bath and saline in the luminal bath, the serosal-to-mucosal flux of HCOi (J..., MC03) across nonnal [CFTR(+)] duodenum was 0.84 :1: 0.15 IJeq/cm"·h and the short-circuit current (1 .. ) was 2.24 :t 0.461Jeq/cm'·h (n=4). Forskolin (10 IJM) treatment increased J., MC03 and 1 .. by 1.84 :1: 0.08 and 2.51 :1: 0.39 1Jeq/cm2·h, respectively. In CFTR(-) mice, basal J., HC0

3 was 0.60 :1: 0.06 IJeq/cm• h and 1 .. was 0.04 :1: 0.36 IJeq/cm"·h (n=3). Forskolin had no effect on J., HC03 but caused a small increase in 1 .. (Aloe= 0.51 :1: 0.27 IJeq/cm'·h). To inhibit the activity of luminal membrane Cl" /HCOi exchange, CFTR(+) duodenum was bathed with cr free solution in the luminal bath. Under this condition, the basal J., MC03

was 0. 78 :1: 0.14 and the 1 .. was 5.13. :1: 0.65 IJeq/cm'·h (n=9). Addition of forskolin increased the J., HC03 and 1 .. by 1.38 :1: 0.26 and 1.88:1: 0.481Jeq/cm2·h, respectively. In CFTR(+) duodenum, preliminary studies indicated that pretreatment of the luminal membrane with NPPB (300 IJM) essentially eliminated forskolin­stimulated He~· secretion and 1 .. in either the presence or absence of luminal Cr. We conclude that cAMP-stimulated duodenal bicarbonate secretion does not involve direct activation of luminal Cl"/ HCOi exchange in the absence of CFTR. However, In CFTR(+) duodenum, cAMP-stimulated HCOi secretion: (1) partially (25%) results from luminal Cr/ HC03" exchange operating in parallel with an activated CFTR cr conductance; and (2) mainly (75%) results from a He~· conductance mediated by CFTR. Supported by NIH Grant DK48816.

232 1997 Cystic Fibrosis Conference

81* EFFECT OF Ct AND HCO,· TRANSPORT BLOCKERS ON SUBMUCOSAL GLAND DUCf MUCIN CONTENT IN ACETYLCHOLINE-TREATED BRONCHL S K loalis. M.R. Corboz, S.T. Ballard. Department of Physiology, College of Medicine, University of South Alabama, Mobile AL 36688, USA.

Pretreatment of excised distal bronchi with a Cl· secretion inhibitor (bumetanide)and three putative HCO,· secretion inhibitors [dimethylamiloride (Na'IH' exchange blocker), acetazolamide (carbonic anhydrase inhibitor), and D!DS (CI"IHCO,·exchange blocker)) prior to acetylcholine (ACh) application causes mucin accumulation within submucosal gland ducts. This response presumably represents an uncoupling of ACh-induced gland liquid and mucin secretion. The aim of the present study was to determine which of the HCO,· secretion inhibitors, in combination with bumetanide, was required to cause gland duct occlusion. Distal bronchi (2-4 mm) were isolated from porcine lungs and incubated for 30 min at 37•c with either (i) drug vehicle (dimethylsulfoxide) (ii) IO~<M bumetanide (iii) bumetanide + IOO~<M

dimethylamiloride (iv) bumetanide + lmM acetazolamide (v) bumetanide + I mM DIDS or (vi) all4 anion secretion blockers. ACh (I Oi<M) was then added. After 30 min, the bronchi were fixed, paraffin embedded, sectioned, and stained for mucins with alcian blue and pyronin Y (pH 2.5). The quantity of mucin in each gland duct was scored on a linear scale from 0 (no mucin present) to S (duct completely filled with mucin). In bronchi pretreated only with drug vehicle, average duct mucin content was 0.66:1:0.07 (n=6). The mucin content of gland ducts pretreated with either bumetanide alone (1.33:1:0.46, n-6), bumetanide + acetazolamide (1.9S:t:0.4S, n•6), or bumetanide + D!DS (1.32±0.4S,n-6) was not significantly different from gland ducts pretreated with drug vehicle alone. In contrast, duct mucin content in bronchi pretreated with bumetanide + dimethylamiloride (3.61:1:0.21, n-6) or all four anion secretion inhibitors (3.60:1:0.09, n-6) was significantly greater(P<O.OS) than all other pretreatments. These data suggest that ACh-induced gland liquid secretion is driven by activeCt and HCO,· secretion and that Na'IH' exchange is required for the HCO,·-dependent fraction of gland liquid secretion. (Supported by NIH HL-48622)

82* ROLE OF CHLORIDE AND BICARBONATE IN ACETYLCHOLINE-INDUCBJ LIQUID SECRETION FROM PORCINE DISTAL BRONCHI. S I Ballard, L. Trout, S.K. Inglis, and M.R. Corboz. Dcpanment of PhysioiO&Y, University of South AlabM!a, Mobile AL 36611 USA.

Previous IIIUdics have shown thl1 the liquid secretion response of distal bronchi to acetylcholine (ACh) is blocked by putalive inhibitors ofbolh Cl· and HC0,·1n1115p0r1.

· The present study was undertaken 10 confirm that ACh induces secretion of these two anions. Distal bronchi were excised from pip and placed in Krebs buffer containing either 10 pM bumetanid! (10hich inhibits Cl· secretion by blocking basolateral Na'-K'· 2Ct cotran~), 100 11M dimethylamiloride (DMA, which inhibits HCO,· secretion by blockin& basolateral Na'/H' exchange), or the drug vehicle dimethyl sulfoxide. Airways wmethen cleared of luminal liquid, cannulated,reimmerscd in the Krebs buffer, and treated with I 0 pM ACh. After 2 hrs, luminal mucus liquid was collected, weighed, and analyzed for ion composition. The rate of ACh-induced mucus liquid secretion in control airways was 0.245±0.0 I 5 ~&Vcm'·min (n•l5). Liquid secretion rate was only sligbtlyreduccdbydimethylamiloridepretreatment(0.203:1:0.035pVan'-min,n•IO)but was significantly(p<.05) reduced by bumetanide prctreatment(0.066:1:0.001 11Vcm'·min, n•IO). Pretreatment with bolh bumetanide and DMA produced an even greater significant reduction In liquid secretion rate (0.023±0.009 J1Vcm2·min, n•5). Ionic composition of the mucus liquid Is shown below. Because of the small volume of secretion from bumctanlde and DMA-pretreated airways, ionic analysis was not ~onned on these umples.

Treatment Na'(mM) K'(mM) Ct(mM) HCO,·(mM)

ACh 133.9:1:5.0 14.2:1:0.4 127.3M.4 27.6±1.4

ACh+DMA 131.8:1:6.3 13.5±0.5 139.1:1:8.9 20.7:!:2.4

A.Ch+Bumet 139.1:1:6.0 15.6±1.0 116.0:1:7.2° 63.2:1:5.0 ..

• litpnflclnt dtfrerence from ACb+DMA 00 aiplifbnt ditr_. from ACh and AOI+DMA

DMA ~CIIIIIIda 8nall iac:rase In Ct concentraliln and a small reduction in HCO,· concentnlion. In COIIII'Ut, bumetanlde pretreatment induced a significant decrease in Ct conc:entnltion and asipifbnt increase in HCO,· concentration. We conclude fiom these datathlt approximately70%ofthe llquidsecretionresponseto ACh Is due 10 bum81anlde-senaltiveCt secretion while the residual liquid secretion is largely due to DMA-sensltive HCO, · secretion. (Supponed by NIH HL48622)

83*

INHIBITION OF ACETYLCHOLINE-INDUCED LIQUID SECRETION IN DISTAL BRONCHI AND ITS EFFECT ON RHEOLOGICAL PROPERTIES OF AIRWAY MUCUS. L. Trout, M. King, W. Feng, S T Ballard,. Department of Physiol~. University of South Alabama, Mobile, AL 36688. USA and Mucobiology Research Unit, University of Alberta, Edmonton, Canada T6G 252.

The combination of both CJ· and HC01· secretion inhibitors causes accumulation of mucins within submucosal gland ducts of acetylcholine(ACh)-treated bronchi (Inglis et al., Am. J. Physiol. 272:L372-377, 1997) suggesting indirectly that these agents block airway gland liquid secretion. The present ·study tested the hypotheses thai ACh­stimulated liquid secretion is driven by ct· and HCo,· secretion and that inhibition of this process leads to secretion of a dehydrated mucus with altered rheological propenies. Distal bronchi were excised from pigs, placed in 37"C Krebs buffer, and pretreated with either a combination of Cl' and HCO; secretion inhibitors [O.QJ mM bumetanide, 1.0 mM acelmllamide, 0.1 mM dimethylamiloride (DMA), and 1.0 mM 4.4' -diisothiocyanosrilbcne-2,2"-disulfonicacid (DIDS)) or the drug vehicle. Airways were then cleared of luminal !icpd, tanlllllacd, reimmerscd in KldJs buffer, am treated with O.QJ mM ACh. After 2 brs, luminal mucus liquid was collected. and we1 and dty weights were determined. The rate o: ACh·irouced lllliCUSiiquid secretion in comoJ airways was 0.2.59±0.044 ,.Ucm'-min (n •12). However, when airways were pretrealtld wilh the Cl' and HCO; secretion inlullitors, the rate of ACh-induced mucus liquid sccmion {0.018±0.005 ,.Ucm'-min, n= 12) WIW martcdly mb:ea (P < .OS). Mucus liquid from inhibilor pretreated airways contained significantly more nonvolatile solids (4.31 ±0.49%, n=10) compared to control mucus liquid (I.SS±O.OI %, n- 10). A magnetic microrheomctricanalysis revealed that mucus liquid from inhibit<r pretreated airways expressed significantly greater G• (rigidity factor) at driving frequencies of I radl~ and 10 radls while tangent ~ (recoil factor) wu significantly reduced at 10 radls. These results suggest (I) that ACh-induced liquid secretion in bronchi is driven by both Ci· and HCO; secretion and (2) that inhibition of ACh-inductd liquid secretion results in the secretion of a dehydrated mucus with altered rheological propenies. We conclude that a defect in airway anion secretion, u occurs in cystic tibrosis, could lead to the production of an abnormal mucus. (Supported by NIH HL 48622 and Canadian CF Foundation)

84* ELECTROLYTES OF LIQUID ABSORBED OR SECRETED BY CANINE AIRWAY EPITHELIA B. R. Grubb. F.R. Schiretz, C. Freiberg, J. I. Gatzy, R.C. Boucher. Cystic Fibrosis/Pulmonary Research and Treatment Center, Univ. of North Carolina at Chapel Hill, Chapel Hill, N.C. 27599

Airway epithelia play a role in regulating the volume and composition of the airway IUiface liquid (ASL). However, the ionic composition of ASLin normal or cystic fibrosis subjects is debated, and little is known about regulation of liquid t.lance IICI'OIS the epithelial borrier. Canine tracheal and bronchial epithelial cells were JI'OWII on the lumenal walls of small diameter, permeable tubes (biofibers). We assessed volume transpon (lv) and modulation of lumenal solution composition by the airway cells grown in the biofibers. Six biofibers with confluent tracheal epithelia thl1 were treated with amiloride (llr M, lumen) and forskolin (lo·• M, outer bsth) secreted liquid at 2.0S:I:O.I7 111 cm·'h"' (means± SEM) (n-6 ) over a S h period. [Na1 and [K1 of liquid in the lumen after 5h did not differ significantly from the bsth solution (Krebs Ringer bic:arbonatc, KRB), but the [CI") was signlrtcantly greater (KBR•II9 :1: 1.7, (n-6); lumen•l33.ll2.2 mM, n-6). This pattern suggests that liquid secretion into the electronegative lumen is driven by Cl" active transport. Secreted liquid was isosmotic (KBR-276±1.4, (n-9); lumen-281M mO.m, (n-9)). Untreated bronchial epithelia spontaneously absorbed liquid (lv- 4.76:1:0.9~&1 cnr'h"'). Liquid that rctnained in the fiber lumena after 5 h was chiJ'aelerized by a significantly lower [Na'): lumen•l20.8 :1:5.6 mM, (n•IO) and [CI"]: lumen-99.4:1:5.1 mM, (n•l)than thl1 of the bath K.BR (Na' • 140 ± 4; Ct • 120 :1:2.1; n-1). Lower lumenal [Na1 and (CI") probably resuhcd from active absorption ofNa', wbich, with Ct, provided the driving force for liquid absorption. The [K1 that remained In the lumenal liquid (18.9 :1:0.4 mM. n•l) was significantly greater than thltofthe bsth KBR (5.2 :1:0.34, n•l). The magnitude ofthe lnlllseplthclial PO was significantly correlated with the lumenal [K'] (slOp or,.._ flow exps), suggestin& thl1 K' distribution IICI'OSS the epithelium is determined by the elcctric:al gradienL Preliminary m-uremonts of lumenal liquid indic:ate that the volume remainin& (and the absoltlatc) were isosmotic. The absence of a stady­"* 01111otic gradient IICI'OSS airway epithelia thl1 are secreting or absorbin& volume Is supported by the high osmotic water permeability (Lp -4.6 x I 0' cmsecr'atm"1)ofCII'Iinc tracbcal epithelia grown on fibers and exposed to 90 m0.111 of raffinose added 10 the bath. We conclude thl1 proximal canine airway epithelia JI'OWII on biolibers can secrete or absorb an isosmodc saline solution, and cannot maintain a measurable transepithelial osmotic gradient. In addition, like the renal dillal tubule, K' distribution IICI'OSS absorbing airway epithelia Is determined mainly by the transepithelial PD. Supported by the CFF.

85* Analyala of Airway Surface Fluid Volume and Chloride Concentration In CF and Non-CF Human Bronchial Xenografta. Yulong Zhang, James R. Yankaskas, and John F. Engelhardt. University of Pennsylvania Medical Center and University of North Carolina at Chapel Hill.

Measurements of fluid transport In CF and non-CF xenografta have been previously reported by this laboratory and demonstrate a four -fold higher rates of fluid absorption In CF (208+/·32 nl min·1 cm·2) as compared to non-CF (52+1·13 nl min·1 cm-2) xenografts. In an effort confirm these differences In fluid transport between CF and non-CF airways, we have developed an ahemative assay for measuring the steady state equilibrated surface airway fluid (SAF) volume of air-exposed xenograft&. This alternative methods also provides the additional attraction allowing for Ionic composition measurements of SAF. The lumenal SAF of fully differentiated 5 weeks human bronchial xenografts (air filled) was collected by perfusion with 100 ul of a 5% (iso-osmotic) dextrose or mannitol solutions containing a known amount of H3·1nulln to calculate dilution of surface airway fluid. SAF volume (V s) were calculated based on the dilution of H3-lnulln and corrected for volume lost (Vi) during perfusion. These calculations demonstrate surface airway fluid volume of CF (28+/-3.0 uVcm2; N•17) Is significantly less than that of non-CF (35+/· 2.4 uVcm2; N=30) xenografts. Calculation of V8 were then used to detemnine the chloride concentration of SAF [CI)s using a solid state AgCI electrode. Comparisons of SAF fluid chloride concentrations using lao­osmotic dextrose perfusate from CF (124+/-6 mM; Ns17) and Non-CF 114+/·5 mM; Na30) were not significantly different (p>0.136). Similarly, comparisons of [CI]s using !so-osmotic mannitol purfusates demonstrated no significant differences between CF (127+1-8 mM; N•10) and Non-CF (114+/·6 mM; N•16) xenografts (p>0.423). Combined mannitol and dextrose data reached significant differences between CF (125+/-4 mM; N=27) and Non-CF (114+1·4 mM; N-48) xenografts (p<0.045). Atthough these resufts suggest a trend toward higher [CQ In SAF of CF airways, differences saen in this model are smaller than previously reported using alternative approaches.

86* IS MOUSE AIRWAY SURFACE n..UID HYPOTONIC? E A Cowley, K. Govindaraju, D.K. Uoyd and D.H. Eidelman. Meakins-Christio Laboratories, Montreal Chest Institute Research Centre, McGill University, Quebec, Canada.

Tho apical IUrfaco of respiratory epithelial cells is covered by a thin layer of low-viscosity Ouid termed airway IUrface Ouid (ASF). Relatively little is known about tho composition and regulation of this Ouid, due to difficulties associated with collection and analysis of IUCh ama1l liquid quantities. It has been reported that in tho normal healthy lung. ASP is hypotonic to plasma and that its ionic composition is altered in cystic fibrosis (Joriset a/. Am Rev Respir Dis 148: 1633), IUCh that Na. and cr concentrations are elevated. We have previously described a technique in which we were able to collect rat ASP and analyse samples using capillary electrophoresis (Transfiguration et a/. Anal Chern 67; 2937). Recently, wo have modified this approach to allow us to collect ASF from mice. Malo CS1BII6 mice (6-10 weeks old) were anesthetised, tracheotomised, and a 6cm length of polyethylene tubing (PE 10 tubing) inserted into the tracheotomy opening so that tho end of the tube was in contact with the epithelium at tho lower end of tho trachea. This sampling capillary remained in situ for approximately 30 minutes, after which it was removed and the Ouid sample (between I 00-200nl) analysed by capillary electrophoresis with indirect UV­detection. Using this method we determined values for the major ions to be: Na• • 88.5 ± 6 mM, K·· 6.2 ± 0.4 mM (n • S mice) and cr • 56.8 ± 3.4 mM (n-6 mice). Mouse ASF appears to be hypotonic in comparison to plasma, suggesting that tho composition of this Ouid is actively regulated. Furthermore, these values are significantly different from those seen in tho rat, IUggesting that important species differences oe<:Ur in the regulation of this Ouid. (Supported by the Canadian Cystic Fibrosis Foundation and the 1 Costello Memorial Fund)

Poster Session Abstracts 233

87* AIRWAY EPITIIELIA AMILORIDE-SENSITIVE SODIUM TRANSPORT CAN BE REGULATED BY ALDOSTERONE, IN VITRO AND IN VIVO. J Zahner, J Launspach, P. Karp, C Chenard, JB Stokea Ill, and MJ Welsh. Howard Hughes Medical Institute, Department or Medicine, University or Iowa Colleee or Medicine, Iowa City, Iowa USA.

Salt depletion and aldosterone have been shown to increase activity of the amiloride-sensitive epithelial sodium channel in several organs. To study the effect of aldosterone in human airway epithelia, we used primary culture& of human airway epithelia grown at the air liquid mterface. The culture medium was a mixture of 49% DMEM, 49% Ham's Fl2 and 2% Ultraser G. To measure transepithelial elecbical properties, epithelia were studied in Ussing chambers. Amiloride (10 J.LM) was added to the mucosal solution to inhibit Na+ channels and cAMP agonists, 10 JIM forskolin and 100 JIM IBMX, were added to stimulate ttan.sepithelial a· current Removal of Ultraser G from the media for four days did not alter cAMP-stimulaled a· transport, ~ithelial resistance. or morphology. However, amiloride sensiuve current decreased by 93%. Aldosterone ( 10·' M) increased the amiloride-sensitive current from 1.2 ± 0.33 J.lA/cm2 to 3.6±.2 J.lA/cm2 (p<O.OI). The effect of aldosterone was not blocked by 10·' M spironolactone but it was inhibiled by the steroid receptor antagonist RU 38486. These data suggest that the amiloride-sensitive Na+ transport can be regulated by aldosterone. To test this hypothesis ill vivo, nasal wltage measurements were perfonned in 6 nonnal volunteers randomized in a crossover study to be on a 0 NaCI diet for 6 days or 10 g NaCI for 6 days. Urine measurements, confll111ed the suppression and stimulation of aldosterone secretion. The change in nasal voltage afler the addition of amiloride was significantly higher on the low salt diet (S.7S±o.1 mY) vs. the high salt diet (3.6±0.5 mY) p<O.OS. The change in voltage with terbutaline in low chloride and with A TP were not differenL These data suggest that airway epithelia Na+ transport can be regulaled both ill vitro and ill vivo by aldosterone through the glucocorticoid receptor.

88 LACK OF AIRWAY EPITIIELIA AMILORIDE­SENSITIVE SODIUM TRANSPORT RESULTS IN IMPAIRED BACTERIAL KILLING. LZillmel:, J Smith and M1 Welsh. Howard Hughes Medical Institute, Department of Medicine, University of Iowa College of Medicine, Iowa City, Iowa USA.

Pseudobypoaldosteronism (PHA) is an uncommon inheriled disorder characterized by salt-wasting and unresponsiveness to mineralocorticoids. Recently, mutations in the alpha, beta and gamma subunit of hENaC have been identified as the genetic basics of PHA. Four patients with severe pseudohypoaldosreronism have been described with recurring lower respiratory infections that resemble cystic fibrosis (CF). We hypothesized that similar to CF, a decrease in amiloride­sensitive Na • transpon in airway epithelia, would alter the Naa concentration of airway surface flwd (ASF) and inhibit bacterial killing by ASF. We used primary cultures of human airway epithelia grown at the air-liquid interface. The culture medium was a mixture of 49% DMEM. 49% Ham's F12 and 2% Ultraser G. To measure ttan.sepithelial electrical properties, epithelia were studied in Ussing chambers. To decrease amiloride-sensitive Na • transport we used two interventions. First, we added 10 JIM benzamil to the serosal side. This bloclced amiloride sensitive Na• transport, probably by diffusing to the mucosal side. Second, we removed Ultraser G from the media for four days. This did not alter cAMP-stimulated a· transport, ttan.sepithelial resistance, or morphology by scanning electron microscopy. However, amiloride-sensitive current decreased by 93%. Thus these interventions provide a model for PHA. We found that 23 of 24 control epithelia killed an innoculum of P. Mruginosa. In contrast only 3 of 18 epithelia grown on USG-free media, and only the 2 of 18 epithelia incubaled with 10 JIM benzamil killed bacteria. In vitro quantification of the ASF killing activity, obtained by washing the mucosal side, was identical in the three groups. These data suggest that inhibition of Na • transpon by nonnal airway epithelia may result in modifications of the ionic composition of the ASP similar to CF and may predispose to bacterial infections.

234 1997 Cystic Fibrosis Conference

89*

DEXAMETHASONE INCREASES SODIUM TRANSPORT IN HUMAN NASAL EPITHELIUM IN VIVO. T Hofmann. C.E.Foy, J.M.Roblnscn, V.Homolya. R.C.Boucher, MR.Knowles. CF-Center, UNC at O!apel Hill, North Carolina, USA

In human airway epllhella, Na • ablorplioo is the dominam basal active loo transport system. However, the regulalion of Na• transport in the lung is not fully understood. We reponed previously that eJIOFIIOUS mineraloconicolds do not affect epithelial Na• transport. The present study was designed 10 test the effect of topically applied Jlucocortlcoids oo airway epithelial Na• transport We aiJo tested the effect of the mineralocorticoid­antagonist, spironolactooe. In a blinded fashioo, de:wnelhasooe (I <rM, in iscxooic saline) versus vehicle, llld spilalolactone (Sx lifM, in 0.2% cdlanol) vemJS vehicle, were ldminislered 10 the nasal epithelium of nonnal subjects (n-8, each) under direct vlsioo by nasal spray (0.2Sml every ISmin) for 8hrs. The effect of drup were mooilo=l by moelectric Indices of Na • transpon, i.e. basal nasal transeplthclial porenlial difference (PD), and inhibitioo with amiloride (10~. a Na• channel blocker. Naaal PD was measured at basellne and 11 2,4,6,8, and 24hrs, and epithelia were sampled 10 estimate EN&C mRNA levels by RT-PCR. Rellllts: After Bhrs of doslnJ, mean PD under the turbinate increased significantly from baseline with dexamelhasooe, but not vehicle (~-11.4 ±2.SmV, dex; ~-3.4±2.2mV, veh. p<O.OS), but amlloride lellsitivity was unc:lunged (~+2.8±1.4mV, veil; ~+8.2:i4.7mV, dex, p=O.OB). PD tended 10 lncreaae oo the medial surface of the turbinate (~-S.8±2.2mV, dex; ~-1.6±2.8mV, veh., poo0.13). Spironolactone did not Inhibit basal Na•transpolt as canpared 10 vehicle, as ISIIelled by basal PD under the turbinate (~+ 1.8±3.0mV, veil; ~.().6±2.0mV, spiro) or oo the medial surface of the turbinale (~-1.8±1.7mV, veh; 4-2.5±1.4mV,spiro). Amiloride sensllivity was WICbmged (4+3.4±2.9mV, veil; 4+2.7±3.0mV, spiro). We conclude that basal airway Na• transport can be upregulared by exogenous glucocorlicoids, but is not modified by mineraloconicoids or mineralocorticold-arttagooists. The uncoupling of airway epithelial Na• transport from body vdume homcostalic (MC-medlated) mechanisms may proteCt airway surfaces fran dehydradon. Support: NIH-HL34322, RR00046

90* IDENTIFICATION, FUNCTIONAL CHARACTERIZATION, AND INITIAL LOCAUZATION OF THE HUMAN LUNG PKA- AND pH-ACTIVATED C1C-2G(2a) Cl" CHANNEL. A.M. S!JwrY, K. Stroff~ LM. Knapp, E.Y. Kupert, P.M. Barker, S.E. Gabriel , 1. Cuppoletti, li D.H. Malinowska. Dept of Mol & Cell Physipl, Univ ofCinclnnati Col of Med, Cincinnati, OH 45267-0576 and -cp Research Center, Univ of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7020.

J. partial eDNA for C1C-20(2a) a- channel was cloned from a human fetallunclibrary and lipted to mutaaenized expressible rabbit C1C-2G(2a) eDNA to construct a fulllen&th expressible human ClC-20(2a) eDNA. This hC1C-2G(2u) eDNA was identical in amino acid ~e~~uence to that found in T84 cells (Cid et al, Hum Mol Gen 4: 407, 199!J). The objective of this study was to characterize hClC-20(2a)'s elcctropbysiolo&ical properties at the sincle channel level, rqulation by PU and pH, and tissue localization. hClC-2G(2a) was expreued in Xenopus ooc:ytes and reconstituted into planar lipid bilayers for functional studies. PKA added to the cytosolic face of the channel increased the channel open erobability (Po). With PKA present, hClC-2G(2a) Cl" channels exhibited a linear (r•0.99) 1-V curve with a alope conductance of 22.1 ± 0.8 J)S in symmetric 800 mM TEACl, pH7.4. A reversal potential of +21.5 ± 2.8 mV was measured under !J fold padlent cOnditions of TEACl, demonstratin& Inion to cation discrimination. Similar to rabbit, hClC-2G(2a) was active at extracytosolic (trturs) pH's of both 7.4 and 3.0. pH1NlJI determined the extent of chanild activation b:f PKA. Thus, Po increaJed 4 fold with pH 7.411D11.r and 8 fol with pH 3.01rtUIS. Sequential effects of PICA and pH on the same channel sugested that PKA- and pH-dependent increases in channel Po were separable and cumulative. Thae Cl" channel characteristiCJ were not si&nificanlly different from those previously described for the rabbit or the human/rabbit chimeric ClC-2G(2a) Cl" channels. Northern blot anal)'sia showed hClC-2G(2a) mRNA to be present in human adult and fetallunc and adult stomach. Using quantitative RT-PCR, adult luna contained about half the mRNA level of this channel compared to fetal luna. Preliminary data sugests that in fetal mouse lunc the chanllel is localized to airWay epithelial cells. These data suggest that

the hCIC-2G(2a) cr channel may play an important role in cr transport in the fetal and adult human lung. Supported by NIH DK43816, DK433n, CFFR4S7 & CFFCUPPOL96PO to JC & OHM; and CFF to SEG. AMS was supported by DK07727.

91* PHARMACOLOGICAL CHARACTERIZATION OF NUCLEOTIDE RESPONSES OF AIRWAYS FROM P2Y2 RECEPTOR (-I-) MICE. L Homolya. B. R. Orubb, E. R. Lazarowski, R. C. Boucher, B. H. Koller. Cystic Fibrosis Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

P2Y 2 receptor ( -1-) mice were generaled to defme the role of the purincrgic rccepton in regulating ion tnnsport in murine airways. Purincrgic receptor stimulated ea2+ signalin& was studied in nasal and in tnlcheal epithelial cells isolated from P2Y2 receptor (+I+) and(-/-) mice. In parallel, a· secretory responses to nucleotide agonists were studied in freshly excised tracheas from wild-type(+/+) and knockout(-/-) animals. In terms ofea2+ mobilization, UTP was effective only in P2Y 2 (+(+)cells. UTP induced a· secretioo in tnlchcas was reduced by 83 % in P2Y 2 (-I-) as compared to wide-type responses. Major reductions (75%) were also observed in both Ca1+ and a· secmory responsca to ATP in the P2Y1 (-/-) tnlcheal epithelia. Similarly,the Ca2+ response to A TP was significantly smaller by 66.0 % (p<O.OS) in the P2Y2 (-/-)nasal cells. The residual response to ATP in cells from P2Y1 (-/-)

mice was insensitive to lldcnosine dcaminase, and adenosine was ineffective in the P2Y 2 ( -1-) cells, indic.ting that A TP induced responses were not a COJIIeQuence of IICtivation of PI receptors. The P2X receptor agonist, u.P­meATP, did not stimulate ea1• mobilization or a· secretion in the P2Y1 (-/·) epithelium. The ea2+ response to A TP was essentially unchanged in the ablence of external ea2+. showing that elevated cytosolic ea2+ reflects mainly release from internal stores. Thus, we conclude that the residual ea2+ response 10 A TP in the P2Y 2 (-I-) cells is due to the expression of another P2Y receptor. A series of desensitization studies suggested that besides the dominant P2Y 2 receptor, only one other purinergic receptor type is expressed in the tracheal and nasal epithelium. The following rank order of potencies was revealed in the P2Y 2 ( -(-) cells: 2-McSADP > = A TP >= 2-McSA TP > ADP » UDP, UTP. This pharmacological profile is consistent with the expression of a P2Y 1 receptor. Adenosine 3' ,S' -diphosphate, a recently reported P2Y 1 antagonist, blocked the ea2+ response to A TP in P2Y 2 (-I-) cells. In summary, we conclude that P2Y 2 is the major receptor mediating nucleotide induced a· secretion in airway epithelia. Supported by R026 from Cystic Fibrosis F oundtulon.

92*

STIMULATION OF Cl' SECRETION BY CHLORZOXAZONE, AN ACTIVATOR OF BASOLATERAL MEMBRANE Kc. CHANNELS. A...K. ~ D. C. Devor, A C. Gerlach, J. M. Pilewski, R. A. Frizzel~ and R. J. BJ'idaes. Cell BioJoay ud PltysiolOIIY, University of PilllburJib, Pitlsburlb. PA IS26l,USA

We previously delmastrated tbat 1-EBIO (1-etbyl-2-beazimidazolooe) directly activates the buolateral menilraDe Kc. (K.-490 11M) tbercby stim.JlatiJia Cf secreDOIIIClllllll several epithelia, iDcludiD& bwJan coloaic cells (T84),1nmanairway cells (Calu-3) as well as priJmry cultures of Inman airway cells. In our punuit Ill ideDti1Y more poteDt Jllldllaton of cr secrctioa, we aearehed several databues, iDcludiD& tllole wbich list aU tbe FDA approved dtuas for CCllqJtJUIIds lltrUciUrllly siJDlar Ill 1-EBIO. Tbls search identified a CCllq)OUIId called Cblorzoxazooe (S-chloro-2(3H)-beazoxazolooe, Parqfon Forte®), which il a ceatrally-8CtiJ!a aaent used cliDically as a IIUICie relaxaat We used several epitbelia iDcludiD& T84 cells, Calu-3 cells as well as priJmry cuhurel of bumut airway cella to dentoallrate tbat cblorzoxazooe bas a similar respoase profile ud mechaDilm of actioa as 1-EBIO tollimllate cr secreti011. For !belle studies, we used 200 11M cblorzoxazooe; a typical plasml CODCelltlltioa reported in petieDts takiDa cblorzoxazoae. Chlorzoxazoae stim.Jlated cr secrelioa iD T84 and Calu-3 moaolayers as well as primuy cultures ofbumut airways. As true oftbe respoase tol-EBIO, thiB cr secretory respoase was blocked by cbarybdotoxiD (CI'X). FollowiDa penneabilizatioo of tbe apical madnDe of T84 celll ud primuy cultures of Iaman airways with Dyslalia ud establilluneat of a JJ~KU~-to-- K• pldieDt. cblorzouzooe stim.Jlated a buolateral meaaane Ox. which was iabibited by CI'X. Ia meniJraDe vesicles Jll1lpll'ld ftoom T84 cells, cblorzoxazooe stim.Jlated -.u, • uptake iDa CI'X-8CI18itive 11111111«. Ia excised, iDiidc-out .. tcbell fhlm aU three cell lilies IIBIIioned above, cblorzoxazooe aclivated aa iawanlly-rectifYiDa K•

channel, Kc.. which was inhibited by CTX. 6-Hydroxychlorzoxazone, the major metabolite of chlorzoxazone, did DOt activate the basolateral merrbrane Kc.. whereas, zoxazolamine (2-amino.S..:hlorzoxazole), which bas a stronger

electron donating group at the 2-positioo was equipoteot as chlorzoxazone in activating this channel in excised merrbrane patches. Since chlorzoxazone is already an FDA approved drug, it could potentially be used off-label as a therapeutic agent for patients with CF. [Supponed by the Cystic Fibrosis FOUD<Iation (1974, E841)and the NIH (OK 45970, OK 31091)).

93* MAXIMIZING G!!!llD CFfR Cr CURRENTS WITH mMX, GENISTEIN AND NS004. ~ Schultz, C. Liu, D.C. Devor, H.L­Balombiny, R.A. Frizzell, and R.J. Bridges. Department of Cell Biology and Physiology, University of Pittsburgh. Pittsburgh, PA, USA.

GSS JD CFTR has been shown to traffic to the cell membrane normally, but to exhibit reduced open probability when compared to w/CFTR and reduced sensitivity to stimulation by IBMX when expressed in Xenopus oocytes. We evaluated the effects of compounds reported to increase CFTR P • either by altering the channel phosphorylation state (IBMX/forskolin, genistein) or by direct interaction with the channel protein (NS004). Double electrode voltage clamp recordings were made 2 to 8 days after injection of Xenopus oocytes with wl ( 4 ng) or GSS ID (27 ng) CFTR cRNA. Results show that neither genistein nor NS004 significantly affected wl or GSSID CFTR Cr conductance when used alone or following maximal stimulation with IBMX (I mM). However, exposure to a threshold concentration of IBMX (100 JJM) revealed a subsequent stimulatory affect of genistein (I 00 JJM) from 14.4±8.4 1JS to 24.8±11.8 1JS (nzS) indicating that genistein requires a submaximal level of PKA activity and/or CFTR phosphorylation for effects to be observed. G551 0 CFTR-expressing oocytes exhibited significantly reduced cr currents in response to maximal stimulation by IBMX (S mM) and forskolin (I 0 j!M; GSS ID, 9.2±0.9 1JS vs. wl, 79.2±8.5 J.iS; n-42 and 34, respectively). Subsequently, GSS ID CFTR cr conductance was increased by approximately 2-fold by the addition of either genistein (I 00 !1M; 2 I. 7±4. 7 J.iS; n-6) or NS004 (30 JIM; 20.8±5.8 IJS; n•S). The effects of genistein and NS004 appear to be additive since, when used in combination, conductance was increased to 32.9±6.5 ~tS (n-9). These results indicate that, in contrast to wiCFTR, G5SID CFTR is not maximally stimulated by saturating concentrations of IBMX. We conclude that, compared to wiCFTR, an activated (phosphorylated) state of GSSID CFTR is more likely to inactivate/dephosphorylate or that GSS ID CFTR enters a non­conductive state that is refractory to IBMX (and hence 'annot be maximally stimulated by PKA). Genistein or NS004 can interact (indirectly or directly) with this state to induce or stabilize a conductive state and thus increase GS51D CFTR cr conductance bringing it closer to that of wtCFTR.

Supported by the Cystic Fibrosis Foundation (SCHUL960).

94 TRANS EPITHELIAL BLOCKER INDUCED NOISE ESTIMATES OF CFTR CHANNEL AMPLITUDE, DENSITY AND OPEN PROBABILITY IN T84 MONOLA YERS B D. Schultz. A.!{_ Singh, D.C. Devor and lU. Bridges. Department of Cell Biology and Physiology, University of Pittsburgh, PA 1S261.

Transepithelial noise studies (fluctuatioo analysis) were undertaken to obtain estimates of CFTR channel activity in the context of a chloride secreting monolayer_ Data were acquired u described by Van Driessche and Zeiske (J. Memb. BioL 56:31) and analyzed u described by Helman and Baxendale (J. GCIL PhysioL 9S:647). Patch clamp studies showed the diarylsulfonylures, DASU-1, to be an open channel blocker of CFTR with favorable microscopic rate coefficients for transepithelial aoise studies. T84 monolayers, with a serosal to mucosal chloride gradient, were stimulated with forskolin (2 !1M) and 1-EBIO (I mM). 1-EBIO is a basalateral membrane potassium channel opener (Devor et.al., Am.J.PhysioL 271:L77S) that was required to obtain an improved driving force and a favorable apical membrane fractional resistance_

· The I 0 to 20-fold increase in short circuit current (lsc) upon stimulation was accompanied with a proportional increase in the current power density spectrum (POS) over the frequency range of 0.2S to 300 Hz. Both the Isc and PDS returned to baseline values with bumetanide (20 liM). DASU-1 caused a concentration dependent inhibition of lsc that was well descn'bed by a simple Michalis-Menten inlubition function (K. • 118 liM; v_ • 87%). In addition

Poster Session Abstracts 235

DASU-1 (10-to-120 j!M) caused the appearance of a prominent Lorentzian function in the PDS that displayed a concentration dependent shift in the corner frequency (fc) and power plateau (So). A plot of 2nfc versus DASU-1 concentration was linear and gave an on rate of S.8~tM' 1s'1, an otT rate of296 s·• and a Ko of S I liM. values that arc in good agreement with the patch clamp results. Further analysis yielded: (i) an estimated single chaMel amplitude of 0.1 pA, (ii) a DASU-1 concentration dependent channel density with 2.5 x 109

channels cm'2 (i.e., 2SOO channels per cell) at 0 11M DASU-1 and (iii) a channel open probability of 0.4. The channel amplitude value of 0.1 pA is in good agreement with that expected for CFTR under these experimental conditions. The results indicate 87% of the Isc can be accounted for by current passing through CFTR channels with an open probability of0.4. Supported by NIH and CFF.

95* THE GENISTEIN ANALOGUES APIGENIN AND QUERCE· TIN ACTIVATE CFTR IN VIVO AND IN VITRO, Bca1e lllck_ Horst Ascher and Jonathan Widdicombe. Chlldren's Hospital Oakland Research Institute, Oakland. CA 94609, USA.

We have previously reported that the lsoflavone genistein (4' ,5,7-trlhydroxylsoflavone) stimulates CFTR-medlated Cl secretion by a cAMP-Independent mechanism. In the present study we tested the effects of related flavones on Cl secretion In airway cells (Calu-3, bovine tracheal primary cultures), on acdvatlng 41'508-CFI'R In trans­fected NIH 3T3 fibroblasts and on human nasal potential differences (PD). We used apigenin (4' ,S,7 trlhydroxyflavone) which Is an Isomer of genistein, and quercetin (3,3' ,4' ,5,7 pentahydroxyflavone) which Is available as a dietary supplement. In Usslng chamber experiments, mucosal addition of apigenin or quercetin (10-300 llMl stimulated transeplthellal CI secretion In airway cell cultures In a concentration­dependent manner. With basolateral membrane permeablllzed by a­toxin, apigenin additionally stimulated CFTR-medlated currents across the apical membrane when the cytosollc cAMP and Ca concentration were clamped at maximally effective values- In cell-attached patches apigenin Increased 4FS08.CFTR activity In NIH 3T3 fibroblasts In the

presence of the phosphodiesterase Inhibitor 3-lsobutyl-1-methylxan· thine (IBMX). In vivo effects of quercetin were tested In nasal PD measurements on healthy volunteers. PO measurements were per­formed on the Inferior surface of the Inferior turbinate. The mucosal surface was treated with amllorlde and perfused with a-tree solution. Under these conditions quercetin Increased the nasal PO_ Pretreatment with Isoproterenol did not affect the quercetin-Induced change In PD. We conclude that, In addition to genistein, the flavonolds apigenin and

quercetin can activate aonnal and mutant CFTR by a cAMP- and Ca­lndependent mechanism.

Supported by CFF and DFG-

96* A NOVEL ACTIVATOR OF CHLORIDE TRANSPORT IN CF CELLS AND TISSUES. J R Jones, M.D. DuVall, EJ. Sorscher, J. Maddry. Department of Organic Chemistry, Southern Research Institute and Departments of Medical Genetics, Medicine, and Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL, USA.

Using CFPAC-1 monolayers mounted in Ussing chambers, we have screened 308 compounds from a panel of 1,101 drugs for their ability to activate transepithelial chloride transporL We have identified a novel nitrogen heterocycle, JMS2931, which is capable of increasing short-circuit current (Isc) consistent with an activation of chloride secretion. This activation is abrogated when chloride is omitted from the bath solutions. Similar activation of chloride secretion by 100 uM JMS2931 was observed in primary human airway cells taken from CF patients and grown on permeable supports. The lsc activation in human airway cells was markedly prolonged compared with what was observed using either ATP or UTP (I 00 uM) under otherwise identical conditions. When distal colon from M508 or cftr -/- mice were studied, JMS2931 at 100 uM again led to prolonged Isc activation

236 1997 Cystic Fibrosis Conference

(lasting up to 35 minutes) without a change in tissue resistance. This compound also strongly activates iodide efflux in CFPAC cells and in primary airway cells from CF patients grown on 35 mm culture dishes. We are currently investigating the mechanism(s) by which JMS2931 activates chloride secretion in CF cells and tissues, including studies of the effect on intracellular calcium and cAMP. The effects of BAPT A, an intracellular calcium chelator, and staurosporine, a protein kinase C inhibitor, on transepithelial chloride transport activated by JMS2931 will also be examined. (Supported by CFF).

97

Localization and quantitation of ENaC subunit expression in fetal, newborn and adult mouse lung. P.M. Barker. C.L. Talbot1

, D.O. Bosworth, E.L. Briley, S. E. Gabriel, and R.C. Boucher. School of Medicine, University of North Carolina at Chapel Hill, and Dept Biology1

, California State University, San Bernardino CA. Liquid absorption by the pulmonary epithelium is required for

perinatal transition to airbreathing, and for normal hydration of postnatal airways. Liquid absorption is mediated by active Na• transport via the epithelial Na• channel, ENaC, which is comprised of3 subunits, a, p, and y. Abnormal Na• transport in the lung is implicated in respiratory distress syndrome and in cystic fibrosis. We quantified ENaC mRNA expression levels of fetal, neonatal and adult mouse lung by Northern blot analysis, and studied regional localization by in-situ hybridization. In whole lung, all 3 subunits were detected at low levels by Northern analysis at 1 Sd and 17d gestation. aENaC was expressed strongly at 19d. peaked at ld of life and had a secondary peak in the adult lung. j}ENaC expression increased gradually in fetal and postnatal lung. yENaC expression was similar to that of aENaC. In situ hybridization studies showed a comparable early appearance ofyENaC in the developing fetal lung bud tubules (1Sd), and subsequent uniform intense expression in all regions of the distal fetal and newborn lung. aENaC and j}ENaC were seen from 16d gestation onwards, and were expressed most intensely in small airways. There was almost no expression of j}ENaC in the alveolar region. In postnatal lung, all 3 subunits were expressed intensely in small airways, and aENaC and yENaC were expressed in a pattern consistent with an alveolar Type II cell distribution. The quantitative changes in ENaC subunit expression are consistent with the known role of Na• transport in liquid clearance of the perinatal lung. Intense expression of all 3 subunits in small airways epithelium suggests that this region is a primary location for liquid absorption in the postnatal lung. Variations in intensity of expression and location of ENaC subunits may alter ENaC function in different regions of the lung. Supported by CFF and NIH-HLBl

98 Identifteation or fetallung-speeHic nuclear facton interacting wltb tbe ClC-2 chloride channel promoter Sbjjjan Cbu. Carol B. Murray, and Pamela L. Zeitlin Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD

The CIC-2 epithelial chloride channel is expressed in fetal lung and down-regulated at birth (similar to CFTR). Little is known about specific nuclear factors responsible for this developmental pattern in lung chloride cbannel expression. We have analyzed an 8-kb flanking sequence S' to the rat CIC-2 gene to determine the activity of transcription initiation and the sites for regulation by DNA binding proteins. A series of constructs were generated in the pGL3 plasmid containing the luciferase reporter gene. Transfections were performed in fetal and adult rat type II cell lines. A plasmid containing the CMV promoter-driven 1acZ gene was included to correct for transfection efficiency. A 67-bpcore sequence from -237 to -171 (relative to the ftrst Met codon) was found to promote the highest transcriptional initiation activity. More than 90% of the promoter activity was abolished if this core sequence was deleted from the s· end of the

promoter construct. Inclusion of sequences upstream from -23 7 decreased the promoter activity, indicating interactions of this region with inhibitory factors. In this 67-bp core sequence, three SPI consensus binding sites were present and the third site overlapped with a stem-loop structure. Nuclear extracts were prepared from adult type II cells and fetal pre-type II cells. Using electrophoretic mobility shift assays, we identified several nuclear protein factors interacting with the second and the third SP I sites in the fetal rat pre-type II cell line that were absent in the adult type II cell line. This suggests a potential explanation for the differential transcription efficiency between fetal and adult lung. These results provide initial information on DNA­protein interactions in the ClC-2 promoter which may play a critical role in developmental regulation of CIC-2 expression. (Supported by the CFF and the NIH)

99 HETEROLOGOUS EXPRESSION OF ROMK2 K + CHANNELS IN MAMMALIAN CELLS. David Riochet, Raha Moharnmad-Panah, Vl!ronique Leblais, Steven C. Hebert, Jason Xu Isabelle Bar6 and penjs Escande. INSERM CJF 96.01, Nantes, France and Harvard Medical School, Boston, MA, USA.

ROMK2 is a widely distributed inwardly-rectifying epithelial K + channel that is regulated by intracellular ATP. Previous studies have focused on the expression of ROMK2 channels in Xenopus oocytes (in particular, it was shown that the sensitivity of ROMK2 to the blocker glibenclamide is modulated by the presence of wild-type CFTR). However, a description of the ROMK2 current in mammalian cells that could be used as a template for comparative studies has not been achieved yet. COS-7 cells were intranuclearly injected with 20 J.lg/ml ROMK2 eDNA. Injected cells exhibited a large whole-cell current (86.5 ± 26.5 pNpF at +60 mY; n=6) selective forK+ ions that was never observed in non-injected cells. The amplitude of the ROMK2-related current ran-down during cell dialysis with the pipette milieu: it was reduced to .. 50% of its control value 5 minutes after breaking the patch membrane. ROMK2 current was time-dependent inasmuch as it slowly relaxed with time (inactivation) upon depolarization. Inactivation kinetics were voltage-dependent (the time constant for inac­tivation twas 46 ± 13 ms at +90 mV and 124 ± 23 ms at +40 mV). Upon repolarization, the ROMK2 K+ current slowly increased (removal of inactivation). The slope conductance related to ROMK2 expression increased with increasing extracellular K + concentration. Inward rectification exhibited an instantaneous component and a time­dependent component that corresponded to the inactivation process observed upon depolarization. ROMK2 current was insensitive to: (i) cAMP stimulation with forskolin (10 j!M); (ii) increased intracellular Ca2+ with ionomycin (ij!M); (iii) and charybdtoxin (10 nM) blockade. In inside-out patches, ROMK2 expression led to almost permanently opened K+ channels (Po >0.9 at potentials between -80 and 80 mY) with an unitary conductance of 42,0 ± 1,0 pS at -80 Y (n=5) in sym­metrical 145 mM K+ conditions. We conclude that ROMK2 expression produces K+ channels with biophysical characteristics reminiscent f?r the inward K+ conductance that ts regulated by CFTR expresston m CFPAC-1 cells. However, ROMK2 channels exhibit a regulatory pro­file very different to that of CFTR-regulated inward K + channels in CFPAC-1 cells. S11pport~d by the Alsocitllion Franroise de Lull~ C0111re Ia Mucovilcitl<>se.

100 THE KVLQT I K+ CHANNEL IS EXPRESSED IN HUMAN RESPIRATORY EPITHELIAL CELLS. Sophie Demolombe, Raha Moharnmad-Panah, Pascal Barbry, Jacques Barhanin Karl Kunzelmann, Isabelle Bar6 and Denis Escande. INSERM CJF 96.01, Nantes, IPMC-CNRS, Sophia-Antipolis, France, and Department of Physiology, Freiburg, Germany.

KVLQTI is a recently discovered gene which encodes a cAMP­modulated voltage- and time-dependent K+ current. KYLQTl is expressed at a high level in various organs including the lung. W,e investigated the characteristics of the KVLQT !-related K + current m COS-7 cells as well as the expression of KVLQT I in various epithelial cells. COS-7 cells intranuclearly injected with a KVLQTI eDNA plas­mid exhibited a fast-activating slow-deactivating K + current (activation time-constant: 37.2 ± 3.6 ms and current amplitude at +40 mV: 4.8 ±

0.2 pA/pF; n=29) with an almost linear current-voltage relationship. Cells co-injected with KVLQT I together with the channel regulator lsK cDNAs exhibited a slow-activating outward current (activation time­constant: 166.5 ± 12.0 ms; n=20) with an increased current amplilude (12.8 ± 0.7 pA/pF at +40 mY). At the single channel level, the KVLQTI elementary conductance was below I pS. Reverse transcrip­tase-polymerase chain reactions (RT-PCR) demonstrated that KVLQTI mRNA was present in various confluent epithelial cell lines including T84 and HT29-19A colonic cells, and in CAPAN pancreatic cells but not in CFPAC-1 (M'508 pancreatic epithelioma) or A549 (alveolar type II epithelioma) cell lines. The channel regulator lsK mRNA was never detected in epithelial cell lines. By contrast, respiratory epithelial cells freshly dissociated from nasal polyps and cultured to confluence on permeable filters demonstrated KVLQTI and lsK expressions. Finally, m COS-7 cells, we showed that the chromanol compound 293B blocked recombinant KVLQTI channels with an IC50: 16.5 !IM in the absence of the lsK regulator and 8.0 !IM in its presence (n=4-12 cells per concentration). In Ussing chamber experiments performed in reconstituted human respiratory epithelium, 293B dose-dependently blocked the cAMP-stimulated electrogenic CJ- transport. Our results demonstrate that KVLQTI K+ channels are eltpressed in various human epithelial cells including respiratory epithelium. Their precise role in water and salt secretion remains to be elucidated.

Supported by the Auociation Franraise de l.MIIe Contre Ia M11~oviscidose.

101 Candidate genes for Ca2•-activated cr channels identified by differential display of normal and CF airway epithelial cells. U ~ R.I. Steagall, A. Sannuti, E.L. Briley, L.G. Johnson, S.C. Olsen, and R.C. Boucher. Cystic Fibrosis/ Pulmonary Research and Clinical Treatment Center. University of North Carolina, Chapel Hill, NC. U.S.A.

The differential display technology is a powerful tool used to identify genes that are expressed at differing levels in two closely matched cell populations. We have used this approach to identify candidates for the alternate Ca1•-activated Cl" channel (CIA). Human CF airway epithelial cells, when grown on permeable supports, demonstrate an absence of cAMP-mediated Cl" secretion and the presence of a substantial Ca1

• -mediated Cl" conductance. Over­expression of either CFTR or B-galactosidase (LacZ) as a control, using an adenoviral vector in CF human primary airway epithelial cell cultures results in two closely matched populations that show a difference in the magnitude of CIA activity. LacZ treated cells maintain a CF phenotype and a substantial CIA response, -80 ~cm2

whereas, CFTR corrected cells have a significantly reduced ionomycin-stimulated current, -4 ~cm2• Application of the differential display protocol has revealed more than 30 eDNA clones that are up-regulated in LacZ vs. CFTR treated cells and are therefore, candidates for the CIA Cl" channel. Several of these eDNA sequences show significant homology to previously cloned genes. Among these candidates we identified the cystic fibrosis antigen (CFA) as up­re~ulated in CF epithelia. The CF A is composed of a pair of small Ca + binding proteins and has been shown to be over-expressed in CF epithelial cells. There is, however, no known function associated with this protein. We have verified overexpression of CFA by Northern blot analysis and correlated expression of CFA with CIA activity for a number of different epithelial cell lines. We have also generated 1

stable transfection of the CF A into Caco-2 cells that previously showed no CIA activity. We are studying these cells for Ca1 -activated cr secretory activity and for affects on the Ca2

+ -mediated second messenger pathway. Suppor1ed by the Cystic Fibrosis Foundation.

102 JMMUNOLOCALIZATION OF p ANDy RESPIRATORY EPITHELIAL Na+ CHANNELS IN HUMAN AIRWAYS DURING FETAL DEVEI,OPMENT. *D Gaillard. *J. Hinnrasky, *E. Puchelle, ••P. Barbry. *INSERM U314, IFR .53, Developmental Biology, CHU, Reims, ••institut de Phannacologie Molkulaire et Cellulaire, Valbonne, France

The amiloride-sensitive epithelial Na+ channel (ENaC) subunits have been reported as regulaton of the ion and water composition of the

Poster Session Abstracts 237

respiratory tract fluid and are controlled by the cystic fibrosis transmembrane conductance regulator protein (CfTR). The expression of the human ENaC subunits has been identified by in situ hybridization in the surface epithelium and in the glands of human adult airway tissue (Burch et a/, Am J Physiol, 1995; 269:C511 ). However the localization of the ENaC proteins has never been analyzed in fetal and adult human airways. Polyclonal antiserum against the p and y ENaC subunit protein was obtained after rabbit immunization and the specificity of the antibodies was assessed by immunoprecipitation. The cellular distribution of p and y subunits was. studied by immun~histochemistry using airway paraffin embedded sect tons an~ frozen sections from 8 fetuses, ranging from I 0 to 35 weeks of gestation (WG) and 4 adults. lmmunogold labeling for ENaC subunits was carried out in ultra-thin airway sections collected from 2 fetuses.(21 and 33 WG) and I adult. The p and ysubunits could not ~ shown m young fetuses before 17 WG. At midgestation, p and y subumts were observed m the cytoplasm of ciliated cells (CC) especially in their apical aspect, in a few basal cells and in the cuboid cells lining the glandular ducts of the fetal trachea and bronchi. These two subunits were also present in CC and Clara cells lining the bronchioles. Over 30 WG, the distribution of P and y subunits was similar in fetuses and in adults. In the trachea and bronchi, the two subunits were detected at the apical domain of CC, in the epithelial cells of the ducts and in the serous cells of the glands. In the bronchioles, both CC and Clara cells were immunostained. At any age, the immunoreactivity was always higher with the anti-y subunit than with the anti·P subunit, and no p or y ENaC subunit could be observed in the secretory cells neither at the surface epithelium nor in the mucous cells of the glands. When CFI"R protein is detected in human undifferentiated airwar epithelium, as early as 7 WG (Gaillard eta/, Pediatr Res,l994; 36:137), p andy ENaC subunits appear later, during midgestation, when the airway epithelium is nearly mature. These results suggest that p and y ENaC subunits may regulate airway ion thransport during fetal development. Supported m part by !'Association Fran~aise de Lutte pour Ia Mucoviscidose (AFLM).

103 HORMONAL REGULATION OF ENaC AND CYCUC­NUCLEOTIDE GATED CATION CHANNELS IN SODIUM TRANSPORTING EPITHEUA. Weiping Qiu and Sandra E, ~. Department of Medicine, Sohns Hopkins University, Baltimore, MD.

Cystic fibrosis appears to be an imbalance of multiple ion channels including CFTR, ORCC and ENaC. Since the airway is sodium hyperabsorptive in cystic fibrosis we are studing regulation of sodium channels to determine ways of decreasing this absorption. The amiloride-sensitive sodium channel, ENaC, has been studied extensively in epithelial tissues including the distal tubule of the kidney, the colon and airway epithelia. The message for the (j subunit of ENaC has been shown to be upregulated in the colon of rats fed 1 low salt diet. Under this condition protein for aENaC is expressed on the apical membrane. But, in high salt fed rats the message for tlENaC is not expressed and protein for aENaC is not present at the apical membrane. We repeated these experiments in rat lung and verified similar results. In addition, we found the message for (jENaC in descending colon and lung, was up regulated by aldosterone and down regulated by spironolactone. In airway epithelia, ileum or in the colon of the high-salt fed rat, there is also 1 sodium-mediated short circuit current which is 11Q1 amiloride sensitive. Using ribonuclease protection assay, RT-PCR, and in situ hybridization we have shown that the cyclic-nucleotide gated cation channel (CNGl), is present in airway and intestinal epithelia of the duodenum, jejunum, ileum and colon. This channel, which is blocked by dichlorobenzamil and L-eis diltiazem, is responsible for a sodium-mediated short circuit current in primary cultures of rat tracheal airway epithelia. The message for CNGl is upregulated in lung, ileum or colon of rats injected with aldosterone or fed low salt diet. The message for CNG 1 is downregulated in lung, ileum or colon of rats injected with spironolactone or fed high salt diet. We conclude that CNGl may account for amiloride-insensitive transepithelial sodium transport in lung, ileum and colon and that high salt diet may relieve sodium hyperabsorption in CF.

238 1997 Cystic Fibrosis Conference

104 106 SUBUNIT -sUBUNIT INTERACTION IN a,p,y-rENaC AND REGULATION OF EPITHELIAL Na• CHANNELS BV CFTR lllamoi!oy. B.K.Berdiev, V.Gh.Shlyonsky, C.M.Fuller, "B.Stonton, D.J.Benoa. Deportment of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL, and *Deportment of Physiology Dartmouth Mad. School, Hanover, NH, USA.

The structural basis for the functional interaction between a,p,y. rENaC and CFTR was studied In planar lipid bilayers using a,p,y-rENaC composed of combinations of WT and truncation mutants of a-, p.., and y-rENaC and WT CFTR. Vesicles of Xenopus oocyte plasma membranes served liS 11 source of the channels following heterologous expression of specific combinations of cRNA. We found that a,p,y. rENaC composed of WT a- subunits and P..and y- subunits truncated at their C-terminal regions (R564X-P..rENaC, and W574X-v-rENaC) still can be downregulated 1-30%) by CFTR. On the other hand, CFTR failed to exert Ita inhibitory action on the hatarotrimeric channels composed of either C-terminal or N-termlnal truncation mutants of a­subunit (R613Xa-rENaC and a 61•101 T1 09M-rENaC) in combination with WT p.., and y-rENaC. While C-terminal truncation of a-rENee did not change the properties of the heterotrimeric channels (in combination with WT p.., and y-rENaC), N-terminal truncation of a-rENaC produced dramatic changes in gating of the a,p,y-channel, suggestive of an altered interaction between individual subunits. These results, taken together with our previous observation that CFTR does not affect properties of the channel formed by the a-subunit alone (lsmallov at a/., Am.J. Physlol., 1997, 272:C1077-C1086), indicate the importance of subunit-subunit interaction in the a,p,y-rENaC-CFTR regulatory relationship.

Supported by the CFF grant lsmail9710, and NIH grants DK37206 and DK45881.

105 FUNcriONAL EXPRESSION OF A TRUNCATED Ca'•·DEPENDENT Ct· CHANNEL Hong-lpng Jj M.D. DuVall, C.M. Fuller and D.J. Benos. Dept. of Physiology and Biophysics, Univenity of Alabama at Birmingham, Birmingham, AL, 35294-0005, USA.

We have previously isolated and cloned a ca>+-activated Cl· channel (CaCC) from bovine trachea (C111111Ingham S.A. eta/. J Bioi. Chem. 1995; 270:31016). The native protein migrates at 3 8 kDa on reducing SDS.PAGE. However, the primary eDNA transcript (2712 bp) codes for a protein of approximately I 00 kDa that is not susceptible to reduction by dithiothreitol (011). In order to teat the hypothesis that the I 00 kDa polypeptide is a pre-pro form of the 3 8 kDa native CaCC subunit, we have senerated a mutant construct (CaCCX) truncated at theN (base 838) and C (base 2016) termini (M,-43,000). The whole cell currents expressed in Xenopus oocytes by iJtiection of CaCCX and wt. CaCC cRNAs at +80mV were 2053.7:!: 296.5 (n-7), and 1956.5:!: 282.6nA (n•20), respectively (P>O.OS), markedly higher (P<O.OOI) than those of H10-injected oocytes (152.2 :1: 33.7nA, n•7). All current records were obtained in the presence of niflumic acid (100 J.iM) to inhibit the endogenous CaCC of oocytes as previously reported ( Cwmlngham. S.A. et al. Ibid ). Activated currents in the presence of 2 J1M ionomycin or A23187 were recorded from oocytes expressins both CaCCX (4395.8 :1: SSO. 7nA, P<O.Ol vs control, n•7) and wt. caec (5831 '7 :1: 424.9nA, P<O.O I VS control, n•S) in the presence of I.BmM CaCI2 in the perfusing ND-96 medium. The effects of both D!DS and DTI were tested on the ionomycin-induced ct· currents. 200 J1M DIDS inhibited the currents byagreaterextent(CaCCX: 87.1 s 7.3% inhibition, n=3; CaCC: 86.8 :1: 3% inhibition, n•3) than did 2mM DTI (CaCCX: 72.2:!: 5.82% inhibition, n-9; CaCC:72 s 3% inhibition, n-1 0), indicating that CaCCX was al10 sensitive to DIDS (P<O.OOI) and DTI (P<O.OOI). The dlord conductancea at +80mVofH20, CaCCX, and wt. CaCC injected oocytes were 2.12:!: 0.67, 26.13:1: 3.62, and 31.11 :1: 4.03!'8, respectively. Additionally, the CaCCX and wt. CaCC expressed conductances were not blocked by S mM BaCI2, an inhibitor of the enqenous ca>+ -sensitive K• channel but were dependent on the extracellular CI· concentration. We conclude that the truncated CaCC cRNA forms a Ct· conductance pathway of identical characteristics to the wt. CaCC cRNA when expressed in Xenopus oocytes. Our findings support the hypothesis that the full-length CaCC may be post-translationally processed to a smaller, fimctional form. Supported by NIH Grant DK48764 and funds from the CFF.

PURIFICATION AND FUNcriONAL RECONSTITUTION OF AN OUTWARDLY RECI'IFIED CHLORIDE CHANNEL (ORCC) FROM BOVINE TRACHEAL EPITHELIA. Bi!iana Jovoy Iskander I. lsmailov, Vadim Gh Shlyonsky, and Dale J. Benos. Dept. Physiol. &: Biophys., Univ. Alabama at Birmingham, Birmingham, AL 35294-0005 USA

We have previously reported the identification of three ORCC candidate proteins (II S, 85 &: 52 kDa} from bovine tracheal epithelia (Jovov, et al., .1. Bioi. Chem., 270: I 521, 1995), and raised polyclonal antibodies against these isolated proteins. Incorporation into planar lipid bilayen of material semi- purified from bovine tracheal apical membranes using one of these antibodies as a ligand (anti-pi 15) resulted in the incorporation of an ORCC of identical biophysical characteristics to one we previously described (Jovov, et al., ibid). As we have previously identified the 11 S kDa protein as an ORCC candidate, we have developed a new purification procedure to increase the yield and purity of this polypeptide. We found that optimal solubilization of the liS kDa protein was obtained using 2% CHAPS. The purification scheme that gave the best results in terms of overall protein yields and purity was a combination of anion and cation exchange chromatography followed by immunopurification. Using this purification scheme, 7 11g of the II S kDa protein was purified from 20 mg of tracheal apical membranes. Incorporation of this highly purified material into planar lipid bilayen resulted in chloride channel activity with biophysical characteristics similar to those previously observed with semi-purified ORCC protein, except that the incorporated channel had a linear IN relationship. We have previously demonstrated that treatment of the ORCC incorporated into planar lipid bilayen with PTX resulted in a Joss of rectification of the ORCC. Using specific antibodies, we have tested for the presence of a Gi protein in the semi- and highly-purified preparations. Western blots revealed the presence of a Gi protein only in the semi-purified material. This Gi protein was precipitated with anti-Gia2 antibodies and the remaining material was reconstituted and incorporated into the planar lipid bilayer. Incorporation of this material resulted in the loss of rectification ofthe ORCC, identical to what we had observed with the highly purified ORCC protein. These dsta suggest that the loss of rectification of the ORCC was due to uncoupling ofa Gia2 protein from the ORCC protein, and susses! that the II S kDa polypeptide is an ORCC. Supported by N.J.H. Grant DK48764 and by the C. F. F. Grants: F981, and 9710.

107 A TRANSGENIC KNOCK-OUT MOUSE LACKING THE AIRWAY WATER CHANNEL. Tonghul Ma. Baoxue Yang and A.S. Verl<man. Cystic Fibrosis Research Center, University of California, San Francisco, CA, USA.

It has been proposed that the abnonnal hydration state of fluid lining airways has an important role in the pathophysiology of Cystic Fibrosis. Functional measurements suggest that water transport across airway epithelia is mediated by molecular water channels or 'aquaporins' (Folkesson et al. JCI 97:664·71, 1996). We reported cloning of the mercurial insensitive water channel (AQP4) from rat and human lung (Hasegawa et al. JBC 269:5497-500, 1994; Yang et al. JBC 270:22907-13) and its expression at the basolateral membrane of epithelia In large and small airways (Frigeri et al. PNAS 92:4328-31, 1995). To define the role of AQP4 in airway physiology, a transgenic knock-out mouse lacking AQP4 was generated by homologous recombination. The targeting construct contained a 7 kb mouse genomic DNA fragment and Neo/TK selection cassettes to give an AQP4 intron 1/exon 1 deletion. Analysis of 16411ve births from lntercrossing of AQP4 heterozygote& showed 41 [+/+], 83 [+/·]and 40 [·/·]genotypes. The [·/·] mice expressed small amounts of a truncated AQP4 transcript and lacked detectable AQP4 protein by Immunocytochemistry. Survival, growth and gross appearance of the[·/·] mice were indistinguishable from wild-type mice. Initial phenotype analysis indicated a mild urinary concentrating defect, consistent with AQP4 expression In kidney collecting duct. The mice should be useful to test the hypothesis that high airway water penneabillty has a role In regulating the hydration state of airway fluid. If so, then modulation of airway water penneability might provide a novel strategy to treat the lung pathology In Cystic Fibrosis.

(Supported by DK35124, HL42368, DK47766, HL51854 and CFF).

108 The effect of cAMP· dependent K• channels on chloride secretion In nasal epithelia from normal and CF mice. L.L MacYinjsh, H.I. Durrington, A.W. Cuthbert, Dept. of Pharmacology, University of Cambridge, UK

CF airway epithelia (nasal and tracheal) show reduced cAMP­mediated chloride (CI") secretion (stimulated by forskolin), in the presence of TBHQ and ionomycin, compared with their littermatc controlsl.2• In this study the effect of forskolin on wr and CF nasal epithelia was investigated without preceding exposure to Ca,.1 raising agents using measurement of short-circuit current (SCC). In tissues pre­treated with amiloride (I OO!!M. to block sodium absorption) forskolin (lOJ.IM) gave sustained increases in SCC in Wf as well as CF nasal tissues (51.2± 6.8 I!Acm·2, n = 21, versus 56.3± 8.4 I!Acm·2, n=21, respectively, N.S.), which was sensitive to furosemide (lmM). Since CF mice possess no functional CFfR, due to the disruption of the gene encoding the protein, then chloride secretion in the CF nasal epithelia must occur by an alternative channel, probably via calcium-activated chloride (CAC) channels, which are known to be up-regulated in CF airways u. The phosphodiesterase inhibitor iso-butyl-methyl-xanthine (IBMX. l!XJILM) was also able to stimulate SCC in Wf and CF tissues thus confinning that the forskolin effect relied UF,n cAMP generation. The potassium (K•) channel blocker barium (Ba2 , SmM), applied to the basolateral surface, significantly reduced peak forskolin-sensitive responses in WT tissues from 41.5 ± 4.5 I!Acm'2 to -3.1± 8.7 I!Acm·2, n .. 8, (p<0.002) and in CF from 47.0 ± 12.6 I!Acm·2 to 7.3 ± 5.7 I!Acm·2, n=l4, (p<0.02). In wr nasal epithelia and in the presence of raised Ca2+1 forskolin caused a mean SCC increase of 43.4 ± 10.1 JLAcm·2 which was significantly reduced by Ba2

• to 3.7 ± 3.91!Acn12 (n = 5), p < 0.008. From this evidence it appears that a basolateral K• channel(s) may exist in murine nasal epithelia which is sensitive to cAMP. When activated this channel causes a· secretion (via CFrR channels in WT and CAC channels in CF) by increasing the driving force for cr across the epithelium. This effect may, in part, explain the lack of lung pathology in CF mice while activation of this channel may be a novel target for CF therapy. 1. MacVinish et al. J. Physiol. ( 1997) 499, 677-687. 2. MacVinish et al. Am. J. Physiol (In Press). 3. Johnson elal. J. Clin. Invest (199S) 95, 1377-1382. Supported by lhe Cyslic Fibrosis Trusl and lhe Medical Research Council.

109 KERATINOCYTE GROWTH FACTOR INCREASES CLC-2 EXPRESSION IN FETAL AIRWAY EPITHELIAL CELLS. Carol Blaisdell Murray, Pamela L. Zeitlin. Eudowood Division of Res­piratory Sciences, Johns Hopkins School of Medicine, Baltimore, MD.

CIC-2 is a fetal airway epithelial chloride channel which is downregulated after birth. It is expressed along the apical surface of airway epithelial cells and activated by changes in volume and pH. Factors which regulate CIC-2 at the transcriptional level have not been identified. Keratinocyte growth factor (KGF) stimulates chloride and fluid secretion by developing fetal lung cysts in vitro by a cystic fibrosis transmembrane conductance regulator (CFfR)-independent palhway. We hypothesized that KGF stimulates CIC-2 chloride channel expression. Litters were delivered at 18 days gestation from timed pregnant Sprague Dawley rats. Lung were harvested, pooled, and treated with trypsin and collagenase. The isolated cells were seeded at 2.5 x I 05 ceUs/cm2

, Primary felal rat dislal lung epithelial cells (FDLE) were grown in the presence or absence of 10 ng/ml KGF in Ham's FI2 for 48-72 hours. The rates of growth of these cells in control and treated conditions were comparable. Five to IS ~g of total RNA prepared from FDLE cultures were separated on 1.5o/o agarose ge~ and transferred to nylon membranes. Northern blots were hybridized with a 615 nt rat CIC-2 32P-labelled riboprobe. CIC-2 mRNA levels were increased in FDLE cells grown in the presence of KGF compared with control cells. SDS lysates from 18-day rat FDLE cultures were separated on an SDS-PAGE system, and transferred to nitrocellulose. Using a polyclonal chicken antiserum to a fusion protein of the C-

Poster Session Abstracts 239

terminal region ofCIC-2, immunoblots demonstraled that CIC-2 protein expression was similarly increased in KGF-treated cells. CFrR protein expression by inmunoblot was not increased in FDLE lysates grown in KGF, compatible with in vitro studies in lung cysts from CFrR knock­out mice. In conclusion, KGF increases CIC-2 mRNA and protein expression in fetal primary airway epithelial cells. Further work will elucidate KGF-stimulated CIC-2 chloride secretion in the airways when CFrR is defective. Supported by the NIH and CFF.

110 APICAL CJ· CHANNELS ON NON-TRANSFORMED PANCREATIC ~I!CT CELLS. T.D Nj:uyen, and M.W. Moody. Department of Medictne, Seattle V.A. Medical Cenler & University of Washington, Seattle, WA, USA.

a· channels are essential for the secretory function of pancreatic duct epithelial cells (PDEC) by allowing CJ·, imported into the cell in excl:tan~e fo_r HCo,·, _to be recycled extracellularly. In cystic fibrosis (CF), unpaired funcuon of the cAMP-stimulated CFrR Cl' channel !"=suits . in decreased HCO; secretion and secondary pancreatic msuffictency. In a model of canine PDEC, cultured to confluent monolayers on Vitrogen-coated Transwell inserts over a feeder layer of human gallbladder myofibroblasts, we recently demonstrated expression of two types of Cl' channels: CFrR and a distinct Ca2•-activated Cl' channel (AlP 272: 0172, 1997). To examine the polar localization of !'Jese channe_Is, the 1251' effluxes from confluent monolayers of PDEC mto the lummal or serosal compartments were compared. Forskolin (I 00 ILM>· acting via cAMP, stimulated an efflux that was directed into the lununal compartment, supporting the apical localization of CFrR Cl' c~annels in these cells. Studies of the differential ml' effluxes stimulated by 1 11M A23187, the calcium ionophore, were inconclusive. We therefore examined, in isolation, the activation ofCI· channels on the api~ surface of PDEC, by mounting confluent PDEC monolayers in Ussmg chambers and permeabilizing their basolateral membranes with 0.36 mglml nystatin. With a serosal ... luminal gradient of 140 mM a· across the monolayer, activation of apical CJ· channels will thus be manifested as an increased shon-circuit current (Isc). In this SY.Stem 100 JLM forskolin stimulated an Isc increase of 67.5 ± 3.3 j!A/cm2 (n .; 6) that was inhibited by 500 11M NPPB and 2.5 mM DPC, but not by 500 11M DIDS, suggesting that it was mediated by the CFrR a· channel. A23187, at lJLM, stimulated a smaller Isc increase of 8.1 ± 1.9~J:Aicm2 (n • 7). This increase was inhibited by 500 11M DIDS, in addt~on to 500 11M NPP_B and 2.5 mM DPC, suggesting that it was mediated by the Caz. -activated a· channel. Previous treatment with forskolin ~so potentiated the subsequent response to A23187 two-fold. Of not~, m CFPAC cells, this potentiation was not observed. In conclUSIOn, while both CFrR and the ea2•-activated a· channel arc expressed on the ~ical surface of canine PDEC, a larger Cl' co~1uc~ce was mediated by the CFrR c1· channel. Potentiation of the Ca -acuvated a· channel by the cAMP pathway could be mediated through CFrR. Research funded by the VA and CF Foundation.

111 ASYMMETRIC RESPONSES OF CELL Caz- AND Cl' TRANSPORT IN NORMAL AND CYSTIC FIBROSIS (CF) HUMAN AIRWAY EPITHELIUM. A.M. Paradiso and R.C. Boucher. Cystic Fibrosis/Pulmonary Research and Treatment Center, The University ofNorth Carolina, Chapel Hill, NC 27599

Epithelial cells exist in a complex setting in which responses to serosal or mucosal environments are mediated by receptors expressed on specialized cellular domains, such as basolateral versus apical membranes. We investigated whether airway epithelia can react selectively through purinergic receptors to A TPIUTP in the serosal or mucosal environments by simultaneously measuring cytosolic CaZ+ and c1· secretory responses in polarized human nonnal and CF nasal epithelia. In nonnal and CF, serosally applied ATP and UTP were equally effective in mobilizing cytosolic Ca2•. However, in contrast to nonnal nasal, serosally applied agonists failed to induce CJ· transport in CF. In nonnal and CF, mucosally applied A TPIUTP increased cytosolic Ca,. and Cl" secretion in both preparations, but both responses were of greater magnitude in CF. Although we

240 1997 Cystic Fibrosis Conference

expected mucosally applied A TPIUTP to mediate c1· transport through cytosolic Ca1

• activity, additional experiments showed that these agonists further stimulated c1· secretion in normal tissues pretreated with BAPT A to maximally clamp cell Ca1

• to low levels. Taken together, these data suggest that: I) basolateral purinergic receptor (P1U-R) activation stimulates Cl" secretion, likely via CFTR; 2) in normal nasal epithelium, apical P 1U-R activation by A TPIUTP appears to regulate separate Ca1

• -sensitive and Ca1• -insensitive c1·

conductances; 3) the larger responses of cytosolic Ca1• in CF versus

normal nasal epithelia may reflect in part larger Ca1• stores associated

with apical membrane of CF cells, increased receptor number, and/or a greater efficiency of coupling between P1U-R activation and phospholipase C. Studies are in progress to elucidate the apparent adaptive mechanisms that promote greater Ca1

• release and Cl" secretory responses to apical nucleotides in CF airway epithelia. Supported by CFF R026.

112

HYPERPOLARJZA TION-AcriV A TED cr CURRENT IN CILIA TED RESPIRATORY CELLS: REGULATION BY Ca2

•,

0 AND pH. R.I11I1n. B.E.Argent &. M.A. Gray. Dept. of Physiological Sciences, University ofNewcastle upon Tyne, UK.

Ciliated respiratory cells, isolated from the murine nasal epithelium possess I hyperpolarization-activated Cl" current (JH,p-A.t) which is spontaneously active under basal conditions. This current is interesting because it is observed at a much lower frequency in respiratory cells isolated from CF nuU mice (1). The aim of this study was to investigate the regulation of~ in wild-type cells. Whole cell recordings were made with NMDG.CI present in both bath and pipette solutions (pH.-7.4; pH; •7.2; 30 °C), and with intracellular Ca1

• ([Ca1•];) buffered to

I nM. Under these conditions IHJp-AI:I was seen in 28 /93 cells, and the current densities measured at the reversal potential ::1: 60 m V were 82 ::1: 18 and -139.0::1: 18 pA/ pF (mean ::1: SEM). Increasing [Ca1

•]; to 100 nM had no effect on current density; however, further increases in [Ca1

.]; caused a dose-dependent inhibition of the conductance which wu maximal at SOO nM (80.S ::1:6% of current abolished, n • S). Conversely, IH!P-Aot was insensitive to both acute(- S mins; n = 6) and chronic(- 24 h; n • 4) exposure to cAMP. Reducing [Cl"]; from 124 to 44 mM reduced inward current by S0.9 :1: 12.6 %, but had no affect on outward current (n • 9). In contrast, decreasing [Cr]. over the range ISS to 6 mM decreased both inward and outward current by a maximum of -70% in a dose-dependent manner (n • 6). IHJp-AI:I was insensitive to changes in external pH over the range 9.0- S.S (n • 6), but reducin& pH. to S.O irreversibly inhibited(- 90 %) both inward and outward currents (n • 4). This inhibition at pH S.O wu blocked by the acid proteue inhibitor ENPP (n • 4). In conclusion, we have shown that IHJp-Aot is regulated by [Ca1

.];, [CJ1., and by pH.. We speculate that IHJp-AI:I may be involved in basal Cl" absorption in the respiratory epithelium and that an agonist-induced rise in [Ca~1 may serve to inactivate this current.

(I) Tarran eta/., 199S. Pediatric Pulmonology. Sl2:68. RT is funded by a BBSRC studentship.

113 CLONING, CHARACTERJZATION, AND FUNCTIONAL EXPRESSION OF p34, A NOVEL CHLORIDE CHANNEL Bw Tu!k and 1obn Edwards, Renal Division, Bamea-1ewish Hospital and the Department of Cell Bioloay and Phyliology, Washington Uniwnity Scbool ofMedicine, St. Loui1, MO USA

It ... .,_-- thllt CFJ'Il ...... the 8CtMty otORCCa ill epltbellal .... To elate, the IIIOiecalar ldcadty oldie CPTR..-cilled ORCC llllllkMwa. In­llbanlloly, "" .._ .,_ llllllyiDJ the a cbuDel p64, uc1 .._ demonlllllocl thllt ill activily II OUlW8Idly ~ ill pllllar lipid ~ Hae, we npon the cloala& dwlclcriJallon, ud ftmc:llcmll cxprealoa ofp34, alwmu bomolosuo of p64. p34 - ilollted flum I Puc I eDNA liblllry IIIia& p64 eDNA as 1 pnJbe.

Nonhcm blot analysis bas demonstrated that p34 is ubiquitously expressed as an approximately 1.8 kb message. Scqueoo: analysis revealed an open reading frame enc:oding a 223 amino acid polypeptide, with a calculated molecular weight of 25.8 kDa. Regions of amino acid homology belwecn p34 and p64 are 64% identical (76% similar). p34 was expressed in bacteria as a GST fusion protein and purified by affinity ~hromatography. The GST tag was reniO\'ed with thrombin, and both purified protein and control protein (prepated from bacteria expressing GST alone) were reconstituted into phospholipid \'Csi~lcs in lhc: presence of KCI. Cl uanspon assays were performed by diluting vesicles into isotonic sucrose solutions and measuring the rate at which ~hloride was released using a chloride sensitive electrode. Initial rates of Cl efflux were 60 +1- 21 vs. 153 +/- 112 nmolcs Cl · min·t for control vs. p34 vesicles, respectively (n=5). Anlipcptide polyclonal antibodies raised apinst the C-tcnninus of p34 recognize a Mr - 33 kDa polypeptide on western blot following SDS.PAGE. Proteins ofMr- 42 and 35 kDa are also recognized by affinity purified anti-p34 antibodies in human skeletal muscle. The I!Uiiority of p34 is soluble. However, a significant portion of p34 is associated with membranes, and resistent to reDI0\'81 by a 100 mM Na calbonatc (pH 10.5) wash. The molecular weights (MW) for soluble p34 and membrane­bound p34 were determined following size exclusion ~hromatography on Sephacryl 5-300. The MW of soluble p34 was dctcnnincd to be 35 kDa, while membrane­bound p34, solubilized with octyl glucoside, was dclected in two peaks corresponding to MW of 280 and 56 kDa. Immunofluorescence was used to localize p34 to lhc: apical membranes of both human kidney conex tubules and TB4 cells, a pattern distinct from that for p64. Thus. it appears that we have cloned a lwmu homologue of p64 which is an apical membrane ~hloridc channel in c:crtaln cells. The role of p34 in CF, perhaps as the CFTR-associated ORCC, is currently under investiption.

114 EXPRESSION OF aC-liN INSECT Sl9 CELLS CONFERS A CHLORIDE CHANNEL FUNcnON. H....Xi!!u. E. Garuni, Y. Wang. K. Galley, M. Ramjeaiaab and C. E. Bear, Diviaioa of Cell Biology, Research Institute, Hospital for Sick C!Udrea, Toronto, Canada

ac-2 bcloap to a novel poe family of putative a·chaancls. Expreaaioa of ac-21eads to tile appearaacc of volume-regulated a· currents ill x_,.., ooc:ytes (Griiadct ct al., Natun: 360:759, 1992). Wbilc lbc inlrioalc: cbaaacl func:Uon of tile related protein ac-o bu been confirmed in planar bilayer ltudica of purified prolciD, tile cbaaacl adivity of aC-2 bu not yet been formally dcmonslrated. 1ntrinsic: dtloridc cbaaacl adivity of ac-2 Ia a pn:rcquisilc for lhia protem to fuaclioaally COIDpcasale CFTR U bu been 111ggcslcd by IODIC

reoean:bera. In preparation for reconstitalion ltudics of purified ac-2, we have examined the functional properties of ac-2 following expression in Sl9 cells, a 111ilable el!prcssion system for blab leYCI productioo of recombinant proteial. A high level expression of OC-2 ill 819 cella following traasfcctlon with recombinant Nc:ulovirus containing the ac-2 ORP (gift from T. Jenlscb) was ronfirmed by lmmunoblotting. In elcclropbyaloloJical llludica, imposition of hypotonic shock (JrrS, 75'11> of normal 0111toluity) on aC-2 transfccted Sl9 cells rcc:ordcd ill a wbole-a:ll confi&uration caused appearance of large a· currents ill lbc praence of 0.2mM DIDS, an inhibitor of swclliag-actlvalcd a· currents cndogenou to 819 cclla. The lteady-llate current amplitude wu 393.9 :!:SS.!l% (:tS.E., n-27, p<O.OJ) of control. This DIDS-inscnsitivc, HTS-activatedcurrcal wu a- dependent, revened Ita poluity at the potential c:Josc to Ea and wu lnblbitcd by a dlloridc c:bamlel bloclcer NPPB (O.JmM). In contrast, HI'S failed to activate DIDS-Inlensltlvc cwrenta ill 819 cella transfectcd with wild type baculoviruel (n•S). Anion selectivity of ac-2 UIOdalcd currents showed a permeability scq.ac:e of a- > Br" > f" , wbicb II diatincl from anion selectivity of swellill&-activated a· c:bamlels endo&enoua to Sl9 cella (I" > a"). 1bcse cbaracterlslics - CODiiatent with previously publiabcd dcsaipliou of ac-2 by Jcnlscb and collepea. More direcl evidence ill support of tile proposal that tile HI'S-Induceda- cwrenta deleded ill OC-21raasfectcd Sl9 celll were conferred by OC-2cxpreaion wu the blocbde of tbcle HJ'S-actlvatcd,DIDS-inlcnallive currents by a polyclonal anti-CIC-2imllbody (n•JO) generated agalnlt lbc amino termiJIIIIIIIiDo adds of OC-2 (34-71).but not pn:lmmuJtC lgG (n•7). In contrast, the lllli-CIC-l antibody failed to inhibit endoJcnoua, HTS-induced currenta in lllltransfec:led ccJJa (a-8). Tbcsc RaUits aaJlPCXIthe bypotbeais that ac-2 may llpeCificaiJy fimt:tloa u a a· CGIIChactancc and provide tbe bala for ODJ future examinations of the cbaaacl ac:tivity of purified, reconatilulcd ac-2 protein. SUJIPOIIed by Canadiaa Cystic flbrosla FOIIJidation and MRC of Canada.

115

A ROLE FOR APX IN THE FUNCTIONAL EXPR~ION OF ENAC J.B. Zuckerman•, Y J. Aim", F. Kosar!•, P.R. Smllh , T.R. Kleyman• *Pulmonary &. Crttlcal Care Division, ~nal Division, Dcpts. of Medicine, PhyatoloJY. Biochemistry & "Biophysics, University or Pennsylvania. VA Medical Ceater. 'Dept. Physiology, Alle&heny University, ~pMa.PA

Apx Is a -160 kDa protein cloned from Xenopus laevis. We have shown that Apx associates with ENaC (§pithelial Na Qlannel) in the Xenopus epithelial cell line A6. Previous work in :Xenopus oocytes injected with A6 total RNA demonstrated that Inhibition of Apx expression with Apx antisense oligonucleotide leads to inhibition of amiloride-sensitive sodium transport. We tested the hypotheSis that Apx plays an lmponant role In sodium transpon by regulating functional expression of ENaCs. We used the Xenopus oocyte expression system to test this hypothesis by Inhibiting constitutive expression of Apx with antisense oligonucleotide In oocytes co-Injected with a. p, 1 mENaC cRNA. An antisense oligonucleotide was synthesized against the Initiation sequence of Apx and a corresponding sense oligonucleotide was synthesized as a control. Oocytes were co-Injected with one of the following 3 mixtures: mENaC cRNA (a, p, "(subunits), mENaC cRNA + Apx antisense oligonucleotide, or mENaC cRNA + Apx sense oligonucleotide (control). Oocytes were Incubated for 24-48 hours prior to electrophysiologlc measurements with two-electrode voltage clamp. After measurement of baseline current, the amllorlde-sensitive sodium current (ASC) was determined by the addition of 10 j1M amiloride. The ASC at -100 mV holding potential Will recorded for each oocyte and data were pooled as mean ASC + SEM . Oocytes co-Injected with sense oligonucleotide (n=l4) showooASC of 1651 + 365 nA. similar to oocytes injected with mENaC cRNA alone. However, a marked reduction in ASC was seen in oocyteS co-injected with antisense oligonucleotide (n•l3), 70 ± 15.2 nA. p <0.001. Taken together with previous work showing Apx association with ENaCs, this data suggests that Apx is critically imponant for functional expression of the mature sodium channel complex in Xenopus oocytes. The mechanism by which this regulation occurs requires further investigation.

Supponed by the CF foundation

116 ACTIVATION OF THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFI'R) CHLORIDE CHANNEL BY CYCLOPHILIN A. Jiying Zhao, Junxia Xie•, Pamela B. Davis•, and Jjaniie Ma. Department of Physiology and Biophysics, and Pediatrics·, Case Western Reserve University, Cleveland, OH 44106.

Cyclophilin A is a cis-trans peptidyl-prolyl isomerase that acts as a cytosolic receptor for immunosuppressant drug cyclosporin A (CsA). The R domain (a.a. 590-830) of the human epithelial CFTR contains eight proline residues, four of which (P638, P740, P750 and P759) are highly conserved among the CFTR molecules in different animal species. We have shown previously that mutation of these pralines into alanines (P3A, P740,P750,P759A) led to increase in open probability of the CFTR channel, without altering its regulatory propenies, i.e. gating of the P3A-CFTR channel remained tightly controlled by intracellular ATP and PKA (Yi et al. Pediatric Pulmonology (Supp.) 13: 221). To funher study the function of these proline residues, we tested the effect of cyclophilin A on single CFTR channels using the bilayer reconstitution system. Cyclophilin A (0.1 I'M, applied to intracellular solution) increased open probability of the wild type CFTR challllC1 from 0.318±0.022 to 0.812±0.042 at -80 mV test potential. Pretreatment with CsA (10 j.~M), which blocked the isomerase activity of cyclophilin A, prevented the stimulatory effect of cyclophilin A on the wild type CFTR channel. CsA itself had no effect on the activity of the CFTR channel. In contrast, the stimulatory effect of cyclophilin A was not present in the P3A-CFTR channel (Po • 0.664±0.034, -cyclophilin A; 0.660±0.045, +cyclophilin A). In addition, cyclophilin A had no effect on an R domain deletion mutant of CFTR, AR(708-835)-CFTR. These results suggest that cyclophilin A activates the wt­CFTR channel probably through its isomerase action on the proline residues in the R domain of CFTR. As residues 740, 750 and 759 are proximate to S737 and S79S which undergo PKA phosphorylation in vivo, our data suggest that the configuration of the R domain strongly influences the function of the CFTR channel. Supported by NIH (DK.Sl770, DK49003, and DK27561) and Cystic Fibrosis Foundation.

117 REGULATION OF CHLORIDE SECRETION BY SUBCELLULAR TARGETING OF cAMP-DEPENDENT PROTEIN KINASE ACTMTY Fei Sun, R.A. Frizzell, and Neil A. Bradbury Department of Cell Biology &. Physiology, University of Pittsburgh School of Medicine, Pittsburafl PA 15261, USA.

Poster Session Abstracts 241

Increases in cellular cAMP lead to activation of cAMP-dependent protein kinases (PKA) which result in regulation of membrane trafficking and increased chloride secretion across polarized epithelial cells. Moreover, using site-selective cAMP analogue pairs, we have demonstrated that specific activation of type II PKA can give rise to transepitheiiai chloride secretion. Immunogold electron microscopy and cell fractionation have shown that although type I PKA is randomly distributed throughout the cytosol, type II PKA is highly localized to the trans Golgi network, endosomes, and the apical plasma membrane; precisely those membranes Involved in regulated membrane trafficking and CFTR-dependent chloride secretion. However, since type II PKA has no intrinsic affinity for membranes, we further sought to isolate and characterize the protein(s) involved in targeting type II PKA to these domains. This is of interest since disruption of type II PKA targeting results in the loss of cAMP-dependent regulation of L-type Ca2• channels in myocyte. Cell lysates from T84 and Calu-3 cells were resolved by SDS­PAGE and transferred to nitrocellulose. Affinity purified regulatory domains from type II PKA were labeled with "P and used to probe the transferred fractions Using this Rll overlay assays we have identified A Kinase Anchoring .f!roteins (AKAPs) in clathrin coated vesicles and membrane fractions from T84 cells, u well u Caiu-3 cells. Although several bands were detected in T84 and Caiu-3 lysates, only one band was present in T84 lysates, T84 ciathrin coated vesicles, and Calu-3 Jysates. "P-Iabeled regulatory subunits were also employed in an interaction cloning strategy, utilizing a T84 library. Results from these studies identified two independent clones encoding separate AKAPs. Although novel proteins, both of the clones show homology to several previously cloned AKAPs. Since PKA is a non-specific serine/threonine kinase, AKAP-dependent targeting of PKA activity adjacent to CFTR would provide specificity for CFTR phosphorylation.

Supported by grants from the Cystic Fibrosis Foundation and the National Institutes of Health DK 50829 (RAF), 47850 (NAB).

118 ATP AND METABOLITES ACI'IVATE HALIDE PERMEABILITY IN COs-7 CELLS EXPRESSING WILD-TYPE AND MUTANT <;:FTR'S BY AN .U ADENOSINE RECEPTOR PATHWAY. I P CI•DC)!, F.B. Rulz, EJ. Sorscher, Departments ofPediatrle~~ and Medicine, University of Alabama at Birmingham, Birmingham, AL 3S294 USA.

CFTR functi0111 u a regulator of Ct, Na •, and possibly A TP transport. A cemnl role b A TP and metabolitea in govemin& membrane loa transport baa been sugested; bowever 1be complex mechanisms underlyin& these sipllng pathways are DOt well understood. We have demoostrated with SPQ analysil tbe acquisitioa of an A TP, ADP, AMP, and adenosine (ADO) regulated halide permeability in COS-7 cells foUowfn& transient expression of wtCFrR usiD& a vaccinia-based expreasioo I)'Stem. COS-7 ceilllack endoeeiiOIII cAMP, Ca •, or nucleotide regulated balide permeability, and are tberefore an idealll)'stem to characterize novel mechanism~ for wild-type or mutant CFrR activatioa in tbe absence of compelin& endoge110111 pathways b halide eftlux. Nucleotide­clepelxlem halide permeability at\cr wild-type CFTR expressioo in COS-7 ceill ia blocked by tbe adenosine receptor antagonist 8-pbenyl theophylline. Similar activation and blocker llpecificities were oblerved in 'f8.4 ceill expressin& endogenous CFTR. The pathway is inhibited by Jtaurosporine and II associated with a modest inlracellular increue in cAMP. PKC activation by phorbol esters fails 10 activale balide penneabiiity by this mechanism, suggestin& that lbe adeJIIJSinHctlvated pathway is PKC-independent. The A I -adenosine receptor agonist R-PlA also fails to activate compared with ADO from 5 nM - 5 j&M. Togelber, these findin&s Indicate that adenosine nucleoddes activate wild-type CFTR in C08-7 cells by an A2-AR pathway. We also IIUdied clinically relevant mutant CFTR constructs in lhil system, includin& A4S5E, GS51D, 61501, M'Sa!, and G1349D. We found that balide permeability of lbe IUfface localiz.ed mu1antS A455B and G 13490 are activated by adenosine. The A2-AR has been SU&aeited to be present in primary respiratory epithelial cells from human subjects. In IIWIIIDII)', tbeae experiments Indicate that adenosine IIUCieotidel activale CFTR In cells expreaoiDc A2 adenosine receptors, and that adelll6ine IIICieoCide analogues might provide a useful approach for stimulalin& llltface localized CFrR containina cllnicaUy important mutations. Monover, 1he results raise 1he possibility that A TP or UTP not only activate auxiliary cr aecrwry pathways in CF airways, but also mijlht mediate cr IICCredOO lbrougb llltface localiz.ed, mutant CFrR via an AlAR-dependent pathway. (Supported by lbe CFF and NIH. l.P. Clancy II a Leroy P. Matlhewa Award recipient).

242 1997 Cystic Fibrosis Conference

119 121 IDEN'IlFICATION OF A FUNCTIONAL CaMK 0 SITE IN A Ca''­ACTIVATED Ct CHANNEL CLONED FROM BOVINE TRACHEAL EPITHELIUM S.J. C!!!!elapd. 1.1. lsmallov, B.K. Bcrdiev, V.Gh. Sblyoaaky, D.J. Bcoos, and C.M. Fuller. Dept. of Physiology and Biophysics, Unlvenity of Alabama at Blrminalwn, BirmiDalwn, AL 35294-0005, USA

Our laboratory hu previoualy ldculiflcd a novel Ca" -activarec! c1· c:lwmel (CaCC) from boviDe tncbeal epllbelium with an open J'eldlna frame of 2712 bp., predictiu& a polypeptide of 903 amino ICida that IDcludea twelve alia for N­linkcd aJycoaylllion (Ouuai"'irllm, S.A., tr al. J. Bioi. arem. 270: 31016, 1995). We have prevl0111ly shown that when the CaCC cRNA is expreaaed in XIIIOpiU oocyta, and oocyte membrane vesicles ue suboequeDdy fulecl to planlr lipid bilayen, tbe observed c:lwmel is aeasltlve (i.e. iDcrcued open probability, PJ to Ca" II biJb COIII:CIIIrllio (S-10 14M). However, upon addition of calmodulin, A TP, and Ca2' /calmodulin protein kiDue U (CaMK m to the c:lwmel incorporaled in lbe bilayer, the dole-responae curve wu lblfted to the left aucll that the hi&hest P, wu observed at I I'M Ca2' (/smailov, /./., tt al. PNAS USA, 93:10505, 1996). We have Uled lite-i!irected miJUieDesil to iDdividually cbange tbree of tbe ten predicled CaMK II aila in the open J'eldlna frame in order to determine wbich lila may be phosphorylation WJCII for tbe enzyme. Serinea 478 and 482 and 1hreoniDe S94 were mutated to tbe c:orrespcmdloa alanine midues. Expmaion of cRNA lrlllleribed from tbc TS94A mutant wu uaoc:laled with a 3SS reduction in c:lwmel P, at I 11M ea•• in tbe presence of CaMK U (0.4 I'Jiml), calmodulin (I 11M>. and ATP (100 14M), u compared to tbc P, of the wild type cbanael (all reaaeuta were added to both aides of tbc bilayer c:bamber). We have previously lbowD that tbc wild type clwmel P, is aiJniflcandy lnbibiled bY tbe recluciD& IFill dithloclueltol (DTI'). In the ,_ experlmems, addition of 100 11M OTT to tbc c:lwmel incorporated in tbc lipid bilayer aboliabed activity of the TS94A mutanl. All experimems were done in tbe presence of 100 11M niflumlc acid wblcb Inhibits tbe eJido&ellous CaCC of tbc oocyte membrane (Whlll, M.M. and Aylwin, M. Mol. PhtJmrocol. 37: 720, 1990). These data suuest that TS94 may be a wpt for CaMK II pboapborylation and clwmel activation at phyaiolo&lcal ea>+ COIICCJIIrationa. Tbc effecta of tbc S478A and S482A muwloaa 011 CaCC aeasitivity to Ca" in tbc presence of CaMK II and calmodulin ue yet to be determined. Supponed bY Nm Grllll DK48764 and CFF Granllamall9710.

120 UTP INHIBITS Na' ABSORPTION IN NORMAL AND CF HUMAN AIRWAY EPITHELIA. D.C. Devor and J.M. Pilewski. Department of Cell Biolo&Y and PbysioiOI)', Univenity ofPitiSburgb, Pit1lblqb. PA.

We delamiDed tbe elfcc:t of JIIIICOIII UTP on tralllepitbelial Na• tralllpOit in primary cuhunl of bumln broDcbial epilbelia. Initially, we determined the effect of JIIIICOIII UTP on tbe bual lac RSJ10111C1 IIIia& slllldard NaCI bath solutions, in the ableace of llllliloride. UTP induced an initial transient inc:reue in lac followed by a 11111aincd inblbitioD. Subsequcal to I 00 11M UTP, amiloride t\u1ber inhibited lac by 7.2±0.5 jiA/cm2 (JP6). In an additional 19 experimcnll. addition of anliloride prior to UTP reduced lac by 12.5±1.4jiA/cm2 (P<0.05). Tbele resul1s IUIFII tbat JIIIICOIII UTP inhibits the lllllilori.._itive Na• tnnsport in bumln airway. Howwer, these results may be confounded by tbe known cr secreto1y response to UTP in human airway. Thus, we cletermiDed the effect of mucosal UTP on Na• transport in tbe absence of JIIIICOIII and sernsal cr (INc). Similar to our above lllltlhs, mucosal UTP iDduc:ed a transient increue in ~ followed by 1

11111aincd inlubltion. In 11 expcrimalls. UTP (100 11M) inhibited 73±5% of tbe amiloridMensitive l~~oo A similar inbibition of IN. was observed In tbe presence oftbe PKC inbibltor, bisindolylmaleimide I (200 nM; 79±4%, n=3). To determine whether Ibis inhibition of IN. was specific for mucosal UTP we evaluated additional CaZ+ -depcDdeat qoaists, includin& serosal UTP (I 00 14M), bistamllle (30 11M) and bndykiDin (100 aM). Similar to JIIIICOI&l UTP, eadl of these aaoaJat5 iDduced a transicllt inc:reue in IN. followed by a llllll8iDed inbibition. Tbapsiprain (I j.IM) iDbibltecl ~ to a similar extent (84±3%, 11"5) illdic:atin& tbat tbe inhibition ofNa• tralllpOit is iDdepcadeat of 1 receptor-mediated tc8pOIIICI. FiDally, we delamiDed the eft'eet of awcosa! UTP on Na • tra111p01t (Ill&) in primary culturel of human CF airway epithelia. Similar to wbat we observed in wild type CFTR expnuina epitbelia, JIIIICOIII UTP iDduc:ed a transient increue in IN. followed by a llllll8iDed inblbltioll of tbe amiloriciHeusitive ~ in CF airway. This iDbibitioa avcrqed 110±4% (a-12). Based on these reau1ts, we c:onclude tbat UTP may have a dual tbcnpeutic effect in CF airway: (a) The stimulation of a cr aec:ntory I'CipOIISCI and (b) the inhibition or Na• tr1111p0t1. (Suppt by CFF arant Devor 96 PO).

Hydrogen Peroxide Enhance• the cAMP Induced Chloride Conductance In Xenopus OocyMI Expreuing CnR. K. Bosiouny, S. Matalon and M D DuYQII Department of Anesthesiology and the Gregory Fleming James CF Center. University of Alabama, Birmingham. AL 32594

Hydrogen peroxide, formed by the dismutatlon of superoxlde anions which are released by lnftammatory cells. has been shown to play an Important role In the lnlttattan and propagation of cellular responses during lnftammatory processes. In this study we have examined the effect of H:P2 on chloride channel activity In Xenopus oocytes expressing CFTR. OOCytes were Injected with 1 ng of cRNA and cultured tor -40 h. Whole-cell currents were measured from a holding voltage of -20 mV to -100 mv through 100 mv using two-electrode voltage clamp. Conductances were calculated from the slope of the current-voltage (1-V) relationships. When oocytes expressing CFTR were treated with a cAMP cocktail (cAMP) containing 10 11M forskolln and 1 mM IBMX. the whole-cell conductance Increased from 5.4 :1: 2.1 to 43.9:1: 23.7 jiS. Subsequent treatment with H202 (500 14M) further Increased the conductance to 191.5:1: 24.4 jiS. This stimulatory effect of H202 was concentration­dependent over the range of 10 ).1M to 500 )JM. When oocytes were treated with lt02 (500 )JM} In the absence of the cAMP. the whole­cell concuctance was unchanged. However. subsequent treatment with cAMP Increased the whole-cell conductance to 165.8:1: 21.6 1'5. Unlnjected or water Injected oocytes were unresponsive to both the cAMP and H:P2· To evaluate the chloride dependency of the H:P2 Induced effects. the both (CI") was changed from 96 mM to 48 mM while the oocytes were current clamped to zero. This produced a parallel right shift In the 1-V relationship and changed the reversal potential from -28 mV to -3 mV. These results Indicate that H202 octs to potentiate the cAMP Induced activation of CFTR. Two possible mechanisms to explain these results are :1) H:P2 Inhibits Intracellular phosphodiesterase octMty to stabilize (cAMP) or 2} H202Inhlblts phosphatase octlvlty to stabilize the phosphoform of the CFTR. Supported by the Parker B. Francia Fol.ndatlon.

122 Modulation of ENaC mRNA gene ~~during pulmonary infection with Paeudomonas aerugtnosa. ueljo. A. Dagenais, Y. Berthiaume, D. Rldzicx:h. Centre for the Study of Host Resistanco, McGill Univenity, Relearcb Center CHU, University of Montral, Montr6al, QuA«:. Canada. The lung eli- in cystic fibrosis (CF) patientJ i1 clwacterized by reduced chloride secretion and aodium hyperabaorption, reiU~ in dehydration of the airway aufice 8uid. The abnormal fluid composition in CF is UIOCiated with high IUICeJ)bbility to several micro-orpnilml IUch uP. Mnlgino.Ja. tit the ptfterlt study, we have inveltipted whether the expreaion ofENaC, a sene coding for amiloride sensitive sodium chanriel, could be retp~lated in response to P. aerugino&a infection. This wu done by monitoring a, p and y ENAc mRNA levels in the lu~ of resistant (BALB/c) and iUSCeptible (CS1BU6, A/1, DBA/2) nuce infected with P. aerugtnosa entrapped in agar beacb. Infection of 1Usceptible strains of mice resulted in a severe acute bronchopneumonia which peaka at 3 days post-infection and resolves after 7 days, whereu resiltlnt mice (BALB/c) developed a leu severe pneumonia which is deared within 3 days. Dual levels of a, P and y ENaC mRNA were detected in the lunp ofuninfected resistant u well u IUsceptJble 1tr1in1 of mice, but no correlation wu found between mRNA fevel1 and the reailtance to infection. Interesti~>:z all ENaC subunits were sliahtly upregulated within the first day of'uuection, but were severely tlo~ at 3 and 7 days post-infection. The mRNA levels of a, p ar:J.::nita progreuively returned to normal levels fiom 14 daYJ post ' The moilulation ofENaC expression observed over the coune of P. Mnlgint»~a did not comlate with the strain-dependent variation in the IUsceptibility to the infection. The ENaC sene regulation found in P. aerugtnosa-1nfected mice could not be attributed to the presence of the beads, since injection of agar beads containing no bacteria had only mild effect on ENaC gene expression. Overal~ the reiUltJ reported in the present studf IUggest that P. a1rugtnosa or inflammatory molecules produced m raponte toP. anvgino.ta infection, modulatu ENaC mRNA expression in the lunp of mice. This observation may represent an important mechani1111 involved in controlling sodium chloride concentration in the milieu of infected lungs. Supported by "Canadian Cystic Fibrosis Foundation".

123 MODULATION OF MEMBRANE TRAFFIC AND ION CONDUCTANCES BY CFTR. M J Hug, I.E. Thiele, R. Greger. Physiologisches lnstitut der Albert-Ludwigs-Universitat Freiburg D-79104 Freiburg, Germany

The cystic fibrosis transmembrane conductance regulator (CFTR) has been reported to be a cAMP-mediated regulator of plasma membrane recycling in epithelial cells. To assess its role in membrane transport, we monitored membrane voltage, V111, membrane conductance, G .. , and membrane capacitance, C111, by whole cell patch clamp analysis using the dual-frequency method. Untransfected CHO cells (K1) and cells stably expressing mutated (AF-508) or wild type (BQ2) CFTR were stimulated by forskolin/IBMX (FSK/1, 0.1 IJmol/l/100 1Jmolll), ATP (100 1Jmolll), or exposure to a hypoosmotic bath solution (Hypo, 150 mosm/1). FSK/1 was without effect on V,., Gm and Cm of K1 and AF-508 whilst it depolarized BQ2 cells by 6 :t: 1 mV and markedly increased G., by 30.1 :t: 4 nS (n=65). C., was not significantly changed from 15.5:1:0.8 pF to 15.6:1:0.8 pF (n=50). Stimulation by ATP led to a strong hyperpolarization by 23 :t: 2 mV in K1 (n=35) and by 12 :t: 2 mV in AF-508 (n=42), respectively, whereas It depolarized BQ2 cells by 7:1: 1 mV (n=122). The effect of ATP on V m was paralleled by increases in Gm as well as in c ... lon replacement experiments revealed that ATP activated a K• • conductance in cells lacking wt.CFTR and a Cf -conductance In BQ2 cells. In the latter cells the analysis of the outward conductance for halides Indicated a permeability sequence of Br" > cr > r for the ATP stimulated conductance. Hypoosmotic cell swelling reversibly depolarized K1, AF-508, and BQ2 cells and activated a cr ·con­ductance. This was accompanied by pronounced Increases in Gm and in c ... Taken together these data indicate that (i) activation of the membrane conductance in BQ2 cella by cAMP Ia not paralleled by a detectable Increase In C.,. (ii) Stimulation of CHO ·cells by ATP or Hypo leads to marked Increases In G, paralleled by increases in C, independent of CFTR • expression. (iii) Overexpression of wt-CFTR alters the anion permeability sequence of the conductance activated by ATP. Supported by DFG Gr 480/11-2

124 DEFECI1VE PHOSPHORYLATION ACTIVATION OF THE AF508 CFrR. T. -c. Hwang. Department of Physiology, Dalton Cardiovascular ResearCh Center, University of Missouri-Columbia, Columbia, MO, USA.

Wild-type (wt) and .\1'508 CFrR channels were studied in cell­attached or excised Inside-out patches from NIH3T3 cells stably transfected with wt or mutant CFTR eDNA. In cell-attached patches, both wt and AF508 CFTR chaMels open in bursts with ioog closed times between bursts (i.e., the interburst duration) and short closed times within a burst. With 10 jiM forskolin, the mean open times of wt and AF508 CFTR were not significantly different, but the mean interburst duration for AF508 CFTR was prolonged (> 30 1 vs. 3 s for wt CFTR). This prolonged lnterburst duration accounts for the reduced open probability of .\1'508 CFTR (Po • 0.03 vs. 0.27 for wt CFTR). The interburst duration likely represents phosphorylation/dephosphorylation of CFTR by protein kinase A (PI<A) and phosphatase& because a prolonged interburst duration (> 20 s) was also observed for wt CFTR when the channels were activated with 100 nM forskolin. Application of 1 jiM phorbol 12· myristate 13-acetate, a protein kinase C (PKC) activator, in the continuous presence of 10 11M forskolin failed to increase the Po of AF508 CFTR (n • 4). Thus, regulation by PKC Is perhaps not affected by deletion of phenylalanine 508. However, 20 - 50 11M genistein could potentiate forskolin-dependent AF508 CFTR activity by -19 fold. In excised Inside-out patches, the time course of macroscopic channel activation upon rapid application of PI<A (62 U/ml) and ATP (1 mM) was fitted with a single exponential function. The time constants for wt and AF508 CFTR were 22.9 :1:4.8 1 (n • 5) and 131 :t: 44 1 (n • 5) respectively. Testing effects of genistein on phosphorylation activation in excised Inside-out patches is currently in progress. These data suggest that the PKA-dependent phosphorylation activation of .\1'508 CFTR is defective. Since phenylalanine 508 is physically located in the first nucleotide binding domain (NBD1) while phosphorylation by PKA occurs in the regulatory (R) domain, our results suggest that structural changes in

Poster Session Abstracts 243

NBD1 can affect ~iochemi~ reactions in the R domain. (Supported by the NIH. Cystic FibrosiS Foundation and the Research Board University of Missouri-Columbia) '

125 A SENSITIVE METHOD FOR THE DETECTION OF UTP IN AIRWAY EPITHELIAL CELLS. E. R. Lazarowskj, L. Homolya, T. K. Harden and R. C. Boucher. Cystic Fibrosis/ Pulmonary Research and Treatment Center. The University of North Carolina, Chapel Hill, NC, USA.

The presence of the P2Y 2 receptor on the apical surface of airway tissues supports the idea that endogenously released UTP may function as a regulatory hormone by promoting I Ca2+ -dependent Cl" secretion. However, the existence of 1 potentially releasable pool of UTP in airway tissues is not known. Lack of a sensitive assay for quantification of UTP has constituted a major obstacle in determining the role of this nucleotide as an extracellular signaling molecule. In this study, we describe an assay for detection of UTP in the sub­nanomolar concentrations, i.e. < 1 nM. Taking advantage of the high selectivity of UDPO-pyrophosphorylase for UTP, the conversion of ("C]glucose-lP to (14C]UDPO was quantified by HPLC as a function of UTP concentration. We have applied this methodology to accurately determine both the intra- and extracellular UTP concentrations with an airway epithelial cell line. The CF/43 cell UTP content was calculated in 180±30 pmol per million cells. The concentration of UTP in the bathing solution (0.5 ml) of resting CF!r43 cells {grown to confluence on plastic 12-well plates) was 4.5±2 nM. In sum, we have developed a sensitive assay that quantifies trace amounts ofUTP, and we report for the first time not only the cell content of UTP with a limited number {104-10') of cells but also the presence of endogenous UTP in the extracellular medium of non­stimulated airway cells. The methodology developed here should be valuable in determining the role of extracellular UTP as an autocrine signaling molecule in airways and opens the possibility of exploring potential mechanism leading to the release of UTP and activation, via P2Y2 receptors, of c1· secretion in the diseased airways of individuals with cystic fibrosis. Supported by R026 from CFF.

126 Antisense oligodeoxynucleotlde to PKCII downregulatH PKCII mass and activity and auppresaH a,-ad,.nerglc activation of Na-CI·K eotranaport In human tracheal epithelial calla. c M Liedtke, T.S. Cola. Department of

· Pediatrica and Physiology & Biophysics, Case Western Reserve Univ., Cleveland, OH USA

Previous studies from this laboratory established a role for protein kinase C (PKC) In the a1-adrenergic-mediated activation of Na-CI·K cotransport In human tracheal epithelial cells. More recent studies established that human tracheal epithelial cells express five PKC lsotypes, PKC-«, -1\11, -11. -c, and -(. Methoxamine, an a,-adrenergic agonist. induced a transient increase in PKC activity with maximal levels at < 1 min and caused a transient shift In PKC-5 and -( mass to 1 particulate fraction. Methoxamine selectively induced a sustained Increase in PKC-( activity but only transiently increased PKC-5 activity. In the present study, a specific role for PKC-5 and-( in a,-adrenergic regulation of human tracheal epithelial Na-CI-K cotransport was studied by using isotype specific PKC inhibitors and antisense oligodeoxynucleotides to PKC-11 or-( mRNA. Pretreatment with 10 ~tM rottlerin, a PKC-11 Inhibitor, blocked 72 % of basolateral-to-apical, bumetanlde-sensltlve 30CI nux in nystatin-permeabilized cell monolayers stimulated with methoxamine, an a,­edrenergic agonist Methoxamine Increased PKC activity In cytosol and a particulate fraction; the response was insensitive to PKC-« and -fill lsotype specific Inhibitors, but was blocked by general PKC inhibitors and rottlerin. Rottlerin also Inhibited methoxamine-induced PKC activity in immune complexes of PKC-5, but not PKC -(. At the subcellular level. methoxamine selectively elevated cytosolic PKC-5 activity and parliculate PKC-( activity, but only cytosolic PKC-5 activity was inhibited by rottlerin. Pretreabnent of cell monolayers with antisense oligodeoxynucleotide to PKC-11 for 48 hr reduced PKC-11 mass, diminished PKC-11 activity, and blocked methoxamine-stimulated Na-CI-K cotransport. Antisense oligodeoxynucteotide to PKC-5 did not affect methoxamine-stimulated PKC-( activity. Sense oligodeoxynucteotlde to PKC-

244 1997 Cystic Fibrosis Conference

II and antisense oligodeoxynucleotlde to PKC~ did not alter methoxamine­Induced cotransport activity. Antisense oligodeoxynucleotlde to PK~ did not affect methoxamine-stimulated PKC-6 activity. These results demonstrate the selective activation of Na-CI-K cotransport by cytosolic PKC-6. Supported by NIH SCOR grant Hl-50160.

127 ION TRANSPORT IN CULTURED TRACHEAL EPITHELIA FROM RHESUS MONKEY. C M Penland and J.J. Wine. Cystic Fibrosis Research Laboratory, Stanford University, Stanford, CA, USA.

Large animal models of cystic fibrosis would be useful if they. unlike mice models, could mimic human CF lung disease. To that end, we are canying out comparative studies of ion transport propenies of rhesus monkey tracheal epithelium. Ussing chamber studies reveal that native Rhesus tracheal epithelia are primarily Na+ absorptive with a limited ability to secrete a- [Penland, C.M. and 1.1. Wine (1997) The FASEB Journa/11(3): A562). The present study evaluated ion transport in primary cultures of Rhesus tracheal epithelia. Epithelia were enzymatically removed and maintained in serum-free, honnone supplemented nutrient media on collagen coated insens. Transcpithclial resistance and short-circuit current (/ sc) were dctennined to be 233 ± 67 O·cm2 and 9 ± 3 IJ.Ncm2, respectively (mean± SEM, n • 4 animals). lsc analysis revealed that 54% of Na+ absorption was amiloridc-sensitive (10 ~M) and another portion inhibited by phloridizin (200 IJ.M}. Addition of bumctanide (10 j!.M) to amiloride-pretreated cultures slightly decreased I sc· The secretagogues forskolin (10 j!.M}, lhapsigargin (300 nM), and ATP (10 j!.M}, effective in native tissues, also stimulated an amiloride­insensitive, bumctanide-sensitive current in cultured epithelia (l!J sc • S1 ± 23%). We conclude that both cultured and native Rhesus tracheal airway epithelia are primarily Na+ absorptive, with a modest capacity for a- secretion. Comparison of these results with srudies of human tracheal epithelia indicates that the simian upper respiratory tract may serve as an accurate model of human tissues.

Supported by CFF PENLAN96FO (C.M.P.) and NIH DK51776 (U.W.)

128 COMBINED PROGESTERONE (P) AND ESTRADIOL (E) TREATMENT AUGMENTS ENaC mRNA LEVELS AND AMILORIDE-SENSITIVE SHORT-CIRCUIT CURRENT (lsc) ACROSS MONOLA YERS OF RAT LUNG EPITHELIAL CELLS. Neil Sweczc:y S. Gapon, S. Tchepichev, and H. O'Brodovich. The Hospital for Sick Children, Toronto, Ontario, CANADA.

We have previously reported that levels ofmRNAs encoding subunits of the epithelial aodium channel (ENaC) are differentially regulated in rat lung by exogenous glucocorticoids (Tchepichev eta/., Am 1 Resp Crit CareMed, 151(4) A182, 1995). Here we report injection i.p. ofP and E in a ratio of 750: I (w:w) to sexually immature, non-cycling female rats (-100 g) was foUowed 8 h later by increased whole lung levels ofmRNA encoding the aENaC subunit, which returned to baseline by 24 h after injection. Control animals were injected with vehicle (sesame oil). Neither P (1.5 mg) nor E (21lg) alone altered aENaC mRNA levels. The effect of combined P:E treatment on ENaC functional activity was assessed by changes in the amiloride-sensitive short-circuit current (a-s Isc) across monolayers of lung epitheUal cells in Ussing chambers. Cells were isolated 14 h after injection, and then maintained in culture. The a-s lsc of cells &om treated animals was not different from control after 4 days [(2.2 :t 0.4) vs control (2.02 :t 0.46) 11A I cm2; mean :t SEM, n • 6, p > O.OS) or after S days in a11ture ((3.18 : 0.17) vs control (2.77 = 0.28) 11A I cm2; n • 6, p > 0.05]. Howewr, when cells isolated from uninjected animals were incubated for S clays in a serum-free medium containing 750 P: I E. a dose-dependent augmentation in a-s lsc was observed. Low

dose (P 64 nM, E 85 pM) treatment did not change Isc [(2.51 ± 0.18) vs control (2.39:!: 0.16) 11A I em'; n = 10, p > 0.05], but Isc was elevated after high dose (P 2.8j1M, E 3.7 nM) treatment ((3.41 ± 0.19) vs control (2.64 ± 0.10) 11A I em'; n = 13, p = 0.002]. We conclude that exposure to exogenous P and E augments ENaC at mRNA and functional levels, with half-lives significantly less than 24 h and 5 days, respectively. These findings may be of relevance to the poorer prognosis of female than male CF patients. Canadian CF Foundation, MRC Canada

129 CYTOSKELET AL DISRUPTION INCREASES EPITHELIAL CONDUCTANCE. Stutts, MJ, Blonigcn, PE and Boucher, RC. Department of Medicine and Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill. NC 27599-7248 USA

Coordinate regulation of apical membrane chloride and sodium permeability may involve indirect interactions of CFTR and ENaC. Several recent studies have implicated the actin cytoskeleton in the regulation of CFTR and ENaC, raising the possibility that CFTR and ENaC could communicate indirectly through their separate interactions with cytoskelton. Notably, the actin depolymerizing toxin cytochalasin D (CD) was reported to increase CFTR single channel open probability in isolated cells, and to interfere with the regulation of sodium channels by PKA. We tested the effect of CD on polarized epithelia. In normal human airway epithelium and in T84 cells grown on permeable supports, CD failed to stimulate Cl' secretion over 0.1-30 I'M· The function of CFTR was demonstrated by the brisk response to I 0 11M forskolin. In each CFTR expressing epithelium, and in cultured CF nasal epithelium, CD increased conductance; in some cases the increases were large and probably represent increased paracellular permeability. In MDCK cells heterologously expressing rENaC and expressing no detectable CFTR function, CD variably increased conductance in tissues bathed in bilateral Cl'-fi'ee solutions. In these cells CD alone slightly increased amiloride sensitive 1,. ( 4. 4 ± I. I 11Amp/cm2

). After forskolin pretreatment, the stimulation of amiloride sensitive I.. by CD was increased (8.4 ± 0.9~tAmp/cm2). We conclude that the actions of CD on polarized epithelia reveal potential involvement of cytoskeletal elements in regulation of ENaC and tight junctional permeability, but provide no evidence that CFTR Cl' conductance is affected by cytoskeleton. (Supported by HL42384, HL34322).

130 Coloalc: III1ICr.al byperproliferatl011 IIIICCOIDpanied by Rlectlve chanps In PKC llolorm ldivatiOD, elevated CFrR abundallce and enhaDced cAMP· depeiiCieut CJ' aecreti011. .s.J.lmii:"·',C. LconarcP, J. Sclliri-2·, A.P. Morrii·'. Div.Gastroentcrolo&Y1

and Dept. lntearative Bioi. 21 Univ. Texas Medical School, Houston, TX. The colonic crypc contains a bale (hiJb) to apex (low) aradient for CFTR

mRNA and protein expression and is lhouJht to be the major site of cAMP­mediated fluid secretion within lhe larae intestine. Proliferative activity, which is an index of cellular differentiation, has been proposed to reaulate CFTR expression within this axis. We investipted the role cell proliferation plays in the comrol of transmiiCOIIBI secretory current aeneration by alterina the balance of mitotically active verses senescent cell populations within the crypc in vivo. Short-circuit current measurements were recorded across four I.S em colonic sepnents encompassina the caecal (reaion I I) and rectal (re&ion 14) colonic boundaries of normal and citrobactcr infected (CBI) Swiss-Webster mouse colon. In this model CBI caused both hyper proliferation and hyperplasia, predominantly within the distal colon, without an inflammatory component. Bilateral addition of the cAMP-aenerating aaonist fonlcolin (FSK, 10uM) to normal mucosa elicited between + 14 and +30 ,.A/cm2 of secretory current (I.,, region 11 and 14, respectively, n•6), which was abolished by the removal of bath ct· (n•8). Subsequent serosal addition of carbachol (CCII, S uM) 10 these sepnents elicited a further + 10 to + 18 p.A/cm2 of I. (n•6). In contrast, FSK addition to CBI colonic seaments elicited a sianificantly larger I,..(P<O.OOI) which averaaed +67± 17 ,.Afcm2 within distal reaions 13 and 14 (n•6). In some mice (3

out of 6), a smaller increase in 1,.. in region #2 was observed. However, none exhibited enhanced FSK I. across the most proximal colonic segment (n=6). Subsequent addition of CCh elicited either a transient positive (region #1, 2) or negative (regions #3, 4) I,... All J,. 's were abolished by the serosal addition of 300uM furosemide (n=l2). CBI induced changes in I,.. were paralleled by increases in crypt mitotic index (Brdu labelling increased from 2.3±0.5 to 17±0.8) and length (x2.4 fold, n=SO). Accompanying these changes were specific alterations in the cellular location/activation status of 4 of 10 PKC isoform 's and increased cellular CFfR protein levels. These results demonstrate that invivo changes in proliferative capacity positively effect cAMP-stimulated c1· secretion. Paradoxically, in hyper proliferating crypts CCh failed to synergistically potentiate CFTR mediated I,.,, suggesting that Ca2• -dependent signalling processes differ in mitotically active and terminally differentiated crypt cell populations invivo. Supported by the CFF.

131 Characterization of two distinct P2Y receptors In human tracheal gland cells. M Menen. A. Saleh, W. Karnmouni, and C.Figarella. G.R.G.E., Facu116 de M6decine, 27 Bid Jean Moulin, 13385 Marseille, France.

Human submucosal tracheal glands are now believed to play a major role in the physiopathology of cystic fibrosis. A TP is used as a therapeutic agent for the disease but action of A TP on tracheal gland ceils are poorly known. To answer this issue we investigated the A TP-binding characteristics and the A TP·induced formation of cyclic adenosine monophosphate (cAMP) in a cell line (MM39) of human tracheal gland cells. The binding of 1 radiolabeled nonhydrolysable analogue of ATP ([3'SJ-Adenosine·S'-thiotriphosphate: ATPylSS) was rapid (within 30 min at 4"C), stable and reversible. The calculated kinetic constants were; t..-1: 66 JJ.M sec., and k .1: 3 sec. Scatchard analysis revealed two classes of ATPf'S binding sites. Low affinity binding sites had a Kdl • 20±5 JJ.M ( Bmax • ISO nmoles/lo6 cells) and the high affinity binding sites had 1 ~2 • 2.S±0.2J.LM (Bmax• !52 nmoles/1()6 cells). Competition experiments showed a potency order of competition corresponding to that expected for the P2u receptor with the respective Logit for the foilowing analogues: ATP (2J.LM) > ADP (6JJ.M) > 2-Me-S-ATP (20JJ.M). However, no competition was observed with UTP. A TP caused an increase in cyclic AMP content of the cells, reaching a maximum after one minute. A TP-induced cyclic AMP formation was concentration dependent and inhibited by suramin whearas UTP did not affect the cyclic AMP levels. The presence of both P2Y2 and P2Y4 receptors was revealed by RT-PCR amplification. In conclusion, in human tracheal gland cells, both P2Y2-ATP·receptors and P2Y4-UTP-receptors seem to be distinct in their binding propenies and in the involved intracellular second messenger

systems.

Suppontd by grants from the Association Fran,aist de Luttt contrt Ia Mucoviscidost.

132 REDUCED ACTIVE AND ENHANCED PASSIVE ABSORPTION OF GLUCOSE ACROSS JEJUNAL MUCOSA OF cftr -1· MICE. H B de Jonae'. A.G.M. Bot', I. Bronaveld', B. Schone•, J. Bijman•, M. Sinaasappef. Oepts.'Biochamlatry and'CeH Biology, Erasmus University, 'Gastroenterology, Sophia Children'• Hospital, Rotterdam, The Netherlandl.

Prevloua atudln of glucose transport In CF jejunum were confined to electrical measurements of Oillucosa-lnducltd ahort-clrcuft currents (ASCC.J In tha Uaalng chamber or tailed to separate transcellular, active absorption from paracellular, passive transport. In lha present study stripped jejunal mucosa from 1 3-20 wk old cftr +I+ and cftr -1- mica was mounted In Usalng chambers, preincubatltd for 20 min with Indomethacin (1 0 JJM) and TTX (111M), and ax posed luminally to 'H-3-0-methylglucosa (3-0-MG), 1 non-metabo~zed marker for active plus passive trensport, and ' 4C-1.1llucose (l-Oiu), 1 marker tor pasllva abllorptlon, raplanlshiKI with 10 or 100 mM Oillucosa. Absorption ratn of both Isotopes (J In nmoVcm•. min) raachad 1 plateau value after 5-10 min and than ramainltd constant tor at laaat 1 hr. After 30 min, either tha cAMP ~~gonlst forskoln (FSK), or tha Inhibitor of tha Na'1llucose cotranaporter Q.a. SGL T1) phlorizin (1001'M)

Poster Session Abstracts 245

was added. Active transport of glucose, defined 11 J 04..., minus J.....,. , corresponded with the phlorizin-inhibitable portion of 3-0-MG uptake (•85% at 1 OmM). Absorption rates of Oillucose measured under open circuit conditions, and t.SCC (IJAicm') wera:

J-.(10mM) J-(100mM) ASCC.,.(10mM) -FSK +FSK ·FSK +FSK -FSK +FSK

cftr +I+ (n•8) 55:1:12 50:1:8 33:1:13 35:1:15 106:1:18 105:1:15 cftr -1- (n•8) 35:1:8 37:1:11 83:1:23 70:1:30 72:1:19 80:1:13 Furthermore, the ratio of 'H-Dillucosei"C-tructose transport at 10 mM glucose/fructose was almilar In cftr +I+ va. -1- mice (3.9:t0.3 va. 4.1:1:0.4; n•3). It Is concluded that: (I) active, transcellular glucose absorption is reduced by 25-30%, and passive, paracellular glucose absorption Is anhenced by 2-fold by the CF condition; (II) neither active, nor passive absorption of glucose was modulated by FSK, arguing against a regulatory role of cAMPICFTR In the cell surface expression/ kinetics of SGL Tt, or In the tight junction penneablfity for glucose; (Ill) In view of the parallel CF· Induced changes In active glucose transport under open and short circuit conditions, and In Na' -<lependent glucose w. Na • -Independent fructose transport, the CFTB-Cr channel apparently does not serve as 1

transcellular shunt conductance facilitating coupled Na'1llucose absorption. The reduced tranacellular movement of monosaccharides In CF mouse jejunum may have developed •• 1 compensatory mechanism that lmlts luminal dehydration end helpe to prevent distal Ileal obstruction.

133 Salt and Fluid Uptake In Rabbit Mandibular Main Duct Epithelium u Model lor CF Epithelia. 1 1. Bronsveld, 'H.J.Veeze, 'B.J.Scholte, 'C.H. van Os, 'H.R. de Ionge, "'.l.....lliinwl· Depts of 'Cell Biology, 'Oasuoenterology, 'Biochemistry and 'Clinical Genetics, Erasmus University, Rotterdam and 'Dept of Physiology, University Nijmegen, Nijmegen, The Netherlands.

Reduced salt uptake in the cystic fibrosis (CF) sweat duct is well established. In CF airway Ouid the conccnuation of NaCI is increased resulting in inactivated epithelial defensins and impaired bacterial killing. These findings suggest that salt uptake in the CF airways, like in the CF sweat duct, is decreased; however, a model for hypertonic salt uptake as in sweat duct has not been presented for the airways. Our transepithelial potential difference (PO) measurements in human nasal epithelium suggest that this epithelium is tight, comparable to human sweat duct and rabbit mandibular main duct epithelium. These epithelia exhibit a c1· diffusion potential which is absent in CF, and in the CF condition, an enhanced PO to amiloride <E.....>- Leaky epithelia involved in salt and water uansport, like gallbladder and proximal tubule, do not display these Na'· and CI· diffusion potentials (Augustus If al (1977) Nature 268: 6!57-658). We further characterized salt uptake in tight epithelia by measuring, in rabbit mandibular main duct epithelium, at open circu;t conditions. PO, net uptake of Na'(J.,.). and Cl' (J0 _..),net PD-change in response to amiloride <E...->. short circuit current (I,J, and E...- (see Table). Perfusate PO E...- R 1,. 1- 10 _..

mmolll mV mV Ohm.cm' nEq.cm' nEq.cm' nEq.cm' 12SCI" ·17.8±1.1 11.6±4.3 12.0±1.8 1.5±0.2 I 1.2±0.7 11.9±0.9 2SCI· -87.0±0.5 53.9±10.2 20.2±3.0 4.3±0.6 4.7±0.3 4.7±0.3 We show that lowering luminal ct· (mimicking the CF-condition) results in i), an increase in E...-. similar to CF tissue, resulting from a decrease in luminal Cl"· diffusion potential, ii) a decrease of 1_ and 1a.-as a consequence of the lowered Cl·-conccnuation in the lumen and iii) an increase in I,. as a result of reduced net uptake of ct·-ions, and therefore, increased number of basolateral active Na' uansportcrs that are not shunted by c1·-ions. Furthermore, at any perfusion condition the net sum of charge uanported (1 • ..., + Jo.-) is zero, a condition set by Kirchhoffs law for elecuolyte uansport at op•n circ"it condition. Possible implications of these findings for net salt- and Ouid uptake in (CF) airway epithelium will be discussed.

134 A CRYOTECHNIQUE FOR COLLECTING AND ANALYZING THE ELEMENTAL COMPOSITION OF NATIVE AIRWAY SURFACE LIQUID. S. Baconnais, J,M, Zabm. A. Percbet, L. Kilian, E. Pucbelle, G. Balossier. INSERM U314, IFR 53, Laboratoire de Microscopic Electronique, Reims, France.

The surface of the airway epithelium is covered by airway surface liquid (ASL) which physical properties are determined by active ion and water transport. In cystic fibrosis, it has been shown that the chloride and sodium content of airway surface liquid plays a major role in the regulation of the antibacterial defense of the lung. However. the knowledge of the elemental composition of airway surface liquid is limited, due to the difficulties in collecting it in native conditions. We therefore developed a technique to collect and analyze the Na, Mg, P, S, Cl, K and Ca elemental composition in native airway surface liquid. This technique was applied to the study of airway surface liquid in germ-free mice. After anaesthesia, the trachea of the mice was incised longitudinally on S mm. To collect the airway surface liquid, we used a cooled probe with a tip ergonomically adapted to the internal curvature of the mouse trachea. The probe was first cooled by immersion into a liquid nitrogen

246 1997 Cystic Fibrosis Conference

bath and then applied for 15 s on the mucosal surface of the mouse trachea at a pressure ranging between 1000 and 2000 Pa. We verified that the ciliary beating frequency recorded before and after airway surface liquid collection on the trachea was not significantly modified. In addition, scanning electron microscopic observation of the tracheal mucosa after collection did not reveal any alterations of the epithelial integrity. After collection, the airway surface liquid as well as murin plasma were cryofixed, freeze-dried and analyzed at low temperature ( -I72°C) with a scanning transmission electron microscope equipped with an X-ray EDAX energy dispersive spectrometer. The elemental concentration of elements was expressed in mmollkg dry wei ht ± SEM.

Na M• p s Cl K c. ASL 263±16 60:1:20 70±39 0±16 60±17 53±20 ~6±12 n-!l Plasma 3094±37 150±20 88±3 1523±163 1661±3()( 217±4 t6±S n-2 These results suggest that the techn.que ~nat we <levetop Cl can. be used to analyze the elemental composition of nattve airway surface liqutd. We will further apply this technique to the in situ analysis of the elemental composition of native airway surface liquid from cystic fibrosis airway tissues. (Supported by Association F~se de Lune contre Ia Mucoviscidose)

135 NASAL J:PITHEUAL POTENTIAL DIFFERENCE IN CVSTJC FIBROSIS : RESULTS FROM A SIMPLE TECHNlQUE APPUCABLE TO DIAGNOmc USL Wona LTK'•, Liana H, Dtyjdsgp AOF'•. Cystic Fibroois Clinic'llld Deputment of Pediatrics', B.C.'1 Childral'a Holpital.nd Univenity of British CoiiDDbio, VIIICOIIVel', B.C.,CIMC!a.

Abnonnll ~ tnaapcrt ICIOIIthe MUI rapiratory epithelial cell membrene in Cystic Fibroois (CF) -~~~ increued (neptive) ll'llllml:mJn. clectricl1 potential dift'erence (NPD)1• This il 1 direct COIIIeqiiCDCO of the aboormal Cystic Fibroaia TI'IIIIDICIIIbnn Conduclance Regulator (CFTR) protein in CF.

A simplified klchnique for meuuring NPD in adulll hu boen deocribed by Alton et II. 2• A mic:rovoltmela' il.-1 to meuurethe eleclriW potential dilference belweell 1 okin ref- e1eclrode llld a MUI elox:trodc. The okin electrode ila cliapouble ECO electrode applied to the inlier IUI'face of the forearm, with electricel conductica eatabtished uaina ECO electrode cre1111 aft« gentle abrasion of the epidermal aurfac::e. The MUI electrode i11 Foley catheter filled with electrode cram puaed Ilona the MUI cavity below the inferior null turbinate.

We have uaed thil syllem to test80 CF children llld inflllls. 8 non-CF adulll, llld 60 pedillric non-CF controi1Ubjecls 1aed one week to I 3 yen. Final cli1polis for the controls included bowel obstruclion (S), flilure to thrive (14), dianbea and/or milk IIJeray (S), recurrent chest infection and/or patroeooplutaeaJ rellux (IS), liver diaeues (S), pancreatic diaeue 1101 due to CF (2), an-IIIJleOUI (S). '1'hRe of the five potients with bowel obllnlclion bad oeoaalll meconiiDD ileul 1101 due to CF (110111111 DNA, 110111111- elcUolytel.110111111 pancreatic ftmction).

NPD forthecontroiiUbjocts(mean +/-ISO), wu-17.4 +1- 8.5 mV, which was sianificantly dilrerentli'om the CF patienta (mean +1- I SO) -41.5 +1- 8.0 m V. {pCO.OOOS). ,_ resulll were similar to thole obtained by Alton et al in adult CF llld control aubjeelarespectively'. Fourcontroll bad NPD >-JOmV. OneoCiheaebadan NPD oC -4S m V durin& 111 epilode oCbypematremic clebydmion clecreuin& to -IS m V wben be was n:tested eilbl months later aft« recowry. Conditions noted to all'ect meuurematl ofNPD included the presence oC I nuopotric tube llld concurrent rhinitis (olltqic or virol).

Our raultlllbow that the -.at ofNPD uaina thil simple l)'llem il applicable to childreo u wellu adulta.llld can be uaefld in the diaiJIIC)Iil of cyllic fibrosia, particularly in mr- who 1ft: yet too amall for utiaCICioly -lellina uaina llandard methodl, or when these melhodo .., 1101ovail1ble. 'W~Cmwtootloi,N8a.. .... Diol2-4:-.t911 'tWPW Allae,tlll., 'lllaru42:11Ut7,t917.

136

CFTR ACTIVATES A GLYCEROL PERMEABLE WATER CONDUCTANCE IN XENOPUS OOCYTES R.Schrelber, R.Grlger, K,Kunze!maoo Physlologlschellllltltut, Hermann-Herder..Strde 7, 78104 Frelburg, Germany

Multiple propertlea Mre lttrlbuted to the cyltlc fibroll1 nnsmembrane conc1uc:tance regulnlr (CFTR}, the gene product which II mut8tecl In cystic ftboll1 (CF). Very recent reports demonatreted clowrngulmlon of epltheiW Na+ c:hannela (ENaC) by CFTR. Moreover, 11C1iv811on of I(+ c:ut'relltl by CFTR was Identified recently, thul damonltnltlng regul8tory properties of CFTR. Among other proper1111 of CFTR a water conductanc:e was reported. In the present ltucly we demonltrlte th8t expreulon of CFTR In Xenof'l# oocyt11 and ltimulatlon by ~1-rnelhylxllnthine (IBMX (1 mM) actlvatu a Ct conductance which Is paralleled by the activation

of a water conductances as measured gravimetrically. In water injected control oocytes or oocytes expressing a mutant form of CFTR (G5510-CFTR) IBMX had no effects on either CJ- or water

· conductance. Phloretin (350 liM) and p-chloromercuribenzene sulphonate (pCMBS, 1 mM) inhibited water transport but did not inhibit CJ- currents when measured in double electrode voltage clamp experiments. Vice versa, glybenclamide (10011M) inhibited CFTR CJ- conductance but did not inhibit water conductance in IBMX stimulated oocytes. Moreover, gravimetric and 14C-glycerol uptake measurements indicated enhanced glycerol uptake by CFTR expressing oocytes after stimulation with IBMX. Enhanced glycerol uptake could be inhibited by phloretin and pCMBS but not by glybenclamide. Taken together, the data suggest that activation of CFTR by increase of intracellular cAMP is paralleled by activation of a gylcerol permeable water conductance. Both water and c.­conductive pathways appeared independent thus water permeation through CFTR probably Is located outside the putative pore forming domain of CFTR or as an alternative, CFTR might be a regulator of an endogenous water channel in oocytes.

Supported by DFG Heisenberg Ku 75613-2 und Ku 75612-2, Zentrum Klinische Forschung 1 Freiburg, Fritz Thyssen-Stiftung.

Genetics

137* VARIABIUTY OF CUNICAL PHENOTYPE IN TWINS AND SIBLINGS WITH CYSTIC FIBROSIS: FIRST EPIDEMIOLOGICAL RESULTS OF THE EUROPEAN CYSTIC FIBROSIS TWIN AND SIBUNG STUDY. M.Bal!mann ', I.Bronsfeld", J.Bijman", U.Laabs', F.Mekus', H.Veeze", B. TUmmler' and lhe European Cystic Fibrosis Twin and Sibling Study Group. • Clin. Research Group Medical School Hannover, Germany and "Depts. of Cell Biology and Pediatrics, Erasmus University Rotterdam, Netherlands

Cystic Fibrosis (CF) Is an Inherited disease with a very variable phenotype even in patients with the ume genotype. The European Cystic Fibrosis Twin and Sibling Study aims to raolve the relative contributions of anviromental factors, residual CFTR and/or othar chloride channels activity and othar genetic factors outside the CFTR gena on the clinical phenotype of CF. In an initial step clinical data of 72 twins and 422 sibling pairs with CF from 158 european CF-c:entres were COllected. The intrl-pair and inter-pair variability of paramelers relevant for clinical status and prognosis were detannlned. Methods: Clinical phenotype was discribed by pulmonary function (FEV1 %) and nutritional status (weight for height). To minimize the effect of age, FEV1 and weight for height (wth) values were convened to oentilas for age and sex-dependent pen:entilas of FEV1 and wth of the Westeuropean CF popul• lion. Patient pairs were than ranked by discordance related to pulmonary function and nutritional status, I.e. relative difference of FEV1 percentiles and wth percentage ~n twins or sibling pairs. Clinical outcome is further in­fluenced by CFTR mutation genotype, exocrine pancreatic status and pul~ nary colonzation with Pseudomonas aeruginos~. These paramaters were also considered. Resultl: The reaulled group of patients with CF was rep!e5enta­tlve tor unselected patients with CF. AF508 homozygous patients had more severe pulmonary involvement and lower wth than non-AF508 homozygous patients. Monozygous twins were more conclo!dant than dizygous twins and siblings. This was more pronounced In nutritional status than In pulmonary Involvement. The younger and alder lib In a family were not different In dlnical s.tus If age and sex adapted CF related percentiles were used to compare dlni<:al s.tua. The statlstk:al analpll of twins and sibling pairs with CF emphasized the etrong Impact of genetic modifiers In CF. WhereiS only a few major genes seem to modulate the nutrlional statui, the genatlc predis­position to severity of pulmonary involvement seams to be governed by multiple genetic datennlnants.

Supponed by the Deui8Che Forsdlu11Q811emeinschal'l and Garman CF­Founclatlon

138* MULTIPOINT MA1UCER ANALYSIS FOR lliE PRESENCE OF A (ll MODIFIER GENE LOCUS ON CHROMOSOME 19. J ZlaJelllkJ M. Corey, R. Rozmahel, D. Markiewicz, T. C8aala, B. Mercier, G.R. Cutting, M. Macek, A. Palacio, C. Ferec, X. Eatlvlll, P. Durie, L.-c. Taul. The Hospital for Sick Children and

University of Toronto, Canada; Cancer Research Institute, Hospital Duran I Reynals, Barcelona, and Centro de Analisis Geneticos, Zaragoza, Spain; COTS, Brest, France; Johns Hopkins Univenity, Baltimore, USA; Univcnity Hospital Motol, Prague, Czech Republic.

It Is dear that clinical heterogeneity in CF can be caused by secondary, modifier genes. As suggested by our mouse studies, one candidate region where such modifier(s) may reside Is human chromosome 19, region q13. In our present study, we analyzed six polymorphic DNA markers from a 6-Mb region in 19q13.2-q13.4: APOC2-D195112-D195412-D19S902-D195246-KLK1. Their Inheritance pattern was studied in 129 sib pairs recruited from six CF centers. The data were analyzed with respect to the concordance and discordance of two clinical parameters, namely, meconium ileus (MI) at birth, presence or absence, and pulmonary function (PF) by difference In FEVl using standard deviation of age adjusted percentage of predicted values. The method of identity by descent was used. The study confirms the modifier gene effect on MI for all 6 markers tested (ie., the entire region) and that the strongest effect was observed with the first 3 markers (p<0.005); in 6 sib pairs where both patients are affected by MI, increased allele-sharing was observed. In contrast, we failed to detect any modifier gene effect on PF in this region (p>O.S). This observation is consistent with the lack of concordance of MI and PF status in CF families. Moreover, there Is no apparent difference among the samples from different geographic regions, suggesting that the modifier gene effect is present In all populations. [Supported by grants from NIH and CCFF]

139* THE PARTIAL PENETRANCE OF THE T5 POLYMORPHISM AS A CBAW MUTATION IS EXPLAINED BY DIFFERENT POLYMORPHIC ALLELES THAT ACT IN CONCERT WITH EACH OTHER. Harry CUPPENS', Wei LIN2, Martine JASPERS', Bruno COSTES3, Anne VANKEERBERGHEN', Michel GOOSSENS', Bernd NILIUS2 and Jean-Jacques CASSIMAN'. Center for Human Genetics'· Department of Physlology2; KULeuven, 8·3000 Leuven, Belgium; ~nd Laboratory of Molecular Genetics', lnsenn U468, 94010 Creteil, France.

In addition to the more then 620 disease mutations, more than 120 polymorphism• heve been described In the CFTR gene. Analysis of five polymorphic loci with frequent alleles In the general population showed that in addition to the Tn locus, the (TG)m and M470V loci did also affect the quantity and quality of CFTR transcripts and/or proteins. On 1 T7 background, the (TG)11 allele Increased the proportion of exon9- transcripts 2.8-fold, end (TG)12 even 6-fold, compared with the (TG)10 allele. The •.lleles present at the M470V locus affected the rate of CFTR maturalton. More Importantly, M470 CFTR proteins were found to have a1.7-fold increased Intrinsic chloride channel activity compared with V470 cFTR proteins. The combination of perticular alleles etthese differentlntragenlc loci significantly decreased the amount and/or chloride channel activity of CFTR. The effect of these polymorphism• on the penetrance of the T5 allele as a CBAVD mutation was next studied. Five different haplotype backgrounds for T5 CFTR genes were found. Only the (TG)111T5/M470 haplotype, which will generate the highest proportion of exon9+ transcripts that will be translated into CFTR proteins with the highest intrinsic chloride transport activities, was found In fathers of CF patients. The other four T5 haplotype backgrounds, having a higher number of TG repeals and/or the V470 allele, were found In 18/19 patients. Different polymorphism• that act In concert with each other thus explain the partial penetrance of the T5 polymorphism as a CBAVD mutation and explain why fathers of CF patients, who also csrry a T5 allele, do not have CBAVD.

Poster Session Abstracts 247

140 CHARACTERIZATION OF MUTATIONS IN THE SECOND TRANSMEMBRANE DOMAIN OF CFTR. Hul TENG', Lin WEI2,

Patrick DE SMET2, Bemd NILIUS2, Jean-Jacques CASSIMAN', Harrv CUPPENS'. Center for Human Genetics', Department of Physiologf, KULeuven, LEUVEN, BELGIUM

The CFTR protein Is a symmetrical protein build up of two similar repeated motifs, both linked by a highly charged regulatory domain. Each motif is build up of a transmembrane and nucleotide binding domain. So far, a considerable number of transmembrane domain(TMD) 1 mutations have been characterised, while TMD2 mutations did hardly receive any attention. We therefore characterised eight TMD2 mutations that were found In patients. Five of these (L997F, M1137V, M1137R, 11139V and t.M1140) are actually located in predicted transmembrane helices. Pulse-chase experiments in COS cells revealed that L997F, G1047D, M1137V,I1139V, t.M1140, D1152H, D1154G did mature up to complex glycosylated mature CFTR proteins. Deletion of amino acid M1140, located In the middle of the last predicted helix of TMD2, thus still allowed full CFTR maturation. While M1137V .. was found to mature, M1137R did nol mature. Maturing mutant CFTR proteins are currently characterised at the electrophysiologicallevel in Xenopu$ /aevi$ oocytes. So far, electrophysiological characteristics of L997F, M1137V,liM1140 and D1152H CFTR proteins were obtained. Compared with wild type CFTR protein, activalion of M1137V, t.M1140 and D1152H CFTR did result in significant lower membrane currents, while membrane currents of L997F CFTR were not significantly different. Moreover, a different penneability sequence was observed for t.M1140 (I">Br'>Ct) compared with wild type CFTR (Br'>CI'>I'}. These studies are important for our understanding of the role of TMD2 In CFTR function.

141* RETROSPECTIVE ANALYSIS OF PANCREATIC SUFFICIENT PATIENTS WITH CYSTIC FIBROSIS WITH AND WITHOUT PANCREATITIS. l:..Jlw:llll. M. Corey, E. Tullis, L.C. Tsui, J. Zielenski, P. Durie. The Resean:b Institute, The Hospital for Sick Children, The Wellesley HospitaliDd University of Toronto, Toronto, Canada.

Bacqround: Puc:reatilil is a known complication in patients with cystic fibrosis (CF) who are pan=atic sufficient (PS). The pancreatitis seen in patients with CF ill not well chalacterized, especially with respect ita relationship to natural history IDd CF aenotype. Pancreatitis can be the initial presentation of CF in pati011ta with mild CFTR mutationl but the prevalence is unknown. Methods: We identified aU patients with a diagnosis of CF on the Toronto Cf database who had been seen between 1966 and 1996. A chart review wu performed and clinical data usessed for evidence of pancreatitis. Data were 011tered into the CF database to permit com:lation with other clinical parameters, sweat teob and genotypes. Reoultt: The database identified 1023 pstieDII with CF: 885 (86.5%) PI; 108 (10.6%) PS; and 30 (2.9%) initially PS but subsequently pancreatic insufficient (PS-tPI). Pancreatitis wu documented in 19 patients from a cohort of 138 with 1

history of PS or PS-tPI (13.8% prevalence). The mean age at the fmt diagnosis of pancreatitis wu 22.7 yean (SD 10.3, median 21.0 years). The mean age at diagnosis of CF wu significantly greater in the patients with paacreatilil compared those without (13.9±8.2 VI. 7.6±9.0 p<0.005). In aix of the patients with pancreatitis (31.6%) the CF diagnosis followed the diagnosil of pancreatitis. The mean number of epiaodel of pancreatitis wu 4 (range 1·15). Serum trypsinogen wu significantly higher in the patieDb with pancreatitis compared with the PS patients who did not develop pancreatitis (75.8±53.8 VI. 41.9±27.1, p-0.02, modified t-tcst) and more variable (p<O.OOI, F-teot). Sixteen of the patients with pancreatitis had genotype anai)'lis. None had 2 CFTR mutations associated with the PI phenotype. Eleven patients with pancreatitis bad genotypeS usociated with the PS phenotype. In the two PS-tPI patients; one carried 1 mutation usociated with PS while no CFTR gene mutation wu identified in the other. four other phenotypically PS patients with pancreatitis had AF508 CFrR with an u yet unidentified second mutation. Conduslou: In over 30% of CF patients with a history of pancreatitis, the diagnosil of pancreatitis preceded the diagnosil of CF. Prevalence of pancreatitis in the PS and PS-tPI CF population wu 13.1%. A omall RJbgroup who are initially PS appear to develop PIICCODdary to recum:nt pancreatitis and not due to their underlyina genotype. The trypsinogen levels were significantly higher and fluctuate more in the subgroup who developed pancreatitis.

248

142*

1997 Cystic Fibrosis Conference

cnR MUTATIONS AND IVS8-5T PREVALENCE IN IDIOPATHIC CHRONIC AND RECURRENT PANCREATITIS. CllllfilWII c•, BoNIUalo A •. Clpolll M", Sgarbl IY', CavalliN/ GCA, Bas• I c•, P.Mnoll P", Mtutella 0° -OCyJt/c FlbroJ/1 Celllu, O•,.dale Civile Magglon, l'u011a, ltoly; AGI1!tra.lllerolog)l UN/I, UN/wn/1)' of Yer011a, Italy; "Swglcal /)eparllftltl, UNiwrlll)l of Yu011a, Italy.

A hl&h lt-equency of mutations In the Cystie Fibrosis TriiiSmembrane Conduetm~ee Reaulator (CFTR) gene hu been detected In patients not showing the full spectrum of Cystic Fibrosis (CF) clinical manifestations, but affected with disorden that shate some featutes with CF (I.e. Congenital Bilateral Absence of the Va Def'erens, Chronic Obstructive PulmoniJy Disease, Btonchiectasls). An inmaecl CFTR mutlllon lneldenee hu also been recently shown In Chtonle Pancteatitls with vatlous etiology. We checked this findlna In a selected Italian population of 46 patients (21 males, II females) sufferlna ITom Chronic and Recurrent Pancreatitis (CP and RP), In whom no elear cause (namely alcoholism and bllllfY 1r1ct disease) had been previously detected; they had thetefore been claslned a Idiopathic:, despite in only a few or them a sweat test had been performed and CF ruled out. Each subjec:t wu seteened for the 15 most frequent CFTR mutations In out population (4FS08, 41S07, RII62X, 2113AA-t0, NI303K, 3149+10KbC-tT, OS42X, 1717-IG-+A, RS53X, Q"2X, GI5E, 7li+SG-+A, 3132deiTO, 2789+50-+A, WI212X), whleh covet 15% of CF ehtomosomes In our area, plus the S-thymldlne allele In intron I of the CFTR gene (IVSI-5T). Amon& the 46 patients we found: 4 hetetozygoCes (onee RII62X and tine times 4FSOI), I compound hetetozygoce (R 1162XI2719+50-tA), S subjects with IVSI-5T &enO(ype Sn and one SIS. The earrier !Rquency of II II (414S) wu conspicuously hi&her than the expec:ted 1132 for the tested mutations; the prevalenee of IVSI-5T wu Instead slmllu to the 10% of the aeneral population. As for the compound heterozygoussubjeet, 11110re thorou&h eheek up showed mild mplntory symptoms and sweat elel:lrolytes above IOOmEq/Ka. A CF diqnosls followina panmatltls sympComs Is an unusual but not exeeptlonal event: 4 of the 927 CF patients (lneludlna 113 paneteatk: suffielent) followed by the Centet of Vetona In the Jut seven years had previously followed the same dlqnostk: Iter. Even thou&h no definitive eoncluslons ean be drawn from our data, a te11011able hypoehesls eould be that at laa some patients with Idiopathic: CP or RP and a CFTR mutation aetually suffer livm mild forms or CF. euryina on the oCher ehromosome an undetected mutation or even a DNA vviant. Further studies on wider populations and/or a more extenllve -lysis of the CFTR aene In these subjects should be able to elear the ....... Supported by a ptt tom Italian Public: Health Ministry.

143* MUTATIONS OF THE CFTR GENE PREDISPOSE TO IDIOPATHIC CHRONIC PANCREATITIS. J A Cgbn, K.J. Friedman, L.M. Silverman, P.G. Noone, M.R. Knowles, P.S. Jowell, Dept. of Medicine, VA & Duke Unlv. Med. Ctr., Durham, NC, & Depta. of Pathology & Medicine, Unlv. of North Carolina, Chapel Hill, NC.

Idiopathic chronic pancreatitis (ICP) is a prevalent disease, accounting for 35% of chronic pancreatitis. Disabling abdominal pain is typical in ICP and complications of pancreatitis are often life-threatening. It is unknown whether genetic factors predispose to ICP, even though such a predisposition might be anticipated based on evidence that individuals vary widely In their susceptibility to pancreatitis. Because pancreatitis occurs In approximately 1 o/o of cystic fibrosis (CF) patients, this study tested whether CFTR mutations predispose to ICP In individuals who do not have recognized CF. Among 25 tested ICP patients, eleven abnormal CFTR alleles were detected. This Included seven CF-causlng mutations (observed:expected • 10:1, p < 0.00001) and 4 patients with the intron 8 5T allele (2: 1, p • NS). Three of the ICP patients had genotypes affecting both CFTR alleles (80:1, p < 0.00001). These three ICP patients did not have typicll CF pulmonary disease based on radiographic, clinical or physiologic parameters. However, each showed abnormal CFTRoflltldimed ion trllnaport In the nasal epithelium. In addition, the one male In thll group had CBAVD. Thus, thla ltudy Identities alubMt of ICP patients in whom paDCI'HtNft Is ai80Ciated with abnormalities of both CFTR aller.. CFTR II the ftrat commonly affected gene shown to prediapoM to pancredtll. (aupported by the NIH and the CF Foundation.)

144 The Occu,_ of CFTR ~hlsmt1 end CF Mutllllo1111 on the Slime Ch-. Michelle M. Egan and Garry 8 Cuttilg. Department of Pediatrics, The Johns Hopkils Unlverslly School of Medicile, BaHinore, Maryland, USA.

The possblly of mun.,le sequence variations on the same allele has often been overlooked. When two mutations are found, genetic analysis Is usually consilerad complete, _, wha1 dilcrepancles 8lllst between the anticipated and obseMid phenotype. However, a number of 'muHiple mutants" have been desctbed, suggesti"lg that addMional variations on the same allele may be more common than assumed. We report three patients who were found to have one CF mutation In each CFTR gene In addlion to a previously reported pol)morphim on one ollhe genes. Two cases -.. genotyped by allele-speclic ollgonucleolide ~as deiF508/cleiF508. However, both cases had mid clinical symptoms Inconsistent with the symptoms of a majorlly of cleiFSOB homozygotas. In both patients, DNA sequencing revealed the deiFSOB mutation on one allele and the 1508V polymorphlam on the other. Analysis of the entire gene by OOGE (25 8lCI)OS) and DNA sequenciJg (2 allOns) iclentlied D1168G in one palilntand 3121·2A->G in the other. Pedigree analysis revealed that each mutation was segregated with the 1508V polymorphism. We presume both of theM mutlltlons- diiMse cauahg. D1168G causes a change from a charged aspartic acid lo aDOfH:harged glycile n a highly conseMid region of the protein. Screenilg of 174 CF heterozygolea (parents of CF patients) did not reveal thil mutation on any of the non-CF chromosomes. 3121-2A->G 14Nids to abnonnal splicing of &liDO 17a. Loas of thil alliin would cause a frameshlt and introduce a premature tenninatlon signal. In e third case, we considered the genotyping complete when 2 CF mutations were found, deiFSOB and 3849+10kbC·>T. H,_, when used as a control lor a dllerent -y. the previously cleacrbed R668C mutation was found in SSIICX:Iation with the 3849+10kbC·>T mutation. R668C has lllo been f<Km on the Bile allele as 0443Y iJ CBA VO patients and n ilolallon n pelllnts with bronchillctasls and emphysema. Screenilg of 20 CF heterozygotes (parents of CF patients) revealed the R668C mutation in one female parent. Thil reau~ suggests that R686C II actually a polymorphism. Therefore, the presence of dilease-SSIICX:Iated mutations on the same gene as neutral ~lams may be more common than realized. The polymorphism may have IItie functional consequence, or could modly the deleterious effects ala dlsaasa aseoc:laled mutation as noted wlh 8 117H and the intron 8 splice ale variant.

145 Heteroaenelty ID the severity or Wollllan Duct abnormalities In Men whh Ableoce oftbe Vu DdereJD: tbe Role ofCFfR Gene Mutatlonl. K. Jarvi, S. McCallum, J. Zlelensld, P. Durie, M. MatJOIIs, M. Asch, B. OiosberJ, M. B. Buclapan, U. Tsui. Toronto, Ontario, Canada

Conptal bilaleral absence of the serotal vu defetens (CBA VD) is 1

well deseribed CIUIC or male inrertlllty, oecurin& worldwide in 2" or men be in& investi&ated for male Infertility. The uscx:iation between CBA VD and mutations in tbc cystic: fibrosia trllll-membrane concluctanee reJII)a10r (CFTR) acne. hu become well established. In autopsy series on male inraau dylnJ with the c:ompliations or CF. abient or hypoplutlc epidldymes and vu defetens wu round in aiDIOit all patients, while semiDal vesiele or ejaeulatory duet pathoiOJY was noted in elose to SO" or the infants. The principal defeets are in the struetures derived rrom the Wollflan Duct (epididymis, vas deferens, seminsl vesicle, ejaculatory duets). Slnc:e the severity of Wollrtan Duct patholoay appears to be telated 10 the type of CFTR poe mutatioo in men with CF, one eould upeet some IJeteroaeneity in the palhoJoaies or the Wollflan Duct struo:tute1 of men with CBAVD, but without oCher lip of ellnical CF. We examined men with CBAVD to detennine if the severity of Wollflan Duct abnormalities could be eortelated 10 CFTR aenotype. A lrllll-rec:tal ultruound (TRUS), whleh offers superior imaging of the distal rep!"O&Jctlve tract, and a CFTR aene analysis was performed 00 the DICII.

Of the SS men with CBAVD who IJteed to acnetic testina. we wete able to perfonn 1 TRUS in 29. Nooe of these men had symptoms or elauic:al CF. Only 22" of the aemlnal vesicles and 24" or the unpullas of tbc vu defetens wete uiiJ"IIOnOIIIPhleally normal, 12" of tbc seminal vesleles itad cysts (laraer then lcm) while 65" and 76lt'. of the aemlnal veaicles or the vu deferens wete abient or atrophic. Thete wu a dramatic: difference in the TRUS riJidinas or the separate Jf0U111 of men subc:lassilled ac:cordlna to CFTR mutation status. While 57" of tbc seminal vesieles wete normal in those men without CFTR acne mutations, only II lt'. of the seminal vcsk:les were normal in those men with a CFTR acne mutation or variant. In contrut, the vu deferens wu normal by TRUS in 43" or lbe men with no lllltationl, while thiS wu the cue for only 6" of the men with I or 2 CFTR Jelle mutatlonl. Thole men with any CFTR mutat1ona a11 lad aboormallda or the seminal vaicla and 111e ampulla or 111e vu deferens, while eloM Ill SO" of lbe men with 110 CFTR acne mutationl had normal seminal vesk:les and so" had normal ampulla or the vu deferens bilaterally by TRUS.

The TRUS tlndlap above indic:ale dlat lbere Is a awrelalion between the diJtal Wolff"tan Duct pbeaocype in men with CIA VD and the CFTR aenoeype.

146 Higb frequen<:y of CFTR gene mutatioos ill men wilh illfertility. K. Jarvi, J. Zielensld, P. Durie, E. TulliJ, S. Martin, T. Longley, M. B. Buck.span, A. Zini, L-C. Tsui. Toronto, Onwio, Canada

Approximately 7 .S 'I of all men ill Norbl America ate iDfertile or sub­fertile. Recently, CFTR JeDe mu1ations have been detected in infertile males wilh Wolffwt cb:t (epididymiJ, vas deferens, seminal vesicles and ejaculatory duels) abnormalities but oo ocher manifeslatioos of Cf. The extent of CFTR mu1atioo involvemenl in male iDfertility wu funber broadened by von der V en et: al. 's (1996) report !hat 17.S'lli of lhe men wilh iDfertility due to sperm abnormalities (but no1 azoospermia) bad CFTR mulAtions.

We studied lhe frequency of CfTR &ene mu1atioos in men presenlin& for infertility invesligatioos. Infertile men were classified u eilher having congenilal absence of !he vu deferens (CBA VD -by scrolal exam), obstructive azoospermia (epididymal obsuuctioa proven IUrgically), azoospermia wilb testicular failure (no sperm production) or sperm abnormalities (men with sperm in !he ejaculate bul abnormaliiCIIlCII analysis). The men all bad a CFTR gene analysis.

CBAVD Obstructive Sperm Testicular D•47 uoospermia Abnormal failure

n•78 n•6Z n•4S

'I infenile a 4'1 90'1 2'1 men

'IICFTR SS'I 21" 21" 11'1 Mut

'I 2 CFTR 28'1 3'1 0'1 0'1 Mut

The CFTR mulAlioos usociated wilh male iDfertility and sperm abnormalities alone were exclusively mild (R7SQ. sn. wbile those associated with CBAVD were often more severe (26'1 of all CFTR acne mulAlions were della fS08). The frequency of lhe fmding of CFTR gene mulAlions Is significantly bigber in these three croups than in lhe men with testicular failure and bigber than expected in lhe populatioo.

It is becomin& clear that CFTR mulAlioos represent one of lhe most common abnormalities auociated with male infertility. As a cooservalive approximatioa, some 20'1 of all infertile men will have a CFTR mulAlion or variant.

147 A NEW CFTR MUTATION (Q781X) IN TRANS WITH THE 5T JNTRON I POLYMORPHISM CAUSES UNUSUAL CLINICAL DISEASE. z...zbQyl, K. friedman!, M. Blalock I, L. Silverman I, S. Man:us2, Y.~. M. Knowlesl, J. Yankukul. Lutheran General Children's Medical Center2, Part Ridge,IL, Depanments of Pathology I and Medicine3, University of North Carolina, Chapel Hill, NC, USA •.

The CFTR ST intron I polymorphism (IVS I) is partially penetrant and has been associated with CBAVD, chronic pancreatitis, and disseminated bronchiectasis. Its role in causing CF is not fully understood. We evaluated the CFTR genotype in four siblings (S) who have clinical CF and intermediate or elevated sweat CJ·, and in their parents (P). Two mutations were identified in this family, 1717-IG>A and Q781X, as well u the IT variant allele in intron I. CF was diagnosed by clinical criteria, sweat tests, and genotype analysis (IVS I polymorphism/CFTR mutation, Table). The genotype analysis demonstrates different compound heterozygous mutations in the siblings, and in the father, who has no clinical disease.

Sex!Aae SweatCt Enzymes Cultures Allele I Allele2

PI Pl 51 Sl 53 54 M/30 F/29 M/10 Mil M/5 Mil NT NT 47 63 102 108 No No Yes Yes Yes No NT NT Sa, Hi Sa, Hi Sa. HI Sa. Hi 7T-1717 7T-WT ST-WT ST-WT 7T-1717 7T-1717 ST·WT 9T-781X 9T-711X 9T-711X 9T-781X 9T-781X

[NT•Not Tcsted, S.•Staplry/ococCJU tllll'ftl, HI•Hemoplrilru lnj/wnza, 1717•1717-IO>A, WT•Wild Type, 711X-Q711X)

We conclude that Q711X is a newCFTR mutation. Q781X in trans with the ST IVS I polymorphism causes clinical CF with intermediate sweat Cl· levels. Orpn-specific gene expression and functional studies are underway. The discordant phenotypes exhibited by the affected siblings may be substantially explained by the different genotypes. Supported by tlw Cystic Fib,os/8 Foundation.

Poster Session Abstracts 249

148 IMMUNOREACTIVE TRYPSIN LEVELS IN FILTER PAPER CORD BLOOD SPOTS OBTAINED NATIONWIDE FOR NEWBORN SCREENING IN URUGUAY: A Aznarez, S. Cabeza, F. Coppe, B. Alvarez, E. Silver. Nuclear Medicine Center, Clinics Hospital, Faculty of Medicine, Montevideo, Uruguay, and Cystic Fibrosis Association of Uruguay.

Screening for Cystic Fibrosis lhrough measurements of Immunoreactive Trypsin (IRT) levels on heel prick blood spots obtained after 24 hrs of life is a well established method. Since 1989, over one thousand affected newborns have been detected, and CF was confirmed by diagnostic laboratory tests. IRT has been assayed in sera during fetal life and on cord blood from mid- trimester fetuses, producing conflicting results (markedly depressed, identical or significantly higher than adult values). Studies of concentrations of IRT in cord blood at nonnal delivery showed a mean concentrations of 40.7+/-27.4 ng/ml, and a progressive increase to 66.4 + /-29 until the 5th day of life. A pilot study was conducted with the aim of investigating if IRT measurements on filter paper cord blood spots, obtained mandatory by law for Congenital Hypothyroidism screening, would be suitable for screening newborns for CF. A figure of I, 727 consecutive samples were assayed by radioimmunoassay's commercial reagents. Median of IRT dose was 60 ng/ml, and centiles 98, 99, and 99.7 correlated with doses of 165, 200 and 300 ng/ml respectively. Upper two percent of samples were re­assayed routinely; among them, 8 neonates (0.5%) showed IRT levels above 300 ng/ml, and were derived for confinnatory tests and clinical follow-up. Results from IRT assayed in filter paper cord blood spots does not differ significantly from values obtained in heel prick 24-72 hrs of life blood samples. Though a larger sample is needed 10 confinn our results, this approach seems primarily suited for screenin& CF at many developing country's programs based on cord blood sample collection. Supponed by lhe International Atomic Energy A&ency, VIenna Austria and !he Central Commission for Scientific Research, Univenity of the Republic of UruJUIY· EMAIL: [email protected]

149 CfTR ALLELE FREQUENCIES IN A COHORT Of SUBJECTS WITH BORDERLINE SWEAT CHLORIDE CONCENTRATIONS . .&JL..em!l. J. Osterman, C. Gerard. Ina Sue Perl muller Cystic fibrosis Resurch Laboratory, Children "s Hospilal, Dept of Pediatrics, Harvard Medical School, Boston, MA.

Picocarpine iontophoresis for sweat chloride determination is performed in subjccll wilh chronic respiratory, Gl or infenility symptoms, or positive family history for CF, when CF is considered in the differential diagnosis. When classic Cf symptoms ore present in conjunction wilh swell [CJ"] ~ 60 mEq/L, the diagnosis of CF is clear. When symptoms concur with sweat [CJ") < 40 mEq/L, the clinician may be aomewhat ~foned in ruling out CF, although o~~tceptions exist. The most frustratina scenario is when sweat [CJ"] is in the 40-60 mEq/1.. range concurrent with suspicious symptoms. In lhis case, a mild Cf variant cannot be ruled out. GenotypinJ performed on these patients may reveal 11 least one mulalion commonly found in affected Cf populations and may raise lhe suspicion for true CF. Absence of the common mutslions should not necessarily reassure lhe clinician, u variant CF forms may be caused by ''mild" mulalions that ate either rare. or not yet characterized. from 3194, we prospectively collected DNA from subjects with swell [CJ") > 40 and < 60 mEqiL assessed in lhe Children's Hospitsl, Boston Sweat Lab, where approximately II 00 sweat tests are performed annually. Of 58 subjects, presenlin& mainly with chronic respiratory symptoms or recurrent pneumonia. but sometimes for m or malabsorption, we performed genotype analysis, utilizing a reverse ASO technique (Roche Molecular Systems, Alameda CA) for 16 CFTR mulalions (41'508, .11507, 621+10-+T, R334W, R347P, 05510, OS42X, WI282X, N1303K. RS53X, RII7H, 1717-10-+A. S549N, 3849+10kbC-+T, RS60T, A455E) and the intron 8 ST allele associated with CBA VD). The ST allele was detected in 8158. five palienll carried 2 CfTR mutslions (4FS081ST(2), GS42X/ST(I), AFS081 Rli7H(I), and AfSOS/3849+ I Okb( I)). Twelve patients (21%) were helerozyJOUS for one of the 16 CFI"R mulations. Of the 116 alleles, 22% were abnonnal (including sn: 4FS08 (9), RII7H, (2), 3849+10kbC-+T(I), RI17H,(2), m42X (3), NI303K(I). We found a significant increase in lhe frequency of abnormal CFTR alleles, and in the number of heterozygote• u cornpated to the expected carrier rate (P<IlO 1). The presence of one allele in this croup may imply significance beyond carrier stale, because eilher an as yet undetected second mild mulation exisll on the "normal" allele, or carrier slate in conjunction wilh an abnormality in some Olher modifyin1 gene mi1ht lead to a mild Cf variant phenotype. We conclude lhe genotypina of this ''borderline" population may provide clinically useful information, and presence of one detectable allele may support the diagnosis of a mild Cf variant, with an unidentified second aenetic abnormality.

250 1997 Cystic Fibrosis Conference

150 152 CFfR OENOTYPINO OF A COHORT OF PUERTO RICAN CF PATIENTS: AN UNEXPECfEDL Y LOW MUTATION DETECTION FREQUENCY. R.B. Parad. A. Comier,I.R. Rodriguez­Santana, L. Pedraz, J. Osterman. Ina Sue Perlmutter Cystic Fibrosis Research Laboratory, Children's Hospital, Department of Pediatrics, Harvard Medical School, Boston, MA., Dept. of Pediatrics, University of Pueno Rico School of Medicine, San Juan and Ponce School of Medicine, Ponce, P.R.

The Pueno Rican population is derived from a Southern European background where lower .Y:SOS frequencies have been reponed than in the Caucasian Nonhem European populations. Prior estimates of AFS08 frequencies have ranged from 38-58%, based on small cohons. We repon the CFfR allele frequencies from Sl patients (at least 213 of known patients) collected from the sole CF clinic on the island of Pueno Rico . Those patients were diagnosed with sweat [Cl"] :1: 60 mEq/L. A reverse ASO (Roche Molecular Systems, Alameda, CA) for 16 mutations (AFS08, MSO?, 621+10--.T, R334W, R347P, OSSID, OS42X, WI282X, Nl303K, RSS3X. RII7H, 1717-10--.A, SS49N, 3849+10kbC-.T, RS60T, A455E and the intron 8 ST allele) was performed. We were able to identify only 25% of the alleles with specific mutations detected as follows: .Y:SOS (14), R334W(7), RSS3X(2), OS42X(I) and 621+10--.T(I). Only I patient had 2 identified mutations (.Y:508JRS53X), whereas 24 had one mutation. This low detection rate is particularly imponant in light of new NIH screening guidelines, given that current screens may perform poorly in detecting carriers of CFTR mutations in this population.

151 TilE USE OF NASAL PO MEASUREMENTS AS A DIAGNOSTIC TEST FOR QUESTIONABLB CF. M. WiJw;hanakj H.FUIBDi, Y. Riv1in, L. Bentur, A Tal, M. Aminm, C. Sprinser, A 1onu, P. Durie, B. Kerem, E. Kerem. Dept. of Pediatrics, Shure Zedek Medical Center and CF Centen of lsnel, and Hospital for Sick Children, Toronto, CIDIIIIa. In the~ of the typic:ll iylllptomatCIIo, elevated aweat cbloride (Cf) or identifiCition of2 CFTR mutations, it ia difticult to determine if thele patieats have CF. Patients with atypical CF do not have charlcteriltic symptoma despite extensive clinical evaluation and Man:b of known CFTR mutations. In IODle ethnic Jf0UP1 in 1.-ael, lela than 60% of CFTR. nutltionl have been identified. We performed nua1 potential di1ferellce meuurements (PO) on 26 such patients (age • I4.S±IIyn, aweat test-64±22 meqn) and compared them to known CF patieats (n-14, age- 17±4.6, aweat tat-83±30 meqn) and controls (n-24,ap-2S.S:t7).(PD meuurementa in mV, Coat-control, Ami-Amiloride, %-percent1p reduction of basal PD, "CC" '"110

cblorh e. ISO-D a..l 4AIIII " A"O" AlSO AISO+"CC"

Coat 24 ·2.0N 11±5 51±17 oo6.4t6 ·7±5 ·l4:t.9 CF 14 ... ~ 30.4±21 74:1:16.5 2:1:3.7 0.4:1:2.4 U:ll4.5 1 l6 -21:1:10 7%9.5 60:1:21 ·2:1:3.2 ·2.6:1:3.5 5±5.6

AU the result~ of the CF patients and coatrol lfOUP were agnificlmly dift'ennt (p<0.001). On the bail of the PD reaulU, the quesdonlb1e patieatl were clividod iato 2 lipiflcant1y dift'erent 8f0Ups. 14 were complllb1e to CF nsulu (sweat t.-66*13) and 12 to reaulu of controls 1 (IMit t. -62*33\. (Nor. tocontroll I lnla..IIAAIDII " A"O" I AlSO J AISO+"O" I

Nar. 12 -21:1:7 I 11.5:1:7 5U:22 -4:1:3 I -5:1:3.4 ·IW.S l I CF I 14 I ·34:1:11 I 20:1:10 58:1:20 ..O.S:tZ I ..o.w J ·1:1:3.5 J

In coaciUIIOJI, PD may be Ulefta1 m atablishi"' a clilpolil ofCF, apeciaiJy in populations where the preYIIeace of kDown nwtations ialow.

INCIDENCE OF AFS08 MUTATION IN INFANTS WITH CYSTIC FIBROSIS WHO ARE FALSE NEGATIVE FOR IMMUNOREACTTVE TRYPSINOGEN BASED NEWBORN SCREENING.

~ Hammond XB. Accuno FJ. Dqartmenu of Pedilltrics and Preventive Medil:U.e and BIDJUtrlc.r. u,.;,_.;ty of O>lorado Healtlt Scie11cu Cmter and 111e Cltildmt 's HospitDI., Denver, CO. USA.

Inc:reuma interest in euly treatment of cystic fibrosis (CF) bas prompted furdler eumiDatioa of opprooc:bes to early cliaposis. Immunorcoctive trypsinoam (IRT) baed newborn sc:reeuiDs propams for CF have identified signific:mt IIWIIben of infanll however the factors leading to false neptive status are incompletely understood. To detmnine whether the incidence of the AF$08 aenotype is different in infauts falle oeptive for IRT based sc-..ing and other CF infanta, we eumiDed senotypes of 477 individuals with CF acrou four diapollic groups: Dewbom oc:reeued (NBS), falae aeptive on DeWbom IICI"eell (FNBS), meconium iJeul (MI), and conventional cliaposis (CON). ConvcntionaUy diaJIDC!ICd subjec:tl were identified through clinical presentation and were born in Colorado before sc:reeuiDs began, or were born outside of the state. Since the statewide newborn began iD 1982, 147 NBS paticntl aad 12 false aeptive patients have been gcnotyped. Tbe raean qe of diqDolis for e.c:b of the diqnostic group~ were (yean±SEM): NBS-Q.II.±(l.02, FN•2.2±1.1, MJ-().16.±(l.06, CON-4.3.±().$. Subjec:ts were divided iDto one of tluee group1 baed on 1enotype: AFSOB homozygous (FIF), AF~08 belerozygOUI (FIN), aad DO AFS08 mutation (NIN). Tbe diatributiona of 1enotypes for e.c:b of the diapostic group~ were:

NBS (n-147): FIF•SS%, FIN•37%, NIN-a% FNBS (n•l2): FIF•33%, FIN•$0%, NIN•17% Ml (n-69): FIF_,B%, FIN•3S%, NIN•7% CON (n•249): FIF•S2%, FN-40%, N/N-8% .

lnciclence of the AFSOB Daltation iD patients false negative for IRT screenin& wu 58%, significandy different (p-0.05) from the iDcideace iD the rest of the palicnll (73%). We conclude that the AFSOB nartation is leu frequent in patienll false neaative for IRT bued newbom sc:nenina than in the acneral CF population. This sugesll that the falle neptive ra1e for JJtT sc:reeuiDs may be increued iD populations with a low &equency of the AFS08 mutation.

153 EPIDEMIOLOGY OF CYSTIC FIBROSIS IN RUSSIA Nl Kaoranoy. NJ Kaahlrakaja, JK Ginter, NV Petrova. Dept of Cyatic Flbroala, lnatltute of Clinical Ganetics RAMS, Moacow.

Cystic fibrosis (CFI Is the commonest lethal recessive autosomal disease among caucasian populations. The frequency of CF and CFTR gene mutations (especially AF508 deletion) Is variable In different countrlea. Over the laat two yeara the frequency of CF In the Russian population has been studied. It waa dlacovered that CF affects1:6.000 (E Potapova, 1995) or even 1:12.000 (N Petrova, 19951 newborns. The major AF608 mutation waa found In the chromoaomas of 68% of CF patients In the Russian population and 41% In CF patients In Moacow.

Af608/&F608 Af508/-- I -

Russia

34% 48% 18%

Moacow

21"' 40% 39%

The moat common mutation• In the Rusalan population were different to thou ducrlbed In Europe. N1303K, 214deiT, 21841naA, with frequency of 3·7%. Only 70% of CF chromoaomea could be Identified. So genetic screening programmes aiming to Identify all CF mutatlona are lmpo11lble In thla heterogenous population

Model Systems

154*

BIOELECTRIC PROPERTlES OF HUMAN CF AND NON-CF FETAL TRACHEAL XENOGRAFT& IN SCID MICE R TlfOUVIIlZi.n'. B. P8ault1, E. Pucheile' llld T. Chine!'. 1CNRS UPR9064, Nogenl-lli'-M-, 11NSERMU314, Reimsm 'lllboratoira de Biologie 111 Phlmlacologie dee Epitheli~m~ Reepiratoirea, Univeniti Paris V, Boulogne, France.

Pationa of tunan fetal tr.a- '""'anted aubcutaneoualy Into SCID mice deYelcp and n maintained on the long tam 11 closed ftiJid.filled xenografta lined with 1 malin paeudostratified ciliated lnl aecntlory lll'face epitheli~.m m IWnucoaal glands (P8ault etal. Hun Gene Ther. 1994; 5: 1131-37). Mature IJ'Iinfected CF and non-CF fetal tracheal xenogratta llhow no elf!~ In muc:oeal armtecbn. Tha bioelectric properties of 33 xenografta from15 dflerant non-CF fetal tr.a- llld 14 xenogralta from 4 dfl811111 CF AF508/AF508 fetal tracheal went evaluated In w~ using cirect lntralumenal rneaauamant of potential dfferance (PO) rnd/a in wtro In Uasing c:hlrrber. CF llld non­CF giOI4)a ol xenograftl had timilar IMIIl gestational 1ge1 (non-CF: 18.6:1:0.8 weeks, range: 13-26; CF: 18.1:t1.2 weeka, range: 11-22; p>0.7) llld cllrationa of engraftment (non-CF: 14.0:1:2.0 weeka, range: 3-54; CF: 11.0:t3.1 weeks, range: 4-50; p>0.5). Within CF llld non-CF groups, all recorded bioelectric parameten W1118 Independent of gestational age end duration of engraftment of the xenogratta, even on the long lllrm. In w110PDin non-CF xenografta (·9.5:t0.9 mV,range: ..C.& to-13.4 mV,n=17) -tignificanlly more electronegative (p<0.04) than In CF xenogratta (-5.9:1:0.7 rrW, range: -3.2 to -8.8 mJ, n=10). Mean baseline In wtro bioelectric properties, e.g. PO (non-CF: ·2.9:1:0.5 mV; CF: -1.3:1:0.3 mV;p<0.02), llc(non-CF: 39.2:1:6.7 j~Aotm2; CF: 14.3:t3.3 j~Aotm2; p<0.02) llld R (non-CF: 76.1:1:8.4 Q.an'; CF: 146.6:1:20.6 Q.an'; p<0.02) Wlll8 liso consistent with 1 lower basal electrogenic activity in CF xenogratta (n=6) u ~red to non-CF (n=10). In ralatiw Vlluea, lddtion of .nlloride (inhibitor of epithelial sociL.m channel ICtivity) ptOYOited tignificent dacraue of lac In both non-CF (·39.4:t7.4%, p<0.006) end CF (-60.0:t10.3%, p<0.03) xenografts. In the praaence ol amiloride, lddtion of fcnkolin (cAMP agonist known to activate CFTR) 111sulted In a tignlficant Increase of lac in non-CF xenogratta (+24.1:1:5.8%, p<0.01) but had no tignificent el!ect on CF xenograft& (p>0.5). By contrast. 11b~ent lddtion of A TP lnlnaiently Increased lac In both non-CF (+43.7:t11.1'Mo) llld CF (+68.8:t15.7'Mo) xenograft~. In COI'flllement low-chloride conditione tested on 8 non-CF xenogratta 111sulted In n~apecliw 71% n 85% lnhibitiona of torskolln-induced n ATP-induced lnc:rellsa of lac u C0f'1'4)11red to oontrol ooncitiona, whereas the el!ect of emlloride wu unchanged. Overall, eleclrOgenic activity In CF xenogratta waa significantly recluoed In lbaoluta values u ~red to non-CF lilher In basal oondtionl (PO, lac) a regarding the ei!ICII of .niloridl a ATP. Never1helen, In ralatiw Vlluea, malin ll'linfected tunan fatal tracheal xenografta In SCID mice exhibit bioelectric properties which 1111 similar to those I1ICOided In tunan postnatal lirwaya, lncludng bllsallllliloride-aenaiti'lll sodL.m lbSOfll(ion. AlP-dependant chloride aec111tion, both higher In CF xanograltl than In non-CF, end cAMP-dependent chloride aecration which II absent from CF ...-.ogralts. Erwtronmanlal ooncitionl IOllld In fetal tracheal xenografta In SCID mice (at.tity, cloaad milieu, no 1ir expoiUIII) may ailed 1hl complete expresaion of ion h1lpolt lbnolmliitiu loiJ'Id In postnatal CF lirwaya. Yet. theSI IIIIUIII fla1her validate the 1M of thia in WIIO model for the uperlmental .-.men~ of human normal n CF limy functionL Supported in pllt lly SySiamix Inc. end lly AFLM

155* SUSCEPTIBILITY TO, AND SEVERITY OF P. /&ERUGINOSA INFECTION BY CF RESPIRATORY EPITHELIUM,~. T. Phillips, M. Kaplan, G. Marlin, R. Ramphal, S. Lory and A.L. Smith. Department of Molecular Microbiology It lmmunoiOI}', University of MissoUri-Columbia, Columbia, MO, USA.

To gain insight into the unique IU!Ceptibility of the CF respiratory tract 10 infection by P. Mruginosa we adapted the xenograft model used for gene transfer experiments. Respiratory epithelial ceUs from non-CF and CF patients were expanded in culture and passed once or twice before I 0" were seeded into devitalized rat trachea Implanted subcutaneously in a nu/nu mouse. Twenty-five daya after aeedina, the xenografts were challenaed by the intraluminal Instillation of araded inoculae of P. Mruginosa strain PAK. Twenty-four houra after inoculation the xenograft wu irrigated with 1/3 PBS and the effluent quantitatively cultured on media aclective for CF pathoge111: irrigation was continued until five daya after inoculation, when the xenograft wu removed. A crou-~eetion obtained for histological evaluation, and the xenograft homogenized in PBS and quantitatively cultured. We found that the CF respiratory epithelium wu more IUICCJ>tible to colonization. The ID,. for PAK wu 316 cfu (range 62-1600; N•20) for CF; 2487 cfu (range 270-12,800; N •23) for non-CF tissue. Five days after inoculation of P. Mruginoso the lumen of the CP xenografts is packed with mUCOUJ and contains numcr0111 PMN'a. To IIICSI acverity of infection we quantitated xenograft bacterial and PMN density five days after inoculation. We found more bacteria in the CF xenograft and 1 more Intense leulcocytc infiltration: with an inoculum of 1.6 • 2.3x10' PAK in the mean bacterial density at 5 days in CF xenografts wu3.14 x 10" cfu (N•7), while in normal epithelium is was 2.71x10' cfu. PAK with mutations in either of two adheslns were unchanged in their ability to elicit infection, in comparison 10 the rpoN

Poster Session Abstracts 251

mutant: using an inoculum of ,,0 to B.lxlO' cfu, we found the xenograft density of the following PAK mutants five daya after Inoculation was: rpoN I.Bx!O' cfu in CF (N •3), l.Bx!O' cfu (N •3) in normal; piiA 6.12x!O" (N•2) in CF and 1.03x10' cfu in normal (N•2); jlaC 4.2x!O" cfu iD CP (Nz2) and 8.6x10' efu in normal (N•2). We conclude that CF respiratory epithelium is more susceptible 10 colonization by P. uruginosa and hal a greater acverity of infection. The RpoN mutant of strain PAK produces 1 leu severe infection in normal and CF xenografts, presumably due 10 the absence of one or several gene products controlled by this sigma factor.

156* EXCESSIVE INFLAMMATORY RESPONSE OF CYSTIC FIBROSIS MICE TO PSEUDOMONAS AERUGINOSA BRONCHOPULMONARY INFECTION. A.nn Heeckeren, T. Bonfield, R. Wallenga, M. Konstan, P. B. Davis, and T. FerkoL Department of Pediatrics at Rainbow Babiea and Childrens Hospital, Case Western Reserve University School of Medicine, Cleveland, Ohio.

Although the pulmonary Wection with Pstudomoruu dtruginosiJ contributes to the morbidity of patients with cystic fibrosis (CF), the Intense host Inflammatory response largely eccounts for the progressive, suppurative pulmonary disease. Mouse models of CF, however, do not develop pulmonary disease spontaneously. We examined the effects of bronchopulmonary Inflammation m transgenic mice homozygous for the S489X (null) mutation, using an animal model of chronic Psrudomonds endobronchial Infection. The mortality of CP mice Inoculated with Pstudomolllls-laden beads was significantly higher than the wild type animals, with 82% (9/11) of Infected CF mice, but only 23% (6/26) of normal mice, dying within 10 days of infection. The absolute neutrophil cowtll measured In the bronchoalveolar lavage (BAL) fluid from infected CF mice (n • 6) 3 days after Inoculation was significantly elevated (p • 0.01) compared to infected normal mice (n • 11). This Inflammatory cell response correlated (r • 0.65, p • 0.03) with the weight loss of both CF and normal mice after inoculation with Psrudomon11s-laden beads. Concentrations of various inflammatory mediators meuured 3 days after inoculation were markedly elevated In the BAL fluid from Infected CP mice compared to their Infected normal littermate1. In particular, concentrations of lNP-a (p • 0.007) and IL-111 (p • 0.03)1n the ELP from CF mice were significantly higher than those meuured in normal littermates after inoculation Psrudomonas-laden beads. Murine IL-6 levels were higher in the Infected CP mice, but the levels detected did not achieve statistical significance compared to infected normal mice (p • 0.06). In addition, LTB.Jevels measured In the ELF from CF mice was higher than that detected in their normal counterparts after Intratracheal inoculation with beads embedded with P. dtruginostL Thus, this model of endobronchial Wection with P. lln'llginOSIJ may permit examination of the relationship of bacterial infections, inflammation, and the cellular and genetic defects in CF.

Supported by National Institutes of Health DK48996 and DK43999.

157* COENGRAFTMENT IN sao MICE Of HUMAN FETAL AJFMAY AND AUTOLOGOUS HEMATOPOtEl1CnSSUE PERMITS THE LONG-TERM ESTABUSHMENT OF TYPICAL BRONCHUS-ASSOCIATEDLYMPHOtDSTRUCTURES R Tirouvanzi.n, I. CUll n B. P8aUt. CNRS UPR 9064, Nogenl-ll.f·Mame, FrllllCe.

111'1111J10inltanmatcry ct¢unctions contrilute llbatantially to CF airway tiaeasa. Recent deta haw suggested that pivotal aitarationa of mucosal irnnu1ty may even OCC1J' VfiiY earty In CF pathogenaall, I.e. bef0111 CMirt airway Infection. lnmre medlaniama in the bronchial mucoea involve in the first line lll'face epithelial cella and llbmucosal glands (antimicrobial peptide aecration, pathogen recognition, cytokine prodlction) but liao apeclallzed ~tic cella which 1111 lither rasident (mast oella, T end B lyrrflhocytal m to aane extent IIIICIIlphagea) a llbnitted to rapid turnover (denditic oalla, grenulocytea). We have previously lhown that tunan CF and non-CF fetal upper lirwaya reach lUI epithelial mallntion m ltilcutaneoua '""'entation In SCID mice. ltl'111U'dlistochemical enalysia of the hematopoietic COI!1l8flmenl in thel8 lirway xenograft& llhowllhlt (1) holt imrTu1oinllanvnatory 01111 1111 abient or VfiiY acame within the xenograftl; when present theSI oella1111foll'ld in the lamina propria bli never In lib- or inlrHpilhelial potition; (2) hL.mln mast oalls 1111 naya present homogeneously dssemi111ted within the lamina p!qlria; (3) dispeiHd tunan lyrrflhocytalara fol.nd in low numbell In lib- a lntrlapilhelial potilion, llough only in late fetal airways which Wlll8 oolonized bef0111 engraltmenl SCID mice 1111 otherllisa widaly used for atudiel on normal llld pathologic human hemat~etic devel~ E~ at tunan fetal bone rnarow leadl to austained endogenous hL.mln hematcpoleais; fetal thymus alao llJRlOIII long-term ~slain SCID mice, W ooengratted with letalliva' u 1 IOII'CI of hematopoietic slam oalla. When engratted alone in SCID ho&la, fetal lhymul11 well u IY!'llh nodes 1111 depleted within two monilia. Bued on theSI lectl, Wit questioned whelher ooengrattment of hematopoietic tiiiUI could enrich airny xenogratta In human

252 1997 Cystic Fibrosis Conference

11111lU10inftllllll18klry cella. 0111818111 autologous llllue combinations were lesl8d. includng aiiWII'f/lhymul, aiiWII'f/lhytllllibone II1IIIOW and aiiWII'f/lyn'pl node. The latter combination leltad wllh ti- as earty as 16 weaka of gestation, gave lhe most atrlking ..Uti. F;,.. lhe filii lme, K allowed lyiT'flh node struc11n and hematopoietic ac1Mty to be maintained on the long n (5 monlha) In SCID mice. On lhe other hand, there was global enriciYnenl of aiiWIY xenograft In hlmlln hematopoielic cells, as K ,_ dlplayecl. In addtion to melt calls: (1) lg-~~Cn~ting plasma cells In lhe lamina propria, located In functional polition around glands; (2) numeroua T lyrl1lhocytea in llbeplthellal (ananged or not In flllllc:le-llka slructinl) or in lntraepilhelial IOCilion; (3) lntraepilhellal pockell replete with hlman hematopoietic cella, typical of M cella (epithelial lflucllna 8p8Cialized In antigen -.). This In 11/vo modal d uninfectad hlman CF and non-CF airways associating malin aurface epithelial lining and glands and lhe sustained praaance ofiiJUctulu medating bronchial mucosa ilm1IJ1Iy could be uaelul to study and experimentally alter IITIIIU10inflarn1Dry proce1181 In CF. Suppcllted In part by a grant from SySinx Inc. and by Altoclallon Franpaa de Lulla aontre Ia Mucovtacldou

158*

CALU-3 CELLS AS A MODEL SYSTEM TO STUDY HUMAN AIRWAY DEFENSE MECHANISMS. c..J.ill, L. Lambert, R.J. Bridges, and R.A. Frizzell. Department of Cell Biology & Physiology, University of Pittsburgh, Pittsburgh, PAIS261, USA.

A characteristic feature of the pathology of CF is the presence of persistent lung infections. Serous cells in airway submucosal glands, which express the highest levels of CFrR in human lung, play an important role in mucosal defense by secreting proteins or peptides that act as endogenous antibiotics, as well as protease inhibitors which protect the airway surface from destructive bacterial and neutrophil proteases. Calu-3 cells, a cell line derived from a pulmonary adenocarcinoma, express high levels of CFrR and several cellular markers characteristic of submucosal serous cells. They form a polarized cell layer with tight junctions and secrets cr in response to cAMP stimulation. Using Calu-3 cells as a model system for serous cells, the goal of this study was to identify antimicrobial substances that may play an important role in human airway defense. The secreted mucus from Calu-3 cells grown on permeable filters at an air-liquid interface for 28 days was collected by washing the apical surface three times. Three bacteria (a Gram-positive strain Staphylococcus aureus and Gram-negative strains Escherichia coli and Pseudomonas aeruginosa) were used as indicator strains in a killing assay to determine the antibacterial activity. Antibacterial activity was detected in the mucous secretions, and was only released from the mucus with either S% acetic acid extraction or boiling in I% acetic acid. The killing activity was not found in the medium bathing the basal epithelial surface. Surprisingly, no detectable antibacterial activity was found in the supernatant of the apical wash after separation from the mucus by centrifugation. This result suggested that the antibacterial substances were tightly associated with the mucous secretions, presumably due to charge interactions between sialic acids linked to mucin ~d the positive residues contained in the antibiotic molecules. A port1on of this antibacterial activity was lysozyme, which was neutralized by an antibody specific to human lysozyme. Western blot confirmed the existence of lysozyme in the Calu-3 secretion. The remainder. of ~he bactericidal activity appears to be contributed by defen~m-hke substance, as judged by its broad spectrum against Gram-positive and Gram-necative bacteria, salt sensitivity, and heat stability.

Supported by the Cystic Fibrosis Foundation and NIH.

159* CILIARY-PROPULSION OF MUCUS DRIVES FLUID TRANSPORT OVER THE SURFACE OF THE AIRWAYS. Hirotoshi Matsui, Scott H. Randell, C. Wjmam Dayjs, and Richard C. Boucher. CF/Pulmonary Research and Treatment Center, University ofNorth Carolina, Chapel Hill, NC 27S99 USA.

The ciliated airways of mammalian lungs are covered by a liquid comprised of a watery periciliary liquid (PCL) layer and an overlying viscoelastic mucus layer. Theoretical analyses of mammalian airway cilia predict that PCL is transported at ~00..4 of the mucus transport l'lte or it even stationary on airway surfaces, but direct measurements have not been made. We have measured the real-time behavior of these two components durin& "mucociliary clearance", using fluorescent microspheret and phototlctivated fluorescent dyes to label the liquid on the surface of well differentiated human bronchial epithelial cell cultures. Data from conventional and confocal fluorescence microscopy show that PCL is transported at

approximately the same rate as mucus, and that the transport of both layers is abolished following the removal of mucus with dithiothreitol and washing in PBS. We conclude that the PCL is transported axially in the airways, and that this transport is due to frictional interactions with the overlying mucus layer driven by ciliary activity. In contrast to the expectations for pure shear-related flow, no vertical gradients in velocity within the PCL were observed. From this observation, and the similarity of the mean velocity of PCL transport to mucus, we also conclude that ciliary mixing of the PCL may be important in promoting the transfer of momentum between the two phases. Axial PCL transport along airway surfaces has important implications for, (I) lung physiology/pathophysiology, including the requirement that airway epithelia absorb a PCL volume load as it moves cephalad up narrowing airway surfaces and the likelihood that abnormal ion transport in cystic fibrosis reflects, in part, abnormal PCL volume regulation; and (2) lung defense, by providing a mechanism for efficient removal of hydrophilic toxins. Supported by the National Institutes of Health and the Cystic Fibrosis Foundation.

160 ASSESSMENT OP 1HE MOLECULAll BASIS POR a· PERMEATION ACilOSS 1HE Cl EPITHELIUM OP "MMLDLY AFFECTED" CP MICE K GJpmgrey R. Rozmahel. K.A. Galley, E. Garaml, M. Ramjeeslns}l, J.M. Rommens, L.-c. Tsul and C.E. Bear. Division of Cell Biology, Hospital for Sick Chlldren. Toronto, Ontario, Canada.

A subgroup ol Cftr odHIIl'ooJHIC CF mice exhibits amelioration ol Gl disease In the jejunum and Ileum (Rozmahel, 1996). In the present study we ldl!d a functional as well as a molecular approach to test the hypothesis that a non-CFTR a· oondudana! Ia upregulated a=- the apkal membrane of the intestinal epithelium In these mice. Ullling dwnber meeawementa using carbachol (lOO!JM), histamine (tmM), bradykinin (Sj&M) and forskoHn (lOIJM) showed that cr• · or cAMP-activated a· aecretlon Ia not upregulated in the jejunum and Ileum of mildly affected Cftr ..vtSCIMIHJC CF mice as these eecretagogues did not elidt an Increase In transeplthellal potential difference (PO). The basal PO In the jejunum and Ileum of the mildly affected CP mice (-2.2±0.2 mV and ·1.7±0.1mV) was lower than normal (-3.2±0.2mV and ·3.0±0.2mV), however it was elevated when compared to the severely affected Cftr ••UHC~oo•wo:: CF mice (.{).9iO.lmV and -1.1±0.2mV).In order to aseesa the contribution of a a· conductance path to the basal PO, we determined the effect of various CJ· channel blockers. The basal PO In normal and CP mice wu significantly inhibited by NPPB (SOOjiM) but not by DIDS (200j!M). The NPPB-sensltlve bual PO was similar In magnitude In the Je;.run and Ileum of CP mice with mild dlseue (APD t.8±0.2mV and 1.4±0.3mV) to that seen in nonnal mice (4PD 1.9i0.3mV and 1.7±0.3mV). This value was algnlflcantly lower In the jejunum and Ileum ol CP mice with severe disease (APD 0.8±0.4mV and 0.3±0.1mV). These resulta lllJ88'I!II that basal a· aecretion II upregulated In the Gl tract of mildly affected Cftr •JifiC/woJHIC CP mice. As the Inhibitor sensitivity of this a· amductance shows similarity to that demonstrated for the putative a· channel ac-2, we aue.ed ac-2 expression In the Gl tract of normal mice. RT·PCR and Northern analysis revealed CIC-2 mRNA expn!llion In the Ileum and J!jlnJm. Preliminary lmmunohiatodlemlcal atudlea further revealed aC-2 protein expreaalon on the plasma membrane of epithelial cells In these intestinal segment& Theae results IUgeall that transeplthelial a· aecretlon through non­CFI'R channel(a) may contribute to fluid aecretlon In the ileum and jl!junum of mice. Further, the ac-2 expl'l!lllon appean conailllmt with a possible role In basal Intestinal a· aecretlon. Thia work Ia IUpported by grants to C.E.B. from MRC (Canada). CCFF and NIH. K.G. Is IUpported through CCFF studentahlp award.

161 A MODEL FOR THE NEODIFFERENTIATION OF AIRWAY EPITHELIUM IN SCID MOUSE-ENGRAFTED FETAL TRACHEAS •A. Delplanque, *R. Tirouvanziam. *B.P~ault, **C.Coraux, **E.Puc:helle, up Gaillard. *CNRS, UPR 9064, Nogent/Marne, U[NSERM U314, IFR 53, Developmental Biology, CHU, Reima, France.

Human fetal lung and bronchial rudiments undergo considerable growth upon microsurgical ectopic implantation in the xenograft­tolerant SCID mouse (I>Uult et al, Hum Gene Ther,I994;.S:II31). The extremities of fetal trachea enpfted in SCID mice are rapidly closed by murine membranous 1iuue forming "operculum"·like structures, progressively covered by airway epit~lial .ce~ls .. The kinetics of development and ep1thehahzat1on of the membranous

operculum was studied in 16 fetal human tracheal grafts, including 2 tracheal rings. These rudiments, ranging from IS to 36 weeks of development were implanted for I to 4 weeks under the skin of SCID mice. The grafts were collected at days 4, 7, II, 14, 28 and S6. Development of the operculum was examined on serial semi-thin sections. Immunohistochemistry for cytokeratin (CK) 7, 13, 14 and integrin a 3 and 81 was used to discriminate the epithelial subpopulations lining the fibrous membrane. This membrane was also studied after iterative engrafternent. As soon as day 4, the extremities of engrafted tracheas were closed by a membranous and fibrous operculum lined with flattened epithelial cells, except in the middle part where the tissue was loose and included a few inflammatory cells. Opercula closed completely the trachea at day 7 and the surface facing the lumen was covered by epithelial cells migrating from the native respiratory epithelium. These cells were cuboid and columnar at the periphery and flattened in the center and were immunoreactive for C K 7, 13 and 14 as well as a3 and 81 integrin subunits, AI day II the epithelium was pseudostratified and the first ciliated cells could be observed. Columnar differentiated epithelial cells were only immunoreactive for CK 7, and not for CK 13 and 14 which are markers of basal cells. Cells in close contact with the operculum expressed CK 13, 14, and the a3 and 81 integrin subunits. At day 56, the operculum was lined by a mature pseudostratified epithelium. When reengrafted into a secondary SCID mouse, each closed extremity of the trachea implant supported the development of a new parallel epithelialized operculum. The development of this operculum mimics the processes of reepithelialization. Iterative transplants of fetal trachea in SCID mice can represent a new model to study the development of human airway epithelium and can be used to identify, sort and assay epithelial stem cells. Supported by EC-Network n•BIO-CT 95-0284.

162 GENERATION OF AIRWAY, PANCREAnC AND SALIVARY GLAND MOUSE EPITHELIAL CELL LINES WITH AND WITHOUT CFTR. M. Takacs-Jarrett and C. Cotton. Department of Pediatric:e, Case Western Reserve University, Cleveland, OH, USA.

We have developed conditionally Immortalized ep~helial cell fines from airway, pancreas and aalivary gland of CF and non-CF mice to study the effect• of the lose of CFTR on the biology of ep~helial cells. Moreover, conditionally immortalized cell Jines may have the added advantage of a more "primary" -like phenotype when grown in non-permissive condrtions. UNC mice, heterozygous for the S489X mutation (CFTR1"'1 -re bred with an JmmortoMouse (H-21<" -ta ASS) to generate a colony of CF and non-CF mice harboring the temperature-aansrtive large T antigen trensgene. Eprtheliai cella from several tlasuea alfec1ed by CF pathophysiology -ns isolated. Subsequent expansion of these cuhures from airway, pancnsaa and aaiivary gland tissue produced CF and non-CF eprthelial cell linea. Each of the these cell Jines has been maintained in cuhure lor at least 10 passages. Cell lines from these mice show condrtional growth characteriatica and a decrease In Jar~e T antigen expression when switched from • permissive temperature of 33 C to a non-permissive temperatura of 39°C. PCR analysis of CFTR DNA from cell Jysatea of the six cell linea generated confirmed the genotype of each. Cells from both non-CF and CF ceil Unes form tight junctions, becoming polarized epnheiial barriens when grown on permeable supports. Confluent monolayera of each cell type wens placed in an Ussing chamber to measuns basal bioelec1rlc properties (Rr, transep~helial resistance; 1,., short clrcuH current) and the eflec1 of elevation of intracellular cAMP w~h lorskolin (10jlM).

~ Al.om Non-CF CF

f.lnmU Non-CF

Br(Q cnl)

785±203 181 ±45

165 t 11 100 t22

2.2±0.5 2.4 t0.7

4.3±0.7 2.8±1.1

0.8±0.1* 0.3 t0.1

17.4 t 3.o• 0.2±0.4 CF

b!l:tm Non-CF 28tl±81 3.1 ±0.2 11.5±0.8* CF 230±28 3.5±0.2 0.0±0.1

Each c:eft Una expresaea the expeeted phenotype with nsspact to cAMP­induced cr secretion. Ce--activated cr aecnstion wu nstalned in all ceil linea. Th- cell linea, derived from tiaauea alfec1ed by CF, should be useful lor detailed atudy of the role of CFTR in eprthelial cell biology. (Supported by the Cyatie Fibroaia Foundation).

163 Altered Mineral Content In the Incisors or CFI'R(-) Mice. L.R. Gawenill1, J.S. Morris1, B. Derenzy1, C.L. Wolf\ P. Spencer", and L.L Clarke1. Dept. of Oral Biology, Univ. ofMi880uri-Kansas City School of Denistry"; Dalton Cardiovasc. Research Center and

Poster Session Abstracts 253

Dept. of Vet. Biomed. Sciences1, and Research Reactor2, Univ. of Missouri-Columbia, MO, USA

Previous studies of abnormal hard tissue mineralization in cystic fibrosis patients include the finding that deciduous teeth from CF children have reduced Ca2+ and normal P content (not tetracycline­related; Cua, Biol. Trace Elem. Res. 30:277, 1991). The incisor teeth of CFTR knockout mice also have abnormal coloration and wear as compared to normal murine incisors. Therefore, we investigated the spec1fic changes in CF tooth mineralization by measuring the mineral content of incisors from CF and normal mice using neutron activation analysis (NAA). Incisors from normal [CFTR(+)J mice were compared to teeth from their gender-matched CFTR(-) littermates (n=5). Calcium was significantly reduced, whereas P was unchanged, in the incisors of CFTR(-) mice as compared to CFTR(+) incisors (Ca~+ content: 26.20 :t 0.62 vs. 29.36 :t 0.69% weight; p<0.002; P content: 11.66 :t 0.41 vs. 11.62 :t 0.35 % weight; ns). In contrast, magnesium was significantly elevated in the CFTR(-) incisors, suggesting a degree of divalent cation substitution [CFTR(-) = 8459.92 z 1226.83 vs. CFTR(+)= 7519.91 z 1139.47 ppm; p<0.02]. Interestingly, chloride was markedly reduced in CFTR(-) incisors when compared to CFTR(+) incisors (690.61 z 76.32 vs. l321.82 :t 147.73 ppm;p<O.OOI]. Sodium was also slightly reduced m the CFTR(-) incisors [CFTR(·) = 6450.06 z 152.31 vs. CFTR(+) = 7024.48 :t 204.02 ppm; p=0.054]. No significant differences were detected for either fluoride [CFTR(-) = 193.25 z 27.52 vs. CFTR(+) = 197.52 :t 25.52 ppm; TIS) or potassium [CFTR(-)= 2144.41 z 331.95 vs. CI-'1'R(+) = 1981.65 :t 280.19 ppm; TIS]. Current studies will compare the mineral content of isolated incisor enamel. We conclude that incisor teeth of CF mice have reduced Ca2+ and normal P content, consistent with findings in CF patients. In addition, increased Mg2+ and reduced NaCI content in the CF teeth suggest that multiple mineral defects may contribute to hard tissue abnormalities in cystic fibrosis. Supported by NJ/1, UMC and CFF.

164 THE ELECI'ROGENIC, PHLORIZIN-SENSITIVE Na+/GLUCOSE TRANSPORTER IS NOT UPREGULATED IN CF MICE ~.P.R. Durie, C.E. Bear. Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.

CFTR has been implicated in the regulation of other membrane proteins, namely the arniloride-sensitive Na + channel. Various reports have linked CF intestine with altered Na•-glucose absorption and it has been hypothesized that CFTR may also act to regulate the

intestinal Na•-glucose co-transporter (Baxter et al,l990; Frase et al, 1985). In order to test this hypothesis we compared the magnitude ~~the elec~genic response to Na•~upled glucose uptake in the JeJuna ofwtld-type (WT) and CF (Cftr UNCI-I·)) mice. Jejuna obtained from 6 week old mice were mounted in Ussing chambers for assessment of transepithelial potential differences (PD).The !Da~itude of the change ~n PD evoked by 30mM glucose was larger tn JeJunal segments obtamed from WT mice than from CF mice, 1.39±0.23mV (n=22) vs 0.78±0.09mV (nzJ8) respectively, (p<O.OS). The component of this response which could be inhibited by phlorizin (I OO~tM), a specific inhibitor of the glucose transporter, was not significantly different between WT and CF mice, 0.40±0.04mV vs 0.47±0.llmV respectively, (p=0.53). Furthermore, activation of CFTR by forskolin (I 011M) pretreatment failed to modifY the magnitude of the phlorizin-sensitive component of the glucose evoked APD in WT jejuna. These results suggest that there may be no direct effect of CFTR on Na•·glucose co-transporter itself. However, there may be an indirect effect of Na•-glucose uptake on the activity of CFTR or another conductance path which accounts for the increased magnitude of the phlorizin-insensitive component of the glucose evoked MD in WT mice relative to CF mice. The ionic basis for the phlorizin-insensitive response to apical glucose administration in jejunal epithelia will be identified in our future studies. Supported by the Canadian Cystic Fibrosis Foundation.

Baxter PS et al (1990) Gut 31:817-820 Frase LL et al (1985) Gastroenterology 88:478-484

254 1997 Cystic Fibrosis Conference

165 GENERATION OF CONDITIONALLY IMMORTAL TRACHEAL CELL LINES FROM KNOCKOUT CF MICE. E.J. Ihomas, S.P. Hardy, M.l. Lclhem Dept of Phannacy, University of Brighton, Brighton, U.K.

Studies of CF in the mouse are limited by the availability and size of the tissues, a problem which could be alleviated by appropriate cell lines. Unfortunately, the high proliferative capacity of many cell lines is associated with a significant loss of differentiation. In an attempt to overcome these problems we have used mice which contain both a knockout for CFI'R and the gene for a temperature sensitive T -antigen to fenerate cell lines in which proliferation can be regulated. Mice whtch were heterozygous for a knockout of CFI'R (UNC mice) were crossed with H-2Jcb-tsAS8 mice (lmmortomouse) which contain a temperature sensitive mutant of SV40 large T-antigen. Dual heteroz_ygotes from the offspring were interbred and subsequent generattons were used for the production of cell lines. Cell lines were isolated from two mice, one homozygous (cell line MTE18) and one heterozygous (cell line MTE7b) for a CFTR knockout, by culture of tracheal epithelial cells at a temperature permissive forT-antigen activity (33"C). Both cell lines exhibit a stable rate of proliferation (population doubling time: 1.5 days) and have reached a high passage number. The T -~ligen status and epithelial nature of the cell hoes has ~n c:onf!rm~ tmm~~mically. The conditional nature of _the tmmortahsanons was mdtcated by a reduction in colony formmg efficiency at the non-permissive temperature (39.s"C), MTE18: 9.1±0.8% (33"C), 0.2±().03% (39.s-C), MTE7b: 13.8±4.9% (33"C), 3.1±0.8% (39.s-C). When cultured at 33"C both cell lines gave

transepithelial resistances in excess of lOOCan2 and phenol red fluxes of 190±57._4 I?ID~ cm·lb·t (MTE18) and 403±36.7 pmol cm·lb-1 (MTE7b), mdicatJng the formation of tight junctions. Basal shon circuit current (lac:) was 4.0;t0.9 !'A cm-2 (MTE18) and 1.8±0.5 !'A cm-2 (MTE7b). lonomycin (l~M) induced an increase in l~e of 5.05±2.4 p.A cm-2 and 1.6;!:0.6 jiA cm·2 for MTE18 and MTE7b respectively. Cells are currently being cultured at an intermediate temperature in order to maximise differentiation. Conditionally immortal cell lines from these mice may prove a valuable model system for studying CF in the mouse.

166 A LOCUS UNLINKED TO CFfR CONlltOLS NASAL POTENTIAL DIFFERENCE IN MICE. K.O ~· T.J. Kelley, and M.L. Dnunm. ~~~ of Genedcs and "atrics, Case Western Reserve UruversLJi ~veland. OH.

reaulates chloride tranaport across the epithelia of numerous tissues. The abnormal ion transport that results from mutations in CPI'R CID be IIICIIUred ill vivo llliD& the IIISal potential diffemx:e (PO) assay. The chanae in IIISal PO in response to chloride-free challenge, combined with the responae to ~J-qonists, bas been sbown to discriminate between CF and normal individuals. CF mice show a nasal PO profile similar to that seen in human CF patients in that neither null nor AFS08 homozysous mice show m increase (more negative) in PO in response to chloride-free solution or to the adenylate cyclase activator, forskolin. While the increase in PO in wild-type lliSil epithelia exposed to cAMP­eJevatina agents is consistent with CFfR activation. the source of the response to low-chloride challenge has only been presumed. Nonetheless, the response to chloride-free solution is lackin& in both humans IDd mice with mutatioas in CFTR, so it is important to understand the basis of this ~· We have found a locus in mice that controls the response to cbloride-free solution IDd segregates with the IJOUli COil color locus. The mouse lltrlin is a mixture of 129/SvEv and CS1BU6J. 8 of 8 qouti mice show ID increase in PO in response to chloride-free solution (4PD • -9.0J:l.OmV) while 12 of 13 black mice lack arapoase to the same solution (4PD • 0.8±1.3mV). 'There was no aipificant dift'emlce in the average PO between the aaouti IDd blaclt mice in response to the fonkolin (4PD • ·3.8±1.8mV and -3.1:!:2.1mV, respectively). ~liminary results 111ing polymorphic marbrs IIJI1'0IIDdln& tbe aaouti locus sugest that the locus controlling the chloride-tree response is IOCiflld in a 4 eM region telomeric to the IJOUli locus. Purtber analysis will be c:arrled out to nanow the locus and identify candidlfe lriDICri~. BecaUie the low-chloride response is llblall or reduced in CF, 11 bas been assumed to be a consequence of reduced CFfR activity. We are c:urrmtly testing that hypothesis pharmacologically, and will carry out PO measurements in the presence of Various chloride cbanDel inhibitors such u DIDS and DPC. As both the low-chloride and cAMP-stimJiated responses are absent in CF, it is

unclear which is more important to the disease process. Therefore, understanding regulation of the response to low chloride may be quite relevant to pathogenesis in CF. A key step toward understanding that process is identifying genes encoding factors which influence it. (This work supported by a gnml from NIDDK and CFF).

New Therapies

167*

DEVELOPMENT OF A SENSinVE FLUORESCENCE ASSAY OF DNA EXIT FROM ENDOSOMES FOR NON-VIRAL GENE DELIVERY. Joachim Biwersi, N. Emans, Y. U, T. Ma and A.S. Veri<man. Cystic Fibrosis Research Center, UCSF, CA. USA.

Recent studies have shown that cationic liposomes do not fuse directly with the plasma membrane, but are endocytosed. Only a small fraction of the DNA is released into the cytoplasm, suggesting that gene exit from endosomes Is an important barrier to liposome-mediated gene transfer. To develop a sensitive fluorescence assay for DNA exit from endosomes, our strategy was to express dextranase in call cytoplasm. Dextranase is produced by bacteria and fungi, but not by mammalian cells, and hydrolyzes the polysaccharide dextran into small trlseccharides. Release of DNA from endosomes is then measured by hydrolysis of a ftuorogenic dextran covalently bound to the DNA. The dextran is heavily labeled with fluorophore so that the fluorescence is self-quenched. Hydrolysis into small fragments in the cytoplasm releases self-quenching and increases fluorescence. Dextran with a high substitution of amino groups was synthesized by reaction of dextran (70 kDa) with chloroacetlc acid and ethyldiamine. To optimize the fluorescence increase after dextranase digestion, aminodextran was reacted with amine reactive derivatives of different fluorophorea. BODIPY-Iabeled dextran showed the highest fluorescence increase (29-fold) upon dextranase additlon. Dextranase from Penicillium mlnloluteum was cloned using primers flanking the 5' and 3' ends of the coding region of a published dextranase (Roca et al. Yeast, 12, 1187-1200, 1996). The cloned 804 amino acid protein had 91% amino acid homology with the published dextranase, probably representing a substraln lsoform. To test whether the dextranase can be expressed In mammalian cells, the dextranase eDNA was subcloned into the pcDNA3.1 (+) in functional form and transiently transfected into CHO and MOCK cells. The cytoplasm of control and dextransae-transfected cells was labeled with BODIPY-dextran by gently rocking the cells with glasa beads (1D0-450 mm). Only dextranase-transfected cells showed bright cytoplasmic fluorescence Indicating the! the dextranase was expressed and functional, and that CHO and MOCK cells do not have endogenous dextranase activtty. The new fluorescence assay should permit the measurement of <2% of DNA-dextranlllposome complex release into the cytosol. (Supported by CFF)

168 EVALUAnON OF FLUORESCENCE METHODS TO ASSESS FUNcnoNAL CFTR GENE TRANSFER. Joachim Blwersi and A.S. Verkman. Cystic Fibroeia Research Center, U.C.S.F.

Two fluorescence strategies were evaluated as potentialaurrogate mar1<ers for CFTR gene delivery: fluoreacent dyes tranaported by CFTR, and chloride senaltlve fluoreecent indicators. Fluoreecence methods are attractive becauae of high senlltMty, ability to quantify functional CFTR delivery on a cell-to-cell balls, and the possibility of making In lit\10 meaeurements. Werllo et al. (PNAS, 93, 1167-72, 1996) IICI'I8ned a serlee of fluorescent dyes for selective uptake by CFTR-expreulng cells and found an Increased uptake of hydroethidium (HET, 2-4 fold) and clhydrorhodamine 6G (ciH-R-6G, 1.5-2 fold) that wee dependent on cell type. We repeated these studies and screened a serlee of synthesized analogs for uptake in control va. CFTR­expreselng 3T311broblasts. Dyes tested included dlhydrofluoresc:ein (diH-F), diH-carboxy-F, ciH-F-dlacetate, diH-dlchloro-F-dlacetate, rhodamine110 (R110), diH-R110, sulfo-R101, diH·sulfo-R 101, and tstramethylrosamlne. The diH-derlvallves were ayntheslzed from the parent dye by reduction with metalic zinc in anhydrous MeOH. They

are very sensitive to oxidation by the air and were used immediately after synthesis. HET (2.0 fold), diH R 6G (1.2), diH-F (1.4), R 110 (1.3) and diH R 110 (1.3) showed a small increase in uptake in eFTA­expressing cells. Stimulation by forskolin had no effect on dye uptake. The Increase was very sensitive to the purity of the diH dyes; small amounts of oxidized parent dye abolished the increased uptake. The use of SPQ and second generation derivatives to measure functional cr transport was evaluated from the kinetics of fluorescence increase in response to er-No; exchange and forskolin addition. The existing indicators (SPQ, MEQ) were adequate to identify CFTR-expressing cells by fluorescence microscopy and cuvette fluorimetry of stirred cells; however, the optical properties (UV excitation, single wavelength detection) did not permit FACS analysis of Cr conductance in individual cells. We conclude that fluorescent dye uptake Is not useful to assess eFTA replacement, but that improved Cl- indicators for ratiornetric measurement should permit quantitative cr conductance determination by FAeS. Work Is in progress to synthesize an indicator containing the er -sensitive quinolinium and the er -Insensitive dansyl ftuorophore linked by a rigid polyproline linker for ratio-imaging. (Supported by eFF)

169* Simultaneous Delivery of Drugs and Genes by Gelatin Nanospheres. S. Walsh', S. Beck', R. Rubenstein', P. Zeitlin', T. Flotte1

, 111d K. Leong'. Departments of Biomedical Engineering' end Pediatrics', The Johns Hopkins University, Baltimore, MD, USA. The Gene Therapy Center', Univenity of Florida, Gainesville, FL, USA.

ResearCh into new clinicallheropies for cystic fibrosis has focused primarily on either phamacological agents or gene delivery IY'!'"'•· This study examines '!'e potential of gelatin nanospheres 10 simultaneOUsly dehver dru~ 111d aenes '? stunulate CFTR expression in targeted cells. We previously descnbed a non-vtral gene dehvery nonoporticle fonned by lhe eornplex coacervation of aelatin 111d. D~A. Gelatin nanospheres were shown to efficiently transfect a hum111 tracheal ep1thehal cell hne (3HTE) and deliver a human specific CFTR eDNA to rabbit airway epithelial cells. Lung tissue sections were examined for expression of CFTR by immunohistochemical stainin& (TrueBiue"', KPL) wilh a human specific 1111i-R domain antibody. Airways administered with the nenospheres showed intense dark blue/purple stainin& compared to saline treated airways. Funhennore, staining was hi&hly localized to lhe apical membrane surface of airway cells, demonstratin& lhat the expressed CFTR is correctly trafficked to its site of function. Airway expression in rabbit lung was con finned using a GFP (green fluorescence protein) aene encapsulated in aelatin n11101pheres. Bronchial epithelial brushings were oblllined from n11101phere 111d untreated airways, cytospun onto slides, 111d examined for GFP expression. Stron& GFP fluorescence was detected in a hi&h percentage of cells (-SO"~) treated with a n~~~osphere DNA dose as low as 651'&· These results demonstrate the efficac:y of selatin n~ospheres ~ an In v~ sene delivery vehicle for CF. One pocential adv111tage of thiS system 11 lhe ab&hty to coencapsulate drugs into the n111oporticles. One anractive candidate for drug lherapy of CF is sodium 4·phenylbutyrate (4PBA). The action of 4PBA has been shown to restore CFTR function on lhe plasma membrane of l&FSOI-expressing bronchial epithelial cells in vitro. N111ospheres with coencapsulated 4PBA end GFP eDNA, which allows for independent detection of dru& 111d eene effects, were incubated with the bronchial epithelial cell line 183-1 (AFSOI!WI282X) for four houra 111d then grown in nonnal media for lhree days. Free 4PBA (lmM) was incubated with the cells for the entire three days. Induction of CFTR expression wu detected by 111tihody stainina in nanosphere 111d free 4PBA treated cells. However, lhe intensity of stain in& was greater in nanosphere-treated cells even lhou&h the estimated dose of 4PBA in n11101pheres was 100 10 1000 times less then the free druB dose. This effect strongly indicates that nanospheres can deliver a high local dose of 4PBA inside cells, thus improving bioavailability of the drua. FacScan data showed normal GFP-nanosphere transfection levels in IBl-1 cells, indicatin& lhat 4PBA coencapsulation does not alter gene transfer capabilities. These results demonstrate the potential for a combined gene 111d drug delivery vehicle 10 more effectively comet CF chloride conductance th111 eilher gene or dru& alone In patients with the t&FSOI mutation. (Supported by the Cystic Fibrosis Foundation).

170* THE CYTOSOL CONSTITUTES A METABOLIC BARRIER AGAINST TIL\NSGENE DELIVERY TO THE NUCLEUS Delphine Lechardeur. Kyoung-Jin Sohn, Barbara Beatty, Jeremy Squire, H. O'Brodovich and ~ergely L. L~s: The Hospital for Sick Children, Research lnslllute and Untverstty of Toronto, Canada

Cellular expression of plasmid-DNA, by non-viral vectors such as cationic lipids, is an inefficient process .as c_ompared to viral infectio~. The tow efficacy of transgene expressiOn IS thought to be caused, m part, by the entrapment of plasmid-DNA in the endo-lysosomal compartment and by the diffusional barrier imposed by nuclear

Poster Session Abstracts 255

envelope. It is highly conceivable that plasmid dissociates from cationic lipid in the cytosol prior to its translocation via the nuclear­pore complex. However, very little is known about the transport kinetics and biochemical interactions of naked plasmid-DNA during its movement from the cytosol to the nucleus. To investigate this step of transgene delivery, plasmid-DNA was introduced into the cytosol by microinjection together with rhodamine-dextran. Localization of plasmid-DNA in microinjected cells was perfonned with light and laser-confocal fluorescence microscopy, following fluorescenl in situ hybridization (FISH) with biotinylated DNA-probes. Despite the high sensitivity of the FISH protocol and the significant level of expression of luciferase activity, we were unable to locate plasmid-DNA in the nucleus of injected cells. To our surprise, single-cell fluorescent quantitative video-imaging revealed that plasmid-DNA rapidly disappeared with a half-life of • 100 min. The rapid turnover of plasmid-DNA was independent of the cell type (COS-I or HeLa) and of the plasmid (pGI2-Iuc, pCMV-CAT or pUC) utilized. Control experiments revealed that neither lysosomal degradation, nor exocytosis or cell division can account for the rapid Joss of plasmid­DNA from the cytosol. In contrast, reducing the incubation temperature to 4 •C or depleting the cytosolic protein content by selective penneabilization of the plasma membrane, inhibited almost completely the disappearance of plasmid-DNA. These observations suggest that naked plasmid-DNA is susceptible to rapid degradation by an enzymatic process associated with the cytosol which presumably diminishes further the overall efficacy of transgene expression. We are in the process of pharmacological characterization of the cytosolic enzyme activity responsible for DNA degradation. Supported by CCFF and HSC Fdn.

171* INCLUSION OF THE ADENOVIRUS E4 REGION IN A PLASMID VECTOR HARBORING A CMV PROMOTER CONFERS MORE PERSISTENT EXPRESSION IN THE LUNG. N..S...:tMi. J. Marshall, M.J. Przyby/skll, R.J. Ziegler, P. W. Rafter, D.M. K)'sokenskl, D. A""entano, R.J. Gregaty, and S.H. Ch#mg. Genzyme Corp., One Mountain Road, Framingham, UA 01701.

Curren! plasmid vectora used for expressing the cystic fibrosis (CF) transmembrane conductance regulator resu~ only in transient expression in the lung. Typically, intranasal instillation of a complex ol the cationic lipid GL-67 and pCF1-CFTR into BALBic mice resuhs in high level transgene expression for up to 4 days, after which the levels rapidly decline to approximately 5% of the maximal value by day 21. Recent studies with adenovirus vectot'll indicate that an E4 product( a) can be supplied In trens to facilitate persistent expre88ion from the cytomegalovirua (CMV) promoter. To determine If these E4 proteins could also act In trana to affect greater persistenl expression from a plasmid vector harboring a CMV promoter, Ad2/BGal..,. (containing an intact E4 region) was complexed with GL-67 and pCF1.CAT end then Instilled into lungs ol BALBic-nu/nu mice. Expression of CAT in the lungs of these animals was undiminished at day 21, and 40% of day 2 expression levels remained after 60 days. In contrast, mice that were instilled with Ad2/BGal-2 (deleted of E4 except for ORF6) complexed with GL-67 and pCF1.CAT exhibited only 5% of day 2 CAT levels at day 21. To ascertain whether other adenovirus proteins in addition to E4 were required for persistent expression of the plasmid vector, the E4 region was cloned into a plasmid (pAd-E4) and then co-instilled with pCF1-CAT into lungs of BALBic-nu/nu mice. Greater persistence ol CAT expression was observed in mice that were co-instilled with the E4·containing plasmid. Approximately 24% of day 2 CAT levels were stili detected at day 21 in animals co-instilled with pAd·E4 compared to less than 2% in animals treated with a control plasmid. The lower levels of CAT observed st day 21 with pAd-E4 (24%) compared to Ad2/BGai-4 (1 00%) suggests that either other adenovirus component• may be required for efficient trans-activation of the CMV promoter or that pAd·E4 may be producing lower levels of E4 proteins than Ad2JBGa1·4. To test this, the E4 region was cloned into another plasmid vector (pCF1-E4) where it is now placed under the transcriptional control of a CMV instead of ita own endogenous promoter. Each of the individual open reading frames in E4 hava also been subcloned into plasmid vector• to ascertain which E4 proteins are required to confer persistent expression. To localize the region within the CMV promoter that is responsive to E4, a number of mutants containing varying deletions of the CMV promoter wera also constructed. A truncated CMV promoter (-140 relative to the transcriptional start) was still responsive to E4. These data suggest that elements contained within E4 can enhance the persistence of plasmid­mediated lransgene expression in the lung and should therefore augment the efficecy of cationic lipid-mediated gene transfer for CF.

256 1997 Cystic Fibrosis Conference

172*

NOVEL CATIONIC LIPIDS FOR GENE TRANSFECTION INTO AIRWAY EPITHELIAL CELLS. N. Oudrhiri,!. Navarro, J.P. Vigneron*, T. Leclerc, M. Peuchmaur, ).M. Lehn* and P. Lehn. INSERM U458, Hospital Robert Debr~. Paris and *Laboratoire de Chimie des Interactions Molkulaires, Coll~ge de France, Paris (France).

Synthetic vectors represent an attractive alternative approach ID viral vectors for gene transfer studies and gene therapy applications. As various amine-carrying cationic lipids have already been shown to be efficient vectors for the transfection of eukaryotic cells, we have directed our efforta toward the design of cationic lipids characterized by novel cationic moieties. Two cholesterol derivatives bearing guanidinium groups bis-guanidinium-spermidine-cholesterol (BGSC) and bis-guanidinium-tren-<holesterol (BGTq - have been synthesized and tested. They combine the membrane compatible features of the cholesterol subunit and the favorable structural and high pK features of the guanidlnium functions for DNA binding. Reagent BGTC is very efficient for gene transfection into a variety of mammalian cell lines when used as a micellar solution. Both BGTC and BGSC present also a high transfection activity when formulated as liposomes with the neutral phospholipid dioleoylphosphatidyl ethanolamine (DOPE). Most importantly, we also have investigated the use of lipid BGTC for gene delivery into primary airway epithelial cells in vitro and in vivo. The results obtained indicate that the lipid BGTC can be used to transfer a reporter gene into primary cultures of airway epithelial cells isolated from human nasal polyps. In ongoing studies, we are at present investigating the differentiation state of the transfected cells. Furthermore, we also found that BGTC/DOPE liposomes are efficient for gene delivery to the mouse airway epithelium in vivo. Transfected cells were detected both in the surface epithelium and in submucosal glands by X-gal staining as well as by Immunohistochemistry techniques. In addition, transfection efficiency in vivo was quantitatively assessed by using the luciferase reporter gene system. Positive data with primary human cells in vitro and in the mouse airways in vivo confirm the potential of cationic lipids for lung-directed gene therapy. Studies in CF mice can at present be undertaken in order to better assess the real usefulness of these novel cationic lipids for lung gene therapy for CF. (Sui'Pf'l'ltd by IM Association Franf'liSt de Lulie conlrt Ia Mucoviscidose).

173*

CONSEQUENCES ON IMMUNITY AND TRANSGENE EXPRESSION BY FURTHER DELETING THE ADENOVIRUS GENOME. A.hYjgaj. M. Lusky, P. Leroy, A .I. Michou, M. Christ ct M. Mehtali. Trsnspne S.A., II, rue de Molsheim, 67000 Sb'Uboura, Frsnce.

In order to ch~ more precisely die molecular and immunoloaicsl mechanisms controllinadle fale oflhe transduced pne, we investlpted in nine immunocompetent and immunodetlcient mouse atnins the Influence of die mouse strain, nature of t1'1111lene (blcterial LacZ VI human FIX), type or immune response (humoral VI

cellular) and 111Jets of immunity(~ products vs adenovirus antiaens) on the In vivo persistence of die tl'll1lpne expression. This comparative study show that: (i) the cellular Immunity 10 viral antiaens plays a minor role in the extinction of transpne expression; in contrut, peniltence oft1'1111Jene expression is fully correlated 10 the stimulation of alai-immune response ~gainst the transgene product; (ii) the mouse ltraln does influence die pattern of expression of the transgene, depend in& on the ability of die animal 10 develop an immune response apinstthe transaene product; (iii) the type or immune response ellcitlld In the treated animals significantly Influences the persistence of the transduced cells: lou of LacZ expression is correlated with an elimination of the transduced cells by anti-LacZ CTLs, while lou of hFIX detection is correlated with an induction of antl·hFIX antibodies. These conclusions were funher supported by a dii'ICI c:omparison of the In vivo persistence of transduced liver cells In mice Injected with finl (EIIEJ-deleted) or second (E IIE3/E4- and E 1/El/ElA-deleted) aa-1on vecton encoclln& no transpnes: persistence or the transduced hepatocytes wu similar in immunocompetent and lmmunodetlcient animals despite the In vU,o demOIIIIratlon that mlclual expression or viral proteins was strongly reduced or fully abolished with second pneratlon vectora. Moreover, no significant differences were obNrvad In the anti-viral immune response elicited by the injection of first or second aeneration vec~MS. Surprlslnaly. lhe expression of lhe transaene appeared to be controlled dift'erendy in EIIEJ· and in EIIE3/B4-deleted vecton: expression cassettes fully functional in a EJIEJ-deleted -were found to be not 1\mctional In an ilopnlc EIIE31E4-deleted -· Interestinaly, expression of the transaene could be ..-..cl In vitro, but net In vivo, by adclina specific cellular re&ulatory sequences. Mono-. the requirement of E4 for transaene expression seems to be cell type­spec:itlc: an eftlcient transaene expression was observed In Vero cells infected by all vec:ton while A549 cells only efficiently expressed transaenes transduced by E IIE3·

deleted vector5. Finally, the nature of the transgene and the route of administration of the vector into mice were also found to influence the level and stability oftransgene expression. The in vitro and in vivo propenies of such E I/E3/E4·deleted vector5 will be presented and their advantages and limitations compared to first generation vectors will be discussed. This work wos supported in part by the AFM and the AFLM.

174*

Persistent expression of human CFTR in the lungs of immunocompetent mice. ~. J.A. StGeorge, J.M. Kaplan, D. Annentano, A.E. Smith, S.C. Wadsworth and R.J. Gregory. Genzyme Corporation, One Mountain Road, Framingham, MA 0170 I, USA.

El-deleted replication-defective adenoviral (Ad) vectors have been used for gene transfer to the respiratory epithelium of experimental animals and of individuals with cystic fibrosis. Studies from several laboratories have suggested that administration of high doses of first generation Ad vectors results only in transient gene expression in the lung, at least in part, due to destruction of vector-transduced cells by host cellular immune responses directed against viral proteins and/or inununogenic transgene products.

We have constructed a new Ad2 based vector encoding human CFTR. (hCFTR.) under the control of the CMV promoter. This vector contains wild type E2 and E4 regions and a partial (1.6 kb) deletion in the E3 region. The new vector, named Ad2/CMV-CFI'RIE361.6, was instilled via the intranasal route into inununocompetent CS7BU6 and BALB/c mice. Although iiLmm cytotoxic T lymphocyte (CfL) analysis indicated the presence of Ad-specific CfLs in treated mice, DNA PCR analysis of lung tissue indicates the presence of vector DNA up to 70 days post-instillation in both strains of mice. Reverse transcriptase-PCR analysis showed persistent hCFTR. mRNA expression for 70 days, which was the last time point analyzed. These results suggest that long tenn expression of hCFI'R in the lung is possible in immunocompetent mice without radical modification of the Ad vector and in spite of the presence of CfLs.

175*

LONG-TERM EXPRESSION AND DECREASED TOXICITY USING LARGE CAPACITY ADENOVIRAL VECTORS. M...Ms1m1. G. ScbiedJier, R. Parb, C. l..anpton, K. Rice, D. Carey, F. Graham, A. L. Beaudet and S. Kocbanek. Dept Molecular/Human Geneticl, Dept. of PstholoaY, Baylor Colleae of Medicine, Howard Huaba Medical Institute, Houston, TX. McMuter Univer1ity, Ontario, Canada, Southwat FOUIIdatlon Biomedical Research, San Antonio, TX

Studie1 performed wltb first aeneratlon adetlovlral (Ad) vectors indicate that residual ellpi'Hiioll of viral proccins elicits immune respooses that can lead to destruction of transduced cells. Ad vectors laddn& all codina sequences should be leu immWIIJCIIlt:, siDce they do not encode viral proteins, and immune respooses will be restricted 10 the proceiD coniCIII of the panicle itaelt'. We have administered IDtravenoualy Into CS7BU6J mice 2 x 1010 panicles of a larae capacity adcnoviral vec10r (AdSTKI09) c:ontainina the aenomlc locus or the human cx,-antitryplin (bAAT), lucludiaa the 111111ral promoter, and compared expression to that with a fant aenerarlon vector (AdhAATAEI). CS1BU6J mice are lmown not to make antibodies 10 hAA T, and expression with the first aeneration vec10r at moderate doles is relatively stable and declilla 1lowly over many weeb. In contrast, expression of hAA T Increases proaressively over die first two weeb and appears entirely stable thereafter for atleut 20 weeks In mice that received AdSTKI09. Hiltoloaical evaluation of liver shows •ians of IJilury at 6 and 12 weeks after administration of a first aeoeration vector, but is lndiltlnpilhable from normal at all tlmea with AdSTK109. lmmWIOIIIiJina for the cell proliferation marker Ki-67 showed lubslantialsWnlaa at 6 weeb after administration of the lint JCIIOfatloa - but wu approxiDWely 2 to S tlmea leu poaitive with the della vector IUIPIIina that Ibis - Ia laa toxic than the tint JCIIIRIIon -. Douae 111Udic1 (IV iD mice) with tint JCIIHallon vector indlcated that closet hlpr than I .8 x 10' pfu c:ause dlnt:t toxicil)lto bepatocytes. COIIIplf8llve studies with the delta vector are iD proareu. Two babooaa were iiiJected IV, and prellmiDary data illdic:ate stable apreuion for I I -o iD one animal whit:b Ia subatanllally better than with lint aeaeratlm vector. In c:oaclusioll, experimcall in mice and babooaa iDdicate thai delta vecton provide IUbllanllally Jonaer duration of ellpi'Hiioll after IV admiDialtatioa. In addllion, the AdSTKI09vectordemonstralelleu hiatoloait:a1 patholoaY at 6 • 12 -o after admiDiatrationwith decreued proliferation anti&ens IUIPIIina the poulbilil)l that 11n1 JCIIHallon vecton cause a hither 10 unappreclahll clirecl cellular toxicity perlutpl due to low levels of viral aeae ellpi'Hiioll.

176* CELL-TARGETED GENE THE,.AIIY VECTORS: ADENO. ASSOCIATED VIRUS VECTOR TRANSDUCTION OF NON· PI!RMISSIVI! CELLS MEDIATED BY BISPECIFIC F(ab•y), ANTIBODY. J S Bartlett, R. C. Boucher, and R. J. Samulski. Gene Therapy Center and CF/Pulmonary Research Center, UrWersity ol North Carolina at Chapel HiU, Chapel Hill, NC, USA.

A unJque vector system hal been developed for the targeted delivery of adeno-asaociated virus (M V) vectorl. With IIIia delivery system we are able 10 trlnSduCe cell lines with l'eCOITDnanl M V vectors lhat .,. normally non-permissive lor M V Infection. R~. a amaH number ol cell Anes havs been shown to be non transducible by MV vectors. We have shown that the failure of M V to transduce thMe cells Is due to the abaence of a functional M V cellular receptor. Presumably, alpecific reoeplor il needed to mediate vector binding and InternaliZation. We have overcome this deficiency and achieved transduction of theM cellliMI by targeting M V binding to a celll!liiTibrane protein which Is specifically expreiHd on lhlae cella. Since this protein has previously been shown to lntemaWze bound ligand, we hypothesized that M V bound to thi8 molecule may also be intemaUzed in a receptor-mediated manner. Targeting MV to the non­permissive calli wu achieved via a bisplcilic (bl) F(ab •). antibody. The blspecilic antibody wu comprised ol Feb' fragment& wbh apecificbiea toward intact MV capaidl and the cell membrane protein. Thus, the bl F(ab•). antibody bound both MV and cella and wu able to spec~ically target M V vector to cell Unes axpreasing the pseudo-receptor rnolecula. Tranaduction occurred through the specific interaction of the MV vector·bl F(ab •). complex and pseudo-receptor and wu dependent on the presence of the targeting antibody. The bl F(ab •). reduced the endogenous viral tropism when cella which expreas the normal M V cellular receptor ware infected. Thus, targeting wu both aelective and exclusive. These results demonstrate that M v vectors can be targeted to a specific can population and that the transduction of cella previously non-tranaduclble, or poorly tranaduclble, can be augmented by circumventing the normal M V NC&ptor and targeting vector binding and lntamallzallon to an altematlva receptor by means of a bispecillc antibody. Implications lor the targeted delivery of MV vectors to the airway eprthelium lor CF gene therapy will be discuased.

177 FACTORS INFLUENCING ADENO·ASSOCIATED VIRUS (AAV) VECTOR TRANSDUCTION OF AIRWAY EPITHELIAL CELLS. J, S Bartlett, R. J. Samulskl, and R. C. Boucher. Gene Therapy Center and CF/Pulmonary Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

Although adeno-associated virus (AA V) vectora appear promising for gene therapy In cystic fibrosis (CF) patients, many features of AA V-mediated gene transfer to CF airway epithelial cells are not well understood. We have Investigated a number of factors and experimental conditions to determine their influence on AA V vector transduction of airway epithelial cells in vitro and in vivo. The transduction efficiency of AA V (AA V~gal) was compared to that of the better characterized adenovirus (Ad) vector (AdlacZ) under similar conditions. Although similar dose-dependent relationships between the vector multiplicity of Infection (mol) and the efficiency of LacZ gene transfer were observed for each vector, a number of differences were observed. Both vectors were more efficient at transducing relatively poorly differentiated (CF/T43) cells and primary CF cultured cells as compared to better differentiated CFT1 cells or well differentiated human primary epithelial cell cultures. Transduction In vivo waa low. The effect of Incubation time, cell proliferation, helper virus, accessory gene products, and pharmacological treatments on transduction efficiency In vitro and in vivo was determined. Attempts were made to correlate differences In transduction efficiencies to differences in the early steps of vector transduction; I.e., vector binding and intemalization. The Influence of secondary events was also investigated. Cell line and differentiation-specific differences were delineated. Implications for the use of AA V vectors for CF gene therapy will be discussed.

Poster Session Abstracts 257

178* ASSESSMENT OF AJRWA Y GENE TRANSFER USING A VSV -G PSEUDOTYPED LENTIVIRAL VECTOR. L G Johnson, J.P. Mewshaw, Luigi Naldini, l.C. Olsen, and R.C. Boucher. CF/Pulmonary Research and Treatment Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC and Somatix Corpora· lion, Alameda, CA.

Lentiviral vectors derived from human immunodeficiency virus have recently been introduced as gene transfer vectors. These vectors are attractive for use in the lung due to their ability to transduce nondivid· ing cells. We used replication defective lentiviral vectors encoding ei­ther the red-shifted green fluorescent protein (GFP) or /acZ cDNAs and pseudotyped with the G glycoprotein from vesicular stomatitis vi­rus to test the potential utility of this vector system in airway epithelia. At an estimated MOl of 20, lentiviral vectors encoding GFP trans­duced a broad range of cell types efficiently (2:800/e of cells) /11 vitro, including CFTI cells (a transformed CF airway epithelial cell line), NIH JTJ fibroblasts, and HeLa cells. Experiments in growth-inhibited CFTI cells treated with aphidicolin (IS ~1g/ml) demonstrated that only the pseudotyped lentiviral vector transduced lacZ efficiently (-100%), whereas no significant transduction was detectable in aphidicolin­treated CFTI cells infected with either amphotropic or VSV-0 pseu­dotyped murine leukemia-derived retroviral vectors. However, lentivi­ral transduction of well differentiated primary human airway epithelia has been inefficient. Instillation of these vectors into the tracheas of anesthetized mice 24 hr after mechanical injury and onto naive and chemically injured (4 ~11 ofpolidocanol lo/e solution) murine nasal epi· thelia /11 vivo also transduced airway epithelia inefficiently. These data suggest that there are multiple barriers to efficient in vil'o transduction of nondividing, well differentiated airway epithelial cells by lentiviral vectors. Efficient lentiviral transduction of nondividing transformed airway epithelial cells in vitro suggest that this vector may overcome one of those barriers. Supported by HLJ83-12(LGJ) and HLS/818 (RCB) from the National Hearl Lung and Blood lnslitute

179*

SINGLE PLASMID REPLICONS FOR CFTR GENE TRANSFER. ~1.4, N. Zoul, S. Cheng5, T. Brokerl, E. Sorscherl.l.4, L. Chowl. Depts. of Physiology and Biophysics!, Molecular Genetics and Biochemistryl,Medicinel, and the Gregory Fleming James Cystic Fibrosis Research Center', University of Alabama at Birmingham, Birmingham, AL. and Genzyme Corporation', Framingham, MA, USA

We have previously reported that co:transfection of expression vecton of the human papillomavirus (HPV) replication proleins El and E2 and a reporter gene containing the HPV origin of replication is capable of a 103 and 104 fold enhancement of reporter expression during lipid mediated gene transfer into airway cells. Using a very sensitive assay, we were able lo verify lhat plasmid replication depends on all three HPV components following in vivo pulmonary gene transfer into mice (FVBIN-CS7BU6). Southern blot analysis will be necessary to determine the magnitude of this episomal replication. We have now constructed s plasmid conlaining replication components from both HPV-11 and HPV-16 which is capable of mediating its own replication in the human 293 epithelial cell line and in CF respiratory epithelial cells (183-1) derived from a patient. This single-plasmid replicon system should improve episomal maintenance in cells over the three-plasmid system. Further modifications of single-plasmid replicons to examine long-term maintenance and transgene expression in vitro and in vivo, including those conlaining the neomycin resistance gene, lac z. luciferase and CFI'R, have been constructed in which the transgene is expressed from a separate transcriptional unit or from the intemal ribosome entry site (IRES) of the encephalomyocarditis virus. Features which limit the usefulness of lipid mediated ~ene transfer include I] relatively low level expression and 2] short duration of expression. Because HPV is capable of latent persistence for years in human airways in vivo, the general strategy described here offen a means by which both of these limitations mighl be addressed. (Supported by the CFF and NIH)

258 1997 Cystic Fibrosis Conference

180 182 ICAM-1 mRNA EXPRESSION IN LUNG TISSUE OF NON­HUMAN PRIMATES EXPOSED IN VIVO TO El-EJ DELETED ADENOVIRUS VECTOR EXPRESSING CFfR. E. Nicolisl, P. Melottil, A. Bout2, A. Paviranil, G Cabrinil. !Cystic Fibrosis Center, Verona, Italy; 2JntroGene BY, Leiden, The Netherlands; 3Transgene, Strasbourg, France.

Previous in vitro studies have demonstrated that infection of respiratory cell lines (A549, BEAS-2B) with replication deficient El­E3-deleted adenovirus derived vectors results in transcriptionally­mediated induction of ICAM-1, which is known to favour inflammatory response through adhesion and activation of neutrophils. To evaluate whether this induction is relevant also in vivo, we infected non-human primates with the first generation E 1-E3-deleted recombinant adenovirus vector expressing CFTR (Ad.CFTR). Healthy Rhesus monkeys were instiDed by bronchoscopic procedure with Ad.CFTR at l.Sxlo•o pfu (n z 4) or with vehicle only (n=3) and sacrificed at day 2, 4, 7, 10 post-infection. Lung portions (trachea, main bronchi, upper and lower lobes) were snap-frozen and total RNA was extracted. ICAM-1 mRNA expression was quantitated by a single-tube homologous competitive PCR assay. Human CFTR mRNA encoded by Ad.CFTR was preliminary tested to distinguish which of the lung portions under study have been successfully infected by Ad.CFTR. Ad.CFTR-encoded mRNA was detected in all the lung samples of the animals sacrificed at day 7 and 10, but only in 4/IS samples ofthose ldDed at day 2 and 4. The average number of copies of ICAM-1 mRNA per 11g total RNA in lung portions of control animals was 93 (SEM = 8, n = 17) while the average value of treated animals with positive expression of Ad.CFTR-encoded mRNA was SOO (SEM = Ill, n = 18), which was statistically significant (p<O.OOI). Lung portions of animals treated with Ad.CFTR but negative for CFTR mRNA expression did not show ICAM-1 mRNA levels higher than controls. The results indicate a time-dependent association between ICAM-1 mRNA induction and Ad.CFTR-encoded transcript expression after in vivo infection of airways of non-human primates with first generation E 1-E3-deleted adenovirus-derived vectors.

181 COMPLEXES OF ADENOVIRUS WITH POL YCA TIONIC POLYMERS AND CATIONIC LIPIDS CAN PREVENT NEUTRALIZING ANTIBODIES FROM BLOCKING ADENOVIRAL INFECTION. M ChUJOn J. Lee, A. Fasbender, and M1 Webh. Howanl Hu&hcs Medical Institute, Depanment of Medicine, University of Iowa Colleae of Medicine, Iowa City, Iowa, 52242

The bWDonl lmmUDe response limits the usefulneas of adenoviral vectors in vivo. Previous work showed that complexea of calioaic compounds and recombinant adenovirus increase bindins, uptalrc and expression of adenovirus-encoded tnnssenes in cells which do not infect easily with adenovirus alone. We bypothesbed that coatinJ the ~vely-dlqDd adcoovirua particle with cationic ~or caliOIUc lipids miaht protect the virus from aeutrallzina antibodies present in the serum of lmmuniJed animals. To rat this hypothesi~ we comp~ adenovirus particlel with various cationic substances and exposed the complexca to neulnlizlna serum from immuniJed animals. VIe tested a variety of cationic polymers and c:adonic lipids. OC those tested, GL-67 fonnullted with the co-lipid DOPE-PEG was most effective. A I: 1000 dilution of ldeaovirul-umnunized nt ICIUIII complcfely neutralizlcd adenovirus alone. However, complexea of lipid:adenovirus required serum concentrationa peatcr than a 1:10 dilution for neutralization. DOPE-PEG wu e&c&ive onl_y when formulaled with OL-67 before complexlns with ldellovirus. The ratio of OL-67 ro DOPE-PEG and the ntio of toCal lipid 10 adeooviral puticlel wu importanL Rcaults of eleclroa mic:rolc:opy and immunoprecipltsdon IUIJeSt leas antiaallantibocly imlirlcdon when adenovirus wu complexed to OL-67/DOPB-PEO. Buecl on these cilia we uked if the complex would proiCCt In vho. 5 formulad0111 were tested by cfWct inlrllracheal adminislndon or till vein injection of lmmunilJed mice. However, thee formulldona liled to ~teet from neutralizins antibodies in vivo. Altboup furtbet work will be required, these results sugest that COllins an adeDovirus might procect from neutralizins antibodies and aDow rq1e1t admiDllualion.

REPEAT ADMINISTRATION OF AdCFTR TO THE AIRWAY EPITIIELIVM OF INDIVIDUALS WITH CYSTIC FIBROSIS IS WELL TOLERATED AND ASSOCIATED WITH MINIMAL SYSTEMIC HUMORAL IMMUNITY. B-G. Harvey, V. Sikand, P. Brion, R.J. Kaner, RG Crystal. The New York Hospital-Cornell Medi­cal Center, New York, NY and GenVec, Rockville, MD

Adenovirus (Ad) vectors have been used to transfer potentially ther­apeutic genes to lungs of experimental animals, and to transfer the cystic fibrosis transmembrane regulator (CFTR) eDNA to the respira­tory epithelium of individuals with CF. Since airway epithelial cells are renewed overtime, repeat administration of Ad vectors carrying the CFTR gene will be necessary to treat the respiratory manifesta­tions ofCF. To evaluate the safety and immunologic consequences of this therapeutic approach, the AdS based vector A<fovCFTR.I 0, an Eta·, partial Etb·, partial E3· vector, carrying the normal CFTR gene, was administered repetitively every 3 months (x3) in ascending doses of 3x I 0' to 2x I 09 pfu to the airway epithelium of 14 individuals with CF. All individuals tolerated the vector well with no adverse events related to the Ad vector. Serum samples from all patients were evalu­ated over time for anti-AdS neutralizing antibodies (Ab). In contrast to the fmdings in animals, in which lung administration of Ad vectors consistently triggers the development of high anti-AdS neutralizing Ab titers in serum, the 14 patients had minimal to no increase in the systemic anti-AdS neutrslizing Ab, with the maximum rise found in a patient in the 3x I 07 5 pfu group, whose Ab titer transiently increased from 20 to 160 by day 30 after the second vector administration. Inter­estingly, in the highest dose groups (I0'-109pfu), no individual had a major rise in Ab titers, with a peak titer seen in one individual in the I 09 pfu group whose titer went from 20 to 80 by day 30 after his third vector administration. These results demonstrate that repeat airway administration of an Ad vector carrying the normal CFTR eDNA to CF patients can be done safely and is associated with minimal sys­temic anti-AdS neutralizing antibody response.

183

Enhanced Adenovlral Tranafectlon of the Pulmonary Epithelium by Exoaurte. S M Manyel, Y. Guo ,and S. Matalon. Dept. of Anesthesiology, Unlv. of AL at Birmingham, Birmingham, AL 35294

Previous studies have shown that pulmonary surfactants enhance adenovirus-mediated transfection of rabbit lung tlasue without addreaslng the mechanisms underlying Increased reporter gene expression. Herein, we report that pretreatment of Isolated, cultured ATII cells with Exosur18 significantly enhances transfection by 1 replication deficient type 5 adenovlral vector that encodes for luclferase (AdCMVLuc) from 2,075 ± 806 to 12,772 ± 2,727 relative light unlta per mg protein expressed as mean± SEM. When the components of Exoaul'f:l were tested Individually In vitro, tyloxapol demonstrated the greatest ability to enhance adenoviral gene transduction. Similarly, prelnstlllation with Exoaurte In rats receiving 1 single Intratracheal instillation of 2 x 1 Q9 pfu AdCMVLuc resulted In marked enhancement In A Til cellluclferaae activity over that of other tung cell types. These studies demonstrate that Exoaurte Increases tranlfectlon of A Til celll/n vitro and In vivo In a cell specific manner and leads to targeting of Ad vec:tora toward alveolar epithelium. Surfactant moleculel may play a role In the distribution of lower airway gene transfer mediated by recombinant ldenovlral vectora. The Impact of surfactant on ATII cell tranafectlon ahould be considered when using aerollzed or inatllled adenovlral vectors for CFTR gene delivery in humane. Supported by NIH grants T32HL07553 & HL51173.

184 BLOCKING HOST T CELL RESPONSES TO ADENOVIRAL GENE THERAPY VIA CELL SURFACE EXPRESSION OF FAS LIGAND. E Mason. I. Rodgers. Department of Microbiology and Immunology, Baylor College of Medicine, Houston, TX, USA.

In the last decade, much effort has been made to develop effective gene therapies. The adenoviral vector, due to its wide tissue tropism, has been used in clinical trials to introduce reporter and co~~ genes to liver and lung tissue. Often, however, gene expresst~n ts s~ort Jived. Loss of expression in animal. model~ infected_ wtth a ~trus containing the LacZ gene is accompamed by mfl~llon assoctated with infiltrating T cells. T cells are largely responstble f~r -~ loss of repaired cells. Therefore, it is our goal to block these tm!lal T cell responses in order to prolong the expression of the therapeutic gene. It has been shown that T cells, upon activation, increase their l~vels of Fas expression and become increasingly sensitive to FasL-m~uced apoptosis. Therefore, we will anempt to. ablate: transgene-spec_tfic T cells by inducing them to undergo apoptosts. It IS our hypothests that co-expressing FasL and the therapeutic Ilene on the surface of the transformed cell will lead to antigen-specttic recogmuon of corrected cells by the T cell and subsequent death of the T cells via the Fas/FasL interaction between the corrected cells and T cells. We have ~ted_a system in the mouse. model to co-express FasL and the ant~gemc protein, hen ovalbumm (OVA) on the surface of both Class I and Class Ir T cells. To test the biological activity of the FasL on th~se cells we have also developed an Annexin V-based method of detecting functional FasL on the surface of these cells. Th~se ~lis w!ll then be used to stimulate T cell responses in vitro and m vrvo usmg OVA­specific T hybridomas and TCR-transgenic mice.

185 FUSION OF LARGE LIGANDS TO THE ADENOVIRUS TYPE 2 FIBER PROTEIN. B A Moore, B.C. Mulach,_ I.S. Hong, E.J. Sorscher, J.A. Engler. University of Alabama at B1rmmgham, B1rmmgham, AL 3S294

One of the issues in the retargeting of adenovirus vectors for ~y.stic fibrosis gene therapy is the choice of the targe~ ~eptor for IDIIlal binding of the therapeutic virus, as a. prerequ1s1te for entry and localization to early endosomes. Larger hgands attac~d to fiber could offer one means of specific targeting of the adenov1ral v~tor. The transferrin ligand has previously been used to target human a_1rway cells for CFTR gene transfer (Am.~- of Resp: Cell & _Mol. 81ol. 9:441-447). The transferrin receptor IS present 10 the ap1cal memb~es _of human airway and other epithelial cells, and could be used to s1mpi_JfY the binding and entry of virus into airway cel.ls that are ~therwtse refractory to adenoviral gene transfer. We destgned expenments !O determine the constraints that exist c~emmg Ad ftber prote1.n assembly and trimerization when 1 large hgand such ~ transfemn IS attached, as part of a more general strategy_ for n:targetmg the vector. We constructed recombinant fiber genes 10 whtch the length of I peptide linker at the carboxy terminus has been inc_reased ~rom four (serine-alanine) repeats. to six repeats. ~ prohne res1due. was engineered at the beginmng and end of each hnker. We also destgn~d and inserted active receptor binding sequences of huma~ transfemn downstream of the 6 repeat linker sequence. The transfemn constructs include roughly half of the full length transferrin molecule (AA 331 !o 691,331 to 696,339 to 691, and 339 to 696 of the human transfemn molecule). The first construct tested (AA 331 to 691) was expressed 81 high levels but only monomeric tiber molecules were detected by Western bl~t. These results suggest that large ligands (e.g. 35 KDa ~f transferrin) will require longer linker sequences . to allow thetr attachment to a functional fiber. We are currently testmg both flex1ble and rigid linkers occu~ng in_ na~ural protei~s in. order to find 1 fiber· linker-large ligand protem wh1ch IS able to tnmenze. Supported by ROl HL58JJ!I,

186 Modification of the Adenovirus Fiber Protein for Development of Tropism-Modified Vectors. 8 L, Mulacb, I.S. Hong, E.J. Sorscher, and J.A. Engler. University of Alabama Ill Birmingham, Birmingham, Alabama 35294. . . .

Our goal is to develop an adenovirus vector wtth mcreased eftictency

Poster Session Abstracts 259

of entry into CF-affected lung tissues, including tJ:le ability to target specific pulmonary epitheli~ cell types.. It has I?rev10uslr been shown that the terminal decapepllde of gastnn releasm~ pepud~ (G~P-1 0) could be added onto the C-terminal end of fiber Without dtsruptmg the secondary structure of tiber and that the added ligand was accessible to a GRP-specific antibody. Using this recombinant tiber, we hope to restrict entry of this adenovirus construct and to dem?nstrate internalization via ligand-specified receptor-mediated endocytosts. We have now constructed five additional recombinants of the Ad2 genome that contain modified fibers in place of wild-type Ad2 fiber using recombinant yeast artificial chromosome (Y AC) technology (from Dr. Gary Ketner). First, in order to eliminate the possibility of incorrect recombination events, an Ad2-YAC was first made in which the tiber gene sequence had been deleted. As a test of the method, the AdS tiber was then placed into this defective Ad2 genome to easily discriminate the recombinant virus from the wild-type virus. Other AdS fibers introduced include recombinant Ad2-YAC containing GRP-14, CSa-1 S, strctavidin-mimic, and biotin-mimic sequences as ligands. GRP-14 contains the C-terminal 14 amino acids of gastrin releasing peptide. We have previously shown that GRP receptors exist on the apical surface of filter-grown ffi3-l cells and on the surface columnar epithelial cells of airways. CSa-15 is the terminal IS aa peptide of the s~!l-<:ompleme~t CS activation fragment, CSa. The CSa is a potent phlogiStiC molecule; It has been shown by immunostaining of human lung tissue that bronchial and alveolar epithelial cells, as well as vascular smoot_h muscle and endothelial cells, express the receptors for C5a. Usmg PCR and southern blot analysis, the identity of each recombinant virus was confirmed prior to funher testing. Currently, these recombinant. Ad2-y ACs are being tested for their ability to form infectious adenovtruses by transfecting the genome into 293 cells. We have. also sho~n ~at ~e CSa-1 S and AdS tiber chimera folds and assembles mto a nattve tnmenc structure. The recombinant AdS knob protein was also used as o_n~ _of the key reagents for identification of normal receptor used for tmllal attachment by adenovirus (CAR; Science, 275:1320-1323). Supported by CFF and by ROI HLS8339.

187 ADVANTAGES OF a-FETOPROTEIN (AFP) AS A REPORTER GENE FOR EXPRESSION IN LUNG AND LIVER IN ANIMALS AND HUMANS . .l&.awli..K. QlkaL Yufcn Wang, Kira Taylor, and Arthur L.Beaudct. Dept. Molecular/Human Genetics, Baylor College of Medicine, Howard Hughes Medical Institute, Houston, TX.USA.

Reporter aenes are widely used in acne therapy experiments, but most have documented or potential disadvantages in tenns of host immune responses. In response to the need for a reporter acne which might escape host immune responses and have value in human clinical trials, we have developed a first generation Ad vector (AdKThAFP) expressing human a-fetoprotein (hAFP). hAFP is secreted from the fetal liver and found al high levels(> I mglml) in fetal serum. PostnaJally, expression decreases sharply and serum levels fall to- 2S nglml above age 2. This expression pauem Ia similar for many mammalian species. These characteristics make AFP an anractive reporter acne with the potential for species-specific utilization. AFP is an endogenous protein that should not be immunogenic, and it is secreted, which facilitates monitorina. hAFP may have potential in the lung, where monitorina gene expression can be extremely difficult The low aduk levels ofhAFP would allow for detection above background levels in the serum and/or bronchoalveolar lavage (BAL) fluid. In lddition, sensitive and accurate tests are available for detection. As a first step toward using hAFP as a human reponer, we have evaluated hAFP expression In vivo in mice after both intravenous and intranasal delivery of AdKThAFP. After intravenous delivery (primarily to liver cells) of I X 10' pfil, high levels (up to 750 pglml) could be detected in the serum three days after injection. Serum levels dropped in immunocompetent mice to undetectable levels by one month after injection, but, after an initial dramatic drop, were maintained at relatively high levels (2000-4000 nglml) in immunodeficient mice for several months. After intranasal administration to the lung, hAFP could be detected in both BAL fluid and serum at days 3 and 7 at moderate to low levels(-SOO nglml). These levels fall by 14 days in immunocompetcd mice. Expression ofhAFP is easily detected immunohistochemically. Decreases in expression detected histochemically closely follow decreases in serum and/or BAL fluid. The initial vector demonstrates some hepatotoxicity, which is beina investigated to detcnnine if this is related to AFP expressim or to the vector backbone. The hAFP vector has been injected at I X to•• pfulkg into one baboon, and hAFP levels of20 pglml were achieved in the serum. These initial experimenls establish that hAFP can be used to follow aene transfer In vivo after delivery to both liver and lung by monitoring serum and BAL levels. It is envisioned that hAFP could be coupled to therapeutic acnes. such as CFTR, such that hAFP levels could be used to monitor expression of the therapeutic sene;, vivo, without the requirement for biopsies. The use of AFP in gene therapy experiments would fit with some of the recommendations of the Motulsky/Orkin gene therapy committee.

260 1997 Cystic Fibrosis Conference

189 ADENOVIRUs-MEDIATED PULMONARY GENE TRANSFER IS SIGNIFICANTLY INHIBrrED BY NONSPECIFIC, VECTOR-INDUCED INFLAMMATION. KazybJy Otaki, David L. Ennlst, Nanette Mltlereder, Kevin Harrod, Jeffrey A. Whitsett, and Bruce C. Trapnell. Dept. Of Virology, Genetic Thsrapy, Inc. Gakhersburg, MD and Dept. Of Pulmonary Biology, Children's Hospkal, Cincinnati, OH, USA.

Replication deficient adenovirus vectors (Av), under development for gene therapy for cystic fibrosis (CF) lung dil8888, have demonstrated unexpectedly low gene transfer efficiency In the highly inflammatory milieu of the respiratory tract of Individuals with CF. These vectors also are known to Induce both nonepeclflc Inflammatory and Immunologically specific humoral and cellular rasponeee In animals. In this context, we hypothesized that nonspecific Inflammation may play an Important role In blocking adenovirus­mediated gene transfer Independent of specific Immune mechanl8ms. To test this hypothesls, an adenovtral vector expreselng a ~-galacto81da18 reporter (Av1 nBg, 4x10'0 partlcleslanlmal) was administrated via Intratracheal Instillation to mice (n • 5/group, Bllb/c, C57BL/6 or nude strains) In the absence or presence of dexamethasone (DEX; 5 mglkgldey, lp, dey ·1 to 3) to block nonspecific Inflammation. Necropsy was performed on dey 3 and blind scoring of pulmonary histopathology demonstrated that vector-Induced Inflammation was significantly reduced by DEX treatment (p<0.001) In both nonmal and Immunodeficient mica. Importantly, quantitative evaluation of II· galactosidale In lung homogenates at day 3 showed 2 to 3-fold higher levels In the DEX-trealed animals (p<0.01, all comparisons). Bronchoalwolar lavage (BAL), also performed on day 3, showed that lhs numbenl of alveolar macrophages (AM), neutrophlll and lymphocytes were lncreeeed by Av Infection In both nonmal and lmmunodeltclent mice. DEX decreued the numbers of AM and lymphocytes, but not neutrophile. DEX did not up­regulate the specific activity of the trensgene (li-gal expreulonlvector DNA content). To understand the molecular mechanisms Involved In this Inflammatory response, prolnflammatory cytoklne expreaslon In BAL fluid was measured. Av Induced expresek)n of IL-111, IL-8, TNF-a. IFN-y, MCP1, and MIP1-a. DEX IIJPI)I1II8ed expresalon of th8l8 cytoklnes concommk&nt wkh the Increase In tranagene expreaslon. These obeervallona demonstrate that non-apeclflc lnflammatton, Independent of epeclfic Immune mechan18ms, is an Important datemlinant of the efficacy of In vMI gene transfer to the lung and may have lmpoltant Implications for human gene therapy In the lung.

190 EARLY CYTOKINE RFSPONSES TO RECOMBINANT ADENOVIRUS ADMINISTRATION IN THE MURINE LUNG. K. Panesar, S. Welty, E.O. Smith, J. Katkin. Department c:A Pediatrics, Baylor COUeae c:A Medicine, Houston. TX. USA.

Recombinantldenovirus vectors are efficient reagents for deliv~rina aenetic material to the respiratory epithelium. Ho\l:'ev.er, T -ce)l ~&ted tmmune respon1e1 in~uc:id by tlle ~ecton have l!nu~ then ulihty ill vivo. The role of 1pec1fJC early Cflokine responses I!' th1s process are !JOI clearlr defined at present. Thia atudy wu des1gned to detennute cytokine retpon~ea to intratracheal delivery of a recombinant .denovinaa vector (Abaal) expreasina 11-aalacro.idue (jl-gal). Adult female CS71BL6 mice were treated with I x I 0" infectious unitslkJ of Abgal, delivered intranually in SO pi of benctant (Survanta•, Rou Ph11111111Ceutical1). Control animals received beractant without the viral vector. Mice were aacrificed at6, 12, 24,48 or 96 houn after treatment. Lunas were lsvapcl with I . .S ml phosphate buffered lllline; luna tis1111e wu homoaeniz.ed in O . .S ml SOO mM NaPhos buffer. Luna homoaenate• were usayed for 11-aat activitr by a standard ONPG usay. 11-~al expression in mice treated with Abaa exceeded that in controls at 48 ancl96 houn after vector delivery (p < .01). Bronchoalveolar lavqe (BAL) fluid, .erum and luna homoaenates were assayed for interleukin-6 (IL-6), interleukin-ljl (IL-IP) and tumor necroa1s factor a (TNFa) uain& . commereially available ELISA• (Bi01011rce International). Cytokine levela in the BAL fluid were normalized to the amount Of alveolar linina fluid (ALF) recovered in each sample b:r. com~na the BAL fluid urea nitroaen with the blood urea nitroaen (BUN). IL-6 was sharply elevated in ALF from treated animals at f2 houn after vector delivery (p < .002), returnina to bueline by 24 houn. IL-111 wu elevated in ALF from treated ani mala at 96 houn (l' < .M), but not at earlier time pointa. There were no siJI!ificant alterauona in ALF TNFa levels, in control or treated animala. In terum, there were detectable increases in all three cvtokines between 12 and 24 houn, but the difference• did not reac& statistical sianificance due to hiah variabili!Y within each aroup. Lcvela of the three cytokinea did not vary m homoaenizccf luna tiasue at any time point. Intratracheal administration of the Abaal vector appean to caue a very early, local increase in ALF level• of fl.-6, and a more delayed rite in ALF IL-Ijl. Thele increases IJ\ay be auociated with subtle ayatemic elevationa in IL-6, IL-IP and TNFa. Since the cytokine clianaes precede detectable transaene expresaion, it aeems likely that they are caused by expoture to viral capsid protein,. The early elevation of IL-6 and later rite in IL-IP are consistent with previou1 findinas in mice infected with wild-type ldenoviru.es. Tran1ient local increases in thele cytokines may play an important role in the pulmonary inflammation seen after delivery of recombinant adenovirus vectors to the respiratory epithelium.

Supponed by CFF pnti96BO.PANESAR

191 EFFICIENCY OF IN-IIfVO GENE TRANSFER IN MURINE NASAL AIRWAY: MODULATION BY DETERGENT PRETREATMENT AND AIRWAY DISEASE. OW Ptmgna'.2, C5 Freiberg', BR Grubb', LG Johnson', RC Boucher' . 1CF Center, University of North Caroll1111 C Chapel HHI, NC, USA, 27599; and 2Department of Pulmonary Medicine, Wornen'a and Chlldren'a Hoapltlll, Adelaide, Sth Auatralla, 5006.

lncrNI&d efllcienc:y of gene lnlnlfer (GT) II needed If the potential of gene-thenapy trutment of llrwlly diaeeae In cystic ftbroall II to be realised. Brief exposure of nasal airways to AdSCMVLKZ i Adl.ecZ") atrtklngly incrNied GT efler polldocenol detergent (Del) pnatreatment of nasal airways (Pad. Pulmon. Suppl 13, 5221, 1998). We therefore a~e~led whether AdSCBCFTR \AdCFTR") GT would be almli8rfy enhanced. Becauselirway diseese will be common when airway gene therapy II offered, the etrect of existing elrway dlaeeae on Adl.lcZ GT efllcienc:y waa also examined. Methode: Transgenic UNC CF mice & C578118 mice were used for CFTR & LlcZ l8riH trilla respectively. In the right nostril of test anlmala 20111 of AdCFTR (7x1010 pfu/ml) or Adl.lcZ (1x10" pfulml) was lnatHied 1 hr efler Del Instillation (ualng 4111. 0.1% \LoDer) In both aeriH; & elso 10111. 1.0% \HIDat") In the AdCFTR aeriH). Functional CFTR expression (n•5 to 10 mice/group) wa measured Iller 5 dlys vii the change In nasal tranaeplthellel potential dlfl'erencl (APD). In other experiments, Adl.lcZ with or wkhout Del ~~~was lnalllled 8 days after esllblishlng 1111111-alrway Infection & int1emrn1t1on (Pad. Pulmon. Suppl13, 5158, 1998). Gene lnlnlfer ef11c1ency was -..c1 efler 2 dlys a the number of XGal-atalned cella ("xac") oblerved In transitional and reapiratory epithelium regionl, In 3 lllndlrd naeel elrway croaa-aectlona (n-4 mice/group). Results: AdCEIB: Del use resulted In ~t enhancement of AdCFTR lnlnlductlon (APD (In mv): untreatedllo().72:t0.8(5EM), AdCFTR-alone group-cl.5±1.1, with-Lo0et•"3.8±1.4, wlth-HIDet-•7.9±1.1, •p<0.05 ANOVA; [nonmal mouse APD is 18.4:t2.3)). Adl.lcZ; Infected mica treated with LoDet/Adl.lcZ (0.28±0.9 xac) or AdLec:Z alone (2.0±1.4 xsc), or unlnfecled mice given Adl.lcZ alone (0.0±0.0 xac) alllhowed algnlflcently lela GT than unlnfected mice given LoDei/Adt..cZ (53.0±8.8 xac, p<0.05, ANOVA). Conclullona: Since a mgte detergent pell'Hin*lt of naal airway epithelium strongly enhancel AdCFTR (and AdiMZ) gene tranaflr 111-IIMI, adjuvant treatments using a detergent-like compound may provide another evenue for Improving the ef11c1ency of ldenovlral GT vectora. When 1111181 airway Infection &

inflammation was present, AdlacZ GT was poor In underlying and surrounding epithelium, suggesting that airway disease may reduce the efficiency of adenoviral gene transfer In-vivo. Supported by the CF Foundation.

192 MOLECULAR ANALYSIS OF ADENOVIRUS-MEDIATED TRANSFER AND EXPRESSION EFFICIENCY OF CFTR eDNA IN INDIVIDUALS Wim CF. Mjcbael A Perricone, James E. Morris, Karen Pavelka, Malinda S. Plog, Samuel C. Wadsworth, Alan E. Smith, and Judith A. St. George. Genzyrne Corporation, Framingham, MA, USA.

Recombinant adenovirus and cationic lipid:DNA vectors are currently being evaluated in clinical studies for CF gene therapy. In the absence of short-term clinical measurements for the assessment of efficacy, molecular analyses represent appropriate surrogates to evaluate the transfer and expression of the CFfR transgene to the airway epithelial cells of CF patients. We developed a strategy for the analysis of airway cells harvested by bronchial brushings that includes nested PCR and FISH to verify the presence of vector DNA as well as nested RT­PCR to detect transgene expression. Cytology brush samples were taken from CF patients 2 days after intralobar or aerosol administration of a recombinant adenovirus (Ad2/CFTR-8) containing the CFfR transgene under the control of the EIA promoter (Clinical Protocol #CF94-0401). DNA and RNA were isolated from the brush samples for subsequent PCR and RT-PCR analyses or the cells dislodged from the brush and transferred to microscope slides for subsequent FISH analysis. Vector DNA was found in 718 samples fro~. pati~nts receiving Ad2/CFTR-8 by either intralobar or aerosol adrrumstrat10n. Furthermore, vector DNA was nuclear-localized in up to 10.~% of~ cells in brush samples from patients receiving 2.SE9 infecuous umts (i.u.) of Ad2/CFTR-8 by the intralobar route and from 0.4% to 3 .. 1% of the cells in samples from patients receiving up to 8E8 t.u. Ad2/CFfR-8 by aerosolization. Only a small proportion of~ vector DNA-positive cells were airway epithelial cells, as ~terrruned by cytokeratin immunofluorescence. We also found expresston of vector­specific RNA in 3/3 intralobar samples and 4/S aerosol samples. We conclude that the combined use of PCR, FISH, and RT-PCR analyses provide an effective means to evaluate clinical samples for the presence of vector DNA, the proportion of and type of cells having vector DNA, and the expression of the vector-specific trans gene.

193 INTERNALISATION, NOT ATTACHMENT IS THE RATE LIMITING STEP RESPONSIBLE FOR THE INEFFICIENCY OF ADENOVIRAL­MEDIATED GENE TRANSFER TO WELL-DIFFERENTIATED RESPIRATORY EPITHELIUM. R.l pjcklcs, D. McCarty, S.H. Randc:U and R.C. Boucher. CP Centre. Univ. of North Carolina II Chapel Hill. NC. USA. , ,

Succeaful sene therapy for C)'ltic fibrosis (CP) tuna cliseue rcquila ctliciena in vivO gene transfer to airway epilhelia. We have previously cleso;ribcd high efficiency adenoviral (AdV)-mecliaiCid sene transfer to poorly cliJferentiatcd (PO) airway epithelial cells in w/1"0 but tow efficiency gene transfer to well cli1ferentiated (WD) airway epithelium in vtvo (Grubb •t a/., Nature llL 102· 106, 1994 ), and speculated that the lower efficiency observed in vtvo is due to a defective step esrly in the WlC!Or-cell interaction. We have utilised a human or rat tracheobronchial airway epithelial cell culture II)'Stem that pcnnill comparative analyse~ of PO cells with WD phenotypes that resemble the pscudoltratified mucociliary epithelium that oecun irr vivo. Exposure of human PO and WD culturel to Ad5Lac:Z and assayed for Lac:Z expression 48h later, rautted in significantly greater ~-gaiiC!Oiidase activity (~-gal) in the PO culturel than in the WD culturel (PO: 8417 :1: 940 compared to WD: 70 :1: 31 11-gal U x I 0 .. per em'). Since thil culture II)'Stem recapitulalel the gene tranSfer disgepancy observed In vivo, we have usecllhis model for comparative analyse~ of the initial interactions of AdV, I.e., attachment and intemalisation, with the two culture-types. Attachment of racliolabeted AdV II 4"C showed that human PO culture~ bound significantly peater amounll of AdV than did the WD culturea (PO: 5055 :1: 938 compared to WD: 12 :1: 4 CPM per em') and also internaliseclaligniflcantly greater amount of AdV (PO: 1167:1:210 compared to WD: 26 :1: 2 CPM per em2). Incubation of the cultures with AdV in the presence of 3000-fotd cxceu purified ftbre-knob protein did not Inhibit AdV bindina to either culture-type whereas a ligniflcant proportion of the expression wu inhibited by the competin& ligand, su&JCIIinl that with PO cultwa the larJe amount of attachment wu predominately due to a 11011-specific interaction. Transmission electron microlcopy lhowa that the majority of AdV auociated with the PO cultureswu attached to Jlycocalyx-like llr\ICtUI'CI on the apical membrane reJiona. 1bete data, combined with data obtained with the nt

Poster Session Abstracts 261

model, suggest that intemalisation of AdV, not attachment is Ole rate limiting step for AdV mediated gene transfer to WD culturel. In support of this conclusion, lllllllCIM:n to increase binding of AdV to WD cultures failed to enhance substquent gene expression. Therefore, efforts should be focused on Increasing the intcmalisation efficiency of AdV. This may require manipulation of the inherent internalisation capacity of the well differentiated epithelium. Supported by the Cystic Fibrosis Foundation.

194 AN Ela-DELETEDADENOVIRAL VECTOR WITH HIGHER TITER AND DECREASEDPOTENTIALFOR REPLICATION COMPETENT VIRUS. Heshap Zhou and Arthur L. Beaudet. Dept. Molecular/Human Genetics, Baylor College of Medicine, Howard Hughes Medical Institute, Houston, TX, USA.

El-deleted adenoviral vectors permit efficient transfer and expression of transgencs, but these vectors typically result in limited duration of transgene expression and are toxic when given to animals in high doses. Two major strategies have been used to develop refinements in adenoviral vectors. One is the developmentofhelper-dependent vector systems that rely on a helper virus to provide the necessary proteins in trans for production of Ad-based vectors deleted for most or all viral coding sequences. Another strategy is the development of vectors with multiple genes deleted, such as the E I IE2a, E IIE2b and E JIE4 deleted vectors. We have developed E2a deleted vectors and a complementing cell line (Zhou et al. J. Yirol. 70:7030, 1996), but this system resulted in lower titers than for the first generation vectors, and there was potential for recombination resulting in vector with E2a wild type sequences. We have overcome these two problems with a new EIIE2a deleted vector and helper cell line. To increasethetiterofthe EIIE2adeleted vector, we developed an inducible EIIE2acomplementing cell line in which a tetracyclineresponsivecis-regulatorye!ement was introduced upstream of the endogenous E2a promoter. The expression of the E2a gene is highly induced by withdrawal of the tetracycline from Ole culture medium. To reduce or eliminate homologous recombination, we have completely removed Ole Ad sequences downstream of the stop codon for Ole E2a in the cell line and deleted the entire open reading frame for the E2a in the vector. Using the new vector system, the titer of the EIIE2a deleted vector was increased I 0,000 fold upon withdrawal of the tetracycline. No virus containing wild type sequences for E2a was detected even after I 0"' fold amplification of Ole E2a deleted vector in the new cell line. This vector system with deletions in E I and E2a is likely to be suitsble for preparation of vectors completely free of replication complement adenovirus and provides an additional option for delivery of CFTR for gene therapy. (Supported by the NIH and CF Foundation).

195 THE EFFECT OF COMPLEMENT DEPLETION ON THE PHARMACOKINETICS AND GENE DELIVERY MEDIA TED BY CATIONIC LIPID-DNA COMPLEXES. L.G. Barron, K.B. Mever, and f.C. Szoka, Jr. Department of Biopharmaceutica! Sciences, University of California, San Francisco, CA.

Our goal was to study the effect of serum complement proteins on cationic lipid-DNA complexes injected intravenously into mice. Previously, in vitro experiments have demonstrated that cationic lipids interact with serum complement. We have shown that transfection with complex composed of OOTAP/CHOL and DNA (5:1 charge ratio (+/-)) encoding the !uciferace reporter gene yields in excess of 40 ng luciferase protein per mg protein in the lung and 0.5 ng luciferase protein per mg in the heart when injected I.V. This complex concomitantly depletes serum complement levels by 35% in mice as measured by haemolytic assay. Furthennore, reduction in complement function was revealed to be the result of complement activation as evidenced by increased complement C3a levels measured with m ELISA in human serum incubated with complex. The increase in C3a was 3 I%, which is comparable to the degree of complement depletion we measured through the functional assay. The intraperitoneal administration of the anti-complementary agen~ Cobra Venom Factor, and antibodies lo C3 reduced complement function by 95%. We found that systemic transgene delivery with our complex was not affected by complement depletion of mice using this protocol prior to complex injection. To study the distribution effects of complement depletion on the complex, we radiolabclled both of the principal components of the complex prior to injection. The lipid label utilised was [131)-1 p-hydroxybenzamidine-DPPE while plasmid DNA was nick translated with [125)-1 dCfP. Complex distribution was not influenced by complement depletion in mice. We found that greater than 75'11> of the lipid

262 1997 Cystic Fibrosis Conference

and DNA was delivered to the lung 5 minutes post-injection while appro~imately 4% was detected in the liver in complement intact and depleted mice. Our conclusion is that calionic lipid-DNA complexes inleract with serum complement proteins upon I.V. injection but this interaction does not influence the lnlnSfection efficiency or systemic distribution of the comple~. (Res~rch funded by the Cystic Fibrosis Foundation. California BREP, DK47766, HL42J68)

196 SECOND GENERATION CFI"R VECTORS FOR GENE THERAPY. A& Dm:ll. H.M. Davidson, H. Davidson·Smilh. A. Doheny, G. McLachlan, J. Widdowson, S.J. Delaney & D.J. Poneous. MRC Human Genetics Unit. Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK.

While the eDNA-derived construct pCMV -CFTR has proved effective in vitro, in CF mouse experiments and in a Phase I (Safety) Clinical Trial assessing prospects for liposome-mediated CF gene therapy. some limitations have emerged. Firstly. the lack of satisfactory antibodies hinders the examination of CFfR expression by immunocytochemistry. Secondly. we have observed that the small intron in the SV40-derivcd 3' liTR of pCMV-CFTR is inefficiently spliced: therefore distinguishina between transfected DNA and its mRNA product by RT-PCR is troublesome. Thirdly, pCMV-CFTR is unstable in E. coli and plasmid yield is low, effects attributable to cryptic promoter sequences. To address these issues. we constructed and tested plasmids derived from pCMV-CFTR (using genomic PACs as source material where required) that incorporate permutations of the following features. (i) Introit 6aASb. Tile aberrant splicing of the SV40 intron in pCMV-CFTR suggested that its lnlnscripts may not be properly processed tbrougb the coupled processes of splicing and mRNA expon. To funher investigate this, we insencd CfTR intron 6al6b into pCMV -CFTR. (ii) Epito~ tags. Sequences encoding three epitope tags were insencd into the exlnlmembrane loop 3 coding sequence. We expect constructs such as these to express functional protein while permitting specific immunological detection and so allow us to monitor CFTR localisation and expression. (iii) Natural J' UTR. To test the possibility that the use of a heterologous 3' UTR in pCMV-CFTR is compromising expression, we substituted the appropriate region of wild-type CFTR, reasoning that we would thereby restore natural mRNA processing signals. MQAE Fluorescence, RT-PCR and immunoprecipitation studies demonstrated specifiC ion channel activity, mRNA synthesis and full length CFfR protein production mediated by all constructs (e•cept that encoding the Pk epitope tag. which presumably must intesfere with protein function) in ltllnsiendy 1n1nsfected cells. In panicular. we observed efficient splicine of the 6al6b intron where present and found that constructs contail)ins it were produced in higher yields in E. coli than pCMV -CFTR. However, 10 far we have not detected epitope tagged protein by anti­HA or anti-FLAG immunoprecipilltion, suggestina that the EL3 tag insertion site is suboptimal for antibody access. A sensitive semi-quantitative RT-PCRISNuPE assay based on exon 6alexon 6b primers is described. It permits facile DNA/RNA and hosl/vector sequence discrimination, is being used for quantitative assessment of new vecton in vitro and in vivo. and has potential for assayins expression levels achieved in clinical trials. We conclude that vectors carryina genes closer in form to natural CfTR offer improved expression and assay modalities compared wilh current vectors.Suppon~d by UK ROPA and MRC grams

197 ITRUCTUAAL AND FORMULATION CONSIDERAnONI FOR CATIONIC LPIOI FOR HIGH LEVEL TRAHIFECTION OF LUNG. J. Mlnhlll, E. I.H, C • ._, S.J. &almM, J. Nletupeld, S. Rudg/MIIy, R. SciHiule, A.E. Smlh, O.J. HMII, antiS H ClwJs. Genzyme Corp,1 MouiUin Roed, MA 01701.

Allhouah typically compriMd of only 3 componenla, the gene tranafer ectlvfly deny - Cllllonlc llpid:neutral co-llpld:pONA c:clqJiu II ct.pendent on -' dllf..nt fectora. Melclnl actMiy II U8UBIIy raelized only after caralul optimization of the many val'faiMa lnherant In thla non-vlralappoach. Wa have continued our effortl to ldantlly IINCiuiHictlvlty ralationlhlpl of cationic lipide with the pi of devaloplng ballc rut.. for the dallgn of lipldl with robull_ gene lranlfer ldMiy In the lung. In thil ragllld, tNW 50 llddlional cationic lipide of cllferant atructlnl ~ ._. bHn aynthMized. Through ana¥111 of _.. IMtchad cationic lipid ~ that cliff• only In the c:onliguratlon of their haqraup, whalhar llnMr or eo-called Tolllapad, we !leva oonllrmed our orlgiMI obearvatlon that anlltlaa halborlng the t ..t.pa are Invariably mora active. lntarutlngly, while the T ·eNpa CCif'I1IIOUIIdl axNbllacl rnai!IWIICIIvly when forrnullted with the neulnllllpld DOPE at a molar ratio of 1:2, their llnMr COIA'IIarparta _. maxlmdy active at 1:1 malar ratloa. lrraepectlva of the configuration of the hudgroup, coqiOUI1da praparad aa a frM baaa - Invariably llgnlflcantly more active than the oorraepondlng aal forma. The Importance of the number and apaclng between protonatabla amlMI In the hMdgroup - alao lnvalllgated. In general, an lncrMM In lranllactlon actlvly In the lungl of lnatlllad BALBic mica - rallllzad with atherwlae matched~ .._ haedgroupe contUiad more protonallbla aminal. However thar. - an upper linit of lhll number (-3) above which no algnllcant addllonal ~ In activity - allalnad. Coqxlunda wlh 1f111C1r9 of 3 or 4 mathytana gtoupa '*­the aminle COIIIlltently provided grMtar gena tranaf• activity than thole

with spacings of 2 or greater than 5. Spacings of 3 and 4 ara more commonly found in naturally occuring compounds such as spermine and spermidine. qgeating that perhaps part of the mechanism by which these cationic lipids mediate g- transfer Is through recognition by soma call surface entity. A variety of different hydrophobic anchors including cholesterol, dihydrochofesterol, cholastamina, and dialkyt chains were also evaluated. In the context of T-shapad headgroups, cholesterol anchors were determined to be the most active. Different linkers were also assessed, including carbamate, urea, amide, amine and amino acids, with compounds harboring carbamate linkers usually exhibiting the greatest activity. A number of aftemative neutral co-lipids ware also screened lor their ability to augment transfection activity. Several cationic lipids displayed enhanced (2-25 fold) activity in the lung when they were formulated with either diphytanoyl PE or dUinoleoyl PE In lieu of the more commonly used DOPE. It is evident from t'-e studiea that significant ~ement In gena transfer activity can stil be attained as our understanding of the structural and formulation considerations of this vector system Increase.

198 BINDING AND UPTAKE OF CAnONIC LIPID:pDNA COMPLEXES BY POLARIZED EPITHELIAL CELLS. 0 M Cbu S.J. Eastman, J.D. Towlgnan~ S. Fang. E. LH, R.K. Schaule, and S.H. CMng. Genzyme Corporation, One Mountain Road., Framingham. MA 01701

lnafficlenl cationic lipid-mediated transfaction of polarized airway eplhelial calli _.,..a rMjor obatacla for CF gena therapy. To battar undarstand the barrie,. aasociated with gena transfer to polarized epithelial calla, an In vitro modal ayatem wu developed to study the binding and uplllka of cationic lipld:plaamid DNA (pONA) c:omplaxu. Thla modal consiata of polarized epithelial cala (FIIchar rat thyroid calla and differentiated normal human bronchial epithelial (NHBE) calla) grown on filter aupporta at an air-liquid lntarface. The efflciancy of binding and uptake of cationic lipid GL.e7:p0NA COfi1Jiax• by lhll cal ayatem waa monitored using fluorescenca rnicroecopy. ~x• went labeled with rhodamlna-phosphatldyl athenolamina as a fluoraecant lipid marlcar and/or TOT0.1 aa a fluoraacent marker lor pONA. Fluorescent probes that were bound to the call atllfaca ware dillingul8had from lntemalized probes by the addMion of trypan blue, which quenched the fluoraacenca of bound but not lntamaHzed probes. The relative amounts of llllface bound and lntarnalized complex-. datannined at 37"C using ATP depletion to Inhibit Internalization. The difference In the amount of call­aaeoclated DNA In the abHnca and preaenca of A TP depletion conditions waa uaad to allimata the amount of Internalized complex. For proliferating cella on filter aupporta. binding and lntamallzation of the GL-67:pDNA ~lax• were detllll'llined to be vary efficient. In contrast, using confll*lt polarized apithetial calla, very little binding or Internalization of GL-67:pDNA complex• waa obealved. However, altar aspirating a amal area of calla from the litter aupport. virtually all of the ca1a adjoining thla newly formed edge bound and Internalized the GL-67:pONA complexes. To determine If their accumulation In edge calla waa related to the abilky of the compiaxaa to ace ... the lateral membrana• of th- calla. the accumulation of GL-87:pONA ~lax• waa monltorad In fully polarized NHBE calla that had bean pra·traated wHh EGTA or calcium-free media, atrataglea known to cialupt gap junctions. Calla lrealed In lhla manner bound and Internalized GL-87:p0NA COfi1Jiaxaa alflciantly and axpraaaad tranag- producla. Control calla wlh intact gap junctions bound virtually no ~laxaa and ware not tral'llfacted. Theae data confirm and extend aarller obaervatlona that the polarized apical membranes of airway epithelial calla are realatant to tranalactlon by llpld:pONA complaxaa. Furthermore, contrery to previoua reports, binding and entry of the~ to theM calla Ia ral•lmHing. Thla In vitro modal ayatem provldaa a toof for taatlng othar llpld:pDNA COf11llax• and vactora for their ability to tranlfact airway epithelial calla In IIMI. The ability to quench the fluoraac~nca of bound Hpld:pONA con.,texea without affactlng the nuor.canc. of lntamalized complex• lhould allo aid attampta to underaland the machanilml of binding, uptake and lntracallular trafficking of cationic llpld:pONA complex ...

199 OOTAPIDNA COMPLEXES FORMULA TED WITH CHOLESTEROL TRANSFECT IN THE PRESENCE OF SERUM. K, Crook, B.J. Stevenson. D.J. Porteous. MRC Human Genetics Unit, Crewe Rd., Edinburgh, EH4 2XU, UK.

For reasons that are poorly understood, but which may include electrostatic inhibition and/or complex destabilisation. cationic lipid reagents transfect extremely poorly in the presence of serum. This barrier presented by serum and other biological fluids probably explains the poor in vivo perfonnance of cationic lipid/DNA complexes and weakens the case for the usc of these reagents in gene therapy trials. We have shown that by including cholesterol in the fonnulation of DOT AP complexes we can significantly decrease the inhibitory effect of serum on in vitro transfection with these reagents. At low

concentrations of serum, DOT AP/cholesterol complexes trans feet as well as in serum-free conditions, while the efficacy of DOTAP complexes drops rapidly. At serum concentrations of 50-100%, DOTAP complexes fail completely to deliver DNA to cells, while DOT AP/cholesterol complexes transfect at levels 25-50% of optimum. Similar results were obtained using broncho-alveolar lavage fluid from humans in place of serum. We have sought to explain this phenomenon by firstly measuring protein levels associated with the complexes, to determine if DOT AP complexes bound more protein than DOTAP/cholesterol and were thus impeded from binding to cells. We next examined the possibility that serum may be causing structural alterations within the DOTAP complex, reducing the integrity of the associated DNA. This was done in two ways: measuring any differences in nuclease sensitivity of the complexed DNA; and assaying any changes in the condensation of the DNA within the complex. So far we have been unable to detect any differences between complexes made with DOTAP and DOTAP/cholesterol. Transfection results from intratracheal and intravenous delivery with DOTAP compared to DOT AP/cholesterol complexes shall also be presented.

200 Investigating the duration and level or CFTR function in rivo after single and multiple deUveries of a DNA· c:atlonle llposome eompln. Catharine A. Goddard. Martin J. Evans and William H. Colledge. Department of Physiology and Wellcome/CRC Institute, University of Cambridge, Cambridge, UK.

Pre-clinical studies using CF mouse models have shown that it is possible to transfer the CFTR gene into the airway epithelium and restore cAMP-dependent chloride channel activity. However, both pre-clinical and phase I clinical trials have only detected CFTR function for a maximum of 4 weeks after gene delivery. Current delivery systems will therefore have to be given repeatedly in order to provide a treatment for CF. Repeat dosing with adenovectors results in diminished CFfR expression with each successive dose due to anti-inflammatory and immune responses. In contrast cationic liposomes have been shown to be non-toxic and non· inflammatory in single dose phase I clinical trials. We have previously studied the effect of a second dose of a CFfR expression plasmid complexed with cationic liposomes OC· Chol/OOPE (CFfRIDC-Chol), after the expression of the first delivery bas ceased. CFfR expression was assayed by measuring the change in short circuit current under conditions where only the CFTR chloride channel was active. The data indicate that a second dose is as effective as a single dose in restoring CFTR activity to wild type levels. Preliminary data suggests that a third dose of CFTRIOC-Chol given after CFTR expression from both the preceding doses has return to pre-treatment levels also leads to detection of nonnalised levels of CFfR function. This highlights an important difference between the suitability of adenovirus versus cationic liposomes as vectors in a repeat dosing regime. Whereas adenovirus repeat delivery results 10 the attenuation of CFTR expression, cationic Iiposomes can be used as a vehicle for plasmid delivery with no compromise in the level or duration of CFTR expression. Ongoing experiments are providing data to further define the duration of CFTR expression after delivery of CFTRIDC-Chol to the trachea of CF null mice after both a single and double delivery. The results of these studies will be presented.

Supported by the Medical Research Council and the Cystic Fibrosis Trust.

201 CATIONIC PHOSPHONOLIPIDS AS NON VIRAL GENE TRANSFER AGENTS IN LUNG MICE • .c..J.llljllaume-Gable (1), V. Floch (1), B. Mercier (1), M.P. Audrezet (1), G. La Bolc'h, J.C. Clement (2), J.J. Va01J8nc (2), H. del Abbayes (2). J.P. Leroy (3), C. Ferae (1). (1) Centre de Biogenetique, CHU. UBO, ETSBO, 46 rue Felix Le Dantec • 29275 • Brest· FRANCE (2) UBO URA CNRS 322, BP 452, 29275 • Brest • FRANCE (3) S81Vice d"Anatomle-Pathologle • C.H.U. ·Brest· FRANCE

Gena transfer Ia currently Investigated wtth two main approaches, viruses and cationic llposomes to deliver DNA Into lung epfthellal cells. A considerable amount of work has been reported uaing cationic llpoaome

Poster Session Abstracts 263

technologies to transfect lung w~h a principle application being to CF therapy. Compared to viruses, their efficiency gene transfer Ia low but they avoid the risk of pathogenic and Immunological complications. Also optimiZation of gena transfer using cationic llposomes Is a preliminary step to gene therapy. We present in this work the resuHs obtained In vivo wHh a family of gena transfer agents, called phosphonolipids, tested In vhro on CFT1 and K562 cell lines. Some of the 30 lipids tasted were more efficient than

commercialiZed reagents (lipofectin• • LlpolactAMINE• • DCChoQ : GLB43. GLB73. GLB84, GLB127, GLB253. The best resuHs were obtained whh GLB43 and GLB253. The translections were performed by intratracheal injection of lipoplex In lung mice. At least 5 mica were tested lor each lipoplex. To evaluate the translectlon efficiencies, we used quanthativa assay : chemiluminescent tests using two different reporter genes <P· Galatosidase and LucHerase) and a cytolluorimetrlc assay allowing the detection of the percentage of transfected cells. We have also used microscopy in order to visualiZe lipoplex In transfacted cells. Cationic lipid GLB73 was shown to be particularly affective In vivo, quantnatively and qualhatively : 4 days after transfaction , GLB73 allows on average 10% of posHive cells wHh maximum reaching 20% to 40% and only 5% for DCC hoi (maximum 1 0%) statistically signHicant. For the GLB253 the best resuHs were observed 7 days after translaction. The study of the kinetics of transgene shows a decrease of translactlon activhy over 1 0 days. We conlinn also that the resutts of lipofactlon efficiency Ia different between In vivo and In vhro on cell lines. We have tried to understand the relationship between structure activity and kinetic expression of the transgena. The resutts highlight the complexity of developing cationic lipids as delivery system. Indeed we could Imagine transfactlon wHh different lipoplex allowing a constant expression during several days.

202

EFFECTS ON REPORTER GENE EXPRESSION FOLLOWING ADMINISTRATION OF INFLUENZA VIRUS IN VIVO. ~. T. Hussell3, S. Cheng2

, D.M. Geddes', P. Openshaw', E.W.F.W. Alton'. ; ton Transport Laboratory, National Heart & Lung lnsthute, London, U.K. Genzyme Corporation, Framingham, MA, U.S.A., 3 Dept. of Respiratory

Medicine, St. Mary's Hospital, London, U.K. The level of gene expression following llposome-medlated DNA

transfer Is currently sub-optimal. It Is known that the Influenza virus can Increase peracellular penneabillty, which In tum may lead to Increased access to the basolateral surface. This may represent a more accessible route lor gene transfer than the apical surface of the lung epithelium. We have therefore assessed whether the administration of a strain of influenza virus prior to the administration of a reporter gene/cationic llposome complex could enhance gene expression In the lungs In vivo. In addition, II has previously been reported that replication-deficient adenovirus, when co-Internalised with plasmid DNA/IIposome complexes, could enhance reporter gene expression In bronchial epithelial cells In vitro (Am. J. Rasp. & Crff. Care Med. lli(4):A20 1995). Therefore, we have also studied whether the administration of Influenza virus. known to escape the endosomal pathway, can also Increase transgene expression If the comptexed DNA Is administered concurrently with the Influenza virus. Plasmid DNA encoding the gene for chloramphenicol acetyl transferase (CAT), driven by e CMV promoter. was complexed to cationic llposomes containing lipid 167, and administered to eight week old female Balb C mice by nasal application. Mica in cohort1 were glvan 50 tJ1 of 10 .. HA of Influenza virus (strain x31 HA4096) on day 0 followed by a single dose of 80 I'll of comptexed plasmid on day 4. It has previously been shown that maximal ephhellal Inflammation without epithelial shedding occurs lour days after Infection. Mice In cohort 2 were given 50 tJ1 of 10 .. HA of Influenza virus, followed by 80 I'll of complexed plasmid both on day 4. Mice In the positive control group of cohort 3 were given 80 I'll of complexed plasmid both on day 4. All animals (n=8 lor each cohort) were sacrificed on day 5, and the lung homogenates assayed for CAT actlvhy. The highest levels of CAT expression were seen In cohort 3 (10.5 x 10 .. :1: 2.9 CAT U/I'IJ protein). In cohort 2, where the influenza virus and the plasmid DNA had been Instilled together, expression levels were 4.3 x 1 0 .. :1: 1.0 CAT U/1111 protein. In cohort 1, where the Influenza virus was administered lour days prior to the plasmid DNA, expression levels were 0.06 x 1 0 .. :1: 0.06 CAT UII'IJ protein. In conclusion, transgene expression Is not Increased either by administering Influenza virus concurrently or 4 days pr1or to the plasmid DNA.

264 1997 Cystic Fibrosis Conference

203 205 CATIONIC LIPIDS IMPROVE UPTAKE AND STABILITY OF SMALL DNA FRAGMENTS IN VITRO. 1 • 2~ ~. 1.2Austin F. Dhorman, and l.l.JDieter C. Gruenert. Gene Therapy Core Center, 2CVRI, 1Dcpt. of Laboratory Medicine, University of California, San Francisco, CA 94143-0911.

Small fragment homologous recombination (SFHR) is a gene therapy technique that has been used to correct the 4FS08 mutation in cystic fibrosis (CF)cclllines and mice. While a number of studies have addressed the in vitro and in vivo delivery of smaller oligonucleotides ( 10-30nt),little work has been done on the transfer and intracellular distribution of these larger fragments of DNA (400-800bp). The ability of a cationic lipid, Lipofectarnine®, to deliver a 49lbp DNA fragment to a CF cell line in vitro was investigated. In panicular, the percentage of fragments which localized to the nucleus of the cells, the integrity of the fragment in the cells, and the subcellular distribution of the fragment following transfcction was determined. A 49lbp DNA fragment encoding for exon 10 of the CFI'R gene was generated by PCR and internally labeled with either a"P-dCTP or FITC-12-dUTP. Cells were transfcctcd with 4 J.IS of either single-stranded (ss) or double-stranded (ds) fragment complexcd with Lipofcctamine at various charse ratios ( -:+ ). As a control, naked DNA was used. Cells transfccted with the radiolabelcd fragment were harvested at S and 24 hours post transfcction, fractionated into cytosolic and nuclear components, and assayed for radioactivity. With bot!' ss and ds DNA, a 1 :4 charge ratio was found to be optimal. Nuclear delivery was better ala 1:12 charge ratio, however this amount of lipid showed high toxicity. At S hours (1:4 charge l'ltio,u DNA), 7.23±0.48% (S.E.M)of the tOtal traMfected counts were found to be associated with the nuclear fraction. SignifiCantly less, 2.29±0.2S%, were found with dsDNA. For both ss ~ ds DNA, approximately 113 of the nuclear associated counts were sU!l pteiCnt 24 hours after transfection. Minimal radioactivity was recovered 10 the nuclear fractions of the naked DNA controls at both time points. !he stability of the radiolabclcd fragment in the cytosolic and nuclear fract.1ons 24 houn after transfection was analyzed using a I 0% polyacry~am11le gel (PAGE) in BM urea. Analysis of both ss and ds DNA transfect1ons showed the fragments to be intact with liule observable ~e~tion in the cytosolic and nuclear frKtions. There was no fragment m either the ~yt~sol or nucleus in the control experiments. The sub­cellular localization of fragment was confirmed using FITC labeled fragment and confocal microscopy. These studies show that cationic lipids are effective in the delivery of small fragments of DNA to the nucleus of cells in vitro and thai asDNA is transfected more efficiently than dsDNA when using cationic lipids. Supponed by NIH Re1earch Supplement for Minoritiu, cuul frtJ/111 DK47766 and DK46()()2.

204 EXTRACELLULAR BARRIERS TO CATIONIC LIPOSOME MEDIATED GENE TRANSFER.~ S.ltolhery', NJ.SeYen2

,

s.a.enas, DM.Geddll1 • EWFW.AltGII1. 1I111 TranlpOI't Unit, Nlllional Heart .t Luna IDititutt, Loadoa, Eapnd. ~ of Cardi8c Medicine, Nlllional Heart A Luna lllllitute, Loadoa, Eapnd. 1Gcmyme Corporation. F~ MA, USA

Cationic: lipolomal ClOIIIIame DNA, bind to coli ~ and elicit acne tranater. A neutral lipid -~~~~ lllldolomal eacape after which plasmid DNA uaftlca to 1111 nucloUI by an u )'Cit unknown mechanitm. In vitro many lipoiOIIIII are equally capable ot produc:iaa acne exprenion. but only one or two have lbown to be eftlcilnt 111 \llvo. In our hindi, 111 vivo acne expreaion il Rill 4-S lop kMw thlllln vitro. To_, tbe I1IIICIII for this dilcrepmlc:y, we have developed an n IIWo model. Usina sheep tracheal epithelium ICC up at an air-liquid interfice and cultured in a minimal media, DNA/Iq,c.ome c:omplex C111 be applied dirwctly to the apicalsurW:c. Applic:alion of 110111plex to trelh tiauc produced little or no gene cxprenion u wenod by the quantitative CAT reporter JCIM. When the tiuuc wu depleted of mucua, JC1M expreuion increaad by 2S fold. No fiutber iDcr-. wu produoed by inhibition or removal of cilia or treatina tbe coli surfilec wilb neuraminidue. To inveltipre flu1ber tbe role ofllll plasma......,_, FITC labelled DNA wu uaed to determine tbe incracelluJar fhlctioa of complex by clual tanpenlllre labellifta. Application of complex at 4"C I1IIUltl ill bindiltl to tbe coli surface whel1lll IJIPiication • 3?-C I1IIUitl in bindilw IDil intemaliutioa. Conballllicrulcopy wu carried out to dllennille 1111 JMIII*Illll of cells ~ ~ ill a 0111 liM (Col7) and in our n IIWo model. 73.3% :t 2.1 ofC.7 ........... ~to IWily 3.7% :t 1.4 ill trach-' apidllliual. Pllllher, 1111 n """' model lhoMd ..... _.. of surflloe libel It bod!......,., IICJC IIIIICMble by acid wuhina. We 1111111t tlllt bodlliiUCUI aad tile l!pical ...,_ are sipitiCIIIt barrien to n 111vo aene t!WWfer. TCJII!her they 1111111t ill 111 importut barrier 1111UltiJrt in poor ... trllllftlr lllinl cationic lipolomel.

OPnMJZAnON OF REPEAT AEROSOL ADMINISTRAnON OF CAnONIC LIPID QL ... 7 AND pDNA COMPLEXES. G. Y. Akita, M J Lukuqo S. Rydbetg, 0. Chu, S. EasttMn, J. Kaplan, S.H. Cheng, and J. A. St. Geoflle. Genzyme Corporation, One Mountain Rd., Framingham, MA 01701.

Aeroaol delivery olalingle doee of the aeroeol formulation of lipid GL-«17 complexed with pONA (117A:pDNA) rNulta In favorable levels of lung lranlll'flll exprellion and ufety profilM. To explore the feuilility ol ~t aeroeot dellYery ol 117A:p0NA, BALB/c mice were exposed to aeroaola In whole body exposure chembera uaing e regime of initial exposures to 117A:pCF1.CFTR followed bye final~ to 117A:pCF1.CAT. This regime allowed -.men~ o1 CAT trenag- expreaaion if an lnvnune reaponae to 117A:pCF1.CFTR or CFTR developed. Mice exposed to the same doH o1 vehicle followed a fl'lal exposure to 67A:pCF1.CAT, or mice exposed to only one doM oi67A:pCF1-CAT Mrved ea controls. Initially an optimallntervel between doses waa determined. Mice were exposed to the first dose of 117 A:pCF1.CFTR and al predetermined Intervals, exposed to the final dose ol 67A:~F1.CAT. A. lingle repeat llllpoaute to 67A:p0NA as little as 7 days apart did not eclveraely affect lung CAT expreasion. There wu no histologic: evidence o1 pulmonary toxicity. Mice _,e then exposed to s5 aeroeol doses oiii7A:p0NA administered at 7 dey intervals and lung CAT expression analyzed following 1, 2, 3, 4, and 5 exposures. Lung CAT expression waa comparable in mice axpoaed to 1 and 2 doHa of 117 A:pDNA, end among vehicle controls. Tranag- exprllllion in mice exposed to 3, 4 end 5 dosal o1 67:p0NA. -e comparable, but approximately haH those ol mice exposed to 1 or 2 do ... ol 117A:pONA. To eueaa the safety and efficacy of long term repeat aerosol expoaur .. to 117A:p0NA, 12 doaea of 67A:pDNA were adrnlnlalered to mice wMh one week between do .... Lung CAT exprealion Will deeermined following the last (12th) axpoaure to 67A:pCF1.CAT. Aft• 2, 4, I, I, 10 and 12 apoeurM to 17A:p0NA, groups ol mice w.e Nhenlzed for hiltologlc -'Yale ol the lungs, llYera, kidneys, and ~P~HM. Sera _,. collected for aNlyM ol entlboclln to GL-«17, a.ONA, d.ONA, and CFTR. Lung CAT exprMCion In mice exposed to 12 doHa ol 17A:pONA. waa approxlmMely half that ol the controls. There wa111 no hlllologic changea auoc:lated with repeeted 117A:p0NA exposure• In the organ1 examined. Serum llllllbodin to GL-e?, u DNA. de DNA and CFTR -e not detected. In llllrMIIIry, a 7 dey Interval bel- two 117A:pONA exposwN did not adveraely afleet ~~~- trenegene exprealion. After 12 once per week expoeuree to 117A:pONA, lung Ira~ exprealion waa approximetely one hel that ol controls. There WM no observllble evidence oiii7A:p0NA releted pulmD~Wry or ayllemlc toxicity. Serum antibodiea to GL-«17, DNA or CFTR were not detected. The.. atudl" demonatrate that repeated aerosol admnstratlone ol117 A:pONA to BALBic mice Ia well tolerated and can result In ax~ ol the tranagene product albeit at reduced Ieveii.

206 PHARMACOKINETICS AND GENE EXPRESSION FOLLOWJNO INTRATRACHEAL DELIVEJt.Y OF CAnONIC LIPOSOMEIDNA COMPLEXES. K B Meyer I, M.M. Thompson2, L.O. BarrOtll, and F.C. Szoka,

Jrl. Dcpamnent of Blophannaceutlcal Sciencea, Univenity of California, San Francisco, California and, Depanment of Pediatric•. School of Medicine, University of New Mellico, Albuquerque, New Mexico, USA.

We have previously lhown that cationic liposoma complexed 10 plasmid DNA cu lrllllfcr ,_. in10 the 1110UM lun1 followin1 intratracheal (IT) ldministralion II levels sipificantly hiper than found followin1 tteatment with DNA alone (Abstract tl83, Tenth An11ual North American Cystic Fibrosis Conference, 1996 and aubmitted for publicllion). The~~e cationic liposomes are fonnullled at a I: I molar ratio with cholesterol, conferrin1 increased stability to the lipos0111es and pouibly explslniRI enhanceme11t in pne delivery. To examine the fate of DNA molecules and liposomcs adminiatered into the lunJ, we atudied the dillribution and half-life of naked DNA and cationic liposomc/DNA complexea. Plasmid DNA was nick translated with [ 125)-I dCTP and used to spike nonradiolabeled DNA, and 1he radiolabeled liposome marker [1311-1 p-hydroxyl benzamidinc-DPPE was included in DOTAPIChol-eaterol liposome preparations. Plasmid DNA (20 Ill) alone or complexed with DOTAP/Choleaterol (3:1 clw)e ratio) was adminiatered IT to mice, and Ieveli of radioactivity were meuured from the lun1 at 5 min, 20 min, 60 min, 8 hr. 24 hr and 48 hr followins tteatment. Parallel reductions in both lipoaome and DNA componenta were observed In the luns with time. Silly percent of the adminiatered dose of both ndiolabeled liposome and DNA was detected in the IUDJ at 8 hrs, thia level declined 10 5()% II 24 hrs, and 2~ at 48 hrs. Radiolabeled DNA delivered in the absence of liposome rapidly declined to leveb I~ of the adminialered dose 81 8 hn and 0.111. at 24 hn. To determine the effect of helper lipid, DOPB was substituted for cholesterol, and levels ol radiolabelcd DNA and liposome were measured at ' min and 24 hn followin1 rr delivery. A more rapid decrease in DNA was -n as I~ of the adminiatered dose woo detected at 24 hn, compued 10 4011. of the admlaiatered liposome dose II 24 hrs. This data lhow1 that DNA remains In the lu•1 lonpr when complexeol with DOT APICholeiWOI lipoliOIMI than with DOT APIDOPB llposo11111. 1'ha inteplty of pl-id DNA COIIIplexecl with DOT AP/Choleswol liposoma - desennlned by Soulherll aaalyaia and PCl of tuns Ulllpln at selectecl timea followi111 IT adminillratioa. lmmunolliatochemical as well u 111 11111 lT-PCit techniques were used 10 identify the location and diatribution ol cells expreuiaJ the tnnspM. Cella expreui111 the tralllpH wete idelllirtecl I• both lirway and piRnchyma cella. (R•"•rclt frutll" by IM Cy1tk Flbrotil F~ c.li/tlmkt IREP, DK47766, HlA2J68)

207 CATIONIC LIPID-MEDIATED TRANSFECTION OF POLARIZED AND DIFFERENTIATED AIRWAY EPITHELIAL CELLS IS INEFFICIENT IN VITRO AND IN VIVO. S p O'Connoc K.X. Wang, S.L Fang, AI.J. PrzybylslaJ, AI. Cunneen, D. Lynch, C. Siegel, C. Jiang, and S.H. Cheng. Genzyrne Cotporation, One Mountain Road, F111mingham, MA 01701.

Although cationic fipida have been lhown to be capable of t111nsfecting a variety of different cell types In vitro and ti- In villo, their utility Ia limited by ineffiCiency and toxicity. Our early aludiea identified a cationic lipid. GL· 87, that ahowed rrarked gene t111nsfer ectivity when ins1illed into the lungs al BALBic mica. However, results of a recent clinical study using the nasal epithelium of CF patients indicated that GL-67-mediated gene t111nsfer was not aigniftcantly mora affective then free DNA alone. Wa have duplicated this study in the nasal epkheNum alt111nsgenic CF null mica and arrived at aimilar conclusions. Although functional correction altha defective CFTR chloride (CI") channel activity could be detected in CF mica treated whh GL· 87:pCF1.CFTR co~J"4)!exas, the msgnhuda altha response was moderate and the frequency inconsistent. Furthermore, the response was not significantly greater than that observed In mica administered free DNA alone. Increasing the residence lima of the co""'lex on the nasal ephhelium marginally i""'roved efficacy but also was associated with toxicity as evidenced by a decrease in the basal nasal trsnsepithelial potential difference. That co""'lexas al GL-67:DNA were not more effiCient than DNA alone at effecting g- transfer to the airway ephhelium in vivo was also corroborated by studies using an In llivo rabbit trachea model. Rabbh tracheas that had been incwated for 80 min whh complex or DNA alone exhibited equivalent levels of transgena expression. These results contrasted those anained when the complexes were instilled into the lungs of mica, where a 100 to 1000 fold greater t111nsfection ectivity waa I'Niized with GL-«17:DNA ~lexn ~r..:l to lrH DNA. However, inatination results primarily In alveolar expression suggesting that perhaps the Type II cella are mora receptive to lipid-mediated transfection than the airway epithelial calls. It also may be that the Type II cells are mora susceptible to cationic lipid-induced darnega and are thereby aelectlvely transfected. This view was supported by In vitro studies demonstrating that poorly differentiated and proliferating nonnal human or dog airway ephhelial cells (auch as when damaged) ware mora efficiently tranafected than fully confluent, polariz-=1 and differentiated cella. Polarized and differentiated dog airway epithelial (DAE) cells exhibited a much lower cellular proliferation rata than their poorly differentiated and non-polarized counterpart• aa auayed by thymidine uptake. Thera was a direct relationship between the profiferative rata of the DAE cella and their transfection efficiency. These reaulta indicate that an intect, quiescent airway epithelium Ia relatively refractile to transfection by cationic lipida. A greater understanding of the barriera to transfecting airway ephhlial cells and approachea to overcome theM barriers w~h this non viral vec:tor II delirable.

208

IMPROVING THE EFFICIENCY OF LIPOSOME· MEDIATED GENE THERAPY FOR CYSTIC FIBROSIS Emily s Scan 1. Richard P HIJbottle 3. Charles Coutetle 3, William H. Colledge 2, Martin J. Evans 1 Wetlcomel CAC lnstnute 1 and PhysiologiCal Ulboratoty 2, Univerllty of Cambridge, Cambridge, UK and Dept. of Biochemistry and Molecular Genetic• 3, lmpertal College School of Medicine at St Mary' a, Norfolk Place, London, W21PG, UK.

The aim of this wert 11 to develop strlllegiea to enhance g-tranlf• In systems with -e pathology in CF: the 8Jrways and intestines. Wa have adopted a receptor mediated gene transfer strategy In order to breach the ceH's first line of defence: the plaama membrana. By incorporating an approprlllle ligand Into alipoaome/plalmid transtection complex we hOpe to target plasmid delivery and increase the number of complexes entenng the can. A candidate ligand Ia the AGO (Arg·Giy·Aap) motif which is bound by many members of the integrin family of cell surface receptors. Such a strategy relies on tha presence of the appropriate receptor on the surface of target cells. To confirm this we have Investigated the distribution of AGO· binding integrtna on human lung and gut eptthelial cell linea (16HBe and T84)1n vttro, aa wetlaa murine airway and gut epithelia. Celt adhllllion assays performed with 18HBe and T84 cellssupportl the preNOC8 of AGO-binding lntegrtns on the surface of th- cella. Confocal microscopy alter ImmunocytoChemistry on polarlled 16HBe cells shOwed the alpha., lntegrin subunit, preMnl in -at AGD·blndlng lntegrlns. on the plasma membrane of theN cells. inctudll19 the apical aurtace. lmmunolocalisat1011 lludiel of these FIGD·blndlng lntegrlns in mouse -•Y tt- have aJ10 supported their prnerx:e on the apical surface of these apilhetta. Theea raeuNs indicate the pr.- of receptOrs capeble of binding the AGO motif on lung and gut apllhatlal calla In 111tro and In .no. Encouraged by this a targeting peptide waa daeigned oontaoning the RGD motif and a non-l!paCffic DNA· binding domain (poty·L)'Iine). In gene tranller experiments on 18HBe cells 1n llltro. pre-Incubating a lucifer- report• plasmid With thts targeting peptide enhanced Hpo801T18 (DC-ChoiiDOPE) mediated o- tranlfer af1iciencY 30-told. wa have a110 lnvntlgat..:la complementary approach for

Poster Session Abstracts 265

improving gene transfer efficiency which iniiOlves pre-incubating plasmid with a peptide containing a nuclear localisation lllgnal sequence and a non· specific DNA-binding sequence. Initial experiments uling a lucfferasa reporter plasmid have Shown a 8-lold enhancement of DC-choi/OOPE· mediated gene transfer to 16HBe cells In vitro.

209 REPEATED NASAL ADMINISTRATION OF LIPOSOME-MEDIATED CFTR GENE TRANSFER REAGENTS; THE CLINICAL AND IMMUNOLOGICAL CONSEQUENCES. KW Southern'. SC Hyde', EM Fitzjohn'. BE Waddell', JJ Eaan'. JY Cole'. K HaMavy'. L Huanr. F SorJi', C Goddard', WH Colledge', HC Gooi'. CF Higgins', AK Webb', DR Gill'. Reaional P..tiatric CF Unit1 •nd Dept. of Cellular l..,.....ltOfot:y", St JMW1'1 U.n.ertiey U.,.et ... L.dc tOll. or Motoculw Modi<iH. Jolul Radclilfo H......,. UoWwnily of O.lonl. O.fonl': a.-., ~ ....... A_"* CF Unit, Wyt

4,.,_, Hoop;IOI. Mu~-· UK': Lobontory of DNa r,.,..,, .. U"""'""Y or Pi.......,._ PA. USA : o., .. of...,...,_. u.-.r~. c.-.... UK.

Twelw &dulls wilh cyslic fibrosis were enrolled in a placelxH:ontrolled atudy r:J liposomc-mediated (DC Chol!DOPE) CFTR acne transfer. Followin1 a pre-11udy assessment period. gene transfer reagents were adminislered topically (melhod; Gene Therapy ( 1997);4: 199·209) to the nasal cavity. Three dolea were siven· (four weeks apart), Subjects M and 15 received 1 placebo plasmid, the rest, a CFTR plasmid (200 j.lg/dose; J.PhyJiol (1997);499:677-687). The subjects remained on standard treatment and were assessed by a !lending physicians (KWS, AKW) and an independent ombudsperBOn (JJE). Subject 82 withdraw from the atudy ahonly aller the first dole. Over the 12 week study period, 6 lllbjeciJ had clinical upper respiratory tract infection (URTI). RhinoviNI wu cultured from nasal lavage fluid in one case. Two subjects had exacerbation of cough requirins treatment (inc. A4) and one, an epi!lode of DlOS requirins admiuion. These complicalions were considered normal for CF. Nasal examinalion was normal on all other occasions. Two 111bjccts required intraoe110111 an1ibiotico for 1ymp101111,

though none had any significant reduction in weight or respiratory function. 11or

any fewr or increase in blood inflammalory markers (WCC, PV. CRP and ESR). A nasal biopsy was taken on the scwnth day aOer the third dole (in one cue this was unsuccessful). Seven biopsies wcte normal. In three. moderate to aewre inflammation was seen. Two of these (inc. 15) had clinical evidence ofURTI at the time. Eosinophil cells wete 1 prominent component of the other biopsy. suggesting a response to seuonal aller,ens. Routine clinical immunolosicallests were normal four weeks aller the final dose. in paniculsr there wete no siJnificant changes in lymphocyte 111bset populationl. There wu no evidence of T cell activation against CFTR (meuured by 3HT lhymidine uptake and IL2 receptor expression) following culture of lymphocytes from the subjects with the CFTR­expressina cell line, Cl27CFTR. No anti-CFTR antibodies were detected in subject aera IISing an indirect immunofluorescence assay, however positive responses wete obtained wilh 1 slandard CFTR anlibody (Mabll-1, dona1cd by GeltZ)"'' ... USA). In summary. no sianificant clinical or llpCCifoc immunotoeical abnormalities were demonstraled in our patienls followina repeated lipo!IOIIIC­mediated CFTR acne transfer. Dr Southern iJ funded by the MRC. United KinJdom.

210 DEVELOPMENT OF A MORE POTENT AEROSOL FORMULATION OF CATIONIC LIPID:pDNA COMPLEX. JQ T0usjgn«nt S. Ea•tman, S. Rydberg, AI. Lulai«NJ. AI. Lana, AI. Charry, J. ""'rshal, A. Smith, R. Scheule, and S.H. Chang. Genzyma Cap, 1 Mountain Rd. F111mingham, MA 01701.

Aeroeolization Is the most practical method to deliver gene therapy Yeclora to the lungs of CF patients. Due to the Inherent Inefficiency al aerosol delivery and the metastable natura of cationic lipid:pONA vectors, M was desirable to develop a highly concentrated and stable formulation o1 cationic lipid:pDNA comptexaa for uea In the clinical aettlng. We have pravioualy described the development of such a formulation containing GL· t17:DOPE:DMPE·PEG1000, in which the addhion ol the PEG lipid raaufted in dramatic lncreaaes In both cationic llpid:pDNA stability and in concantrations al Npid:pONA achievaabla (>12 mM In DNA). Wa now have axtandad these studiea to explore the affects al varying the PEG and neutral lipid components of the formulation on the transfection efficiency of the aerosolized cationic lipld:pDNA c~lax. Components of the PEG ~pid investigated included the lipid head group (PEG-. PEG,_}, lipid acyl chain (dimyristoyt [OM), dipelmitoyt [DP), diolaoyt [DO)), and the mota parcent a1 the PEG lipid in the formulation. Fonnulations ware teat..:! first In trltro on polarized and non-polarized cells followed by In .no tasting using in1111neaal Instillation Into BALB/c mica. Formulations that exhibited high levels of translection in thaaa aaNys ware tasted for their stability through nebulization. Finally, ect~ and stable formulations were tasted in an •aroaol model ayalam using whole body exposure of BALB/c mica. Formulations containing DM·PEG lipids oonaiatenlly exhibited transfection afficianciaa 2 to 5 fold great.- than those al identical formulations containing PEG lipide with longer acyl chains (DP, DO). a-transfer to ceRa in culture or to the lung by intranasal instillation was aqu~lant for formulations containing akhar s PEG- or a PEG... head group, Ia the t111nslaction efficiency a1 • given formulation was indapandant of the PEG lipid head group. In contrast,

266 1997 Cystic Fibrosis Conference

aeroeol atudill dlmonatreted a diatinct dapendence of gene expresaion in the lung on the PEG lipid heedgroup; aubatltution of DMPE-PEG1000 lor DMPE-PEG1000 in the formulation reaulted in up to a 3 fold increaae in reporter gene expreulon. Screening of formulation• with aKamata neutral r~plda by intreneael inetilation into mice showed a 3-5 fold enhancement of trenalactlon alllclancy when diphytenoyt PE (DPhPE) wu substituted lor DOPE. The incluaion of OPhPE In formulations containing PEG liplda raauMed In little enhancement of gena axpresaion in tha lung by intrenaaal lnatillation. However, aeroiOI atudlaa using DPhPE-subslltuted lorrnulationa revealed a rnodlat (20·40%) enhancement of gena axpraasion over analogous formulation& containing DOPE. Thus, several important trends have bean Identified wMh appllcetions to the design of more potent aaroiOI formulation& of llpid:pDNA complex. Moreover, a novalaaroiOIIormulation has been Identified, GL-87:0PhPE:DMPE-PEG1000, that enhances gene expresaion in the lung by 3fold over that of our previous formulation.

211

EXPRESSION FROM AAV VECTORS IS NOT EUMINATED APTER REPEATED DEUVERY TO THE BRONCHIAL EPITHELIUM. ~ S.S. Allen, T.R. Flotte, and W.B. Gugino. Departments of Pedialrica and Phy1iOIOI)', Jollllll Hopkins Unlvmity, Baltimore, MD, USA, and the Gene 1berspy Center, University of Florida, Gainesville, FL, USA.

INTRooucnON: Altboulh M V-CFI'Il vectors pmiat In vivo 6 moolhl after delivery to the bronchial epithelium, repeated dosin& may be n-.ary to ICCOlllplilh whole tuna delivery or to achieve IUI!Iclent levels of expreuioll. 'fnm&ene expreuion may be limited after repeated exposure due to I) the illitillion of humoral bnmune respoun 11plnll M V capsid protei111 Clplble of neutralizln& mbsequent - uptllce or 2) the development of cellular responMa to vinl or traiiiJene proceln produc:ts capable of cellular dellructioll. RATIONALE: To tell the hypolbnis that the immune responae to repeated delivery of M V vectors to the bronchial mucosa will block tra111JeDe expreuioo. METHODS: The MV-GFP - TR-UF5 (111 M V-ITR-fllllkccl, CMV-driven, bumaniZlld peen fl.._t procein reporter Jene, AA V 11r0type 2 -) wu bronchoscoplcally Instilled into the lower lobes of the New Zealand white rabbit. Allanimall received AA V -GFP vector to the RLL 30 days prior to a IOCOIId localiZlld delivery of either M V -GFP vector to the LLL (n-3). or M v-cFTR vector to the RLL (n•3). Sinale closed animals (n•$) and lallne lrelled animall (n•3) served u controla. GFP expressioo wu examined 30 days after the final dole by FACS and GFP-specillc fluorescent microscopy 011 cytoioJic bnuhlnp of the bronchial miiCOSI taken prior to IICriflce. Serum, colleeted prior to each - delivery and It the time of IIKriflce, wu usayed for anti-AA V capsid antibody titm uailla an ELISA ll*iflc for VP3 of M V2. RESULTS: CytoJoaic examilllllon of the mucosal brushinp taken ttom the site of delivery demoallrated comparable GFP fluorescence in the slnaJe and repeated doled anlmall, and confirmed that fluorescence emanlted ttom bronchial epithelial cells. SeroJoaic reaponse to M V capsid proceln (>I: I 00) oc:cwred in 2 of 3 rabbitl after a Npe8led dole of M V vector to the 11me lobe. Inflammatory cytoldne prollles (IL-6 and IL-l ELISAI) and broncho-alveollr lavqe cell counts were comparable to controls indlcatln& that local inflammation to both GFP and to npaled - expoiUI'O wu minimal. CONCLUSIONS: Ropeated bronchial delivery of M V vectors nsulted in peniatent traiiSJene expreuioll. Repeated delivery of M V vector to the ume site In the lower airway resulted in the production of antibody dlncted It the M V capsid protein. In either cue, local inflammation was minimal and expreulon ttom the flnt dole of - was not elimialed. Further bnmunoJoalc ltUdiel may balp detennine the 11n1eJY for repeated airway delivery of M V vectors. Stapportad by an•al'roa tile CFF, NHLBI alld NmDK.

212 In Vitro Aei'OIOI DeUvery of an Adeno-Aaoclatecl Vlrus­CFTR Gene Therapy Vector. D. Debelak•, S. Manley•, K. Ung+, N.Eglilmez-Reynolds+, and E.M. Atkinson•. •Development Department, Targeted Genetics Corporation, Seattle, W A. USA and +Battelle Pacific Northwest Laboratories, Richland, W A, USA.

We have constructed an adeno-associated virus (M V) gene therapy vector containing the CPTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene. This vector bas been tested in Phue I trials in human lung and maxillary sinus. The primary target tissue for CF gene therapy is lung air way epithelium, so an aerosol fonnulation and delivery system is destrable. For further ~linical and clinical studies with tgAAV-CF, we have chosen to deliver the vector via jet nebulizer. Preliminary screening has identified three

nebulizers (PARI LC+, Respirgard ll, Ultraneb ultrasonic) suitable for use in the clinic, while physical constraints have identified a nebulizer (Vortran Mini heart) suitable for use in primate toxicology studies. To further characterize these devices, studies involving correct formulation, aerosol characterization, biological recovery, and particle size distribution were performed. Here we report the results of these studies. Experiments were performed to assay for both total viral genomes as well as infectious virus. We have observed consistent high-level recovery of material, with particle size ranges consistent with mid- to lower lung delivery. These experiments further illustrate that the delivered material maintains an acceptable particle to infectivity ratio and thus is not inactivated by the procedure. This study demonstrates the feasibility of delivering the tgAA V -CF vector to patient lungs via jet nebulizer.

213 SUSTAINED EXPRESSION FROM PLASMID VECTORS CONTAINING THE TERMINAL REPEATS OF ADENO-ASSOCIATED VIRUS. M f'rzybylskl. K.X. Wang, S. Ky6stlo-Moore, N. Wan, S. WadsiiOith, N.S. Yew, and S.H. Cheng. Genzyme Corporation, One Mountain Road, Framingham, MA 01701

An allactive g- therapy far cystic li:lroais (CF) wil require sustained axpresaion of the cystic libroala transmarrbrane conductance regulator (CFTR) In CF calla. Recombinant edano-associatad virus vactora have the potentlal to Integrate into the genome and provide long-term axpreulon but have a lmiled lnaert c:epacily and - dillicuM to produce. In contrest, plasmid vectora have a large IMert capacily and can be produced in large amounts but provide only aholl-term expresaion. To utilize the advantages of both vector system& we have Incorporated the tarmNI repeats of AA V into plasmid vactora axpreuing either CFTR or the secreted alkllfine phosphataaa reporter gene (SEAP) and measured the persistence of axpreaslon in vitro. The genomic aequencaa encoding the AA V Rep and Cap genes ware replaced with an expreuion casaatta consisting of the cytomegalovirua (CMV) enhancer/promoter, a h~rid intron, aMber CFTR or SEAP, and the bovine growth hormone polyadlnylatlon signal. CFT1 calla, an invnortallzed human CF *'-1 epithelial cell Una, ware either trenslacted wMh a CMV plasmid lacking tha AA V tanninal repeats (pCF1 -SEAP) or c:o-transfec:ted wHh an AAV plasmid (pAAV3-SEAP) and with a plasmid that expresses the AAV Rep78 protein (pRep78) that has bean previously shown to be required lor siJ•apeclllc integration of the v1rua into chromosome 19. SEAP axpraasion from pCF1-SEAP declined llleadily to residual levels (1.3 mUiml) by day 27. Expreuion from pAAV3-SEAP aleo declined Initially but than persisted a1 a low level (14.2 II'LIIrn It day 27). ~to pCF1-SEAP, co-translaction with the Rep78 expreaaing plasmid inhibited the Initial levels of pAAV3-SEAP axpresalon but appeaq to be required for the longer term persialence that - obaerved. We have aleo Incorporated an Insulator element from tha chick8n B-giobin locua, ahown pravloualy to lncreaae the axprauion of Integrated tranagenaa In transgenic mice, upetream of the CW promoter of pAAV3-SEAP. The preMnce of tha Insulator algnifiCintly increased the Ieveii of suatalnad expresalon compared to pAAV3-SEAP lacking the insu1a1or aequence. In ordlr to examina the fate of the plasmid vectors in tranalacted calli, a number of lllably axpraaslng single cal clones ware generated after trenetactlon of CFT1 cells wMh pAAV3-SEAP and pRap78. F'relinirwy I'MU!Ia uaing Southern blot -lylia indicated Integration olthe plasmid In some olthe cal clonea; however, there - no evidence o1 siJ• apeclfic integration into chromo1011'18 19. Resulta olthe lntegretlon atudlea and the parsiatance of expreaslon of the pAA V vactora wiU be praaanted.

214 A Blah Throupput Assay for Neutrallzlna Antibodies Spedftc: for AA V. L. Evans•. D. Debelak#l, T. Reynolds#, E.M. Atkinson#, L.A. Jones•. Departments of Immunology• and Development*, Targeted Genetics Corporation, Seattle, WA.USA.

An obstacle to gene delivery with viral vectors could be an immune response directed against the virus. Preexisting immunity or repeated delivery of the viral vector may result in viral specific neutralizing antibodies. These antibodies can inhibit the entry of the virus into the cell and severely decrease the transfer of the therapeutic gene. To follow the immune response generated by administration of M V vectors, we have developed a robust, high throughput assay for

determination of AAV neutralizing antibody titer. We have compared total anti-AA V capsid antibody titers measured by EUSA and AA V neutralizing antibody titers in three species: rabbit, macaques and humans. Vu:al protein was introduced utilizing three different methods: rabbits were immunized with viral protein/adjuvant; macaques were treated with an AA V/CFrR vector delivered to the lung; and humans were screened from a pool of anonymous donors and were presumably exposed to AA V via natural infection. This assay will be useful for following neutralizing antibody titers in in vivo models and clinical trials utilizing AA V for gene therapy.

215 Approaches to overcome Ad vector-aaaoclated pulmonary Inflammation and Immune response. J A St George, K.A. Smith, M.J. Lukason, A.L. Galea, K.A. Claussen, SA Rydberg, C.A. Sacks, L.A. Woodworth, J.M. Kaplan, R.J. Gregory, a'ld S.C. Wadsworth. Genzyme Corporation, Framingham, MA, USA.

Numerous studies on adenovlral (Ad} vector-mediated gene transfer to airway epithelium have demonstrated the promise of gene therapy for CF, but have Identified barriers to a safe and effective therapy. Two Important barriers are the Ad vector-associated pulmonary toxicity and the Immune response that reduce the efficacy of repeat administration. We have characterized the vector-Induced cytoklne response profile In the bronchial-alveolar lavage (BAL) and then used this profile to screen combinations of clinically approved anti­Inflammatory and Immune suppressive drugs for their efficacy In reducing the Immune response and associated toxicity. The BAL from mice exposed to 1 E8·1 E9 IU of Ad vector was collected from 2 hra to 21 days post-exposure. Elevated levels of TNFa, IL·B, GM-CSF and KC were detected and peaked at 4 hrs. post·exposure. I L 1-a and ~. IL-2, IL-4, IL-10 and ytFN were not detected or were detected at very low levels. Steroid (dexamethasone) administration was found to reduce the levels of the 4 cytokines detected after vector administration. Direct pulmonary Instillation of the steroid ( 0. 12 5-0.5 mgJkg) was found to be more effective then IP administration ( 2 mglkg). The antioxidant N-acetylcystelne Injected IP (200 mg/kg) failed to reduce the cytoklne response. Other experiments essesaed the ability of therapeutics to reduce or block the antibody (Ab) response to Pd vector. Although there was no detectable serum antibody response to Ad following 2 low doses (1 E7 IU) given at a 21 day Interval, Ab tilers were detected after additional exposures to the same dose. These Ab there continued to Increase with each successive exposure. Current atudles are testing the ability of Immune suppression regime• to block or reduce the antibody response to low do888 of Ad vectors. The drugs under examination Include the combination of 1terolds, cyclosporln and azathloprlna. These studies will define the optimal combination and doaes of drugs to be tested In nonhuman primates.

216 EFFIOENT AND PERSISTENT GENE TRANSFER OF AA V­CFTR IN THE MAXILLARY SINUS OF CF PATIENTS WITH ANTROSTOMIES. John A. Wa~;ner. Mary Lynn Moran, Anna H. Messner, Richard Daifuku, Julie K. Desch, Alexander M. Norbash, Carol K. Conrad, Ilynn Nepomuceno, Sara Manley, Sandra Friborg, Thomas Reynolds, William B. Guggino, Richard B. Moss, Jeffrey J. Wine, Barrie J. Carter, Terence R. Flotte, and Phyllis Gardner. Stanford University Medical Center, Stanford, CA, USA, Targeted Genetics Corporation, Seattle, WA, USA, and the Johns Hopkins Medical Institutions, Baltimore, MD. USA.

AAV-CFTR was used in a phase I dose-escalation study to transfer CFTR eDNA into respiratory epithelial cells in the maxillary sinus of 10 CF patients. The maxillary sinuses are attractive for evaluating new treatments of CF because they have the same ion transport systems and microbiology as the lower respiratory tract and allow localized delivery of known concentrations of agents directly to human respiratory

Poster Session Abstracts 267

epithelial cells. Dose-dependent gene transfer, assayed by semi-quantitative PCR. was observed at doses of greater than 1 x 1o4 replication units (RU) AA V-CFTR. At 1 x loS RU AA V -CFTR. gene transfer was observed in the range of 0.1-1 vector copy per cell in biopsies obtained two weeks after treatment. Persistence was observed for up to 10 weeks. Functional restoration of the sinus transepithelial potential differences was observed. In contrast to adenoviral vectors, little or no immune response or inflammation was observed, even after repeat dosing. The results of this phase I study suggest that AA V-CFTR administration to the maxillary sinus results in successful, dose-dependent gene transfer to the maxillary sinus with partial correction of sinus TEPD and little or no host immune response. Based on these results, 7 patients to date have been treated in a phase II, double-blind, placebo-controlled, within-subjects study on the effect of AAV-CFTR at a dose of 1 x loS RU on recurrence of sinusitis.

217 DELAYED EXPRESSION OF AA V VECTORS IN THE ABSENCE OF ADENOVIRUS. ~ S. Walsh, W.B.Guggino, T.R.Fioue. Gene Therapy Center, University of Florida, Gainesville, FL and Depll. of Physiology, Pediatrics, and Bioengineering of Johns Hopkins University, Baltimore, MD. Sevenl groups have demonstrated thotthe lddition of ldenovirus (Ad) or the

AdE4orf!i protein is required for efficient leading strand synthesis and maximal expression of AA V vecton 11-ly time points [Fisher, 1996; Femri, 19961. This fmding is 110mewhat pandoxit:al sinc:e during 1 latent infection wt-AA V is rapidly converted to double-stranded fonn In the absence of Ad. Our hypothesis was that the observed effect wu due to kinetic: fac:ton, i.e., to a relative delay In conversion to cis-DNA and subsequent transgene expression rather than to an absolute requirement for Ad. In order to test this hypothesis, parallel cultures of Hell c:ells were infected with either the AAV-cMV-Green Fluorescent Protein (GFP) vector alone [m.o.i.• I i.uJcell) or with AAV-cMV-GFP +AdS [m.o.i.-2.S p.f.uJcell). Expression was assessed by fluoresc:ence-ac:tivated c:ell sorting (FACS) at timed intervals ftom 16 hours to 6 days after infection, while conversion to cis-DNA was assessed by Southern blot of standard agarose and alkaline agarose gels of Hilt-extracted DNA. Total viable cell countJ were also assessed by Trypan blue exclusion and combined with the FACS data to detennine the total number of GFP-expressina cells at each time point. The resulll of these studies are shown In the two tables below:

nrr=~ liGZJ....... · .... .~:

I o I : ••. 'O'.:o I I I I I I :., . ..:. I I 0

The data In table I confinn that at 48 houn Ad infection resulted in a 4-fold Increase in AA V vector expression. When considered u an absolute number of GFP-expressing cells (Table 2), however, AA v-cMV-GFP expression continued to increase for 6 days, white no Ad-infected cells remained viable. This corresponded with gradual convenion to cia-DNA fonns on Southern blot. In parallel experiments perfonned with AA V plasmid DNA, there wu no evidence of vector replication to account for this findings. auggestina thll slow, continued convenion to da-DNA offset any dilution of vec:tor genomes, th111 resulting in a sustained level of expression. We conclude that the molecular eventJ in AAV vector transduction are complex. and that AA V vector transduction is not absolutely dependent on Ad co-infection under all conditions.

Supported by 1rantJ from the CFF, NHLBI, aad NIDDK

218 A FLUORESCENCE VIDEO-ENDOSCOPY (FVFE) TECHNIQUE FOR DETECTION OF GENE TRANSFER AND EXPRESSION IN THE AIRWAY. L.&.E1l!lm. S.E. Beck, M.Potter. Depts. of Peds., Moi.Gen.&:Micro. and the Gene Therapy Center, University of Florida, Gainesville FL 32610 and Eudowood Div. of Peel. Resp. Sciences, Johns Hopkinl University, Baltimore, MD 21287.

OBJECTIVE: A more sensitive and specific method for detection of aene transfer and expression in the airway could help resolve several questions regarding the optimal delivery technique for CF gene therapy,such u the optimal dose of any aiven vector, the dosing interval, and the unifonnity of distribution of the trans gene. We sought to develop such a method based on the Green Fluorescent Protein (GFP) reporter, which has a specific fluoresc:enc:e signature within livina cells. METIIODS: An OlymJIUI EVIS video-bronchoscopy system was modified by

268 1997 Cystic Fibrosis Conference

insertion of a specific excitation filter (Chroma HQ410/40) within the primary filter wheel of the ll&ht source and a barrier detection filter (Chroma HQS IOLP) between the eyepiece and the camera head. Doses of an adeno-associated virus (AA V)­CMV..QFP vector of lxl07 to 5xl07 infectious units were instilled into the ri&ht lower lobe (RLL) of each of S New Zealand white rabbits. Three other rabbits had saline Instilled into the RLL and served u neptlve controls. One additional rabbit had a dose of I o• p.f.u. of an adenovirus (Ad)-CMV -GFP vector instilled in the JU.L. Inspection of the airway surface under bri&htfield and fluorescence optics wu perfonned at 3 days for the Ad-CMV..<JFP and at 30 days for the AA V-CMV..QFP and the saline controls, since these have been the peak times of expression with Ad and AA V, respectively in previous studies. Cells were then brushed from the apparently fluorescent areu and analyzed by standard fluorescence microscopy and flow cytometry. Animals were then sacrificed, and fiXed lung sections were examined by standard fluorescence microscopy, u well. RESULTS: With the AA V -CMV ..QFP vector, discrete patches or strips of epithelium were found to be fluorescent at the 30 day time point. This corresponded with a rate of fluorescence of approximately 20% omonJ cells brushed from those sites or within sections of fiXed tlsaue from those sites. The fluorescence pattern with the Ad vector wu bri&hter and more diffilse ujudaed by FVFE. Correspondinaly, 40 to 50% of cells in those somples were fluorescent. No fluorescence wu observed in saline control animals, either by FVFE or by standard microscopy. CONCLUSIONS: The observed concordance between FVFE detection of GFP expression and detection by standard techniques indicates that FVFE may serve u a non-invuive bioloaical indicator of acne transfer in the airway. This may allow one to usess expression and yet avoid repeated somplina which could alter the bioloaical properties of the vector by causina cell proliferation or inflommation.

S•pported by 1nats f'ro• the CFF, NHLBJ, a ad NJDDK.

219 ASSJ:SSMJ:NT Or GUJ:N' rLUORJ:SCJ:NT PROTJ:JN AS HISTOLOGICAL UPORTJ:R GJ:NJ: IN MUmNE AlllWAY. ~.E. Hillery', P.K.Jelfcry 2 , S. H. Chen&', D.M. Geddes 1 &E.W.F.W. Alton 1• 1 Jon Transport Unit, National Heart & Lung Institute, London. 2

Department of Luna Petholoay, Royal Brompton Hospital, London. ' ()enzyme Corp., MA 01701.

Development d a relilble llletolcJPW aasay d aene transfer in murine lung Coliowin& lipid-mediated 1l'IDifecllon woulcl be d beneftt. Green fluorescent protein (GFP) hu been suuesrect u a candidate for IUCh studies. Initial studies were therefore undertaken in female Balb-C mice (n-6) treated with a single dose (80 Ill) ofpCMV·EGFP comptexed with lipklll67. Animals were sacrificed on day 2, lungs fixed in 4% formalin, and IUbjected to ltandard pantftn proceuing. No GFP lluoracence and c:onaidenble autofluoreocence of the tiaue perticularly in larger airway epithelial cells wu ..n. In vitro lltUdiea In Col cells llhowed that GFP lluoracence wu liable after fixation with 10% formalin. To determine the stage of puallln procesaiDJ at which fluorescence wu loll, GFP expressin& Col cells were oubjected to similar procesaiDJ. These ltUdles llhowed that the IMS steps uaed to delrydntc tl- CIUied prDIICIIivc klla d fluoreacence. To overcome this, frozen leclionl were IIICIIed followina lixatloa in 10% formalin and snap ftcezing. AI before no GFP ftuoreacence and hi&h autofluoracence wu-. To- whether the autofluoreacence prevented detection of GFP IICVerll protocols were UDdertaken to reduce the ~. includiDJ prior ftxatloa in 2% pmformaldehyde or no fixation prior to frozln .alonlng. Furtllcr, 11xat1on with 2% pmformaldchyde. acetone or llir-dryiDJ after fleezlna ,..,. .-d. l'lllt..ftxatlon with llir-dryiDJ proved optimal in reduciDJ ~. Prior to 1\arther In vlw lltUdiea we IIICIIed whether a hiiiiOioP:ai or quutitatlve aasay would provide the ll'lllR aensitive GFP auay. The lcvd of GFP exprea1on wu manipulated by lrlllll'ectlna eo. cells with a COIIItant dole (0.5 11J) of pCMV-I!GFP for pi'CIIrCISively lhorter times. For the quantitative auay Ouoreacence of cell lysltel wu ~ usin& ltandard fluorimetry. The quantitative lipal wu loll between 30 and 10 minutes of incubation with complex, whereu Ouoreant cells - ltiU ICCil with I minute of int:ubatlon. Finally two cohorts of animals (n-6 each, 10 and 240 111 DNA) were treated with pCMV·EGFP. Tisaue wu frah frozen and half the ICClions post·fixed in 2% paraformaldehyde and half air-dried. No GFP fluoreacence wu detected. These results IIJIICSl that at the preaent level of 1ipo101110-mediated poe expresaion. GPP is unlikely to be a lllitable reporter aene for biltololica1 detection of aene transfer in murine luna tissue.

220 Targeting human CFTR expreHion In lung alrwaya wHh 1 novel human epithelial expreaalon ca ... tte )'v-HJa Cbpw JonMhM Plumb, Yanxllo W..,, Kyoung-Jin Sohn, Zlwl Lu•, Mlnulllluchwalcr, Ch-Chung Hut*, Gergetf Wcaca, Keilh T-'1, Hugh O'lnldcwlcll, and Jim Hu, OIYilion of Relpiatory R..-dl, Dlvieion of Endocr'•llllotw* llld DepMmlnt of a.n.tict•, Ho.pltaf for Sick Children, 5115UnlveNily Awnue, Toronlo, Ontllrto, M6G 1XI, CINda

We are interested in lung gene expression and in gene therapy for fatal lung diseases, such as cystic fibrosis. The CFTR gene expression in human lungs after birth is localized predominantly in epithelial cells lining the upper airways, especially in submucosal cells. Utilizing regulatory elements from human cytokeratin 18 (Kl8) gene, including 5' genomic sequences and an intron, we have developed a novel expression cassette that can efficiently express reporter genes as well as the human CFTR gene in cultured lung epithelial cells. Preliminary results of transgenic expression analysis in mice show that the expression casselte can direct highly efficient and cell-specific expression of the E. coli LacZ reporter gene in the airways of mouse fetal lungs. There is no detectable expression in the lung fibroblasts or endothelial cells. To our knowledge, this is the first expression cassette which directs transgene expression in the proximal airway of lung. We have found that the native Kl8 sequences of the expression cassette are not compatible with some of the reporter genes nor with the human CFTR gene. RNA analysis of the human CFTR transcripts expressed from the primary version of cassette revealed two major alternative splice sites. We have sequenced the alternatively spliced transcripts and modified the Kl8 intron and CFTR eDNA sequences without changing the amino acid sequence of the human CFTR protein. This leads to enhanced CFTR mRNA and protein expression and biological function. The modified cassette is also compatible with all the reporter genes we lested, such as CAT, SEAP and LacZ. Since our expression cassette is small enough to be incorporated into any delivery systems, such as liposomes, artificial chromosomes, or viral systems, it has a great potential to be used in gene therapy studies. We are currently using this expression cassette to target human CFTR expression in transgenic mice.

221 THE DEVELOPMENT OF NOVEL RECOMBINANT LENTIVIRAL VECTORS FOR GENE TRANSFER TO AIRWAY EPITHELIAL CELLS TO TREAT CYSTIC FIBROSIS. P. Metharom 1. B Shc!W. R. Q,_.2, E. Gowana3, J. P. Wang 1, and M. WeJ1. o- Therapy Unit1, Sir Albert Sakzewaki Virua Re ... rch Cantre3, Nutrition ~reb Centre2, Department of Peedlatrice and Chid Healh, Uni. of Queenaland, Royal Childr..,'e Hoepilal, Briabane, Qld,AUIIrala.

o- therapy 1a being actively lnveetlgated aa a rn.na of treating CF lung ~ .. but none of the cu11'81'11 vector ayattmW ie 18tiafectoly for the deUvaty of the CFTR g- to non-dividing airway epilhellel calie to obtain .-.lned gene expr181lon. BecauM HIV·beMd lentivil'll vectora can infect non-dividing cella, the development of novel retroviral vectora baaed 1 non-human lentivlrua may ...-111 a 111011 edvantageoua gene delivery vehicle for gene tranefer to airway epithelial celfa for gene therapy of CF. The identification of Jembrana dileaM virus (JOV) u a naw bovina lentivlrua, and the aubHquently available of ita eDNA provided the opportunity for ua to conatruct 1 non-human lentivirua· baeed retrovlral vector. The following cis-acting aequencea were retained In the I1ICOI'I1binant vector, the 5' and 3'LTR, packaging ligna!, extended D8Ckaalna algnalln the 111111 gena and part of the Rev reeponM element (RRE).- Plumid pCIN4 wea ueed aa a backbone to eupply multiple cloning litea (MCS), internal rlloeome entry de (IRES) and a ae1act1on marker, the bacterial neomycin pholpholranlf .... DNA (Neo). To make LXIN (whaM Loo L TR of the JOV, N• Neo, I-IRES),~ L TR of.DV wae PCR ~ and cloned in the Xbaland Xhollite, then the 5' LTR, packaging llgnal, extended~ ligna! In the gag gene and part of the Rev reapoOM element (RR were Independently amplified with eeveral lela of prlrnera, then Un together by apllcing PCR and cloned in the Nn1l and Cllollit18 in pCIN4. New veclora bued on LXIN encoclng 1 marker gene and a therapautlc g- aeparetely are being conatructed In our llborelory. One ie ullng the~ ftuoretcent protein (QFP) u a rnertcar, which glvea bright green colour under fluoreacant rniciOecope, enabling prompt monitoring gene tranafer. Another new vactor encoding the therapeutic CFTR gena ullng an epilope tagged CFTR eDNA would give the edvantage of qualitative meaaurernant of the exprelllon of CFTR by uuying the lagged epltope with conwnerclaJ kl. Final eequ..-~ng and packaging of !h.- viral vactora are under '*-~·

222 CFTR GENE TRANSFER INTO PRIMARY CYSTIC FIBROSIS (CF) AIRWAY EPITHELIAL CELLS WITH LACTOSYLATED POL YL YSINE AS A NON VIRAL VECTOR. W.J.W. Ko!len, X. Wei, AE. Mulbera, M.C. Glick, and T.F. Scanlin. Department of Pediatrics, University of Pennsylvania School of Medicine and The Cystic Fibrosis Center, The Children's Hospital of Philadelphia, Philadelphia, P A. USA.

Lactosylated polylysine is a non-immunogenic and non-toxic vector, that delivers the reporter gene luciferase effectively into airway epithelial cells (I). To detect CFTR gene transfection with lactosylated polylysine, in situ hybridization was performed, using a CFfR!Exon 14 probe labeled with digoxygenin. Primary CF tracheal epithelial cells were transfected with lactosylated polylysine complexed to pAdCFTR or pBQCFTR in a 3/1 (w/w) ratio. To improve transfection efficiency the use of potentiating agents in the transfection medium was examined. With S% glycerol as the only additive to the transfection medium, the efficiency ofCFTR gene transfection was 90 %. In similar experiments, using immortalized CF/T43 cells (M508/M508), a combination of chloroquine and a fusogenic peptide was more effective than glycero~ enhancing transfection efficiency of the CFTR gene to 90 %. Similar transfection efficiencies were obtained with the reporter gene LacZ. In contrast to the enhancement of the efficiency of the primary cells by the addition of glycerol alone to the transfection medium, glycerol did not give the highest transfection efficiency in the immortalized cells. This observation points out the importance of utilizing cells in primary culture. Lactosylated polylysine is an efficient vector for the transfection of the CFTR gene into airway epithelial cells, with transfection efficiency comparable to reported efficiencies with viral vectors. Therefore lactosylated polylysine is a potential non­viral vector for the gene therapy of CF. Supported by the Ter Meulen Fund. Royal Netherlands Academy of Arts and Sciences (W JWK).

1. Kollen, WJ.W., et al (1996) Hum. GcneTher. 7, 1577-1586.

223 TRANSPORTATION THROUGH THE NUCLEAR MEMBRANE OF eDNA COMPLEXED WITH A NOVEL SYNTHETIC VECTOR: POL YETHYLENIMINE. Heltne Pollard, Gild as Loussouarn,

. Jean-Paul Behr, and Denis Escande.INSERM CJF 96.01 Nantes and CNRS URA 1386, lllkirch, France.

In vivo gene transfer using non-viral vectors is a promising approach to treat CF. Polyethylenimine (PEl) is a novel non-liposome agent that ionically condenses DNA and effectivetr transfects various epithelial cell lines. In an effort to gain insights mto the cellular mechanisms leading to PEl-mediated cell transfcction, we used a direct eDNA microinjection approach. COS-7 cells were injected either into the nucleus or into the cytoplasm with a pCMVlacZ reporter plasmid diluted at 0.1-100 IJg/ml in an injection medium supplemented with fluorescein. Twenty-four hours post-injection, cells were stained for 8-galactosidase expression. Intranuclear injection of the reporter plasmid at 100 IJg/ml complexed with PEl (PEl/ DNA ratio • 5) or naked yielded 91 ± 5% (n=465) and 81 ± II% (n=400) blue cells respectively. At the lowest concentration tested (0.1 IJglml), intranuclear injection of eDNA plasmid complexed with PEl yielded only 25 ± 5% blue cells (n:o: 178). Intracytoplasmic injection of naked eDNA produced only 4 ± 3% (n=250) expressing cells at the highest concentration tested (I 00 IJg/ml) and no expressing cells (n=250) at lower concentrations. By contrast, intracytoplasmic injection of 100 IJg/ml plasmid complexed with PEl (PEl/ DNA ratio • 5), yielded 37 ± 9% expressing cells (n=236). Varying the PEl/ DNA ratio between 2 and 7.5 (ionic neutrality assumed for a ratio of 3.5) did not significantly affected the percentage of cytoplasmically injected cells that expressed the transgene. For PEl/ DNA ratios <2, this percentage significantly decreao;ed and at a PEl I DNA ratio of 0.5 was comparable to that obtained with naked eDNA. Intracytoplasmic injection of eDNA-PEl complexes also produced expression of the reporter gene in the airway epithelial cell line AS49 and in the M508 epithelial cell line CFPAC- I. Our results show that the presence of PEl in the nucleus does not interfere with transcription of the reporter gene. They also demonstrate that the synthettc vector greatly facilitates eDNA transportation from the cytoplasm to the nucleus, a process that is not affected by the ionic charge of the eDNA-PEl complex. Finally, they suggest that eDNA transportation through the nuclear membrane is a limiting step for efficacious gene transfer using a non-viral synthetic vector. Supporttd by the A11ociatiD11 Fmnraist ill U.nt Cot~tiY Ia Mucm·i•cidoJt.

Poster Session Abstracts 269

224 ExGen 500 IS A POWERFUL VECTOR FOR GENE DELIVERY TO LUNG EPITHELIAL CELLS IN VITRO AND IN VIVO. S. ferrari, A. Pettenazzo, JP. Behr•, F. Zacchello, M. Scarpa. Department of Pediatrics and CRIB! Biotechnology Center, University of Padova, Italy; •Facultl! de Pharmacie, UniversiU! L. Pasteur,lllk.irch, France.

Non viral vectors might represent a safe alternative to adenoviruses for gene therapy of cystic fibrosis (CF), although new synthetic vectors are needed to improve the actual low gene transfer efficiency. To this purpose, we have tested the new cationic polymer ExGen 500, a linear 22kDa polyethylenimine (PEl) derivative, In vitro and In vivo. ExGen .500 showed to be 2-3 ~rders_ of magnitude more efficient than cationic lipids (Ltpofectm, OOTAP and DOGS) in transfccting CF lung epithelial cells i11 vitro (ca. J09 RLU/10 sec.lmg of protein). This result was oblained by using electrostatically neutral PEl/DNA complexes. in the presence of serum. 111 vivo experiments were performed by injecting neutral complexes into newborn and adult rabbits' lungs. Luciferase activities corresponding to ca. to3 RLU/10 sec.lmg of protein were reproducibly obtained 48 hours post-transfection throughout the 4 lung lobes of newborn and adult rabbits. p. galactosidase expressing cells were visible in the trachea as well as around the lumen of large and small bronchi, no signs of acute toxicity were detected by direct hystopathological analysis. In control animals, naked DNA was unable to give rise to a relevant transfection efficiency. A drastic decrease (97%) of Juciferase activity was observed one week post-injection. When complexes were repeatedly administrated for 7, 14 and 21 consecutive days, no improvement in luciferase activity was detected because of a luciferase specific immune response. Our data indicate that ExGen 500 is a safe compound able to transfer genes efficiently and reproducibly ill vitro and ill vivo. Further experiments are needed to set up conditions allowing prolonged gene expression.

This work was supported in part by a grant of the Association Fran~se pour Ia Lutte contre Ia Muscoviscidose (AFLM), Paris, France.

225 IN m'V DELIVERY OF EXOGENOUS GENES TO THE AIRWAY BY TARGETING THE SERPIN-ENZYME COMPLEX (SEQ RECEPTOR. Asscrn Ziady*, Thomas Ferko!+, David Perlmutter••, Pamela Davil+. Depts Physiol. II: Biophysics* and Pediatric:~+, Case Western Res. U Sch of Med; Dept Pediatrics, Washington U Sch of Med**.

We established In vitro that gene delivery to receptor~pressing cells it possible by targeting the serpin-enzymc complex (sec) receptor. We now report expression of reporter gene in nt tissues following intravenous injection of sec-receptor directed complexes. Complexes contained CIOSY, a 16 a.a. peptide based on the human ex !-antitrypsin sec receptor binding sequence, covalently coupled to polylysine, c:ondensed by charge interactions under high salt conditions with plasmid DNA containing bacterial lac Z gene driven by the CMV promoter. Compacted complexes containing a single plasmid molecule form under these conditions. Rats were injected in the tail vein with complexes containing either 200 ~~ DNA in I ml, or I mg DNA in I mi. Four days later the animals were sacrificed and tissues harvested for histology, histochemical analysis, and enzyme analysis. No inflammatory response was observed in any tissue examined. Little or no !}-galactosidase activity was detected in animals injected with I mg DNA. In animals injected with 200 ~g DNA, the characteristic blue color of bacterial P­galactosidase was detected in liver sections in hepatocytes, in luna sections in alveolar macrophages and also in epithelial cells in larse airways, and in ependymal cells in brain, but not in spleen or heart. Galacto-Light assay of transfected livers averaged 234,013:1: 184,231 (SEM) ILU/mg protein; lungs averaged S32,909 :1: 338,076 ILU/mg; brains averaged 76,384 * 46,096 ILU/mg. All spleens contained <SOOO ILU/mg; all tissues from animals injected \\ith naked DNA contained <I 0,000 ILU/mg protein. Tissues from animals transfected \\ith polylysinciDNA complexes (no receptor ligand) had <10,000 ILU/mg except for lung which had 67,907 :1: 42,0671LU/mg. We conclude that sec-receptor-directed gene transfer is feasible In vivo, and systemic injection results in reporter gene expression predominantly in liver and lung, including airway epithelial cells. We speculate that inclusion of a transgene driven by an airway epithelial cell specific promoter in this gene transfer complex might result in specific transfection of airway epithelium. Supported by NIH grants DK43999, DK 49131, DK276S I, and the CFF.

270 1997 Cystic Fibrosis Conference

226 228 ALTERING THE PROTEIN PORTION OF RECEPTOR· MEDIATED GENE TRANSFER COMPLEX AFFECTS BOTH INTENSITY AND DURATION OF GENE EXPRESSION IN VITRO. A13em.O•I•I Zjady•, Thomas Gerken+, David Perlmutter .. , Thomas Fcrl<ol+, Pamela Davis"+. Dcpts Physiology&. Biophysics• and Pediatrics+, Case Western Reserve U Sch of Mcd, and Dept Pediatrics, Washington U SchofMed••.

Reccptor·mediated gene transfer allows delivery of genes to specific cell types \\bid~ express a IUitablc rcc:eptor with good in vivo efficiency and few ad.cnc effects. We found that a complex which targets the scrpin~ canplcx (sec) receptor transfers genes specifically to rcc:eptor·bearing cells in vitro. Complex consists of peptide rcc:eptor ligand covalently coupled to poly-L-Iysinc by SPDP, condensed \vith plasmid DNA by charge interaction under high salt conditions to give compact complexes containing a single plasmid DNA molecule. We studied transfer of the pGL3 plasmid into cultured HuH? cells, which express high levels of sec rcc:eptor, keeping the target cells and transgenc constant, and monitored compaction of the DNA by electron microscopy (EM). We varied polylysine chain length, while keeping the degree of substitution of SPDP and ligand constant per lysine residue. Although polylysinc of average chain length 36, 100, and 250 residues all gave the same degree of DNA compaction by EM, and all (but the most dilute ligand/lysine ratio) gave high level luciferasc expression (>I 06 ILU/mg protein), the shortest chain length had earlier peak time of cxpressioo and shorter duration of expression than the longer chain lengths. Expression from short chain polylysine \vas <IO'JLU/mg by 10 days after

transfcction, but longer chain lengths gave expression >10' ILU/mg even at 40 days. We also studied the degree of substitution oflysines \vith SPDP, comparing samples in which 5%, I 0"/o, and 33% of lysine residues were substituted (but rcc:eptor ligand substitution was held constant). Polylysine substituted either 5% or 10% with SPDP gave good expression, but 33% substitution gave poor expression. We conclude that chain length and degree of substitution of polylysinc affect both intensity and duration of gene expression in sec receptor-mediated gene transfer. Supported by NIH grants DK27651, DK43999, DK49138, and the CF Foundation.

227 SCREENING OF COMB IN A TO RIAL PHAGE DISPLAY LIBRARIES TO IDENTIFY AIRWAY EPITHELIUM • SPECIFIC LIGANDS. L. Vaysse, A. Vekris, B Aryeiler. Laboratoire de Pathologic Mol~ulaire et TMrapie Genique, Universit6 de Bordeaux II, Bordeaux, France. It is generaly agreed that efforts to improve gene delivery systems should focus on the basic aspects of gene transfer. Such effons relate, first of all, to directing gene transfer to specific cell types by receptor· mediated endocytosis. This can be accomplished by incofF.rating ligands for cell surface receptors in the transfer vehtcle, by modification of the surface proteins of viral particles or by the incorporation of the ligands in DNA complexing molecules. A powerful means for selecting artificial ligands is offered by the screening of large combinatorial phage libraries, displaying billions of different polypeptide sequences fused with coat proteins on the surface of filamentous bacteriophages. Such libraries have been used recently by others to isolate cell specific ligands by either in vitro or in vivo selection techniques, without any previous knowledge of the cell surface receptor (Barry et al., Nature Med 1996, 2, 299-305; Pasqualini and Ruoslahti, Nature 1996,380, 364-366). The primary target tissue for gene therapy of Cystic Fibrosis is the respiratory epithelium. We are using an in vitro selection approach to isolate peptides selectively binding to tracheal epithelial cells (CF· T2) isolated from a cystic fibrosis patient (Lemnaouar et al., Eur J Clin Inv 1993,23, lSl-160). Constrained random oligopeptides (6to 8 amino acids) displayed at the surface of phagemids (pSKAN Hyx libraries, MoBiTec) are incubated with the target cells to allow binding to membrane receptors. The phagemids binding to the cells are recovered, amplified in vivo in E. coli and used for further selection. This is repeated until the complexity of the selected set of phagemids is stabilised. Individual clones are then characterised for bmding to the target cells by phage ELISA, western blotting and sequencing of the peptide encoding region. The specifiCity of the selected ligands will be assessed against a set of non epithelial cell lines. We will present a progress report on this project. Peptides able to specifically bind to epithelial cells will be used to target proteosomes carrying either reporter genes or the wild type CFTR eDNA to the mouse airway epithelium in vivo.

ASSESSMENT OF THE EFFICACY OF IN-VIVO CFI'R PROTEIN REPLACEMENT THERAPY IN CF MICE. M. Ramjeesingh,M., 1...·1. Huan, C. Li, M. Wilschanski, Durie, P., K. Gyomorey, Y. Wang, Beharry, S., C.A. Ackerley, K. Tanswell, G. Kent, C.E, Bear. Hosp. for Sick Child Toronto, CAN

With the development d methods for the large scale expression, purification and reconstitution d CFTR protein, it is possible to mvestigate the efficacy din-vivo replacement d CFTR in the airways of CF mice. To date, we have examined the effect of CFTR proteolipo10111e delivery or liposomes delivery alone on the bioelectric ~es of nasal epithelium of 34 CFTR knock-out mice atr .JUNCt+ . Prior to administration to the mouse nasal epithelium, the quality of

the CFI'R. protcin was confinned by analysis of its channel activity in lipid bilayer studies and by analysis of its A TPase activity. Nasal potential diff'em1cc (PO) measurements; amiloride-sensiuve PD and the PO response to chloride-free solutions, were obtained one week prior to treatment. Following treatment of the nasal epithelium, the nasal Po's were reassessed by an assistant who was blind to the liposome composition. We found that the magnitude of amiloride·sensitive PO's were signifiCantly diminished toward values found in non-a< mice ( • S.S±o.8 mV) by administration ofCFTR protcOliposomes (·9.0±l.S to -S.1±1.2 mV), whereas, there was no significant change induced by liposomes alone (·10.6±1.6 to -10.4±1.8 mV). Furthennore, we found that there wu a signifiCant change toward normal values (2. 7±9.8 m V) in the magnitude of the response to chlaide-free solutions following administration of CFI'R. protealiposomes ( -2.Si0.9 to 0.2±().6 m V). Surprisingly, liposomes alone also induced a signiflCIIIt change in the magnitude of the response to chloride free solutions ( -2.2±().6 to -0.1±0.7 mV), suggesting that these lipids (PE,PS,PC and/or ergosterol) may influence the activity of non-CFrR chloride channels which line the nasal epithelium d (]< mice. Localization of CFrR protein in the ~ical membrane of nasal epithelial cells of some mice, treated approxunatcly 18·20 hours previ~~ was confinned by EM analysis using immunogold-labelled anti- monoclonal antibody. We conclude that CFI'R. Jl!OII:in replacement leads to partial correction of the bioelectric propemes of the airway epithelium and u with gene therapy, greater success will depend on the development of more efficient means for pro~ein delivery to the target tissue. These studies were supported by CCFF.

229 TARGETING THE POLYMERIC IMMUNOGLOBULIN RECEPTOR AS A MEANS OF DIRECTING THERAPEUTIC PROTEINS TO THE AIRWAY. E Ec:kmao. C. Silski, J. Schreiber, P. Davis, T. Ferlcol. Dept Pediatrics. Case Western Reserve U Sch ofMed .. Cleveland OH 44106. The epithelial surface is wlncrable to elastase, which at low concentrations

stimulates IL-l release (thereby promoting neutrophil migration into the airway) as well as macromolecular secretion. Thus, strategies to deliver anli-clastases directly to the apical surface of the epithelium arc of interest. Drup delivered by llei'OIOl may have difficulty penetrating the mucus layer, and are unevenly distributed to favor the better ventilated, less compromised areu of the lung. We cleviscd a llratcgy for delivering protein drugs to the apical surface of the epithelium by capitalizing on transcytosis by the polymeric immunoglobulin rcc:eptor (plgR). Since this receptor delivers its ligand ficm basolalaal to apical surface of epithelial cells, we reasoned that a therapeutic protein, linked to a rcc:eptor ligand, should also be delivered to the apical surface. We selected a 1-antitrypsin (a1AT), an inhibitor of neutrophil elastase, as the therapeutic protein. We used MOCK cells transfccted with the human plgR (MOCK -SC) as a test system and compared them to untransfccted MOCK cells. These cells deliver mouse monoclonal lgG (IgG) directed against plgR from buolateral to apical surface in wctorial fishion. This transpon process is regulated in much the same way as the traffickina of the rcc:eptor itself, and appearance of the ligand in the apical medium is inhibited by nocodazole, leupcptin, and to a small extent, brefeldin A. Anti-plgR lgG was coupled to human a, AT using SPDP. When conjugate •vas placed on the basolatcral surface of MOCK.SC and MOCK cells, immunoreactive a, AT appeared at the apical surfaceofMDCK·SC but not MOCK cells. Conjugate did not traffic from the apical to basolateral surface. Thus, a,AT was delivered in receptor· specific vectorial fashion from basolateral to apical surface. Prior work shows that lllOit epithelial cells much cmtact the ainvay lumen express plgR and thus c:culd tran1p0rt protein conjugates. We conclude that it is feasible to deliwl' thcnlpcutic protA:ins to the apical epithelial surface by targetins the pigR, and that in vivo testing is warranted. Supported by NIH grants DK276$1; DK43999; DK41ll38; T32 HL074l.S, and the CF Foundation.

230 Recombinant human elastase inlubitor (rHEI) protecta rats from hmg injury induced by instilled CF sputum sol or neutrophil elastase. 112. P.cca. 1R. Dowgiert, 1R. Rogers, 1l_ Mandel, ~. RemoJd-Q'Donnell, 1Physiology Program. Harvard School of Public Health, and :Jcenter for Blood Research, Boston, MA, USA.

Recombinant human elastase inlubitor (rHEI) may be a promising candidate for therapeutic use against lung injury caused by neutrophil elastase (NE). To evaluate its ability to protect the lungJ against NE, we measured rHEI'a effect on pulmonary injury caused by CF sputum sol orNE instilled into the lungs of healthy Spraguo­Dawley rats. Four hours post-instillation, lungJ were lavaged and red blood cells (hemoglobin) and albumin in bronchoalveolar lavage tluid (BAL) were measured. NE instilled into rat lungs produced a dose­dependent hemorrhage and increase in epithelial permeability, while NE preincubated with 17.5 jiM rHEI did not. Instilling 17.5 jiMNE (0.1 S ml/1 OOg body weight) resulted in BAL hemoglobin and albumin levels of 10.9 z 1.14 mglanimal and 970 z 180 jig/ml in the absence of rHEI, but only 0.77 z 0.47 mg/animal and 172 z 4S.O jig/ml in the presence ofrHEI. Preinstilling rats with rHEI (JS jiM} I or 24 hours prior to exposure to NE also offered protection, reducing hemorrhage by 1.67".4 and 26.6% respectively. rHEI was also capable of mitigating lung hemorrhage in response to instilled CF sputum sol (6.S6 z 1.12 mg hemoglobin/animal in the absence ofrHEI, but 0.078 z 0.040 mg/animal in the presence). To study rHEI distnbution in the airways, rHEI labeled with Texas Red (rHEI-TR) was instilled into rat lungs (20-60 jig active rHEI-TR/animal). Lung tissue was studied 5 min., 1, 4, and 24 br post-instillation by confocal microscopy. rHEI­TR was present on large airways at all time points studied. At 5 min. rHEI-TR was observed on airways and alveolar surfaces, and at 1 br in alveolar macrophagea. rHEI mitigates NE-induced lung injury and remains present in the lungs for 24 hours post-instillation. Therefore, rHEI may be a suitable candidate for therapeutic use in NE-induced lung disease in CF. (Supported by CFF G980, HL52290, HL41S79, the Center for Blood Research and CBR, Inc.)

231 ACI'IVATION AND INHmmON OF CFrR CHLORIDE CHANNELS EXPRESSED IN HEK 293 CELLS BY ADDmON OF CPX, DAX AND DPC. Arispe, N., Pollard, H. and 1Ma. J. Uniformed Scrvi~:e~ Uniw:rsity. School of Medicine. Bethesda, MD. 20814 and 'Case WCSICIII Rcscrvc University ClcYcland OH. 44106

Expression of wildtype CFTR protein in human HEK 293 cells is followed by the appearance of two weD defined chloride conductances which have been localized in the microsomal membrane fraction by using ion channel reconstitution and recording from artificial lipid bilayer. Both conductances, which have an average of 7.S and 2.S pS conductance values, are equally sensitive to arylaminobenzoates (diphenylamine-2-carboxylate, DPC) and alkylxanthines (8-cyclopentyl-1,3-dipropylxanthine, CPX and 1,3-diallyl-8-cyclohexylxanthine, DAX). DPC, in the micromolar range, produces in both conductance types, a time and concentration­dependence reduction of the amplitude of the unitary opening currents events. It also affects their channel gating kinetics by locking the current activity into prolonged electrically silent or open periods. DPC binding to the channel molecule involves participation of electrical charges since there is a clear voltage dependence in the reduction of unitary current amplitude. This effect is enhanced u tho DPC concentration and the transmembrane voltage are increased. CPX and DAX profoundly activates both chloride conductances. Current records after the addition of the drugs are characterized by multiple open current levels. This current activation is basically tho result of altered gating kinetics of the chloride channels since it is found that unitary conductance and channel selectivity are not affected. The degree of activation is clearly dose-dependent, reaching a maximum within 200 to SOO nanomolar drug

Poster Session Abstracts 271

concentration. In conclusion, activation and inhibition, respectively, by these drugs are observed to occur in parallel to both chloride conductances. This suggests that they probably correspond to tho already reported subconductance states of tho CFTR chloride channel.

232 AN IN VIVO MODEL TO TEST THE EFFICACY OF CHEMICAL CHAPERONES FOR CORRECTION OF THE 6F508 CFTR TRAFFICKING DEFECT. c...Baf, J. Biwersl, R. Brown, A.S. Verkman and M.A. Matthey. Cystic Fibrosis Research Center, University of California, San Francisco, CA, USA.

In vitro studies In transfected cells have Indicated that a 1-3 day incubation In glycerol (0.5-1.2 M) or trimelhylamlne oxide (TMAO, SQ-100 mM) can correct the 6FSOB CFTR trafficking defect. TMAO Is an osmolyte present In high concentrations In fish but not In mammals. To test the hypothesis that chemical chaperones can correct the trafficking defect In vivo, experiments were carried out to establish the feasibility of high dose chaperone administration In CD1 mice. Glycerol and TMAO were administered by Intraperitoneal (IP), subdermal (SO), and oral routes. Serum (glycerol) was assayed using 1 ~L of tail vein blood by a glycerol kinase-coupled reaction giving a decrease In NAOW chromophore. Although IP and SO administration gave serum glycerol concentrations of -100 mM, this level is well below the effective In vitro concentration. Higher doses of glycerol gave unacceptable toxicity. However, a single IP dose of TMAO (6. 7 glkg, 8% solution In water) gave serum [TMAO) of 80-100 mM as assayed colorlmetrically after reduction by TiCI3, toluene extraction, and reaction with picric acid. Pharmacokinetic analysis Indicated a t112 for clearance of -24 hour after a single IP or SO dose. Sustained high serum [TMAO) for 3 days were achieved (21-25 mM, 3.4 mglkg q8h; 43-48 mM, 5.4 mglkg qBh) by SO administration of TMAO In water every 8 hours. There was no apparent toxicity as judged by animal activity and Intake/excretion patterns. These results demonstrate that potentially therapeutic concentrations of TMAO can be sustained in mice in vivo. TMAO will be administered to 6F508 CFTR homozygous mice for biochemical and functional assay of correction of the trafficking ~efect. (Supported by CFF)

233 GENTAMICIN RESTORES ENDOGENOUS CFfR EXPRESSION AND FUNCI'ION BY SUPPRESSING PREMATURE STOP MUTATIONS. D Be~ll1 , A. Kaenjak1

, Z. Bebok2, D. Benos2, J.

Bubien2, J.P. Clancy , T. Jilling2, J. Hong3, A. Tousson3, R.

Lyrene .. F. Ruiz .. and E. Sorscher •, Depts. of Microbiology', Physiology and Biophysics2

, Cell Biologyl and Medicine•, and the Gregory Fleming James CF Center, The University of Alabama at Birmingham, Birmingham, AI.., USA 35294.

Premature stop mutations represent approximately 5% of aU CF mutations and may occur in 10% of all CF patients. We previously reported that low concentrations of the aminoglycoside antibiotics G-418 and gentamicin in the culture medium can suppress premature stop mutations in a human CFIR eDNA, resulting in the synthesis of full length, functional CFTR when expressed in HeLa cells (Nature Medicine 2: 467 (1996)]. We have also determined that G-418 can suppress a CFIR nonsense mutation in mJ-1 cells, a bronchial epithelial cell line that carries the CFTR W1282X premature stop mutation in the nuclear genome. Our results indicate that G-418 restores cAMP-activated chloride currents, CFfR protein at the apical cell surface, and the abundance of the CFTR nonsense mRNA in ffi3-l cells. Although experiments using a cell free translation system indicate that G-418 suppresses premature stop mutations 30 fold more efficiently than gentamicin, we found that gentamicin can also restore CFTR function in ffi3-1 cells. We will present initial data from an ongoing clinical trial testing the ability of gentamicin to restore CFfR function in CF patients canying premature stop mutations in the CFTR gene (supported by the NIH and the CFF).

272 1997 Cystic Fibrosis Conference

234 A ......... DMIIII 111M, I'IHello-CHinlllll Trial er Sodhi• 4-,._y .. tyl'llle ( ....... yl) .. Anii-H...,._ Cystic Flbralll Palinll: Portllll Reoteredeoo er N-11:~1 CPTR F•IICtioll. Rpnald C Rubml!ein and PIIMia L Zeitlin. Eudowood Division or Pedillric: leapinlory Sciences, The Johlla Hopkins Medicallnllltutiona, Baltimore, MD, USA.

Sodillll 4-pheayllNtynlc (Buphenyl, 4PBA) is a new FDA approved dru& for ...-.- of UN& cycle diaorden. We have pnvioualy presented data ouunlin& !hal 4PBA, at clinically achievable concentntions, induces CFTR channol function on tha pluma membrwM of 4F50I .... xpnssina Cystic Fibrosis (CF) airway epithelial cella In vllro (llubenatein, R.C. and Zeitlin, P.L. (1995) l'lcl. Pulmon., Suppl. 12:234 (llilllrect)). Wt hypolhaaizecl IIIII 4PBA would illcluce epithalial CFn liiMtion In -.IYo in indivicluala homoznoua for 4F501-CFn. A IWidomblod, double-blind, placebo-controllecl trial wu conducted in tiah- 4F501-honlozyaoua CF petlenll for one wtek uain& the llllndard approved edull dote of 4PBA, 19 1fM11 p.o. divided t.i.d.. N-1 pocential diffennce (NPD) riiJIOIIM p111em1 and aweaa chloride concentntions were determined before and after IIUdy drultreaement. and 4PBA and metabolites were uuy.d in pluala and urine at tha end of study clrul -ent. Subjects in the 4PBA poup c1etncJtuicnite email, but -iltically lipiflclnt improvements of the NPD reapo1111 to perfuaion or an iloprolennoVemiloride/chloride-fiee aolution; this 111t11111re rellecta epithelial CFn function and is hiply discriminatory lootw- petlenll wilh and without CF. Thero wu a potitive linear correlation (1"().54, p<0.02) lootw- the NPD rnponae to iaoprot«enoVamiloride/chloride­fiee aolution and tha trouah ~ of the ~ pluma metabolite of 4PBA, phenylacatate. Thiloua- IIIII hiah« clru& levels were mponsible for tho improvenlllll towardlanon..CF mponae. Subjects who had received 4PBA did IIIII demon- aipiflcutly reduced aweal chloride concentntions, alterations in their muimal btaal NPD, or their NPD rnponae to amiioride perfuaion. TheM obtervations augeal IIIII 4PBA, at this doll for one week, did not aipiflclntly influence CFn activity in the aweal duct or aodium transport in the nua1 epllhelia. SkM etrects due to drul therapy were minimal and compenlole in the two paupa. n- data are consistent with 4PBA theropy inducill1 pertial CFn function in the nua1 epithalia of 4F501-homozycous CF patients. s.w-w 6y tlw Cy.tic Fil>rolu FOflllllallott lllfli tlw NIH.

235 TJD: lrRCTS Or 4-PIUNYLaUTYUC ACID ON LIVIUI or GINIIXPUSSION An'l:a GIN! TJlANSna IN JIITltO AND IN n..v. 'l,E. B!!l!!l!ju. 'E.s. Hlllety, •s.H. Chen~. SO.c.Gruaaert, 'o.M. Gecldea, E.W.F.W. AlloB. 11on TriJIIII(Irt Unit, Natlollal Hart and LUJt1 lnllitulc, Laadaa, UK, 'o.zymc Corpontioll, CaJnbrid&e, MA, USA, 'CuditMtlc:lllar ~ blldlute, Saa l'rulclaco, CA. USA.

4-plleBylllalyri acid (4PBA) bu pnviaully '-t 1111Dw11 to re110re CF1'R chlmlll l'uclioll in a AP501 expnaiq ~ fibnJIII airway epithelial oell liM (l:CJITB29o-) llf vitro (Pedialrl' Pulmoaolol)', Suppl. 12:234 1995). Gene lrUIIflr il Ill ,_. iDellldcal and maybe calwK:ecl by pblnucoloP:al ~ We h8ve 1llaefore ..-d the eii"KI or 4PBA 011 the cxpnaion or bath CF1'R and the reporter pne CAT after pne lrlalfer. The oellliae tCI'TE29o- wu expoeecl to 4PBA (100 jiM for 72 haun) and CFI1l eDNA camplexecl with the c:.lionic: lipid 1167. In SPQ llaJide.dllux .-y~ 4PBA tnlllled cella did IIIII show a fonkolin llimulated llalidHIIlux .. abcM lllllnllkld cella (0.22 ±0.06 and 0.19 :1:0.09 mM.IK·l n.pec:tlwly, each a-6). 4PBA in COIIiunction with pne tranlfer did not lhow a liplllcut fonkolin ltimuletecl halidHiflux rate abcM tllll or,_, trudlr a1a11e (1.14 ±0.57 and o.7o ±0.22 mM.--1

llllpiCiiwly, each 11'"6). A CMV-cAT pllad complcxld with the catiOIIk: lipid 1167 - ~ 10 female Balli c mice by ...... lpplil:atiOII (10 II& DNA per -). 3 butynle COIICIIIInllionl were ..-c~ co.1, 1, ... 10 .M). At each ~ four c:obans or lllillla1l were __. I'ICIIiviq I, 2, 3 or 4 edminillntlioll by nua1 llllitllq, lllpUIIllll .., 12 llaur iiMnall. Gene UIJIIfer lmmecllately follo'll'eclllle ftnt PIA adallaillratioa All anlmall (n-6) for each cohort were IICiificed Ill 41 11om after pne llllminillnllio and the 111111 ............. .-)'ld far CAT IICiivlty. No co11art demoaltrateclleYell or CAT cxpnaion ....... than that -Ia the t1b1cnc:e of butylate. The data 11U1P111 that 4PBA il not eft'ectiw atlnc:reuinl pne expNIIIion followin& sene tralllfer.

236

DEFI!CTIVIIN'T'MCI!LLULAit TRAfFICKING OF AFIOI CFTR: COMI!CTIOH BY IIHI!NYLBUTYitATI!. JA Cpbo, R. Palil, A.L. Portbuty, J. Kole. Dept. tl Cell Biology & Medicine, V.A. & Duke Unlv. Mid. Ctr, oum.m, NC

CF most commonly results from AF508, a mutation affecting the intracellular trafficking of CFTR. The mutant protein, AF508-CFTR, can be produced in a functional, properly trafficked fonn by growing cells in 10% glycerol or at reduced temperature. It ia unknown whether similar benefits can be achieved using a drug at clinically attainable concentrations. This study examined effects of phenylbutyrate on the production and function of AF508-CFTR expressed in C127 cells. The intracellular traffiCking of AF508-CFTR was studied by immunoblotting, by immunocytochemistry and by assaying cAMP-activated anion efflux. In cells treated with 5 mM phenylbutyrate, the band C fonn of AF508-CFTR increased three-fold, as detected by immunoblotting (p<0.001). Phenylbutyrate increased the ratio of band C to band B without affecting the amount of band B. Endoglycosidase digestion was used to confinn that band B and band C are the core and fully glycosylated forms of AF508-CFTR. Increased production of band C was associated with increased AF508-CFTR at or near the plasma membrane in cells exposed to 5 mM phenylbutyrate. Finally, cAMP-activated anion efflux was increased 4-fold in cells exposed to 5 mM phenylbutyrate {p<0.001). The effects of phenylbutyrate both on the band C signal and on anion efflux were dose-dependent with significant effects occurring at a dose of 3 mM. Thus, phenylbutyrate increases cAMP-activated anion efflux and promotes both the production of fully glycosylated AF508-CFTR and the delivery of the mutant protein to the cell membrane. If phenylbutyrate can similarly increase CFTR function in patients with CF, this might be of clinical benefit.

237 PROIAS'11N SUPPRESSES LUNG DAMAGE, INFlAMMATION AND BACI'ERIAL PROLIFERATION IN A MODEL OF CHRONIC PSEUDOMONAS AERUGINOSA LUNG INFECTION, t Fcetl'JuT. Stru~nell, C. Kooi, D.E. Woods. Unite de Re ere lmonatre, Univenite de Sberbrooke, Sherbrooke, P.Q. and Department of Microbiolo&Y and Infectious Diseases, Univenity of Calgary, Calgary, Alberta, CANADA.

Hi&h levels of active neutrophil elastase (HNE) are present in neutrophil-containing respiratory secretions of patients. We hypothesized that aerosolized alpba1 protease inhibitor purified fiom blood (Prolutinll) could not only prevent elastase-mediated luna damqe but also suppress bactefial proliferation and the inflammatory airway response associated with chronic Pseudomonas~ lunJ infec:tlon. Prolastina was found to inlu'bit HNE activity in vttro in a 1:1 molar ratio. Lung hemorrhaae induced in murine (CS7Bl/6) lunp by 9 um HNE was completely suppressed bv 90 um ProlastinR mstilled at the nares up to 12 h before adding liNE (96 + 1-2% inhibition, p < 0.001 ). In tlie rat apr bead mocfel of chronic P. uruginosa lung infection aerosolized ProlastinR significantly decreased elastase activity ( < O.OS), lung neutrt?Phil counts (p < O.OS) and bacterial colony counts· (p<O.OS). HistopatholoJicill examination of lung tissue revealed' a marked decrease In lung injury in animals treated with ProJutina. 'lbcle studies indicate that Prolastinll (1) is an excellent HNE inhibitor, (2) protects 1unp ._alnst HNE Injury up to 12 h after delivery to the airways, (3) suppresses luna lnfiammation and tissue damqe in chronic P. 00UJ1110S11 infection and (4)) helps decrease P. aouginosa density in the chronically lnfecica luna. These data atroDJly support the concept that aerosolized Prolutina may be usefUl in tfle treatment of CF luna discue. SupportedJ'd; the Canadian Cystic Fibrosis Foundation and the Bayer/CaDLian Red Cross Society R&D Fund.

238 EFFECT OF DEXTRAN MOLECULAR WEIGHT ON CF SPUTUM RIGIDITY: FURTHER EVIDENCE FOR THE MECHANISM OF ACTION. ~.David P. Speert*, Malcolm King. Pulmonary Research Group, Univ. of Alberta, Edmonton, Canada; *Dept. of Paediatrics, Univ. of British Columbia, Vancouver, Canada

We have previously shown (feng eta/. 1997 Am Thoracic Soc mtg) that dextran (m.w. 4000), as a potential mucolytic agent, reduces the viscoelasticity and spinnability of CF sputum and improves its muco­ciliary clearability in in vitro testing. We speculated that these changes were due to disruption of network cross links attributed to hydrogen bonds between neighboring mucin macromolecules. We wished to explore the effect of dextran molecular weight on these effects, hypothesizing that the reduction in crosslink density due to dextran would disappear as the dextran molecular weight approached that of the mucin glycoprotein units. Sputum samples were obtained from 10 CF patients not receiving rhDNase treatment. Aliquots of sputum were incubated at 37" for 30 minutes with Ringer diluent (10% added volume) or with dextrans of three different molecular weights, i.e. Dextran 4000, Dextran 70,000, and Dextran 500,000, to achieve a final concentration of 4% (40mglml). The rigidity modulus (viscoelasticity) was determined by magnetic microrheo­meter, and spinnability by filancemeter. Dextran 500,000 had no signifi­cant effect on mucus viscoelasticity, while the effect of Dextran 4000 was comparable to that previously observed, reducing the rigidity modulus by 0.69 log units or a factor of 4.9 (p<.OOI), and the spinnability by 15% (p<.05). Dextran 70.000 had an intermediate effect. These data provide evidence that the fluidifying or mucolytic effect of dextran on mucus viscoelasticity is primarily confined to the lower molecular weight fractions of dextran. This is consistent with the proposed mechanism that the saccharide moieties in dextran compete for hydrogen bonding sites with other mucous glycoproteins, and that for the low molecular weight dextrans, these new hydrogen bonds are structurally and rheologicall y ineffective, thus reducing the overall crosslink density. This reduced crosslinking of the three dimensional mucous glycoprotein network should make the mucus more easily clearable by ciliary and cough mechanisms. Low molecular weight dextrans thus have the potential to improve airway clearance in patients with mucus hypersecretion.

Supported by Canadian CF Foundation.

239 PREDICTIONS OF LUNG DOSAGES OF NEBULIZED LIPOSOMAL CIPROFLOXACIN. 'W H fjnlay, 2P.Zuberbuhler, 'K. Blaseni, 'J.P. Wong. 'Aerosol Research LaboraiOry, Ocpt.of Mechanical Engineering. University or Albcna. Edmonton, Athena. Canada. ZC.F . .t Pediatric Pulmonary Oinlc, University of Alberta Hospital, Edmonton, Albena. Canada. 'Medical Countermeasures Section, Defence Research Establshment Suffield, Medicine Hat, Albena, Canada.

Uposome-encapsulated ciprofloxacin (2.!1 ml, 22.2 mglml) ~as aerosolized using five nebulizer models known to perform well wllh salbutamol sulphate in previous studies by the authors. Predictions of the amount of encapsulated ciprofloxacin depositing in the lung ~ere made by combining experimental measurements of the nebuhzed aerosol with calculations from a mathematical lung deposition model. Three vented jet nebulizer models (Pari LC Star, Pari LC+, DeVilbiss Perrnaneb), one conventional T-mouthpiece nebulizer (Hudson T­Updraft U), all driven by a DeVilbiss Pulmo-Aide S610C co~press~r, and one ultrasonic nebulizer (Medix Sonix 2000) were used. F1ve umts of each nebulizer model were tested. Ambient humidity and temperature were conttolled at.50±3% RH and 24±1" C. Tidal breathing (with a tidal volume of 0.7!1 l, inhalation/exhalation flow rates of 0.3 Vsec) was simulated experimentally with a breath simulator using a square wave breathing pattern. Total inhaled ciprofloxacin was determined using filter collection and UV specttophotomeuy in methanol, while the fraction of inhaled ciprofloxacin remaining encapsulated was detennined in saline using centrifugation and subsequent UV assay of the supernatant compared to an unnebulized standard. Distribution of the liposomes among the different aerosol droplet sizes was determined using cascade impaction, with methylene blue as a water tracer and subsequent centrifugation and assay. The liposomes were found 10 be homogeneously distributed among the droplet sizes. Significant loss of drug from liposomes was found to occur with the DeVilbiss Permaneb and Hudson T-Updraftll jet nebulizers, but did not occur with the other three nebulizer models tested. As % of dose placed in the nebulizer, the

Poster Session Abstracts 273

average amount of ciprofloxacin predicted to deposit in the lung remaining encapsulated in liposomes varied dramatically from 0.7% to 18.7% among the different nebulizer models.

240 Alterations of the C-terminal Tail of the P2Y1 Nucleotide Receptor Affect Receptor Desensitization and Sequestration. R.C. Garrad. M. Otero, L. Erb, J.T. Turner, F.A. Gonzalez, L.L. Clarke and G.A. Weisman. Depts. of Biochem., Pharmacol., Vet. Sci. and Dalton Res. Center, Univ. of Missouri, Columbia MO USA, and Dept. of Chemistry, Univ. of Puerto Rico.

An alternative pathway of chloride secretion in CF epithelia may be mediated by activation of apically present P2Y2

nucleotide receptors. In response to UTP, these receptors undergo desensitization of Inositol phosphate accumulation and calcium mobilization and sequestration from the cell membrane in a time- and concentration-dependent manner, in both native epithelium and in 1321Nl human astrocytoma cells expressing recombinant P2Y2 receptors. The protein kinase C (PKC) activator phorbol myristate acetate (PMA), desensitizes UTP-induced inositol phosphate formation and calcium mobilization, but has no affect on receptor sequestration. Recombinant P2Y2 receptors with carboxy terminal deletions and point mutations of three consensus PKC phosphorylation sites have been expressed in 1321Nl cells to understand the effects of these alterations on desensitization, sequestration and down-regulation. Results indicate that deletions of the C·terminus can cause a rightward shift of the UTP dose response curve for desensitization of calcium mobilization. Wild type P2Y2 nucleotide receptors expressed in 1321Nl cells sequester in response to 1 mM UTP (but not PMA) with 50% of the receptors removed from the cell surface within 5-10 minutes· (t112) of incubation. In contrast mutant receptors with deletions of the C-tenninus have a t112 of sequestration of 60-90 minutes. Comparisons of the results of desensitization of calcium mobilization and sequestration between deletion mutants of the P2Y2 nucleotide receptor indicate a critical area of the C-terminus necessary for receptor regulation. This research Is supported by the CF Foundation and Nlli.

241 Effect of a Short Course of rhDNase on Mucoclllary Clearance In Patlenta with Cystic Fibrosis. Hemming AL 1, Robinson M1• Moriarty C1• Bautovich GJ2,

Bye PTP'. Departments of Respiratory Medicine (1) and Medical Imaging (2), Royal Prince Alfred Hospital, Sydney, Australia. Alma: To asses the effect of a seven day course of aerosolised rhONase on mucociliary and cough clearance in patients with cystic fibrosis (CF). Methods: Fifteen rhONase naive patients with CF (9M:6F, (mean±SO (Range)) age: 25±7 (18-41) years, FVC: 84±21 (43-117)% pred, FEV1: 64±25 (33-102)% pred and FEFzs.75: 38±27 (13-86)% pred) were recruited for a randomised, double-blind, placelxH:ontrolled, cross-over study, consisting of two seven day arms (placebo and rhONase) separated by a two week washout. At the beginning and end of eacll arm patients were assessed for lung function (FEV1, FVC and FEF25.7,)

and mucociliary clearance (MCC). MCC was measured using a radioaerosol technique (Regnisela/.A.R.R.C.C.M. 1994;150:66-71) for one hour to determine the effect of rhDNase on MCC alone, followed by a 30 minute measurement of cough clearance (CC) in which patients were required to cough 120 times. Two patients were withdrawn during the washout period due to exacerbations requiring hospitalisation. Resulta: MCC was improved in the whole right lung by 7±12% on the active arm compared with ·2±10% on the placebo, however this did not reach significance (p=0.09). CC was not improved on the active arm. FVC was improved by 5±7% (p=0.02) on the active arm while FEV1 and FEFzs. 75 were not significantly improved. Patients who achieved a 10% improvement in FEV1 ("responders') did not show any greater change in clearance.

274 1997 Cystic Fibrosis Conference

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. . ,..... .. .... Conclualon: In this small number of patients, MCC was not shown to be significantly enhanced by 1 week of rhDNase treatment CC was not improved by rhDNase treatment. "Responders" did not show a greater improvement in MCC. Supported by Roche Products Pty Ltd (Aus), Australian Cystic Fibrosis Reseerch Trust end NHMRC.

242 BENZAMIL IS METABOUZED INTO AMILORIDE ON THE HUMAN RESPIRATORY EPITHELIUM IN VITRO. IHofmann. H.Lindemann•, C.RUckes'", R.C.Boucher, M.R.Knowles, H.J.Schwandt•, W.M.Weber'". •CF-Worldng Oroup at the JLU Giessen, Ocnnany; CF­Ccntcr, UNC at Chapel Hill, North carolina, USA.

Cyslic fibrosis Is usoclatcd with increased Na• absorption across a relatively a· -impenncable respiratory cpltheliwn, resulting in an increased transepithelial potential difference (PD). The raised basal PD can be reduced by topical application of amlloride, a Na• channel blocker. Na• inhibition by amlloride in CF airway epidlelium has been shown to be clinically effective in the short term, but the duration of its effect may not be sufficient for long term clinical efftcacy. Rec:enlly, benzamll has been suggested as being at least 2-3 dmcs more po!ellt to inhibit Na• absorption 111 vivo (Hofinann ct al., Ped. Pulm., Supp1.13: 280, 1996) llld 111 vitro (Blank ct al., Cell Pbysiol Biochem 199S; S:38S-390). In this study, we investigated the metabolism of benzamil, a second generation Na• channel blocker and analogue of amiloridc, -M1en apically applied to cultured nasal epltheliwn. Nasal polyp epithelium was grown to confluency on coiJa&en aupport. and benzamll was applied to the apical solution (initial concenttation IOOIM) in modified Ussing chambers while measurlni b'aiiSepithelial short-circuit current (1..), Samples were taken from the apical and basolatenl aolution after lOmin, and analyzed by HPLC for amlloride and benzamil contents. Relulta: Initial benzamil concenttations were between 21.4 and 42.7 ng/ml. No benzamil or amlloride was detcaed in the basolatenl aolutions IOmin after drug adminlslralion. In 9 aamplcs, then: was no conversion of benzamil to amlloride in the apical solution. In 4 aamples, the benzamll to amlloride conversion (% of initial benzamil ooncentratlon) ranged from S.S to 43.8% (30.1±16.9%; mean±SD). Condllllonl: The po1e11t sodium inhibitor benzamil, a possible second generation inhalative drug for Cyslic Fibrosis, is partially metabolized into amlloride by the respiratory epithelium. Since the safety prolilc for sySielllically applied ami1oride is well known, the metabOOsm of benzamil 10 amlloride locally within the lung would enable and simplify the further clinical evaluation of benzamll.

Support: MukoviszldMe-FIJrderverdn Glesse11 e. V., DFG-Ho 174611-1.

243 DNA AND LEUCOCYTE ELASTASE IN SPUTUM OF CF·PATIENTS AnneUc Bock, E.M.~, I Hpfmam, P.Bittncr-Dcrscb, G.HUis, H.Lindemano, Univ. Kindelldinik. Feulgenstr. 12, D-3538S Giessen, •Med. Klinik I, Abt. ~ Kllnlkum Grosshadcm. Marchloninistr. IS, D-81377 Mllnchen,(}ermay

Baetaround: Rodlat et al. R:Celltly prclellted data sugestlng that DNuc lherapy of IS daya in CF patlenta may lead to a pcrsiltent activslion of proceaaca. in partic:ular, Ieucocyre elutale (LE). 1be8c findings CClU1d be clinically relevant. 1be lim of this lllUdy waa to Investigate whether long-term lhmpy with DNaac causes lncrcaacd LE gencndon. Melhods: DNA and LE wen: measuR:d in the sputum of 36 CF patients (7-3S yean) widxut and 15 paUenta (1~35 yean) widl DNuc therapy of >3 monlbs (criteda for 1lelllllent: c:hronlc inflnmadon, n:peatedly inciWed amount of leucocyees in the sputum). The total DNA-corwent of each IPUtwn sample was CJIIIIIIfted after a RNuc A-dlicstlon widl a pbotometric analysis. Elaataae

activity was measuR:d with Suc-L-alanyl-L-valyl-4 nitroanilide as a specific chromogenic substrate. In 7 patients >3 sputwn aamples were analyzed Results: In patients without DNase inhalation (see criteria for treaunent), lower levels of DNA (avg ± SEM: 6. 7 ± 1.2 mg/g sputwn) and LE (2524 ± 362 U/g) wen: found than in patients with DNase therapy (DNA: 9.7 ± 1.2 mg/g; I.E: 3267 ± 539 U/g). DNA and LE values were correlated (r=0.67; p<O.OI). DNA values of patients (on DNase therapy) were variable, and not correlated with higher LE values. Repeatedly perfonned sputum analyses yielded a close individual correlation between I.E and DNA (r from 0.81 to 0.99), which was dependent on the state of inflammation, but not ON ase therapy. Conclusions: No incR:ase of LE in the sputwn of CF-patients was seen after long term DNase therapy. Thus, DNase does not seem to interfen: with the proteinase/antiproo:inase system to a clinically n:levant extent Uteraturc: Rochat I, Dayer PastoR: F, Schlegel-Haueter SE, Fillhuth I, Auckenlhaler R, Belli D, Suter S (1996) Aerosolized rhDNase in cystic fibrosis: effect oo leucocyte proteases in sputwn. Eur Respir J 9:2200-2206 Suppon: Mukoviszidose-FlJrderverelll Giesse11, e. V., DFG-Ho 174611-1.

244 EFFICACY AND SAJIETY OF REPEATED DOSES OF AEROSOLIZED URJDINE-!1-TRIPHOSPHATE IN PATIENTS Wlm MILD TO MODERATE CYSTIC FIBROSIS. ~. KW Hobnekcr, JM Winders, GZ Retsch-Bogart. RC Boucher, MR Knowles. Cystic Fibrosis Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill.

Cystic fibrosis (CF) is characterized by impaired CFTR mediated chloride conductance across airway epithelia. Together with accompanying sodium hypersbsorption. these airway epithelial ion transport defects in CF contribute to abnonual airway surface liquid biorllcology, defective mucociliBI)' clearance, and airways disease. Uridine-S-triphosphate (UTP) is a novel therapy that stimulates chloride secretion across airway epithelia via extracellular P2Y2 receptors. UfP also stimulates ciliary beat frequency and goblet cell degranulation. TakCil together, these multiple sctions may be beneficial in cystic fibrosis lung disease. Aerosolized UfP has been shown 10 improve mucocilialy clearance in normal subjects and, in combination with amiloride, in patients with CF following acute administration, without advene effects. To select the maximally safe dose, and to assess the effects of rqJcated doses of UfP in patiCilts with CF, we csrried out a double blind, placebo controlled. rising dose IOierancc study of repeated doses of UfP in patients with mild to moderate CF lung disease. The doses of UfP selected for delivery were 10 mg. 20 mg. 60 mg. 100 mg. and 180 mg. equivalent to daily doses of 30, 60, 180, 300 and S40 mg daily respectively. Five cohorts of four patients were selected to receive either drug or placebo three times a day for three days: three in each cohort to receive UfP, one to receive placebo (0. 9% saline). PatiCilts with stable clinical disease, and an FEVI greater than 4S% predicted were enrolled and admitted to the GCRC of UNC-Hospitals for the duration of the study. On all study days, detailed safety parameters were obtained. including vital signs, physical examination, spirometry, and oxyhemoglobin saturation. with clinical laboratory tests and lung volumes on days one and four. 4 mls ofUfP or placebo was delivered by aerosol over IS minutes using a Pari-LC Plus nebulizer. Each subject was educated in the correct usc of the nebulizer: inhalation of aerosol slowly from FRC 10 TI.C, with a slow exhalation to FRC over three to four seconds. Oxyhemoglobin saturation monitoring was performed throughout the aerosolization period of the morning doses, as well as observation for adverse events. R.aultl: To date, IS patients have been studied (three at the 300 mg daily dose). No safety problems or siplificant adverse events have been noted. Once the filial dosing cobort has been completed. aU safety and ellicacy data will be compared for UfP vs placebo. Coaclulioal: The dsta to date 1AJ8iC11 that aerosolized UfP is safe when delivered on a repeated basis (three days) at doses of up to 300 mg daily. Supported by Inspire Pharmaceuticals Inc.

245 MODIFICATION OF NASAL MEMBRANE POTENTIAL DIFFERENCE WITH A COMBINATION OF INHALED SODIUM CHANNEL BLOCKERS AND UTP IN THE CF MOUSE S Ghosal, C.J. Taylor. Department of Paediatrics•, University of Sheffield, Sheffield, UK.

We have previously examined the effect of nebuliscd Na+ channel blockers on nasal potential difference (PD) measurements in insertional mutant mice1• We now report the effect of combination therapy with extracellular nucleotides (UTP) in two populations of CF mice. Changes in 24 insertional mutant mice2 and 7 AF-508

homozygous micel wen: compared with age and weight matched controls. Significant differences wen: found in the basal PD

measurements in the three groups. The expected fall in PD was seen after administration of the sodium channel blocking agent Amiloride. A similar fall in PD was seen with Loperamide again suggesting Na+ blockade. The subsequent addition ofUTP (0.1-IOmmol!l) dissolved in chloride-free vehicle increased the PD in all groups (table). Mouse Basal PO 4PD 4PD UTP MD 4PD UTP + type (mY) amiloride + amiloride lopenunide lopenunide control -21 + 7 -5 ~ -3 CF (ins) -28 +12 -41 +IS -12.5 CF (F508) -34 +17 -IS +16.5 -12.5 We demonstrated dose dependant PD changes in the airway epithelium with UTP in the presence of sodium channel blockers suggestive of C)­secretion. The action of UTP was brief and not prolonged by the addition of 10 nmol/1 a-13-methylene-adenosine s· diphosphate. MS08 mice showed significantly greater responses compared with insertional mutant mice (p<O.OS). More stable forms of UTP in combination with long acting Na• channel blockers such as loperamide may have therapeutic potential in CF.

1• Ghosal Setal. Thorax 1996; Sl: 1229-32.

•. Dorin JR et al. Nature 1992; 359: 211-215. '.College WHet al. Nature Genetics 1995;10: 445-52.

246 METABOLISM OF TRIPHOSPHATE NUCLEOTIDES BY EPITHELIAL CELLS, CF SPUTUM AND BLOOD. AD Winders, RC Boucher and MJ Stutts. Department of Medicine and Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hil~ NC 27599-7248 USA Receptors for triphosphate nucleotides stimulate the activities of key functional components of mucociliary clearance, including ciliary beat frequency and cr secretion, in vitro, and aerosolized UTP has been shown to acutely increase clearance of particles deposited in the human lung, in vivo. Duration of potential therapeutic benefits of delivering UTP or UTP analogs to the airway surface will depend in part on how fast they are metabolized by enzymes on the cell surface and contained in airway surface liquid. Nucleotide analogs with slow metabolism may act longer. Potential therapeutic regimens must also address the possibility of absorption from the airway surface and resulting circulating concentrations. We have developed assays to monitor nucleotide metabolism by luminal cell surfaces, by CF sputum and by whole human blood. Surface metabolism was assayed on highly polarized human airway epithelial cells cultured on Transwell Col supports (4.7 cmz). Minimal transepithelial resistance was 200 ohm•cm. Nucleotide• were distributed onto the surface at 10-300 IJM in a volume of 1 SO J.l) for 0-60 min, recovered, quick frozen and analyzed later by HPLC. CF sputum was solubilized in dithiothretiol and DNAse in ISO mM TrisCI buffer, spun and filtered. Nucleotides were diluted into prepared sputum at I 0-300 liM final concentration, incubated 0-60 min, frozen and analyzed by HPLC. Nucleotides were diluted directly into whole heparinizied blood and incubated 0-60 min. Metabolism was stopped by addition of 10 mM EDTA. Blood was spun free of red cells, serum was collected after Centricon filtration, and HPLC analysis was carried out. These assays demonstrate significant nucleotide metabolism on cell surfaces and in sputum and blood. Triphosphate nucleotides (A TP and UTP) at 30 IJM are quickly metabolized by epithelial surfaces (halftime • 10-IS min), by sputum (halflife ofS-10 min) and in whole blood (half life • S min).

247 Regulation Or Pl Receptor Detensltlzatlon by EctoA TPase Activity In Epithelial Cells. M. Otero', R. Oarrad', L Erb', L L Clarice', F. A. Gonzalez', 0. A. Wetsman'. Dep'l of Chemistry, Univ. of Pueno Rico, and the Dept '1. of Biochemistry', and VeL Biomed. Sci. and the Dalton Res. Center', Univ. of Missouri-Columbia, MO. Activation of calcium-dependent chloride secretion by P2Y 2 nucleotide receptors in epithelial cells may prove to be an effective means of bypassin$ the defect in CFfR that underlies cystic fibrosis (CF). The

Poster Session Abstracts 275

utility of inhaled P2Y2 receptor agonists such as UTP for the treatment of CF may depend upon the development of methods to minimize receptor desensitization and to regulate the activity of ectoA TPases that can degrade UTP. Studies with murine epithelial (MOEP) ceUs Indicate that these cells express ectoATPase activity that degrades UTP to UDP. Degradation of the nucleoside S '-diphosphate generated from ectoA TPase activity in MGEP cells to nucleoside S' monophosphate was not significant, in contrast to ectoATPases (apyrases) described In other tissues. Continuous challenge of MGEP cells with UTP over 2 hr causes rapid desensitization (within minutes) of P2Y, receptor-mediated calcium mobilization and chloride secretion followed by recovery of activity (after 30-60 min) and a second phase of desensitization (after 60 min). In contrast, MGEP ceUs did not recover from P2Y, receptor desensitization induced by continuous Incubation with the non­hydrolyzable ATP analog, ATPyS. Preliminary results Indicate that inhibitors of ectoA TPase activity, such as pyridoxal phosphate-6-azophenyl-2' ,4 '-disulphonic acid (PPADS), can enhance desensitization of calcium-dependent chloride secretion Induced by UTP In MGEP ceUs. These studies suggest that ectoA TPase activity in epithelial cells may serve to regulate the concentration of P2 receptor agonlsts at the cell surface. . The results Indicate that ecto-A TPase activity may enable epithelial cells to recover from desensitization due to long-term exposure to UTP, although generation of a metabolite or recycling of receptors may cause a secondary phase of desensitization. It appears likely that modulation of ectoATPase activity may serve as an additional route for improving the efficacy of UTP pharmacotherapy In CF epithelium. This research is supported by the CF Foundation and by NIH grants OM36887 and DK48816.

248 THE EFFECT OF MILRINONE ON MURINE AND HUMAN AIRWAYS. ~ S.Rolleston., A Jalfe, 28. Wainwrigh~ D.M. Geddes, E.W.F.W. Alton. Jon Transport Unit, National Hean .t Lung Institute, London, U.K., 2 Institute of Molccular .t Cellular Biology, Brisbane, Australia.

We previously reported that milrinone (M.IOO 11M), a type m phosphodiesterase inhibitor, failed to genente responses 111 vivo when administered in a low chloride solution (6mM) in the p!CSCnc:c of amiloride (A,IOO 11M) to the nasal epithelium ofG5SJD mutant mice. In order to determine whether the murine nose predicts the human nose, we assessed the effect of M+ isoprenaline (I 00 11M) on the nasal potential difference of volunteers possessing at least one allele for the G5SID muiJition (n-4). In the presence ofamiloride and a low chloride bathing solution no response was seen in any subject. The consistency of the G5SID murine and CF subject nasal responses suggest that in this case the murine nose predicts the human nose. We have extended this further by perfusing M in the p~CSCnce of the drugs described for the CF subject nasal experiments onto both proximal and segmental airways of CF subjects in vivo. Five patients were studied under general anaesthesia, in the absence of lignocaine. No responses to M were seen. One potential reason for the lack of effect is a low level of mucosal access. We, therefore, compared the response to mucosal and serosal administration of M in the murine trachea mounted in mini-Ussing chambers. In wild-type mice (Balb\C) serosal application was more effective than mucosal administration (serosal: 4.0 (±1.4); mucosal 0.9 (±0.7) ,.A/cm2, both n-6, p<0.05). We also assessed the effect ofM plus forslcolin compared toM or F alone added to the serosal surface of the GSS ID mouse trachea. M alone bad no effect but M plus forslcolin enhanced the response in contrast to the response to F alone (F+M 33.0 (±7.2) n-4, F alone 14.0 (±3.0), n•7, p<O.OS). Thus, the effect of M in CF tiSSUCI may be more pronounced when applied to the serosal airway surface.

249 INHIBITION OF HUMAN NEUTROPHIL ELASTASE BY 2-SPIROCYCLOPROPYL CEPHALOSPORIN SULFONE DERIVATIVES. D.E. Woods, T. Stru$nell, C. Koo~ A Cantin. Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta and Unite de Recherche Pulmonaire, Universite de Sherbrooke, Sherbrooke, P.O. CANADA

Active human neutrophil elastase (HNE) is present in high levels in the respiratory secretions of cystic fibrosis patients chronically infected with Pseudomonas aernginosa. The purpose of the present studies was to examine the ability of 2-spirocyclopropyl cephalosporin sulfone (2-SCS) derivatives to inhibit HNE activity in vitro and in vivo. The most effective 2-SCS derivatives were found to inhibit 50% of HNE activity in vitro at an average molar ratio of 2:1 (2-SCS:HNE). Lung hemorrhage induced in murine

276 1997 Cystic Fibrosis Conference

(C57Bl/6) lungs by HNE was reduced by over 50% with 2-SCS:HNE molar ratios of 10:1. In the rat agar bead model of P. ae1UJ.i.nosa chronic lung infection, aerosolized 2-SCS derivatives sigruficantly decreased elastase activity (p < 0.05) and lung neutrophil counts (p<O.OS). The most potent molecules in vitro were less potent in vivo, likely due to theu poor bioavailability. 2-SCS compounds Syn 1390 and Syn 1396 were the most active agents in vivo. The 2-SCS derivatives demonstrate specificity for HNE and have no immediate adverse effects on animals exposed to high concentrations. The potential advantages of these novel elastase inhibitors over other synthetic inhibitors are: small size, water solubility, structure ( cephaJosporin) with an extensive profile of use in humans. These novel irihibitors may have therapeutic potential as effective HNE inhibitors delivered to the lung epithelial surface of cystic fibrosis patients by aerosol. Supported by the Canadian Cystic Fibrosis Foundation and Synpbar, Inc.

Epithelial Cell Biology

250*

CYSTIC FIBROSIS OF THE PANCREAS: INVOLVEMENT OF MUC6 MUCIN IN OBSTRUCfiON OF PANCREATIC DUCTS. C.J.Reid, K.Hyde, AJimia. Paediatric Molecular Genetics, Institute of Molecular Medicine, Oxford University, John Radcliffe Hospital, Oxford. UK.

Cystic fibrosis (CF) is characterized by pancreatic insufficiency due to destruction of the pancreas. The precise cause of the destruction has generally been thought to be obstruction of the pancreatic ducts by inspissated secretions. These secretions may include mucous glycoprotcins and other proteins; however, the composition of the secretions has not been defined. One of the earliest pathological manifestations of CF in utero is the deposition of periodic-acid Schiff positive material in the fetal pancreatic ducts at about 12 weeks of gestation. We have shown previously that the cystic fibrosis transmembrane conductance regulator (CFTR) gene is expressed by 12 weeks of gestation in the fetal pancreatic duct epithelium. We have now identified MUC 6, which was originally isolated from stomach, as a major pancreatic mucin. mRNA In situ hybridization has shown that MUC6 is expressed by 13 weeks of aestation and shows a pattern of distribution that is very similar to that of CFTR from this age through to adult life. The localization of expression of MUC6 mRNA has been confirmed with a polyclonal chicken antibody raised against a synthetic peptide correspondin& to a tandem repeat reaion ofMUC6. MUC6 protein is seen in the acini, mainly in centroacinar cells, and in the epithelium of intralobular and interlobular pancreatic ducts. Further, - have shown in fetal CF pancreas that the MUC 6 mucin is a significant constituent of the material that causes the characteristic obstruction of small ducts. These data are of importance in attempts to develop novel treatments for CF pancreatic disease.

Funded by the Cystic Fibrosis Research Trust, UK and NIH DK46589.

251* MUCLIN EXPRESSION IN THE CFTR KNOCKOUT MOUSE GASTROINTESTINAL SYSTEM. R. C. De Lisle, K.S. Isom, 1. Huff; lnd D. Ziemer. Department of Anatomy lnd Cell Biology, Unlvenity ofKinlu Medical Center, Klnlu City, KS 66160, USA.

A poorly undentood aspect ofCF is the increase in mucin and Jlyc:oprotein leCI'etion in afl'octed OIJIIII. In addition, how lou of CFTll f\mction lead• to the c:hanpl in carbohydrate composition of IUCb protein~ is unlmown. An impediment to raolving thele quaUODI hu been the lade of a model aiYcoconjupte which can be ltUdied in an

snimal model ofCF. We are now using MUCLIN, a sulfated llllU:in­like glycoprotein which is one of two protein products of the CRP­ductin mRNA. MUCLIN is expressed in several gastrointestinal organs including the exocrine pancreas, crypts of the intestinal tract, and gallbladder. In the pancreas, MUCLIN is a major component of the zymogen granule. In addition, MUCLIN is expressed on the luminal plasma membrane in the pancreatic acinus, intestinal crypt, and gallbladder epithelium. In the CFTR knockout mouse (cftt"'1CRI<), MUCLIN exhibits increased expression (2-4 fold) in the pancreas and intestinal tract and, morphologically, is closely associated with protein/mucus plugs in these organs. Thus, MUCLIN expression is increased in orgsns prone to obstruction in CF. The molecular weight ofMUCLIN on SDS-PAGE is increased in CFTR knockout mice in the small intestine and gallbladder, both organs whose cells normally express high levels ofCFTR. This is consistent with a role ofCFTR in normal Golgi function, nsmely regulation of pH in the Golgi lumen. We also investigated the developmental time course of the increase in MUCLIN expression and appearance ofCF-like protein plugging of pancreatic acini and small intestinal crypts. Increased MUCLIN expression lagged behind the morphological changes, suggesting that MUCLIN is upregulated in response to development of these CF-Iike pathologies. MUCLIN should serve as a good model for determining how such genes are upregulated in CF, and how loss ofCFTR can alter post-translational processing. Supported by a CF Foundation grant.

252* CHARACTERIZATION OF EPITHELIAL CELL LINES DERIVED FROM CF AND NON-CF MOUSE PANCREAS. C U Cotton and M. Takacs-Jarrett. Dapartments of Pediatrics and Physiology and Biophysics, Case Western Rasarve Unlvarsity, Claveland, OH, USA

Dlaaase-caualng mutations In CFTR are known to raduca or eliminate apical plasma membrana chlorlda conductance In affected tissues. Additional phanotyplc abnormalitlaa have bean observed in CF aplthelial calla Including: regulation of other ion channals, changes In macromolecule composition, regulation of exocytosla and endocytosis, pH regulation, end bacterial adherence. Well-matched, CF and non·CF epithelial cell linea that axpreaa endogenous levels of CFTR would be useful for studies aimed at ldantlfylng tha llnk(a) between mutant CFTR and these cellular abnormalities. We ganeratad pancreatic epithallal call lines from CF and non·CF mice that carry a temperature sensitive Larga T antlgan tranagana (lmmortoMouse bred with UNC CFTR H-) mouse). The advantage of using a temperature-aenaltlve Immortalizing tranagene Ia that the laval of differentiation of the calla can be modulated. Tha non-CF (NMPEC) and CF (CFPEC) call linea hava bean maintained In culture for 23 and 10 paasagas, respectively. Both call linea proliferate wall at the perml881ve temperatura (33'C) and exhibit raatrlcted growth at tha non-parmlaalva temperatura (39'C). Cella grown on collagan­coatad parmaabla aupporta form confluent, polarized epithelial monolayara with wall devalopad junctional complaxea. Confluent monolayera of NMPEC and CFPEC cella generate e amen lumen-negative tranaapithallal voltega diffaranca. The short·clrcult currants and trenaeplthellal raalatance• were: NMPEC, 1,. • 4.3 :t 0. 7 pA/cm2 and 186:t11 O-cm2; CFPEC, 1.,•2.8:t1.1 pA/crri' end 100:t22 O·crri'. The cell lines expresa amlloride-senaltlva Ne • absorption: NMPEC, t.l,. • • 1.8:t0.3 pA/cm2; CFPEC, t.i,.•·1.1 :t0.2 pA/cm2

• Elevation of Intracellular cAMP 11 OpM forskolln) Increased 1,. In NMPEC (t.l,.•17.4:t3.0pA/cm2

) but not In CFPEC (t.I,.•0.2:t0.4pA/cm2). In

contraat, elevation of lntracalluler calcium by expoaure to ATP ( 100 pM) elicited lerga, tranalent increase• In 1,. In both NMPEC end CFPEC monoleyara (AI,.> 180 pA/cm2

). Thua we hava auccaaafully generated wall-matched CF end non.CF pancreetlc epithelial cell linea that exhibit the expected chloride tranapon phenotype. Thaaa cella will be useful for atudle1 dealgned to Identify cellular procaasea effectad by the 1011 of CFTR. (Supported by NIH·DK 43000 and the Cyatlc Fibrolia Foundation)

253* SECRETIN ACTIVATES TWO TYPES OF CHLORIDE CURRENTS IN GUINEA PIG PANCREATIC DUCT CELLS. M A Gm. J. P. WinJICIIIIY 4 B.E. Arpnt. Deplrtment Gl Pbyliologicsl Scienc:a. University ol NewcutJe upoa Tync. UK.

Pancreatic duct cells (PDCs) secrete the Hco,· ions found in pancreatic juice and are the site of the pancreatic defect in CF. In most species. including humans. pancreatic juice [HCO, ·1 can reach I SO mM with a pH of - 8. The current model for HCO,"-secretion by PDCs bas opical CFTit Cl" channels operating in parallel with Cl"IHCO," exchangers. although it is possible thai some HCO, · is also secreted directly via the CFTR channel. Using whole-cell parch clamp recordings with a pipelte solulion conraining (mM): 110 CsCI. 5 EGTA and I ATP (pH, • 7.2), we have previously shown (I) that exposing guinea pig PDCs 10 a cAMP elevating cocklail of 5 11M forskolin and 100 11M dibulylyl cAMP activates CFTR curreniS in -70 'Yo or cells (26137). However. in a small number or cells (4/37). a cr condUCianCe with quite distincl biophysical properlies w-as also acrivated. This current showed time- and voltage-dependent gsling similar 10 the Ca'' .. clivated Cl" currents (CACCs) that we have previously described in mouse PDCs (2). We no.. show that exposmg guinea pig cells 10 the peptide bonnone secretin (I 0 nM). which is the most important physiological regulaiOr of Hco,· secretion. also leads 10 the activation of both 1ype5 of current. but at significantly different frequencies compared 10 the cAMP cockrail. CFTR currents were found 10 be acrivatcd in only 8124 cells (p- 0.008). whereas the CACC-Iike current was observed at a higher frequency (11124 cells; p-O.OOS). Overall. the CACC-like currenrs were - 7-9 fold larger than the CFTR curreniS under both condilions (current densities at E,.. ~ mV for cAMP cocklail and secretin were: CFTR.. 68 ± 9.5 pA/pF. (n~26) and 62 ± 26 pA/pF. (n-8); CACC-like, 481 ± 103 pA/pF, (nz-4) and S68 ± 100 pA/pF, (n•ll: mean± sem). Both currcnrs were also found 10 be rapidly inhibited by extracellular HCo,· (SO­l SO mM), and from reversal potential measurements were at least - 2·3 fold less penneable 10 Hco,· than to cr. We conclude thai guinea pig PDCa possess two distinct 1ype5 of cr currents which can be activated by increasing cAMP. Since the properlies of the voltage-dependent current are similar 10 CACCs. we speculate that raising cAMP levels in these cells may increase inllacellular (Ca''J. Alternatively. cAMP may alter the Ca2

' -sensitivity of the CACC channels. I. Grdy. MA 11 a/. (1996) Pediatric Pulmonology Suppl. 13. 93. 2. Gray. MArt a/. (1994) Am. J. Physiol. 266. C21J.C221.

Funded by the Cystic Fibrosis Trust

254* THE PANCREATIC DUCTAL TREE- A NEW HYPOTHESIS FOR mE SECRETION OF A BICARBONATE-RICH FLUID. y Sohma. M.A Gray•, Y. Imai & B.E. Argent•. Dept. of Physiology, Osaka Medical College, Japan and *Dept. of Physiological Sciences, Univ. of Newcastle upon Tyne, UK.

Pancreatic duct cells (PDCs) secrete the HC03' ions found in pancreatic juice and are the site of the pancreatic defect in CF. In most species, including humans, pancreatic juice [HCOJ1 can reach ISO roM and the juice pH is- 8. The current model for HC<X­secretion by PDCs hu CFTR cr channels operating in parallel with CI"IHC03" exchangers. However, this scheme suffers from 1 problem in that with near isotonic HC03" in the duct lumen the concentration ofCr in the luminal fluid will be very low(- 10 mM). This means that given reasonable values for intracellular [HCOJ1. the intracellular ~r concentration would have to be very low indeed (< I mM) to dnve HC03" secretion on an apical crt HCOi exchanger. Our approach to resolving this paradox has been to develop a computer model o~ the duct cell using network thermodynamics (1). The model proVIdes information about secretory rate and the HCOJ. concentration in the secreted fluid, u well as transient and steady state data on pH;, [CI];, membrane and transepithelial potentials, and the voltage divider ratio. From our modelling studies we have developed a new hypothesis regarding the secretion of 1 HC03' -rich pancrealic juice. The hypothesis predicts: (i) that the pancreatic ductal system can secrete an isotonic HC03" -rich fluid based on parallel operation of apical CFTR channels and Cll HCo,· exchangers, (ii) that HCOJ. secretion occurs mainly by the exchanger in duct segments near the acini, but mainly via the channels further down the ductal tree, and (iii) that changes in the composition of the luminal fluid u it flows along the ductal system modulate the transport characteristics of the duct cells in such a way that secretion of a HC01' -rich fluid is maintained. 1. Sohma, Y. eta/. (1996). J. Membrane Bioi. 154,53-67.

Funded by the MRC and the Cystic Fibrosis Trust

Poster Session Abstracts 277

255*

UTP Stimulates Transepithellal Bicarbonate Secretion Acroaa Intact Epithelium From CFTR Knockout Mice. M.C. Har1ine', N. M. Walker', G. A. Welsman2 and L.L. Clar1<e1

• Dalton Cardiovascular Research Center, Oepta. of Vet. Biomed. Scl.' and Blochemlst,Y, Univ. of Missouri-Columbia, MO, USA.

The loss of CFTR-mediated transepithelial bicarbonate secretion contributes to the pathogenesis of epithelial disease In cystic fibrosis patients. Although recent studies have Investigated the utility of P2Yz receptor agonists (e.g., UTP) to bypass the defect In CFTR by activating calcium-dependent cr aecretion, little Ia known regarding the value of this treatment to facilitate transepithelial HCOi secretion. We have previously reported that a model epithelium (murine gallbladder mucosa) expresses the P2Yz nucleotide receptor and responds to luminal addition of UTP with Increased short-circuit current (110, an Index of anion aecretion). Therefore, in the present study, - employed the pH stat technique to estimate the effect of UTP in stimulating He~· secretion via the Ca2

• -dependant anion conductance in intact gallbladder from eFTR knockout mice. With Kreb's bicarbonate Ringer in the serosal bath and unbuffered Ringer In the luminal bath, the serosal-to-mucosal flux of He~· (J.,. ~ across CFTR(·) gallbladder was 3.05 ± 0.96 ~eq/cm"·h and the 110 was 2.08 :1: 0.14 ~eqlcm"·h (nz3). Luminal addition of UTP (100 ~M) resulted in an immediate Increase In 110 that decreased to a stable plateau within 5 min. The mean increases In J,.. ..co3 and 1.. measured for 30 min during the plateau period -re 0.93 ± 0.21 and 0.54 ± 0.38 ~eq/cm"·h, respectively. In separate studies, CFTR(-) gallbladders were bathed with cr free solution in the luminal bath to eliminate the contribution of luminal membrane ertHe~- exchange (n=3). However, the mean increase in J ... HeX» during the UTP-stimulated plateau period was unaffected (AJ .. HCOJ• 0.82 ± 0.23 ~eqlcm"·h) whereas the 1.. response was Increased (AI .. • 1.36 :1: 0.25 ~eqlcm"·h). Preliminary studies also Indicated the UTP-Induced increases In J ... HeX» and 1.. in the luminal cr free gallbladders were completely inhibited by luminal addition of OIOS (1 mM). We condude that P2Yz receptor activation stimulates a sustained increase in electrogenic He~- secretion via a Ca2•-dependent anion conductance. This action may be useful to nonnalize transluminal pH across CF epithelia. Supported by NIDDK grant 48816 and CFF.

256* Regulatloa of Chlorlde/Bic:arboaate Enhanaer Activity By Wild· Type aad Mutaat CFTR. S.A. Grubman, R.D. Perrone, D.W. Lee, M.D. Brett, C. Jiang, S.H. Cheng and D M Jefferson Dept. of. Physiology, Tufts University School of Medicine, and Depts. of Pediatrics and Medicine, New England Medical Center, Boston MA and Genzyme Corp., Framingham, MA.

While the lack of functional CFTR in the membrane of intrahepatic biliary epithelial (IBE) cells has been demonstrated, the cellular mechanisms responsible for CF-associated hepatobiliary disease remain to be elucidated. The CI"IHC03" anion e"changer (AE) is responsible for HCOi secretion, a major function of biliary epithelial cells. We have demonstrated that AE activity, as assessed by the rate of increase in pH1 following removal of Cl', in CF-patient derived intrahepatic bile duct epithelial (CF-IBE) cell lines is reduced significantly compared to normal. Following complementation with wild type CFTR, CI"IHC03" e"changer activity in CF-IBE cells returned to normal, suggesting that wild-type CFTR can regulate AE activity. Objectlyc: To determine if mutated CFTR that is trafficked to the plasma membrane can restore AE activity in CF-IBE cells. Mnblllb:. AE activity was determined by measuring the initial rate of alkalinization (using BCECF) following Na-gluconate substitution for cr. Treatment of CF-IBE cells with I OmM butyrate or cooling (23"C overnight) was used to induce the apical membrane expression of AFS08 CFTR, u determined by SPQ halide efflux assay. Adenovirus expression vector containing construct for GSSID (Class III mutation) was used 10 infect CF-IBE cells. GSSI D trafficks to the plasma membrane but does not secrete cr. RnuJta.; The expression of AF508-CFTR in the plasma membrane (generated by either treatment scheme) did not restore AE activity in CF-IBE cells. Additionally, expression ofGSSID­CFTR also did not increase the rate of AE activity in CF-IBE cells. ConcJus(ops: These data are consistent with either of two possible conclusions: I) The level of membrane e"pression or function of AFSOS­CFTR was below the threshold necessary for restoration of AE activity; or 2) the mechanism(s) of interactions between CFTR and AE are disrupted due to the mutation(s) in NBFJ. The current data do not distinguish between these

...

278 1997 Cystic Fibrosis Conference

two possibilities. However, these studies suggest that the therapeutic usc of phannacologic agents that facilitate AFSOB trafficking to the membrane may not be effective for the treatment of CF-associated hepatobiliary disease. Supported in part by P30DK34928 & CFF.

257* Morphometric and Clonal Derivation of Submucosal Glands Ia

CblrnHric and CF mutaat mice

pupcM W Bonhwjck1, Sheila Webb'. Jean H. Aockhan2, Margaret A.

Keighren', John D. Wesl', Julia R. Dorin'· 'MRC Human Genetics Unit, Edinburgh, UK. EH4 2XU and 'Dept of Obstetrics and Gynaccology, Centre for Reproductive Biology, Edinburgh, UK. EH3 9EW

Submucosal glands are a major site of CFTR expression in the human airways and are potentially an important target for Gene Therapy. The inherent difficulties of repeated gene delivery to these glands has prompted us to explore the targeting of submucosal gland progenitor cells using the mouse as a model. Murine submucosal glands are found in the proximal regions of the trachea. We show here that although fewer in number than in humans, murine slands exhibit similar cell morphology, structure and neonatal developmental pattern. In addition we have compared PAS stained trachea whole mounts. The submucosal glands from homozygous Cftr .uv.' mice were seen to eKtend more distally than in wild type mice (p<O.OOS). This submucosal gland hypertrophy is in common with human CF patients.ln order to investigate the development of submucosal glands in vivo we generated asgregation chimaeric mice. Chimaeric offspring contained a contribution of transgenic cells that were detectable either by DNA insitu hybridisation [reiterated beta-globin transgene TgN(Hbb­bi)83Ciol or beta-galactosidase histochemistry [Lac-Z reporter gene TgR(ROSA26)26Sor) . Analysis of the distribution of transgenic cells in chimaeric tissues provided evidence for clonal expansion of submucosal glands from a single progenitor cell. In an attempt to identify a proliferating cell population in the adult mouse we investigated trachea epithelial cell kinetics usins BrdU and an antibody against PCNA. Our work SUJSCSis that only basal cells have the capacity to proliferate and therefore maintain the trachea epithelium. In conlrUI with human CF individuals, no statistical difference was seen in the cell proliferation index between CF and non-CF mice. Thus, we have demonstrated that the mouse submucosal gland is a valuable model of human submucosal gland development and displays genotype specific hypenrophy. Evidence was found to suggest that such tissues arc clonally derived from a progenitor cell population. Work is underway to further characterise this population.

258* LEF1 TRANSCRIPTION FACTOR EXPRESSION DEFINES AIRWAY PROQI!NITOR CELL TARGETS FOR IN UTERO GENE THERAPY OF SUBMUCOSAL OLAND IN CYSTIC FIBROSIS. Qongaheng puan Anll Sehgal, Jle Yao, and John F. Engelhardt. University of Pennaytvanla, PhHadelphla , PA, USA

Cyallc ftbroals has emerged aa a paradigm for aaaeaalng the utility of gene therapy In the treatment of genetic dlaeaaM. However, the CF lung poses several elgnHicanl oblltaclea for gene tharapy with regards to the complexlllea of cellular targeta. The eubmucolal glenda In the airway are one example of cellular targets which have been excluded from current gene thenapy trlal1 due to their lnaccenlbllity from the lumen of adult proximal airway. In utero gene therapy approaches to correct submucoaal gland dysfunction In CF provide an attractive alternative strategy. To rationally approach !'- In utero gene therapy eflorta In the lung, a baalc understanding of the stem ceiVprogenltor cell targets nec-ry lor transgene perslatance and prolonged expreulon Ia Imperative. We describe a newborn ferret model of the proximal airway lor identifying submucosal gland progenitor cell targets and tasting gene therapy strategies lor targeting these cellular phenotypes. To this end, we have Isolated the ferret eDNA to a HMG lraDicriptlon factor called Lef1 and have demonstrated that Its mANA expreulon epecffically defines a sublet of surface airway eplthaUal progenitor cella Involved In the formation of primordial eubmucoaal gland buda and eubeequent gland morphogenule. Such findings euggeat that the tranacriptlonal switch regulattng ICIIvatlon of Lef1 exp11111lon defines the phenotype of early submucolal gland progenitor cella. Furthermore, we have evaluated our ability to target theu aubmuco•al gland progenitor cell• using racomblnlnl relro'llral vectors In a xenograft model eyetem. Flndlnge from these atudlea demonetretad eucceaelul g- targeting to progenitor cella of gland buds which Ia stable throughout eubuquenl gland development. In eurnmary, localization of Lef1 expreulon to apeclflc

progenitor cell phenotypes In the airway involved in submucosal gland development has provided a useful molecular marker for dissecting the complexities of gene regulation which define subpopulatlons of progenitor cells In the airway. Furthermore, gene targeting to this progenitor cell phenotype using integrating viral vectors may provide attractive alternative strategies for In utero gene therapy of submucosal glands in cystic fibrosis.

259 LYMPHOID ENHANCER FACTOR-1 (LEF·1) IS FUNCTIONALLY INVOLVED DURING SUBMUCOSAL GLAND MORPHOGENESIS Dongsheng puan, John F. Engelhardt. University of Pennsylvania, Philadelphia, PA, USA

Submucosal glands may play an important role in the pathogenesis of cystic fibrosis They have also been implicated in other hypersecretory airway diseases such as asthma and chronic bronchitis. Hence, a better understanding of the submucosal gland development will help to elucidate the mechanisms of gland hyperplasia and hypertrophy in these diseases . In present study, we have identified a HMG family transcription factor lymphoid enhancer factor-1 (Lef-1) as a critical regulator of submucosal gland morphogenesis. When anti-sense oligonucleotides against the ferret Lef·1 translational starting site were applied In newborn ferret tracheal xenograft, a 4-fold decrease in both the number and the size of the submucosal gland was observed. Sense oligonucleotides and no oligo xenograft controls showed no difference In submucosal gland development. In vitro experiments confirmed the down·regulation of Lef-1 protein by anti-sense ollgos but not by sense ollgos. These findings suggest that Lel-1 is essential for submucosal gland development in the airway. In order to determine whether Lef-1 alone is sufficient to initiate and support submucosal gland development, we have evaluated the effect of ectopic over­expression of lef-1 in human tracheal xenograft model using a bi-cistronlc recombinant adeno-associated virus expressing both Lef-1 and Green Fluorescent Protein . Although a significant portion of airway epithelium was efficiently Infected by rAAV-Lef-1, no significant change in submucosal gland morphogenesis was observed. In summary, our results suggest that the Lef-1 transcription factor Is necessary but not sufficient to promote submucosal glands development.

260* LIMITATIONS OF RETROVIRAL MEDIATED GENE DELIVERY TO RESPIRATORY EPITHELIAL CELLS. Zs.K. Zsengelll!r, C.L. Halbert*, J.A. Whitsett, and C,J BacburskL Children's Hospital Medical Center, Cincinnati, OH and *Fred Hutchinson Cancer Research Center, Seattle, W A

Recombinant amphotrophic retroviral vectors are efficient vehicles for gene transfer to respiratory epithelial cells in vitro. We previously reported that retrovual vector mediated gene transfer to the lung was inefficient despite stimulation of epithelial cell proliferation by pre­treatment with recombinant keratinocyte growth factor (KGF). In order to define the barriers to retroviral infection of the pulmonary epithelium the effect of pulmonary surfactant on viral titer, and the expression pattern of the amphotropbic retroviral receptor (RAM-I) in the lung were determined. In situ hybridization (ISH) and Northern blot analysis were perfonned to detect expression of RAM· I in the lungs of FVBIN mice. Low levels of RAM-I mRNA were observed in total lung RNA by Northern blot analysis. RAM-I mRNA was undetectable in the pulmonary epithelium by ISH, whereas it was readily detected in the pulmonary vasculature. Since surfactant proteins-B and -C inhibtt transfection br liposomes in vitro and surfactant protein-A can act as a viral opsonm, we hypothesized that the endogenous surfactant lining the alveolus may also present a barrier to retroviral infection. The effect of pulmonary surfactant on transfection efficiency of PA317/LAPSN (a recombinant amphotrophic retrovirus encoding placental alkaliDe phosphatase) was studied in MLE-12 cells, a murine lung epithelial cell line tbat expresses RAM-I. Gene transfer, as measured by alkaline phosphatase staining, was decreased up to S fold when 25-200 J.Lg/ml SurvantaTM or rabbit pulmonary surfactant was mixed with PA317/LAPSN retrovirus immediately prior to infection. These results suggest that both lack of significant receptor

concentration, and the inhibitory presence of surfactant in the airway lining fluid may present barriers to amphotrophic retroviral infection of the pulmonary epithelium in vivo. The efficiency of retroviral mediated gene transfer to the lung, therefore is not solely dependent on cell proliferation. Supported by HLS 1832 and DK47754 Centers for Gene Therapy, and the Cystic Fibrosis Foundation; recombinant KGF was kindly provided byAMGEN.

261* GROWTH FACTORS STIMULATE PROLIFERATION OF DIFFERENTIATED HUMAN AIRWAY EPITHELIA. .G.., wang, E. Appleton, T. Nakamura, K. Matsumoto, B.L. Davidson, P.B. McCray, Jr. Osaka University Medical School, Osaka, Japan and University of Iowa College of Medicine, Iowa City, lA, USA.

Cell proliferation is required for gene transfer with integrating vectors such as MMLV-based retroviruses and AAV. This limits their utility for gene transfer to the mitotically quiescent adult lung. We hypothesized that growth factors might stimulate mature epithelia to divide. Recent studies show that specific growth factors stimulate epithelial cell proliferation in a variety of tissues in vivo during normal development and in response to injury. We tested the mitogenic effects of keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) in vitro on fully differentiated, mitotically quiescent primary monolayer cultures of human airway epithelia. At concentrations of 50 ng/ml, KGF stimulated cell division 3-fold as measured by [3 H) thymidine incorporation. HGF had a similar effect at 200 ng/ml. After 48 hrs treatment, 50 ng/ml KGF treatment doubled the monolayer cell number compared to control. We used BrdU histochemistry to determine the percentage of cells dividing in response to KGF. After 36 hr of KGF treatment (50 ng/ml), 8 to 20 % of cells were labeled compared to 0.3 to 4% of controls (n • 3). These studies document that mature nondividing airway epithelia proliferate in response to specific growth factors in a model that closely mimics the human airways. These results have implications for lung injury and repair responses and gene transfer with integrating vectors. (Supported by the Cystic Fibrosis Foundation, Z987)

262*

GROWTH FACTOR INDUCED PROLIFERATION FACILITATES RETROVIRAL·MEDIATED GENE TRANSFER TO PULMONARY EPITHELIA. G. Wang, J.N. Kline, J. Zabner, M. Bodner, D.J. Jolly, P.A. Thomas, B.L. Davidson, P.B. McCray, Jr. University of Iowa College of Medicine, Iowa City, lA and Chiron Technologies-Center for Gene Therapy, San Diego, CA, USA.

Cell proliferation is required for gene transfer with MMLV· based retroviruses, thus limiting the utility for gene transfer to the mitotically quiescent adult lung. We asked whether growth factor-induced epithelial proliferation would facilitate gene transfer. Neonatal rats received intratracheal keratinocyte growth factor (KGF, 5 J.lg/g x 2 doses) followed by high titer amphotropic retrovirus (DA-Bgal, total dose N1Q7 cfu). KGF stimulated transient epithelial proliferation In the distal lung (30-40% PCNA positive cells at peak). Epithelial gene transfer (X-Gal positive cells) Increased in KGF treated rats, but proliferation exceeded the level of gene transfer. Because KGF/Virus treated rats had Increased alveolar macrophages (AM) compared to controls we performed experiments to determine if AM inhibit retroviral gene transfer. Human AM were treated +I· LPS activation, mixed with DA-Iuciferase retrovirus, and incubated with human bronchial epithelial cells at a ratio of 1 :1 :1. 3 days after transfection, lucilerase activity was detected in cell lysates. Gene transfer decreased by -60% In the presence of unactivated or activated

Poster Session Abstracts 279

macrophages, respectively. Pretreatment of AM with dexamethasone restored gene transfer to -60% of control values. Thus, growth factor induced proliferation facilitates retroviral-mediated gene transfer to pulmonary epithelia. While we have identified that AM may be a barrier to retroviral gene transfer, this may be overcome with pharmacological regimens. (Supported by the Cystic Fibrosis Foundation, Z9B7)

263*

IMPROVED IN VIVO GENE TRANSFER EFFICIENCY TO MURINE AIRWAY EPITHELIA USING PSEUDOTYPED RETROVIRAL VECTORS. L G Johnson, J.P. Mewshaw, R.C. Boucher, and J.C. Olsen. CF/Pulmonary Research and Treatment Center. The University of North Carolina at Chapel Hill, Chapel Hill, NC

To study gene transfer efficiency in vivo, we used a murine inha· lational injury model (500 ppm so2 for 3 hr.) that stimulates airway epithelial cell proliferation. Proliferation rates of luminal tracheobron­chial cells at 24 hr. post injury approached SO%. Moreover, some epithelial shedding occurs making replicating cells more accessible to the lumen at the time of retroviral transduction. In initial experiments, instillation of high titer (-109 IU/ml) murine leukemia-derived retrovi­ral vectors (MuL V) encoding the lacZ eDNA and pseudotyped with the G glycoprotein ofvesicular stomatitis virus (VSV-G) into the tra­cheas of anesthetized mice transduced lacZ in patchy regions of the trachea. While gene transfer within the patches could be efficient, the overall efficiency of gene transfer throughout the trachea was low. We investigated factors that might limit the efficiency of in vivo gene transfer. Freshly isolated mouse serum and lung lavage fluid did not inhibit the infectivity ofVSV-G MuLV. Addition to the vector instil­late or pretreatment of murine tracheas /11 vivo with bovine testicular hyaluronidase (250 U/ml final concentration) to cleave potential inhibi­tory glycosaminoglycans (Batra el a/., J. Bioi. Chem. 272:11736, 1997) also failed to increase transduction efficiency. However, the age of the animal did appear to affect /11 vivo retroviral transduction effi­ciency. In preliminary experiments, the efficiency oflacZ transduction in tracheas of weanling mice (3-4 wk.) was 7"/o (n=5}, compared to 1% in mice S wk. of age (n=4), and <0.5% in mice greater than 6 wk. of age. These data suggest that retroviral transduction can approach the efficiencies predicted to correct the CF Cl' permeability defect. Further studies are warranted to validate these observations. Supported by HL583-12 (LGJ) from the National Heart /.ung and Blood Institute.

264* DEVELOPMENT OF RECOMBINANT EQUINE LENTIVIRA\. VECTORS FOR GENE THERAPY. John C Olsen Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

Equine infectious anemia virus (EIA V) is a member of the Jentiviral group of retroviruses. Though it causes disease in horses, EIA V replication Is highly species-specific, and there are no known cases of human infection. ElA V possesses several features that may be attractive for a CF gene therapy vector, including (i) the ability to replicate to high titer and (ii) the ability to efficiently integrate its DNA into chromosomal DNA of non-dividing and terminally differentiated cells. Our studies of vector development are aimed at (i) optimizing the expression of EIA V proteins that are important for producing high-titer vectors, and (ii) defining the cis-acting sequence elements that should be included into vector genomes. To this end we constructed replication-defective EIA V vectors encoding puromycin-N-acetyl transferase and S-galactosidase. To package the vectors into viral particles we constructed helper plasmids that express EIAV proteins, but are not themselves propagated during gene transfer. Additionally, we created packaging cells by modifying human 293 cells to stably express EIA V proteins. In transfection studies, we found that vectors can be pseudotyped with the G envelope glycoprotein from vesicular stomatitis virus. These vectors act as vehicles to carry genes into cultured human epithelial cells and we found that they are efficiently integrated into host DNA. Our "first generation" EIA V vectors

280 1997 Cystic Fibrosis Conference

demonstrated a transduction rate of greater than 50% in cultures of human CFI'l cells or primary hUIIWl airway epithelial cells. Unlilce retroviral vectol'!l baaed upon murine leukemia virus, we found that EIA V vectors transduce non-dividing (aphidicolin-arrested) cells as efficiently as dividing cells. We suggest these properties make EIA V vectors promising gene transfer vehicles. We are currently investigating EIA V gene transfer using modela of well-differentiated epithelium.

265* DEVELOPMENTAL EXPRESSION OF MUCIN GENES IN THE HUMAN RESPIRATORY TRACT. CJ.lWd and A. Harris. Paediatric Molecular Genetics, Institute of Molecular Medicine, Oxford University, John Radcliffe Hospital, Oxford. UK.

Mucins play a fundamental role in the pathogenesis of many lung diseases, particularly those involving chronic inflammation of the airways or susceptibility to infection such as asthma, chronic bronchitis and cystic fibrosis. Nine human mucin genes have been identified, MUC /, 1, 3, 4, 5AC. 58, 6, 7 and 8, though only MUC /, 1 and 7 have been fully cloned. We examined the developmental expression of human mucin genes to establish which genes arc important in the development and differentiation of the human lung epithelium. We previously reported expression of MUC I and MUC 1 in the fetal lung. We have now examined the expression of MUC 3-MUC 8 in the mid-trimester airway epithelium. MUC /, MUC 1, MUC 4, MUC 5AC, MUC58 and MUC7 mucins genes are expressed in the developing human fetal lung. MUC /, MUC 4 and MUC 58 mRNAs arc seen at 13 weeks gestation which is the earliest age that we have examined. MUC 4 expression is seen in the majority of airway epithelial cells that line the trachea and larger airways though, in contrast to MUC I, is not seen in smaller airways. MUC 5AC transcripts arc not detected before 17 weeks and, like MUC 1 transcripts, are highly restricted in their pattern of expression. MUC 5AC mRNA is seen in individual goblet cells in tracheal surface epithelium and in the necks of submucosal glands. MUCSB and MUC7 arc primarily submucosal aJand mucins. MUC58 transcripts are detected in the surface epithelium of the trachea at 13 weeks but arc mainly seen in submucosal gland epithelium by 23 weeks of gestation, an expression pattern that follows the differentiation path of these glands. The relatively late onset of mucin gene expression in the human airways in comparison to the intestine may be relevant to the prenatal pathology ofCF.

Funded by the Cystic Fibrosis Research Trust. UK.

266* DIFFERENTIAL EXPRESSION OF MUCIB AND MUC7 IN MUCOUS AND SEROUS CELLS OF SUBMUCOSAL GLANDS IN HUMAN BRONCHUS. Sharma, P.M., Dudua, L., Hollingsworth M.A., Nlalaen, P.A., Clauaen, H. and Engelhardt, J.F. Unlvaralty of Pennsylvania Medical Canter, inatltute of Human Gene Therapy, Philadelphia, PA 19104, Eppeley Institute for Reaearch In Cancer and Allied 01188188, Unlverelty of Nabruka Medical Canter, Omaha, Nebraska 88198. Department of Oral Dlagnoatlca, Unlveralty of Copenhagen, DanrnaiK

Muclna are high molecular weight glycoprotein• Involved In the protection and lubrication of reaplratory, gaetrolntaatlnal and reproductive tracte. Mucus overproduction Is obaerved In aeveral dlaeuea auch as Cyatlc flbroala (CF), uthma, chronic bronchltla and cancer. To date, eight mucin ganea (MUC1-MUC8) have been ldentHied. MUC2, MUC3, MUC4, MUC58, MUC5AC and MUC8 are expreaaed In the alrwaya. Alterations In mucin atructura and expraaalon are obaarved In Cyatlc flbroala, however leas Is known about the apeclflc call types In the airway which axpraaa different mucin genaa. The objectlvel of thla atudy ware to ldentHy tha cell typal which aacrata MUC5B and MUC7 In the human bronchu. and to determine whether any alterations In the axpreaalon patterns of MUC5B and MUC7 exist between CF and Non-cF bronchial tlsauaa. To achieve theaa goala we performed In situ hybridization to Identify MUCSB and MUC7 RNA• using spacHic alkaline phosphataaa labeled oligonucleotide probal to the repeat domains of thala mucin genaa. lmmunofluoraecenca, ualng apacHic monoclonal

antibodies were used to identify the MUCSB and MUC7 proteins. Our findings suggest that MUC5B Is mainly expressed in the mucous cells of the submucosal glands whereas, MUC7 is mainly expressed In a subpopulation of serous cells. Interestingly. when MUC7 expression was observed it appeared to be spatially confined to Individual glands, suggesting that heterogeneity In gland phenotypes may exist in the human airway. No difference was observed In MUC5B and MUC7 expression in CF bronchial tissues when compared to non-CF samples. In summary, MUCSB and MUC7 are differentially expressed in mucous and serous cell types in submucosal glands.

267* A METHOD FOR STUDYING PROMOTER/ENHANCER ACTIVITY IN WELL DIFFERENTIATED AIRWAY EPITHELIAL CELLS Sk.Q11 H Randell and John C. Olsen. CF/Pulmonary Research and Treatment Center, The University of North Carolina, Chapel Hill, NC, 27599

Mechanisms governing gene expression within well differentiated airway epithelial cell types are poorly understood but such Information could serve many purposes. For example, It may be useful to systematically manipulate either marker molecules or functional proteins globally, or on a cell-type-specHic basis. Unfortunately, mature polarized airway epithelial cells are notoriously difficult to translect with widely available replication deficient adenovlral vectors, murine leukemia retrovirus (MuLV)-based vectors or llposomes. Gene-gun or electroporation approaches are not currently options lor technical reasons and the In vivo transgenic approach Is cumbersome and only restricted to a few species. Our goals are to 1) establish a method lor studying promoter/enhancer activity In well differentiated human airway epithelial cells, 2) to examine potential cellular heterogeneity In marker protein expression driven by putative strong constitutive type promoters and 3) to determine whether the activity of cell type-specific promoters becomes correctly regulated In culture and In a tracheal xenograft model In vivo. When approximately 50% confluent, primary human nasal or bronchial airway epithelial cells grown on plastic were Infected with various MuLV vectors containing neomycin or puromycin resistance genes as selectable markers. Dose-response survival curves wera performed to optimize selection, which was carried out lor a minimum of 7 days. Selection afflclency was evaluated using lacZ-contalning vectors and was typically >97%. Puromycin or G418 selected primary cells were passaged onto permeable cutture supports In vitro or Into xenograf1s In nulnu mice In vivo and were allowed to grow and differentiate. Interestingly, histologic localization of lacZ expression driven by the Mul V L TR promoter In well differentiated cuttures revealed marked heterogeneity, with high levels In secretory and basal cells and weak activity In ciliated cells. Further studies are needed to determine H lacZ expression Is downregulated at the RNA or protein level In ciliated cells. Xenograft results with the same vector are pending. Vectors utilizing cell-type-specific promoters are under construction. Thus, with a lew caveats, our studies illustrate a method to study promoter/enhancer activity In primary human airway epithelium which will also be useful to experimentally manipulate the phenotype of well differentiated cells. Supported by the CFF, NIH and the ALA of NC.

268* MUCIN GENE EXPRESSION BY PRIMARY HUMAN AIRWAY EPITHELIAL CELLS IN VITRO I Susan H Bernacki, 2Ann Harris and I Scott H. Randell. 1CF/Pulmonary Research and Treatment Center, UNC, Chapel Hill, NC, 27599 USA. 2Paedlatrlc Molecular Genetics, Institute of Molecular Medicine, Oxford University, UK.

Goblet cell and gland hypertrophy and hyperplasia are prominent features of chronic airway diseases auch as cystic fibrosis (CF), but we are just beginning to understand tha mechanisms controlling airway secretory cell phenotype. Mucus hypersecretion likely contribute• to phyalologlc airway obstruction and chronic Infection, however, It has not yet been determined which mucin genes are predominantly expre11ed. Recent Improvement• In human airway epithelial cell culture will enable studies of mucln gene expreaalon. Our goals are to determine 1) the relallve abundance of steady state mucin mRNAsln well differentiated airway epithelial cell cultures, 2) whether there are systematic differences In mucin gene expreaalon In cella derived from naaal versus tracheobronchial epithelium and 3) If the presence of CFTR mutations reaultlln elevated expression of mucin genes. Total RNA waa extracted from normal and CF primary human nasal and tracheobronchial epithelial call culturaa grown either on plastic (undifferentiated) or permeable supports (well dlfferentlatlated) and

from appropriate positive and negative control specimens. RT-PCR, RPA and/or Northern analyses were performed for all described mucin genes depending on available gene sequences and mANA abundance. To date, PCR studies reveal differentiation-dependent expression of MUC1, MUCSB, MUCSAC and MUC8, with few differences noted between nasal and tracheobronchial airway epithelial cells. MG 1, the large salivary mucin, was detected in well differentiated cultures and In bronchus tissue but MUC7, the small salivary mucin, was not. Additional experiments comparing CF versus normal cultures are in progress. Our studies provide a basis for examining the molecular regulation of mucin gene expression in primary human airway epithelial cells with the ultimate goal of better understanding mucus hypersecretion. Supported by the CFF, NIH and the ALA of NC.

269* MUC1 MUCINS ON THE SURFACE OF EPITHELIAL CELLS ARE ADHESION SITES FOR Pseudomonas aerugfnosa. K C Kjm D.l. Lee, S.W. Hyun, X. Zhang. Department of Pharmaceutical Sciences, University of Maryland, Baltimore, MD, USA.

Mucins are high molecular mass glycoproteins that are mainly responsible for the viscoelastic property of the mucus. Recently we have cloned and characterized a full-length eDNA of one of the mucin genes, called MUC1, from our eDNA library for primary hamster tracheal epithelial (HTE) cells. Deduced amino acid sequence revealed a transmembrane glycoprotein with resemblance to a cytokine receptor. This gene is highly expressed in epithelial cells, however, its function in the airway is unknown. We hypothesized that MUC1 mucins are adhesion sites for Pseudomonas aeruginosa (PA), a major bacterial strain almost invariably associated with cystic fibrosis airway. In testing the hypothesis. we have first selected a hamster epithelial cell line which does not express MUC1 gene. and then stably transfected the hamster MUC1 eDNA to examine whether the expression of the MUC1 mucins on the surface of the epithelial cell results in an increase In PA adhesion. The resu~s were: (1) CHO cell does not express MUC1 mRNA but can be stably transfected with the MUC1 eDNA as verified with Northern blot analysis as well as Western blot analysis; (2) expression of MUC1 mucins results in a significant increase In PA adhesion; (3) proteolytic cleavage of MUC1 mucins results in a complete abolishment of the increased PA adhesion. It i1 therefore concluded that MUC1 mucins expressed on the surface of CHO cell serve as adhesion aites for PA. The present result auggests an important role of MUC1 mucins In the early stage of airway infection, and may also provide a basis to study the epithelial responses to bacterial adhesion which may be crucially Important in airway inflammation in general, and cystic fibrosis in particular. (Supported by Cystic Fibrosis Foundation).

270* ROLB OP PIMBRIAB IN ADHESION OF NONTYPEABLB H. INFLUENZAB HUMAN TRACHEOBRONCHIAL MUCIN, M. Kubiet, A. Smith, R. Ramphal, A. Weber. Department of Molecular Microbiology and Immunology, University of Missouri-Columbia, Columbia, MO, USA.

Nontypeable Haemoph!1us !nfluenzae are among the most common pathogens encountered in patients with chronic bronchitis and are the most frequent isolate from BAL of infanta with cF. In order to test the hypotheses that adherence to mucin playa a role in respiratory tract colonization we developed a reproducible microtiter plate assay to study H. !nfluenzae adhesion to purified human tracheal mucin (HTM). Since alpha fimbriae (also called LKP pili) play a role in the adhesion of NTHI to human buccal epithelial cella, we examined the role that these surface structures play in

Poster Session Abstracts 281

adhesion to mucin, as certain mucina are also expressed on the surface of eukaryotic cella. Using a panel of fimbriated and non-fimbriated isogeneic NTHI, we found significant differences in adhesion. The binding of fimbriated NTHI was 20-fold greater than the isogenic nonpiliated strain (171 ± 42.5 x 102

cfu per well vs. 7.6 t 5.9 x 102 cfu per well). this adhesion difference was confirmed with a strain with mutated h!fA. Antipilin antibodies decreased the binding of piliated strain (6.4 ± 2.6 x 102 cfu per well vs. 185.1 ± 55.9 x 102 cfu per well). E. col! OHSa carrying and expressing the cloned a fimbria! operon from a type b H. influenzae had a 7-8 fold increase in binding when compared to the E. col! host containing the vector (pEMBLB) alone. These data suggest that a fimbriae may mediate binding of NTHI to HTM.

271* TilE BINDING OF PSEUDOMONAS AERUGINOSA TO THE SIAL YL-LEWIS X DETERMINANT AND TO HUMAN RESPIRATORY MUONS. Sc:harfman A., •Mazurier J., Hallaert C., Degroote S., Lamblln G. & Roussel P. • INSERM U377, Ulle and •UMR CNRS 111, Villeneuve d'Ascq, France

Pseudomonas aeruginosa (P.a.) binds to the carbohydrate part of respiratory and salivary mucins and its binding to mudns from CF patients is even higher [1). The bronchial and salivary mudns secreted by CF patients show abnomalities in sulfation and glycosylation, but the precise link between these alterations and the increased bincling of P.a. is still unknown. By analyzing the adhesion of P.a. to neoglycolipids, different neutral carbohydrate receptors have already been described [1). However there is very little Information concerning the binding of P.a. to carbohydrate receptors containing sialic acid or sulfate residues.

The present work was designed to compare the binding of different polyacrylamide-based glycoconjugates labeled with fluorescein (Gly-PAA-flu) to a non-piliated strain of P.a. (1244-NP) using flow cytometry. Glycoconjugates containing Lewis a, Lewis x, Lewis y, sialyl­or 3-sulfo-Lewis x, sialyl-N-acetyllactosamine, or Gal(cx1-2)Gal determinants were used. The affinity ot the sialyl­Lewis x containing Gly-PAA-flu for the strain of P.a. was 1 order of magnitude higher than that of the other glyconjugates that were studied. Moreover this binding was better inhibited by mainly sialylated glycopeptides derived from human respiratory mucin than by neutral or mainly sulfated glycopeptides. Reference : [1] Scharfman A. et al Am. J. Respir. Crit. Care Med. 1996,154,5163-5169 Supported by AFLM (Association Franfaise de Lutte contre Ill Mucoviscidose).

272* AIRWAY MUCIN SIALYLATION AND INFECTION. Lamblin G., Davril M., Degroote S., C. "Galabert & .B.ausW f. INSERM U377, 59045 Lille, and •Hopital R. Sabran, Giens France.

Human airway mudns are high molecular weight 0· glycoproteins made of apomucins encoded by different M U C genes and bearing a very large number of carbohydrate chains which can be sialylated, sulfated, sialylated and sulfated, or devoid of aciclic residues. The sulfation of respiratory mucins is increased in cystic fibrosis (CF) and carbohydrate structures such as the 6-sulfo-sialyl

282 1997 Cystic Fibrosis Conference

Lewis x epitope have been reported. In addition to this primary alteration of airway mudns, mudn glycosylation abnormalities might also occur in patients with severely inflamed or infected airways. In order to test this hypothesis, human airway mudns were purified from four groups of patients using CsBr density gradient ultracentrifugation : patients suffering from chronic bronchitis or from CF were subdivided, according to their bronchial DNA content, into infected and non-infected patients. The sialyl-Lewis x epitope was expressed in the mudns from all patients, independently from their Lewis status, indicating that fucosyltransferases different from FUT2 (Secretor enzyme) and FUT3 (Lewis enzyme) are expressed in the human airway mucosa, at least in disease. Moreover there was a significant increase in sialylatlon of the mudns from the severely infected patients as compared to the non-infected patients, espedally in the mudns from the CF patients that are frequently highly contaminated by Pseudomonas atruginosa. These data suggest that inflammatory cytoldns may influence the expression of different glycosyltransferases (sialyltransferase and fucosyltransferase) by the human airway mucosa. Supported by AFLM.

273 ACTIVATION OF NFKB IN CF AND NORMAL RESPIRATORY EPITHELIAL CELLS. Emily QIMango•. Setareh Tablbl*, Adam Ratner, Ruth Bryan, Allee Prince. Departments of Medicine• and Pediatrics, Columbia University, New York, NY.

Chronic Infection with P. aeruginosa Induces an Inflammatory response In the airways of patlenta with cystic fibrosis. Adherence of the organl1101 lo respiratory epithelial cells resuhs In secretion of IL-8, the major neutrophil chamoattractant In the lung. NF1eB factors participate In the transcriptional activation of a large array of Inflammatory g-• Involved In call activation, Including IL-8. These factors are present endogenously In the cell cytoplasm 11 Inactive precursor protelna cornplaxed to the lnhlbhory factor IKB. Followi'lg activation of the cell, IKB Ia pholphorylated, and cleavage of the NFKB precursor with translocation to the nuclaua occurs, resulting In tran~crlptlon from varloua promoters. We lhow that IHAEo- cella, an SV40 transformed airway epithelial call line, undargoea activation of NF1eB, as shown by electrophoretic mobility ahlft asuy (EMSA), In response to Infection with PA01. This activation Is blocked by co-Incubation with S~tM dexamethasone, which acts to lncreue available hcB'a. lmmunostalnlng with antibody to p65 (NFKB· reiA) derncnatratn prnance of NF~tB In the cytoplaam of unatlmulated cella and tranalocatlon of NFKB to the nucleus altar lnfactlon. Pretreatment of epithelial calls with the proteue Inhibitors TPCK (1011-M) and TLCK (1011-M), which block the degradation of llCB, reaults In a marked reduction of IL·8 aecreled from the cell. In the presanca of TPCK (10j.LM), NFKB activation In responu to expoaure to PA01, 11 detected by EMSA, Is Inhibited. IB3 cells (an SV40/AAV tr101forrnad CF respiratory apllhallal call line) and their plaamld corrected couoterparta (C· 38) cella also lhow activation of NFKB In response to lnfactlon with PA01. 183 cells and another CF cell line (CFTE cella) have activated NFlCB In the unatlmulated state, compared with IHAEo­celle and c-38 calla which have no auch conatltutlve NF1eB activation. Thla conltltutlve activation of NFKB In CF aphhellal cells may ba one potential explanation for the airway lnftammatlon aaan In unlnfacted Infants with cyetle flbroele. Supported by CFF, Parker B. Francia Foundation and NIH.

274 EXTRACELLULAR NACL VPREGULATES IL-l PRODUCTION BY RUMAN CP AIRWAY SUBMUCOSAL GLAND CELLS. 0. Tabary, I. Pen, J. HiMruky, P. Comillel, M. Ouenounou, J.P. Couetil, E. Puc:helle, IIICI LlaGAJuii,INSBRM U. 314, IFR n"53, Slll92 Reims, Laboratoire d1mmunoi0Jie, H4pltal M. Blanche, 51092 Reims IIICI HOpiral Broussais, 75674 Paris, Prance.

In CF human airways, lhe defect In CF aene product (CFI"R) has been shown to

lead to an increased NaCI concentration in airway surface liquid (ASL). The role of high NaCI concentration in ASL on the production of the major neutrophil pro­inflammatory interleukin 8 (IL-8) cytokine by CF and non CF airway epithelial cells remains an open question. We used in situ immunofluorescence to evaluate the IL-8 expression in human CF cryofixed bronchial submucosal tissues compared to non-CF submucosal tissues. We observe a significant (p < 0.01) higher percentage of CF submucosal glands expressing intracellular IL-8 protein (78% of examined gland sections. n=l43 from 3 lt.F508 homozygous CF bronchial tissues) as compared with non-CF submucosal glands (12%. n= 166 from 2 non-CF bronchial tissues) . As submucosal serous gland cells are a major site of CFTR protein expression, we studied the in vitro production of IL-8 by cultured lt.F508 homozygous CF versus non-CF human bronchial serous gland cells obtained from lung transplantation. By Nonhero blot analysis. there is no evidence of endogenous IL-8 mRNA from non-CF submucosal serous gland cells. in contrast to high amounts of IL-8 mRNA expressed in CF cells. In a serum-free culture medium. IL·8 secretion measured by EUSA was significantly higher (p < 0.001) in CF cell cultures (1492.0±25.1 pgiL-8/106cellsfhour. n=8) compared to non·CF cell cultures (28.1±2.1 pgiL-8fto6cellsfhour, n=8). Non-CF and CF cell cultures were then exposed for 1-hour to a saline solution containing either high ( 170 mM en or low (85 mM en NaCI concentration. Our results showed that in the presence of 170 mM ct·. non-CF gland cells behaved like CF gland cells : IL-8 production was 3.3·fold increased (7.0±0.7 pg!L-8f106cellslhour to 23.1±2.3 pg!L-81106cellslhour). More imponantly, in the presence of 85 mM c1·. CF gland cells behaved like non-CF gland cells : lowering ct· concentration from 170 mM to 85 mM resulted in a 11.2-fold decreased IL-8 expression (112.0±6.1 pg1L-8/J06cells/hour to 10.0±0.5 pg1L-8/Io6cellslhour). These results demonstrate that I) CF airway submucosal glands represent a major source of IL-8 even in the absence of an infectious stimulus and 2) an elevated extracellular ct· concentration induces the release of IL-8 by airway gland cells which can be corrected in CF glands by reducing the extracellular Cl" concentration. We propose that in the absence of any infectious stimulus. an elevated NaCI concentration in ASL associated with 4F 508 CF mutation may induce, at submucosal level, the release of IL-8 by airway atand cells and therefore. contribute to neutrophil recruitment in CF airways. Thus. upregulation of submucosal gland IL-8 production in CF airways could represent a primary event that initiates the early onset subtained inflammation in CF human lungs. Supponed in pon by AFIM

275 GLYCOSYLATION DIFFERENCES BETWEEN CF AND RESCUED CELL LINES ARE NOT CFTR-DEPENDENT. X.S. Jiang•, W.O. Hill+, J.M. Pilewski•, and O.A. Weisz••, Departments of*Medicine and •cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA USA

Altered glycosylation of mucus and membrane glycoconjugates could explain reported differences in binding of bacterial pathogens to cystic fibrosis (CF) versus normal tissue. However, because bacteria can alter cell surface glycoconjugatcs, it is not possible to assess the role ofCFfR in glycosylation in these studies. To address this issue, we have developed quantitative lectin binding assays to compare cell surface glycosylation in well-matched immortalized CF and rescued cell lines. The CF airway bronchial epithelial cell line 183-1 consistently bound more peanut agglutinin (PNA) than its clonal derivative S9, which stably expresses functional wild-type cystic fibrosis transmembrane conductance regulator (CFTR). Pretreatment with neuraminidase increased PNA binding and abolished the difference between the two cell lines. However, infection of the 183-1 cells with a replication-deficient recombinant adenovirus encodina CFTR restored CFTR function but did not alter PNA binding to cells. By contrast, treatment with the weak base ammonium chloride iDcreased PNA binding to both cell lines as expected. Our data show that even clonally-relatcd CF and rescued cells can exhibit sianificant differences in carbohydrate processing. While the differences we found are consistent with the proposed role for CFfR in modulating intraorganellar pH, our data strongly suggest that they are CFfR-independent. These studies add a cautionary note to interpreting differences in glycosylation between CF and normal primary tissues and immortalized cells. Supported by grants from the Cystic Fibrosis Foundation to OA Wand JMP.

276 ADENOVIRUS-MEDIATED CITR GENE TRANSFER CORRECTS OVERSULFATION OF SECRETED GLYCOCONJUGATES IN CYSTIC FIBROSIS AIRWAY EPITHELIAL CELLS. J.D. Latoche, S.M. Arcasoy, D.C. Devor, and J.M. Pilewski. Depts. of Medicine, and Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA, USA.

Previous studies have indicated that high-molecular weight glycoconjugates (HMG) from CF airway epithelia are oversulfated relative to non-CF epithelia Although various gene transfer vectors have been shown to coiTCCt the ion transport abnormalities in CF airway cells, the ability of these vectors to alter mucus sulfation, and the efficiency of gene transfer necessary to achieve this, remain unknown. We therefore determined whether CF gene complementation corrects the oversulfation of HMG in primary CF airway epithelial cells (AEC). To accomplish this, CF and non-CF primary AEC were grown at an air/liquid interface and metabolically labeled with 3'S04 and [6-'H]-glucosamine (GleN) to determine differences in sulfation of HMG. CF AEC showed a 35S04:[6-'Hl­GicN ratio 2.3 times higher than that of non-CF AEC, consistent with Ereviouslr, published data There was no significant change in the 5S04:[6-·H]-GicN ratio after transfection of >90% of non-CF and

CF cells with an adenoviral vector containing the E.coli LacZ gene, or transfection of non-CF cells with an adenoviral vector containing wild-type CF gene. However, compared to lacZ transfected cells, CF AEC (genotypes AF508/AF508 and AF508/WI282X) transfected with an adenoviral vector containing wild-type CFfR at the same multiplicity of infection showed a 56±14% percent (p<O.OS, n=7) reduction in sulfation of HMG. In contrast, following CF gene transfer to -30% of differentiated CF AEC (genotype ?/G85E), a reduction in sulfation was not observed despite restoration of a cAMP-mediated chloride conductance. These data support the existence of a sulfation defect in CF airway epithelia, demonstrate that correction of HMG oversulfation is feasible in cultured CF airway cells, and suggest that correction will require more efficient gene transfer than required to restore cAMP-mediated chloride secretion. (Supported by Cystic Fibrosis Foundation, Q933 to JMP).

277 EPITOPE TAGGING OF A Ca,..ACTIVATED CJ- CHANNEL (CaCC) DOES NOT MODIFY CHANNEL FUNCJ'ION. fL..Ril!2!l, V.Gh. Shlyonsky, B.K. Berdiev, 1.1. Jsmailov, C.M. Fuller, and DJ. Benos. Depl of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL. 35294-000S, USA.

Airway Ct transport Is mediated by lleYeral separate anion channel proteins includin& those "'gulated by cAMP (CFTR) and calcium (CaCC). We have P"'Yiously identified a protein isolated from bovine trachea that behaves u a Ca'' -sensitive c1· channel when inc:orporatecl into planar lipid bilayers (Fw//w C. M. a a/. J. Bioi. Clterrt. /994; 269: 26642-16650). The native protein isolated from the bovine trachea miJV8111 at 140 kDo when sepmated by SDS-PAGE in tile absence of DlT, but u a smaller muced form of 38kDa In tile presence of DlT. We have cloned a protein fi'om a bovine tracheal eDNA expression library that hu biophysical proportiea identical to those exhibited by the nalive tracheal channel (Cunningham S.A. « a/. J. Bioi. Chem. /995; 170: J/0/6-31026). In contrast to the native protein, the primary eDNA transcript codea for a polypeptide of 100 kDa, that can not be "'duced by DlT when ill y/tro translated. However, when injected in XenO{JIU oocytes, the cRNA expresses a channel of identical characteristics to the native tracheal protein. In order to determine whether the primary eDNA transcript II subject to post-translational processin&. we have constructed an epitope-tagged protein by cloning a 2746 bp Bglll fragment containing tho full length CaCC eDNA into the BamHI (compatible end) clonin& site of the pcDNA3.1/HIS-A eukaryotic expression vector (InVitrogen). This construct codes for the entire CaCC protein plus a 42 amino acid sequence-tag at the N-terminal. ThiJ 42 amino acid sequence containl I) a N-terminal polyhistidine taa for proteiD purificalion, 2) the anti-Xpreu epitope liJ for tile recombinant protein deteCtion with the Anti-Xpress antibody and 3) the enterokinase cleavage site to separate the liJ sequence from tile CaCC protein after purification by Ni1

' .. trmity chromatography. The cRNA of this construct (2S ng/$0 nl) wu injected in Xenopu6 oocytea and oocyte membrane -iclea were fused to pl.,.. lipid bilayers for single channel recording. We obterved a 31 pS channel that had a linear cumnt-voltage relationship. ThiJ channel wu also sensitive to 100 11M DlT but not to 100 11M niflumic acid, a compound which has been previously shown to Inhibit the endogenous CaCC of the oocyte (Whit• M.M., and Aylwin M. Mol. Phonnocol. /990; J7: 720-7U). Expression of this epitope-taggecl conllnlcl tllerefore exhibited identical characteristica to both the native and wild-type channel when studied under these conditiona. These data auaest that the 18gged proteiD il trafficked and inserted into the oocyte plasma membrane. ThiJ wu not observed when veaicles prepared from water injected oocytel were fused to lipid bilayer. This conatruct should provide a useful tool to examine processing of the CaCC in tnnsfected epithelial cell linea. Supported by NIH Grant DK48764 and CFF Orant9710.

Poster Session Abstracts 283

278 THE CA1•-INDEPENDENT PROTEIN KINASE C ISOFORM, nPKC!I, MEDIATES PURINERGIC ACTIVATION OF MUCIN SECRETION IN SPOC 1 CELLS. Lubna H. Abdullah, Jason Bundy, and C. William Davis. CF/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, NC 27599 USA.

SPOCI cells secrete mucin under the regulation of an apical membrane purinergic receptor (P2Y2 or P2u). Previously, we have shown that the cellular messengers mediating this purinergic response appear to be ca•• and DAG: mucin secretion is stimulated by ca•• ionophore and by PMA, but not by cyclic nucleotides. Importantly, Cal+ and PMA appear to be independent in their actions: the response to maximal doses of the two secretagogues is fully additive, and in permeabilized cells PMA promotes secretion under conditions in which Ca2

• is clamped to 10 nM. In this study, the PKC isoforms mediating the purinergic mucin secretory response were studied at both the mRNA and protein levels. RT-PCR primers were designed with ::!:94% identity to conserved regions of the genes within each PKC subfamily. From restriction enzyme maps of the resulting PCR products, five PKC isoforms were identified: the conventional (DAG/Ca2

' -dependent), cPKCa, the novel (DAG-dependent/ea2•.

independent), nPKC6 and TJ, and the atypical (DAG/Ca'•. independent), aPKCl; and A.. No new PKC isoforms were discovered. Western blotting using commercially-available polyclonal antibodies toward synthetic peptides mimicking unique regions of these PKC isoforms verified that each isoforms identified by RT-PCR was expressed also at the protein level. Subsequently, SPOCJ cells were challenged with maximal doses of A TPyS or PMA, following which they were fractionated and studied by Western blotting for PKC isoform translocation. Of the five isoforms expressed in SPOCI cells, only nPKC!I was found to migrate from cytosol to membrane fractions in response to ATPyS stimulation. Thus, we conclude that mucin granule exocytosis in SPOCI cells is regulated along parallel and independent pathways by Ca2

• and nPKC!I. Supported by Glaxo Wellcome pic.

279 AIRWAY MUCIN SULFATION AND CYSTIC FIBROSIS. G Lamblin S. Degroote, J-M Lo-Guidice, G. Strecker & P. Roussel. INSERM U377, 59045 Lille, France.

Human airway muclns are high molecular weight 0· glycoprotein& made of different apomucins encoded by M U C genes and bearing a very large number of carbohydrate chains. An increased suUation of respiratory mudns secreted by CF patients has been reported for a long time. More recently, Zhang tf al have shown that increased sulfation of CF mucins was a primary defect [1). In carbohydrate chains of respiratory mucins, the sulfate residues can be attached to the 3 position of a non-reducing terminal galactose or to the 6 position of a penultimate N­acetylglucosamlne residue, suggesting the presence of at least two different sulfotransferases in the human respiratory mucosa [2]. We have characterized two such enzymes in the human mucosa : {i) a galactose 3-0· suUotransferase adding suUate from PAPS to the terminal galactose residue of a N-acetyllactosamine containing carbohydrate chain and {il) a N-acetylglucosamine 6-0· sulfotransferase adding sulfate to a terminal N· acetylglucosamine which can be further substituted : (i) Gal(J31-4)ClcNAc ... -+ H03S-3-0-Gai(J31-4)GlcNAc ...

(il) GlcNAc. •• -+ H03S-6-0-GlcNAc ... -+ Gal(J31-4)[H03S-6· O]GlcNAc ... Barasch tf al have already observed an increased pH of some Golgi compartments of CF cells [3] and, since the N­acetylglucosamlne 6..0-suUotransferase (6.7) has a higher optimal pH than the galactose 3-0-sulfotransferase (6.1),

284 1997 Cystic Fibrosis Conference

this might explain why the carbohydrate chains of CF respiratory mucins are predominantly 6-sulfated. References : [1) Zhang et al J Clin Invest, 1995, 96, 2997-3004; [2) Lo-Guidice et al, J Bioi Chem, 1994, 269, 18794-18813; [3) Barasch d al, J Cell Sd 1993, 17, 229-233. Supported by AFLM.

280 DIFFEREN11AI.. SENSI1MTY OF EPfTHEUAI.. CEU.S EXPRESSING EITHER IJW..O. TYPE OR MUTANT CF1R TO EPINEPHRJt.E 1,2 Kaarin K. Goncz,1,2Luz Feeney, 4Maurizlo Scarpa and 1,2,30ieter C. Gruenert. 1Gene Therapy Core Center, 2CVRI and 3Deptartment of Laboratory Medicine, University of California, San Francisco, CA 94143-0911; 4Gene Transfer Laboratory, Univeraita Degli Studi Di Padova,35121 Padua, ltalla.

Enrichment of cells originating from gene therapy experiments on C1' epithelial cells by either site-specific exchange of CF1R sequences by small fragment homologous replacement (SFHR) or by the Introduction of eDNA has been complicated by the fact that a mechanism to select corrected cells has yet to be found. Functional correction of the CFTR gene restores the cAMP-dependent chloride secretion in tergeted cells. However, the electrophysiological measurements required to isolate such cells are either inconvenient and labor Intensive or, limited in their sensitivity to detect corrected cells. Recently, a method that allows for selection of corrected CF cells (WICFTR+) over uncorrected cells (WICFTR-) was reported. Treatment of transformed human epithelial cells with epinephrine showed a lethal effect in WICFTR- cells whereas, cells expreaalng WICFTR were more resistant. The observed effect was dependent on epinephrine concentration, exposure conditions, cell clone variations as well as cell density. Here, we report the results of using this treatment on different cell lines: (normai:WICFTR+) 56FHTE8o-, 16HBE14o-, 9HlE; (CF:wtCFTR-) CFDE, :I;CFTE20o-, CFSMEo-; (CF:wtCFTR+) CFDEipREP.CfTR, l:CFTE29c>lpCEP.cFTR. The 56f'HTE8c>. line, as previously reported, was found to be more resistant to 200 jiM epinephrine than the CR:E line. However, overall the cells most resistant to this treatment were found to be the l'.CFTE29o- line and, least resistant were the 16HBE14o- line. This unexpected result indicates that sensitivity to epinephrine cannot be direct1y correlated to the absence of rAMP regulated Cl- transport for all cell types. As epinephrine has been reported to effect the transport of other ions, such as Na+, as well as water, these results suggests that the differential sensitivity that was observed could be due to selective regulation of ion channels by adrenergic receptors In the various cell types. This work is supported by NIH grants DK46002 and DK4n66.

281 ADENOVIRUS EXHIBITS INSTANTANEOUS ESCAPE FROM ENDOSOMES: EVALUATION OF GENE TRANSFER VECI'ORS DIRECTLY CONJUGATED WITH FLUOROPHORES. f.L. ~B. Fenis, S. Worsall, R.G. Crystal. The New York Hospital­Cornell Medical Center, NY, NY

For a sene transfer vector to suc:c:essfully invade a host cell, it must avoid encountcrins desradative compartments including lysosomes. Followins entry into the cell by receptor-mediated endocytosis, adenovirsl vectors avoid desracJation by escaping from endocytic orpnelles prior to endosome-lysosome fusion. The objective of this study is to detennine the subcellulsr endocytic compartment from which escape occurs, i.e., early or late endosomes. To address this topic, we created adenoviral vectors conjuaated to tluorophores thus enablins direct observation ofviral particles throupout their lifetime in the <:ell using fluorescen<:e micro­scopy. Quantitative analysis of digital imases showed linear accumulation of adenovirsl vecton in the AS49 lung epithelial cell line throushout the course of a 60 min incubation. Interaction of fluorescent viral particles with AS49 cells was prevented by prior incubation with purified adenovirus fiber protein, the capsid protein that mediates high affinity binding. In pulse·labellna experiments, adenovirus particles were initially distributed in the cell periphery but within 30 min shifted to a peri-nuclear localization indicative of interaction with the nuclear envelope. Dual localization studios comparina subcellular distribution of adenovirus with endosome-marking transfenin or with lysosome-marking dextrans revealed that the adenovirus vectors were rarely observed in the same

compartment with other endocytic markers, leading to the conclusion that the adenovirus escapes from early endosomes with extremely high efficiency and rapid kinetics. This property is further illustrated by the absence of co-localization of adenovirus marked with different colored fluorophores, i.e., Ad-containing endosomes do not have time to coalesce prior to Ad escape. Whereas previous studies suggested that adenovirus escape occurred following endosome-endosome fusion, our results are consistent with the hypothesis that adenovirus escape often occurs before endosome-endosome fusion and that the degradative function of late endocytic compartments, including lysosomes, plays little or no role in attenuating the extent of adenoviral infection in epithelial cells.

282 ENHANCED REPORTER GENE EXPRESSION IN CELLS TRANSFECTED IN THE PRESENCE OF DMI·2, AN ACID NUCLEASE INHIBITOR. G Ross, M. Bruno, M. Uyeda*, K. Suzuki*, K. Nagao*, J. Whitsett, T. Korfhagen. Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, OH, USA; *Division of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.

Cellular nuclease activity is a potential barrier to efficient, DNA-mediated expression of foreign genes in mammalian cells. Endogenous nucleases rapidly hydrolyze foreign DNA after entry into the cell. In this study, we test the hypothesis that transfection in the presence of a specific DNase inhibitor enhances gene expression after plasmid mediated gene transfer. DMI-2, a polyketide metabolite of Streptomyces sp. strain 560 was used to enhance the expression of bacterial chloramphenicol acetyltransferase (CAT) in the human lung adenocarcinoma cell line H441. DMI-2 has been shown previously (Nagao, et al.) to have inhibitory activity against porcine DNase II, an acid pH optimum nuclease contained in the endosomaVIysosomal compartment. Transfection of H441 cells In the presence of 0.1 - 1 j.tg/ml DMI-2 gave the following results: (1) approximately 10 fold enhancement of CAT activity when the bacterial plasmid was complexed with either surfactant protein A-poly-lysine or transferrin-poly-lysine; (2) 1.5 to 2 fold enhancement of CAT activity in cells exposed to llpofectin-DNA complexes, (3) no effect on transfectlon via calcium phosphate co-precipitation. In control experiments, DMI-2 alone showed no inherent transfection activity. In experiments using SP-A-poly­lysine and plasmid containing the beta-galactosidase reporter gene, DMI-2 Increases the number of transgene positive (X-gal stained) cells. Taken together, the data support the hypothesis that nuclease action Is a significant barrier to gene expression of transferred DNA. Inhibition of specific Intracellular nucleases may enhance expression after transfection mediated by a variety of gene transfer systems. Supported by CFF and HL51832.

283 CORRECTION OF THE CF BACTERIAL KILLING DEFECI' CAN BE ACHIEVED BY TARGETING A FRACTION OF THE AIRWAY EPITHELIAL CELLS. J Zahnc:r· J. Smilh, T. OI'IIJI&t, and MJ. Welsh. Howard Hughes Medical Institute, lleplnment of Medicine, University of Iowa Colleae oC Medicine.lo- City, Iowa, USA

Dewiopmellt of FDC transfer to CF airway epithelia requires knowledae of the percent of airway cells that must be WJCfCd to improve the CF phenotype. To adclreu this problem we UICd an in vitro cell cultuie moclei or CF airway epithella and eumined dcctrolyec ll'll1lpOrt and blcterialldllinJ followina direct application or P. aeru1'- to epithellL We lltlldied primary cultures of airway epithelia In which lncreuinJ proportions of nonCP cells were mixed with CF cella. Affi:Jt two weeks in culture when the epithella had ditTermtiated, the proportion of normal to CP cells was detamined by quantitati~ fliiOI'eiCCIIt PCR 1111y. We found that when 10-2~ of an epithelium wu composed of nonnal cells, the a· transport defect, as evaluated In UuinJ chambers, was concctcd. In para11cl lltlldies we found that s-~ of normal cells was suffiCient to COI'I'ect the CF defect in b1ctcria1 killing. We also examined the effect of

expressing CFI'R in CF airway epithelia using an adenovirus vector. The epithelia were infected with Ad2CFTR-8 or Ad5/GFP expressing green fluorescent protein at MOis ranging from 1-100. By FACS analysis we determined the percent of cells that were infected. We found that when 8-10% of the cells were transduced that 80% of the a· tranSport defect was corrected and killing of P. tufl4ginosa was restored. These data suggest that correction of approximately 10% of the cells is sufficient to correct the defect in bacterial killing. The results also show a correlation between correction of transepithelial Cl transport in CF epithelia and bacterial killing.

Infection/Microbiology

284* PSEUDOMONAS AERUGINOSA INTERNALIZATION IN HUMAN RESPIRATORY EPITHELIAL CELLS DEPENDS ON CELL DIFFERENTIATION AND POLARIZATION. M.C. Plotkowski. S de Beo1zmapn, A.C. Buisson, l.M. Klossek, and E. Puchelle. INSERM U314,1FR 53, REIMS, France.

Internalization of P. curuginosa in human respiratO<)' epithelial cell lines haa been shown to be CFTR-dependent (Pier et al., Science, 1996,271:64). However, because we rarely and uniquely observed P. auugino_sa internalization in human respiraiOry epithelial cells in primary culture whtch do not express CFTR, we addressed the question whether P. aeruginosa internalization may depend on cell degree of differentiation and polarization. Human respiratory epithelial cells obtained from nasal polyps, were grown up to confluency in primary ~ulture .on two differen.t substtates all~wing obtention of either poorly dtfferenttated or well dtfferenttated pnma'! cultures. 16 HBEI4o- cell line was also Jrown up to confluency on plasttc substtate, and in these culture conditions, 16HBEI4o- cell line was poorly differentiated. P. aeruginosa internalization was evaluated after I or 4 houra incubation period with loB cfulml by the gentamicin exclusion teehnique, and compared with internalization of poorly invasive strains , PAKN I mutated in the rpoN gene, and the E. coli DHSa strain. Polarization of confluent cell monolayera was investigated by immunoreactivity of ZOI protein, a cytoplasmic protein associated with tight junctions. Highly differentiated human respiratO<)' epithelial cells uniformally express ZOI throUghout the cell monolayer, while poorly differentiated human respi~ epithelial cell, as well a~ 16HBEI4o- mo~olay~l'l lack. ZOI protetn expression. No P. auugmosa was tnternahzed tn P?lanzed a~d well differentiated respiratory epithelial cell cultures, whereas tntemaltzatton was efficient in poorly differentiated respiratory epithelial cell cultures. as ~ell as in t6HBE14o- cell line (2.3±0.5 and 1.2±0.6 10' CFU/ml, respecttvely). When P. curuginosa internalization occured, it was already maximal after I hour incubation period. In poorly differentiated respiratory epithelial cell cultures, P. a~rugino1a internalization wu significantly increased by a factor of 7 after EDTA tteatment. Control strains PAKNI and DHSa were poorly internalized (1.9±0.7 and 1.8±2.2 H>' cfulml). These resuiiS suggest that P. aeruginosa internalization in respiratory epithelial cells depends. ~n. thetr degree of differentiation and polarization as well as on accesstbthty of basolateral receptOI'I, usually proteeted from bacteria in polarized epithelium by tight junctions. This work was supported by AFLM.

285* PSEUDOMONAS AERUGINOSA INFECTION IN SP·A DEFICIENT MICE. A M. Levine, K. E. Kurak, M.D. Bruno, J. A. Whitsett, T. R. Korfbagen. Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, OH, USA. .

Pulmonary disease in cystic fibrosis is caused by recurrent bactenal infections that contribute to the increased morbidity and mortality associated with the disorder. Distinct innate and immune mediated host defense mechanisms have evolved to maintain the lung clear of microbial pathogens including acute mediators of bacterial opsonization and killing and more complex cytokine or immune mediated pathways. Recent in vitro studies demonstrated a potential role for surfactant protein-A (SP-A), a member of the Ca-dependent lectin family, in host defense in the lung. SP-A binds gram positive and gram negative bacterial pathogens, In vitro, contributing to their uptake and killing by alveolar macrophages. In order to assess the role of SP-A in host defense in the lung In vivo, the murine SP-A locus was targeted by homologous recombination generating mice lacking SP-A (SP-A-/-). SP-A (-/-) mice were intratracheally infected with Pseudomonas aeruglnosa (PA). Bacterial burden and lung inflammation. were assessed in SP-A(-/-) and wild-type mice. PA colony counts m ~he lung were significantly increased in SP-A ( -1-) compared to control nuce

Poster Session Abstracts 285

at 6hr (2.!5E+08±7.8E07 vs 8.3E07±3.6E07) and 24hr (l.3E07±7.4E06 vs 3.7E05±2.4EOS) after administration. Systemic dissemination measured by spleen colony counts was not different between the groups. Pulmonary infiltrates in both wild-type and SP-A ( -/-) mice consisted predominately of neutrophils. Absolute neutrophil counts and myeloperoxide activity of lung homogenates were greater in the SP-A(-/-) mice. Lung histology revealed more extensive pulmonary infiltrates at 24 and 48 hours in the SP-A(-/-) mice. In summary,lungs of SP-A(-/-) mice had a greater bacterial burden and inflammation after PA infection. These findings demonstrate that SP-A plays an important host defense role in the clearance of bacteria from the lung. The finding that SP-A is dysregulated in cystic fibrosis and other inflammatory lung conditions suggest that deficiency of SP-A might also play a role in the pathogenesis of the chronic bacterial colonization and infection in lungs of patients with cystic fibrosis. Supported by CFF and HLS8132, the Center for Gene Therapy.

286 INTERLEUKIN-4 CAUSES ENHANCED CLEARANCE OF PSEUDOMONAS AERUGINOSA FROM MOUSE LUNG. S. Jain-Vora, A M Levine, J. A. Rankinl, J. A. Whitsett. Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, OH, USA; IPulmonary Disease Section, VA Medical Center, West Haven, Cf, USA.

Chronic lung infection with Pseudomonas aeruglnosa ( P. aefl4ginosa) is commonly associated with cystic fibrosis. However, the mechanisms involved in the clearance of P. aeruginosa from the lung in cystic fibrosis are poorly understood at present. lnterleukin-4 (lL-4) is a pleotrophic cytokine that is expressed during lung injury and inflammation. Transgenic mice were produced in which IL-4 was overexpressed in the mouse lung under the influence of Clara cell secretory protein promoter (CCSP-IL-4 mice) causing increased monocytic and leukocytic cell infiltration, mucus hypersecretion and increased surfactant protein accumulation (Rankin et al. PNAS-1996; Jain-Vora et al. AJRCMB-1997). In order to ascertain the role of local, chronic IL-4 production on Pseudomonas colonization, mice were intratracheally (i.t.) infected with S X 107 cfu of P. atfl4ginosa. While the bacteria proliferated in wild type mouse lungs, a rapid reduction in the number of injected bacteria was seen in CCSP-IL-4 mouse lungs at 6 hours post-infection. Acute pretreatment of wild type mice with IL-4 intranasally (i.n.) (10 J.lg and 5 J.lg at 16 and I hour before infection, respectively) prior to P. aeruginosa administration, blocked bacterial proliferation assessed 6 hours later. Cellular responses to P. aefl4ginosa were predominately leukocytic and did not differ among the different groups tested, although the CCSP-IL-4 and IL-4 i.n. mice contained increased numbers of neutrophils and macrophages prior to infection. The myeloperoxidase activity of lungs harvested 6 hours post infection was similar in wild type, CCSP-IL-4 and IL-4 treated wild type mice. Together, these data indicate that acute, chronic and local IL-4 production caused the rapid clearance of P. aeruginosa from mouse lungs. Although the mechanisms involving increased bacterial clearance are not yet known, these results implicate an important role for IL-4 in the clearance of bacterial infections and offer a novel potential for therapy or prevention. Supported by the Cystic Fibrosis Foundation and National Institutes of Health (HL S 1832).

287 PliO aaaoclated glycoaylatlon of P .. udomon.. aeruglno .. gene producta affecta virulence. D Brlght:1 M. Feldman,1 D. Balderes,1 and A. Prince 1, Department of Pediatrics, Columbia University, New York, NV1

The glycosylatlon of prokaryotic gene products, Including P. aerug/nosa pill, has been recently described. Glycosylatlon Is critically Important In Immune recognition and host-pathogen Interactions, suggesting that pliO, associated with the glycosylation of P. aeruglnosa PA1244 pill (Castrlc, Microblol. 141:1247, 1995) may be associated with an Increased virulence of this strain. To test the role of pliO In virulence, a pliO mutant of PA1244 was constructed and tested lor Its ability to adhere to epithelial cells and cause pulmonary Infection In neonatal mice. The pliO coding sequence was cloned from PA1244 genome using PCR and primera constructed from the reported sequence. A pliO null mutant was obtained by deleting 453 bp DNA fragment from pliO structural gene and substituting a gentamlcinR cassette. This construct was used to replace the wild type gene In PA1244, and the expected recomblnational events were confirmed by Southern hybridization

286 1997 Cystic Fibrosis Conference

and PCR amplification of the Interrupted pliO gene. Since pi/0 Is directly downstream of the pllln structural gene piiA, pllliatlon and motility of the pliO mutant 08103 were confirmed to be unaltered. The loss of pliO expression resulted In a mutant significantly attenuated In virulence. intrs-nassl Inoculation of 8AL8ciByJ mice with 08103 was associated with a 67 % decrease In pneumonia (p<0.0001 ), a 63% reduction In bacteremia (p<0.005), and an 86% decrease In mortality (p<0.0001) as compared with the parental atraln PA1244. Adherence of the pliO mutant strain to human epithelial cells was significantly greater (2.5 fold) than the parental strain (p<0.00001). To assess the role of pliO In clinical strains of P. aeruglnosa, sequential Isolates from 6 CF patients collected over 6·12 years were probed for DNA homologous to pliO. Although Isolates from 516 patients were previously shown to have become Fla" In vivo none had pliO deletions. Screening a group of non-CF clinical Isolates of P. aeruglnosa revealed that 50% were plio+. These studies suggest an Important and complex role for the glycosylatlon of pllln and perhaps additional P. aeruglnosa gene products In the pathogenesis of Infection. (aupported by CFF and NIH).

288 REGULATION OF THE STAPHYWCOCCUS AUREUS CAPSULAR POLYSACCHARIDE TYPE 5: C02 INHIBmON IN VITRO AND IN VIVO. 1S. Herbert, 1C. Wolz, ZC.-P. Wlelaad, lp', Glitz 1G. Dllr!nJ. 1Dept of Gen. and Environm. Hygiene, Hygiene-Institute, 21nstltute of Microbial Genetics, Unlvenlty of Tllblngen, TObingen, Germany.

Staphylococcus aureus causes chronic endobronchial infections in patients with cystic fibrosis (CF). The virulence of S. aureus is regarded as multifactorial including hemolysins, protein A, teichoic acid and several capsular polysaccharides (Cps). The role of CPs in the virulence of S. aureus is still not clearly defmed. We investigated CPS expression in lung tissue and nasal polyps of two CF patients, in rats and in vitro using ELISA and immu­nofluorescence. In CF tissues, S. aureus expressed protein A and teichoic acid but only 1 o/o-S% of cells expressed CPS. When rats were challenged with CPS-positive S. aureus in the granuloma pouch model, only lo/o-S% CPS-positive cells were detectable in pouch exudates. CF and pouch isolates, however, re-expressed CPS (700/o-90% of cells) when grown in vitro with air. Addition of 1% C01 or more to air or to O/N2 gas mixtures reduced CPS expression significantly (f< .001) in a dose-dependent manner (6o/o-l% CPS-positive cells). The CPS promotor gene was ligated to the S. hyicus lipase gene in plasmid pPSll and the vector transformed in the lipase-negative S. aureus strain Reynolds. Addition of 5% C02 to air reduced lipase production during growth 2.5 times compared to growth in air. Northern blot analy­sis of strain Reynolds revealed that col induced earlier tran­scription of agr, and profoundly upregulated transcription of the S. aureus hemolysin genes hill and hlb.The results show that S. aunus does not produce CPS in CF airways and in rat granuloma pouches and that col is an environmental signal which regulates CPS expression.

289 BAL SURFACTANT PROTEIN-A LEVELS ARE DECREASED IN INFANTS WITH CF. KE Greene· SH Ye. RJ Mason. F Accurso· Department of Medicine, National Jewish Medical and Research Center, and Department of Pediatrics, University of Colorado School of Medicine, Denver, CO, USA.

Patients with CF suffer from chronic respiratory infections and luns in11ammation, cbaracterized by increased BAL IL-8 levels and neutrophil infiltration. There are reports suggesting the lung disease beains very early postuatally. Pulmonary surfactant proteins are emergins as important modulatora of host defense and

alveolar inflammation, in addition to their role in reducing alveolar surface tension. Surfactant Protein-A (SP-A) has been reported to inhibit the inflammatory effects of lipopolysaccharide and decrease the neutrophil chemoanractant effects of IL-8. SP-A is decreased in other pulmonary diseases associated with lung inflammation and infection. Therefore, we hypothesize that SP­A may be decreased in BAL fluid of infants with CF. We suspect that these deficiencies occur early and may contribute to the progression of chronic infections and inflammation in patients with CF.

To begin studying this, we analyzed SP-A levels in BAL fluid using a double monoclonal antibody sandwich ELISA. We examined BAL fluid from five infants with CF and compared them to eight infants with non-CF pulmonary disease. We found that infants with CF had dramatically lower BAL SP-A levels compared to the non-CF children (1.3 uglml vs. 5.4 uglml).

These results suggest that infants with CF may be deficient in SP-A, an important modulator of lung inflammation and host defense. This could have important clinical implications for early treatment ofCF.

(Supported by the CF Foundation)

290 SURFACTANT PROTEIN-A (SP-A) LEVELS IN ADULT PATIENTS WITH CYSTIC FIBROSIS Paul G Comber•, Ann Marie LeVinet, Joseph A. Kitzmiller•, Keith C. Meyer:, and Robert W. Wilmott•. *Division of Pulmonary Medicine, and tDivision of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, OH; and tDepartment ofMedicine, University of Wisconsin Medical School, Madison, WI.

Chronic pulmonary infection is a significant part of the pathophysiology of cystic fibrosis (CF). Surfactant protein-A (SP-A), a member of theCa-dependent lectin family, is involved in control of the turnover of alveolar surfactant. /11 vitro studies have shown that SP-A binds to gram-positive and gram-negative bacterial pathogens. Recent in l'il'O studies have confirmed that SP-A plays an important role in the host defense against bacterial pathogens in the lung. We measured SP-A levels in the bronchoalveolar lavage fluid (BALF) of 31 adult patients with cystic fibrosis (age 22±5 [SD]) and 34 normal controls (age 34± 12) by ELISA. Eleven of the CF patients underwent bronchoscopy both at the beginning and end of an admission for treatment of a pulmonary exacerbation. SP-A levels were significantly (p<O.Ol) decreased in the CF patients (46.9±10.4 [SEMI j.lg/ml BALF) as compared to the normal controls (294.5±72.1 j.lg/ml BALF). The amounts of SP-A were not significantly different in BALF obtained before (20.3±9.8 j.lg/ml BALF) and after (42.0±22.0 j.lg/ml BALF) antibiotic therapy in the II CF patients, but remained significantly less than the normal controls. These data suggest that the chronic pulmonary infections in patients with CF may in part be due to decreased host defense caused by a deficiency of SP-A. Further, the levels of SP-A in CF patients do not increase significantly following antibiotic therapy.

!Supported by a Cystic Fibrosis Foundalion Leroy MaUIIC'I\5 A\\ard (PGC)J

291 ADHERENCE AND KILLING OF TRACHEAL EPITHELIAL CELLS BY PSEUDOMONAS AERUGINOSA W. Tseng, A. Lee, B. Haua, D. Evans, G. Chandy, D. Chow and T. Machen. MCB Dept. and Optometry School, U. Calif., Berkeley, CA.

Binding and killing of tracheal cells by Pseudomonas aeruginosa (PA) wu studied in living Calu3 cells, a serous-like, high resistance (> 1000 ohm-cm2) cell line that expresses hip levels of CFI'R.

Monolayers grown on filters or coverglasses were mounted into chambers on the stage of both widefield fluorescence and confocal microscopes. Binding of PA to Calu3 cells was assessed using green fluorescent protein-expressing PAO 1-GFP, and epithelial cell killing by PA6206 and PA 103 was detennined from nuclear uptake of the impermeant dye propidium iodide (PI, 1-IOJ.tM). When io6-!0S cfu/ml PAOI-GFP were added to Calu3 cells for I hr and then washed, bacteria remained bound to cells with an "exposed" basolateral surface: cells at the perimeters of cell "islands," surrounding holes in otherwise confluent epithelial monolayers, or, especially, adjacent to regions which had been mechanically damaged. Progressively fewer PAOI­GFP bound to cells farther from regions with exposed basolateral membranes, and no PAOI-GFP bound to regions more than •IOcells from a "free" edge. PAOI-GFP bound to basal and lateral membranes of Calu3 cells, but only infrequently at the apical surface or in the cytoplasm. Basolateral binding was independent of the extracellular matrix because PAO 1-GFP adhered poorly to coverslips on which confluent Calu3 cells had been removed using I hr EGT A treatment. Disruption of tight junctions (5 min EGTA followed by incubation in Ca-free solution) caused PAO 1-GFP to bind to cells in confluent areas that had previously excluded bacteria. PA6206 or 103 caused dose­(io6-10S cfu/ml) and time-dependent (beginning after 1.5-4 hrs) cell killing. The fattem of Calu3 cell killing by toxic strains was similar to the pattern o binding of PAO 1-GFP: Calu3 cells were killed first at the periphery of islands or around small holes or ncar mechanical wounds, followed by sequential killing of cells adjacent to those already killed. We conclude: PA bind to the basal and lateral membranes ofCalu3 cells, and not to the apical membrane (where CFfR is located) or the underlying matrix. Access to the basolateral surface from the apical side of confluent mono layers is normally prevented by tight junctions. Epithelial cell killing by PA103 and 6206 occurs when bacteria interact with basolateral membranes. Support: NIH DK51799

292 PREVALENCE OF NONTUBERCULOUS MYCOBACTERIA (NTM) IN U.S. CF CENTERS.~. A. Faiz, B. Jones, C. Pue, M. Knowles. CF/Pulmonary Research & Treatment Center, Univ. of N. Carolina, Chapel Hill, NC, USA for The NTM in CF Study Group

The objective of this period, cross-sectional study was to estimate the prevalence of, and assess factors associated with, NTM in patients with CF. Three expectorated sputum specimens were collected over the course of a year from consecutively enrolled patients 2: age 10 at 21 sites. At enrollment, demographics and indicators of disease severity were collected via an adapted CFF Registry Questionnaire. Comparison of the study sample was made to the CFF Registry Database (RD). Specimens were processed at the site labs standardized by a proficiency study. All positive specimens were confirmed and stored at a reference lab. Enrollment began in February 1993 and closed at the end of January 1997 with 1200 patients. Prior to completion of specimen collection, 41 patients died, 27 were transplanted, II were dropped for clinical reasons, and S I moved or were lost to follow-up. To date, 849 patients have complete data available for analysis. The prevalence ofNTM among these 849 patients is 12% (102/849). Seventy two patients had one, 17 had two, and 13 had all three of the three cultures positive. Of the patients completing the study, 54% were male (RD • 52%) with an average age of2l (RD • 21) and FEVI% ofSSo/o (RD • 65%). Relative to culture negative patients, patients with positive cultures were significantly older (26 v. 21 yrs p<O.OOOI ), less likely to have P. aeruginosa (OR 0.6, 0.4-1.0), and more likely to haveS. aureus (OR 1.7, l.l-2.6). Patients with a single positive culture had significantly better lung function than patients with negative cultures. However with increasing number of positive cultures, the FEVI% significantly declined, suggesting an untoward effect of the organisms (113+ • 63%, 3/3+ • Sl%, p<0.04). NTM arc prevalent in the lower airways ofCF patients. Increasing burden of organisms correlates with worsened lung function. Supported In part by CFF A9U & NIH RR00046.

Poster Session Abstracts 287

293 FLAGELI.All MOTILITY IS REQUIRED FOR EmCIENT NONOPSONIC PHAGOCYTOSIS OJ' PSEUDOMONAS AERUG/NOSA BY -~CROPIIAGES. D. A, SimPSOn and D.P. Specrt. Department of Pediatric~, UIIIV. of British Columbia, Vancouver, B.C., Canada

PHildomontu ar~~g/IW$tl C8IISCI severe respiratory tractlnfcctlon In patients with cystic fibrosis (CF). Our lab hal bcco cxaminingnonopsonlc pbaJOC:ytosiJ at P. Mrllgl- by macmphages. In order to IIUdy the P. «r~~glno.ra-IIIICIOphagc Interaction II the molecular lcvcl, we have constructed a tnnspoiOII Tn5G bank In P. MrtiJlllfOMI 4020. Thilllraio iJ the Initial P. «rug/....., isolate roc:o¥CRd from a pediatric CP patient. MulaDII oC P. MrllgiiW$tl 4020 rcsistaDt to oonopsonlc phaJocyiOSi• were Identified u foUoq; The transposoo bank wu grown overniJ)lt and added to thloJ)ycollalo­clicited murine peritoneaiiiiiCIOphaJe monolayeR 11 a M.O.I. o1 approximately 1: 10. The bacteria were gcntrlfugcd on10 the monolayw to facilitate the interaction of all mulaDts with the macropbagCI. After 1 bour Incubation 11 Jrc, the 111pematant wu removed and added to the next macrophage monolayw. After passage CMt 3 to 6 monolaycn, the bacteria remaining In the 111pemataot were plated on selcctlve 1gar plates, and grown ovaniJ)lL This procedure wu repeated 5 times with the lonoculum for the next round of enrichment clcriw:d from the unlngcsted organisms liom the prcvi0111 round at sclcctlon. Bacteria roc:o¥CRd after thll enrichment had bcco passaged over a minimum of 18 macmphage monolayers. We have acreened approximately 600 individual mulaDtalsolaled liom thll enriched pool in a nonopoonic pbaJOC:ytosi1assay. We have iclcotlfied 85 Plllative mulaDts that are more resistant to phaJocyiOSi• than the parental 4020 llltain. Here we have ~baracteriud one oC these transposoD mutaoll. P. ar~~giiW$tl 4020 Hl7A wu !"gcstcd II I lcvcl -20% of the parcntalllltain. Thil mutant .,.,._sed a Tn5G insertion in I JC11C eucoding I proteiD poucsslng bomology to the MotA sene at several bac1Cria. Thil-rA::Tn5G mulaDt wu motile on1gar pta~a~ but wet mounts of the bacteria indicated that the mutant possescd an altered awlnuning behaviour. Partial complementation of the mutation wu observed when plasmid& containing the motA gene Isolated liom a cosmid bank of P. «,g;....., 4020 DNA were Introduced llf ll'tiiUinto the parental 4020 llltain. Partial complcmeotation of the mulation indicatel that the P. MrllgiiiOMI MotA protcin likely fonns part of a complex, and dcfcctlve copies at the prolcln are interfering with the fwlctiOD at the wild type protein. 'Tbc8c dala Rlggcsl that normal flagellar motility Is n:quired for the efficient Ingestion of P. IM!rugiiW$tl by macrophagCI.

294 HUMAN TRACHEAL lCENOGRAFTS IN SCIO MICE AS A MODEL TO STUDY TIE MECHANSMSOF NFmAY INFECTION BY PSEUDOMONASAERUGINOSA R nrouvanziam 1, S. de Bentzmann2, B. P&ault 1 and E. Puchella2. 1cNRS UPR9064. Nogent-sur-Mameand21NSERMU314, Reims, Franca.

Bacterial colonization Ia a hallmart< d CF airway pathology. ~. lhe mechanioma which render CF patianta men IUsc:eptibla to Infection, aa well aa 1hoee which positively se1act P. aeruginosa remain llldew, ciJe In particular to lhe lack olin w~ data onlhe lrwtial P. aerugmosa 1nfactian d CF airways. H~.m~n CF n non-CF felal and aalty poatnalallracheal xenog~afts In SCIO mice may 19P18sent a pertinent modal to study the Infection d airwaya by P. aeruginosa.lndeed, ltlese closed in IIi~ rap! less of_t'KJman airways: (1) dsplay a malin epithelial lining lncludng a pseudoatratifl8d aliated and seaetOIY surtace epithalil.m and llbnucosal glands (P&ault et a1. Him. Gene Ther. t994; 5: 1131-37); (2) ha111 ne\111' been exposed to pathogen· (3) secrete eirway surtace li<f.lid _(ASL) into their k.men; (4) exhibit malin bioelectrk: properties (e.g .. amloride-senSiti\11 sodum abSOflllion, AlP-dependent n cAMP-dependent chloride transports, lhe latter being absent from CF xenografts). Studes we111 carried out as follows. Firstly, ASl from malin Wlinfecled CF and non-CF tracheal xenograft& -1811l>ied and tested in llitroforitsbactaricidal IC!Mty. Secondly, airway xenograft& W8l8 Infected in IIi~ using lhe following protocol: grafts wal8 slightly Incised, the excess of ASl was rem0118d -and tested as abo111- and a high P. aeruginosa inoculum (PA01 strain, 50 to 100 111 at 1 0' to 1 0' CFUhnl) was Injected Into lhe graft lumen. !wenty-four hotn later, host mice wal8 analyzed for systemc and/or localized mftammaiOIY 188Ctions; inltaluminal bacteria wal8 <f.iantilated n airway xenograft& ware taated by lmmlmhistochemislly and by scanning and transmission electron ·~for (1). histological modlicsliona, lncludng both epithelial 181110defing and mftammatOIY 188Ctiona; (2) the pre~e at adleranl and intracellular bacteria. Thinly, freshly dissected xenog~alts W8l8 mlected ex WI~ and portions ware analyzed aa ~ alter 1, 3 and 24 hotn in contact with P. aeruginosa, to follow the kinetics of the 11teracllona between the airway epithelium and the bacteria at ealty timepoints. Results show that (1) ASL_from malin uninfected xenog~afta exhibit bactericidal activity; (2) P. aenJgl~oaa in)8Ctlon led to mart<ed. desquamation of lhe surtace epithelium; (3) bactaria wal8 lollld to adlere to exfoliated surtace epithelial cella and to aecondarily exposed basal oells; (4) in this in IIi~ modal, host inftammatOIY response was strictly fin1ted at the aile of i~ection.

The autholl thlrlk Drs C. Finc, M. Catala and F. Narey for supply of tunan tissues. Supported In pllt by lgrlllt hom SyStemlx Inc. and by Alaoclation Fran~IIH de Lutte contre t. Mucovlacldoae.

288 1997 Cystic Fibrosis Conference

295 297 EXPRESSION OF CHEMOKINE AND ICAM-1 MRNA IN LUNG EPITHELIAL CELLS FOLLOWING STIMULATION WITH BURKHOLDER! A CEPACIA FROM CF PATIENTS. R. W. Palfreyman, C. E. Eden, M. L. Watson, A. W Smith Department of Pharmacy and Pharmacology, Univ. of Bath, Bath, UK

Chemokine-induced leukocyte accumulation is implicated in airway inflammation in cystic fibrosis (CF). We have shown that B. cepacia elicits high levels of the neutrophil chemoattractant cytokine interleukin-8 from lung epithelial cells in culture and we have now extended this study to examine expression of intercellular adhesion molecule ICAM-1, which is associated with leukocyte migration, and other leukocyte chemoattractants. Cultured A549 epithelial cells or the CF tracheal epithelial cell line CFT -2 were stimulated with CF clinical or environmental B. cepacia isolates. ICAM-1 mRNA was strongly expressed 2-6 h after stimulation with whole cells of the clinical isolate. Cell-free supernatant alone did not strongly induce ICAM-1 mRNA, whereas induction was seen upon coincubation with interferon-y (100 U/ml). mRNA for the chemokines ENA-78, MCP-1 and eotaxin were also upregulated 2-6 h after stimulation, however RANTES and MIP-lalpha mRNA were not strongly expressed at these times. The levels of expression were higher in cells stimulated with the clinical isolate compared with the environmental strain. FACS analysis of surface ICAM-1, indicated greater constitutive expression on the CFT -2 cells, which was increased further upon stimulation with B. cepacia. These results suggest that bacterial induction of ICAM-1 and chemokine expression contributes to the characteristic inflammation in CF lungs. Supported by the Cystic Fibrosis Trust, UK

296 THE EFFECT OF INTRAVENOUS ANTIBIOTIC TREATMENT ON CHANGES IN SPIROMETRY IN CF. JM Bradley11, ES Wallacell, JS Elbom0 , JL Howard", MP McCoy•. Departments of Sport and ExerciiC Science111 aNI Physiotherapy", Univenity of Ultler, 1ordallllown. aNI tbe Rcpoaal Adult Cysai<: Fibrolis Centre, Belfast City Holpitsl0

, Northern lrela11d.

Pulmonary func:tion IIIC8IUred by spirometJy is an important indicator of rapiniOiy cxaa:lb8lions, and is tbe main outa>mc I1ICIIIWC c:urrendy used 10 monitor tbe eftlc:lcy of intmciiOUI (IV) antibiotic:s on acute respiniOI)' iDf<:e:tiona in palieats with C)'llic: fibrolis (CF). The objocti..e of Ibis IIUdy was 10 c:omp~~e dlanpll in spiromcUy f'ollowin& bome, hospital, and a combination of bomc and bospital II'CIIIIDCDt, in CF patients with an acute rapiliiOry iof<:e:tion. In addition, tbe outcomes of bospital and bomc based treatment were compared in -<:rely affected patients with baseline FEV1<40% precllc:led nonnal. A 10181 of 51 IV antibiotic lralments were analyacd. Spirometric IIICUIII'CS of luns function were compared between groups 11 tbe bellinnins (TI), and 11 tbe end (T2) of an IV course of antibiotics. Tbe perc:entase improvement (T2·TI)xl00 In each of tbe Iuns timc:tion paramciCII was also compared. IntravenOUS antibiotic lraiJDeot ligniftcandy illlpi'O\'Cd spirometri~ JDCaiUJ'CI of tuns function in all three groups. In eadl of the three tpOUps FEF,..,,. (absolute value and % pnxlided normal) lbowed tbe pate1t iiii)JI'CIYCIIIet in responiC ID IV antibiotics. Tbe mean )ICitCIIIale improYemcDt in Iuns ftmclion in tbe hospitaiJfOUP wu 29% (l'EV1), 25% (PVC) and 48% (FEF,..,,.). Tbis wu llipificantly sr- (p<O.OI) than tbe mean % improYemellt in luns timc:tion in tbe home II'CIIIIDCDt paup (l'EV1, I 1%: PVC, 10%; FEF,..,,., IS%), and in tbe combined bomc and bospital uatmenl poup (l'EV, 17%; FVC, 9%; FEF,..,,.., 36%). Patients with a PEV,<40% predicted normal who lllldcrwent hospital lraiJDeDI (n-18) bid a mean% Improvement of 43% in FVC, and 42% in PEF,..,,.. Tbis wulipiflc:antly peatcr (p<O.OJ) than tbe mean pen:ealllle improvement in FVC (17%), a1ld PEF,..,,. (27%) in patients with a FEV1 <40% predicted normal who underwent bomc trea1mc111 (n-6). In condulion, patients with CF ba\'e a peatcr improYement in FEV, FVC and FEF2,.,,. when treated with IV antibiotics In bospital compared 10 borne lralmeDt.

A COMPARISON OF VENTED VERSUS UNVENTED NEBULIZERS FOR THE DELIVERY OF TOBRAMYCIN . ~. CF MacNeish, LC Lands, D Meisner, S Kelman, EB Vadas; from the Divisions of Respiratory Medicine of the Hospital for Sick Children, Toronto and the Montreal Children's Hospital and Merck Frosst Inc., Montreal, CANADA

Tobramycin (tobra) is frequently given by wet nebulization in cystic fibrosis (CF) despite data suggesting that less than I 0% of the nebulizer dose is deposited in the lung. The Pari LC Jet+ vented nebulizer has a theoretical advantage over the Hudson 1730 T Up-Draft 11 unvented nebulizer in that the rate of drug output increases during inspiration in the vented nebulizer thereby reducing the relative amount of drug wasted during expiration. Six examples of each nebulizer were charged with 80 mg of tobra in 4 mL and driven by a PulmoAide compressor. A simulated inspiratory flow (Vi) at 0, 5, 10, 15 and 20 Umin with a relative humidity of 40% was directed into the vent in the Pari and through the t-piece in the Hudson. Drug output was calculated from changes in weight and concentration of tobra. Particle size distribution was measured by laser diffraction analysis. Total (Tot) drug output and output in the respirable(< 5 Ill") fraction (RF) were compared as were the tobra output rates. Total drug output was similar (51±2 mg vs 55±5 mg, Pari vs Hudson, mean±SD) and it was not affected by Vi. The rate of output was measured during the time prior to intermittent aerosol output. When Vi=O, it was similar (3.35±0.48 Tot and 1.72±0.23 RF vs 3.88±0.52 Tot and 1.47±0.09 RF (mglmin) (Pari vs Hudson). While the addition of Vi had no effect on the Hudson, it increased both the total output and that in the RF for the Pari. At Vi=20 Umin the output was 9.87±0.79 Tot and 6.11±0.74 RF (mg/min). This enhanced output during inspiration with the vented nebulizer would reduce the relative amount of drug lost during the patient's expiratory phase. Hence, for a CF patient with a mean inspiratory flow of 20 Umin and inspiratory/expiratory times of 2/3 seconds respectively, the vented nebulizer would be expected to result in double the pulmonary deposition compared to the unvented nebulizer.

Supported by the Canadian Cystic Fibrosis Foundation

298 DELAY OF REC'lJRRENCE OF PsEUDOMONA.S A.ERUG/NOSA. IN PATIENTS WITH CYSTIC nBROSIS WITH INHALED COLISTIN AND ORAL CIPROXIN: A COMPARISON BETWEEN 3 WEEKS AND 3 MONTHS OF TRJ:ATMENT. B. Frederiksen, A Han1eo, C. Koch, *N. Hsiby. Dep. of Pediatric and "Clinical Microbiology, The National University Hospital (Rigshospitalet), Demnark.

We have previously lhown that treatment of early Pseudomonos aerugtnosa (P.a.) colonization in patients with cyllic fibrosis (CF), using 3 weeks inhaled colistin, 1 Mill iu b.Ld. plus oral ciproxin, 25-50 mglkglday divided in two doses, polltpones onset of chronic P.a.­infection. In view of this result, all non-infected patients have since 1989 been treated according to an intensive ''3-step-protocol": lltep I (first isolate): 3 weeks colistin, 1 Mill. iu b.Ld._+ ciproxin, step D (the following isolate( 1 )): 3 weeks colistin, 2 Mill. iu t.i.d. + ciproxin, step m (third isolate within 6 months): 3 months colistin 2 Mill. iu_t.i.d. + ciproxin. After several yeara using this regime, a "test for trend" showed that step m aeemed superior to 11tep1 I and D. Thia waa thm studied in a controlled prospective manner in S 1 CF patients, only intermittently colonized with P.a., by randomization to 3 weeks or 3 months of colistin, 2 Mill. iu t.i.d. + ciproxin. Cuhures of sputum are done every month, and the endpoint was recurrmce of P.s .. We could not use time to ouet of chronic infection, since the routine "3-step protocol" baa led to a reduction in annual incidence of chronic P.a.­infection to <2%, with 80% of patients treated with the "3-lllep protocol" having more than 7 years between the firlil isolate of P.a. and oBJet of claronic infection. Nine patients were excluded, leaving 20 in the 3-week group and 22 in the 3-montha group. There were no dift'ermces in age or sex between the groupa. The median time inter­val from ouet of treatment to recurrence ofP.a. was 9 months in the 3-week group, venus 18 months in the 3-months group (p<O.OS). Copdga!oP: 3 months treatment of early P.a. colonization with in­baled colistin plus oral ciproxin is superior to3weeks treatment in de­laying the time until recurrence of P.a. in sputum.

299 MOLECULAR INVESTIGATION OF HIGH-LEVEL TOBRAMYCIN RESISTANCE. p.L. MacLeod. R.M. Shawar, L.E. Nelson, L.G. Lockwood, B.E. Lin, J.E. Dirks, J.L.Bums ', B.W. Ramsey •, R.L. Garber, PathoGenesis Corporation, Seattle, W A 98119; 'Children's Hosp. &. Med. Ctr., Seattle, WA 98105, USA

During a recent phase lii TOBI'"" clinical trial that tested aerosolized delivery of tobramycin or placebo to over 500 cystic fibrosis patients, approximately 14,000 isolates of Pseudomonas aeruginosa were archived. A detailed molecular microbiology study was carried out on sputum isolates from a patient who was randomized to tobramycin and who at baseline (prior to initiating drug therapy) had the highest MIC strain (5121'g!mL). Susceptibility testing of this patient's isolates found tobramycin MICs ranging from 0.25 to 512 Jtg/ml. Strain genotyping using RAPD PCR fingerprinting identified a total of five different genotypes over the 6 month trial. One genotype included all the high MIC isolates (MICs ranging from 32-5121'g/mL). A second genotype included the majority of susceptible isolates. Three genotypically unique isolates appeared only transiently during the course of the trial, specifically at the time of a pulmonary exacerbation. High level resistance to tobramycin correlated with the presence ofacetyltransferase modification genes located on a large plasmid. This plasmid was capable of stably transferring tobramycin resistance to drug-sensitive E. coli and P. aeruginosa in the laboratory. Despite starting the trial infected with a P. aeruginosa strain whose MIC level would be considered too high to respond to parenteral therapy, this patient showed improved lung function on TOBI therapy. More importantly, this strain did not become the dominant genotype of P. aeruginosa infecting this patient, nor did it transfer its resistance capacity to co-resident nonresistant strains. In conclusion, MIC testing may not predict clinical response to TOBI therapy.

300 MOLECULAR EPIDEMIOLOGY OF Pseudomon11s 11eruglnos11 STRAINS FROM CYSTIC FIBROSIS PATIENTS RECEIVING AEROSOLIZED TOBRAMYCIN. 8 M Shawar, B. B. Lin, J. E. Dirks, D. L. Macleod, L. G. Lockwood, J. L. Burns', B. W. Ramsey', and R. L. Garber. PathoGenesis Corp., Seattle, WA 98119, 'children's Hosp. & Med. Ctr. 2, Seattle, WA 98105, USA.

The aim of this study Is to evalua.te the genotype «?f serially collected P. aerugmostl sputum tsolates from Cysttc FibrOSIS patients and to correlate genotype wtth morphotype and mintmum inhibitory concentration IMIC) to tobramyctn. Patients were receiving 28 day on/off intermittent therapy with tobramycin for inhalation (TOBI"'I during a phase Ill clinical trial. The MIC to tobramycin was determined by microdilution susceptibility in Mueller-Hinton ~roth. Over 25 isolates with various morphotypes were studted from each patient. Patients were chosen because of recovery of at least one P. aeruginos11 isolate with a tobramycin MIC of ~ 128 Jlg/ml at eny time durinp the trial. The series of isolat~s studied rerresented a wtde range of MICs to tobramyctn (from s 0. 2 to 612 l'g/mL). Random Amplified Polymorphic DNA (RAPDI analysis was used to characterize these strain!'. DNA amplification was conducted by polymerase chatn reaction using a single primer, and products were separated by agarose gel electrophoresis to yield characteristic patterns of multiple bands. Soma genotypes were represented by up to 4 morphotypee. Each patient harbored 2 main genotypes throughout the trial, and up to 3 additional transtent genotypes were seen. Genotypes often correlated with MIC, although some genotypes spanned . a wide ~ange ~f MIC levels. In conclusion, RAPD analys11 made tt posstble to determine when a particular ~train was acquired by.~ patient and whether strains having different drug suscepttbthty.arose from acquisition of a different strain or from c.hanges tn the susceptibility of the existing strains of P. IIBrugmosB.

Poster Session Abstracts 289

301 COMPARISON OF THE ANTIMICROBIAL, NEBULIZATION, AND

AIRWAY TOLERANCE PROPERTIES OF COLISTIN AND POLYMYXIN E,. V n0evanter1

, E. Tolentino', J. Van Oalfsen', B. Lin', A. Montgomery', T. Standae , and T. Gordon•. 1- PathoGenesis Corporation, Seattle, WA, USA, 2-Cystic Fibrosis Center, Children'• Hospital Medical Center, Seattle, WA, USA, and 3-Nelson Institute of Environmental Medicine, N- York University Medical Center, Tuxedo, NY, USA.

The intravenous (IV) formulation of Colistin (also known •• Coly·Mycin) 11 a powdered prodrug of the broad spectrum gram-negative antibiotic Polymyxin E frequently administered as an aerosol to cystic fibrosis patient• with chronic lung Infections. IV-formulated Colistin Is Inactive against gram negative pathogens, but hydrolyses slowly to release Polymyxin E, formaldehyde and sulfites, suggesting that aerosolized Polymyxin E may be a aafar, mora effective therapy than aerosolized Colistin for cystic fibrosis patienta. The objective of this study was to compare the In vitro and In vivo properties of substantially pure Polymyxin E1 and the traditional tV-formulated Colistin as aerosol therapies. Isotonic, pH balanced Polymyxin E1 (-95% pure) was compared with freshly dissolved tV-colistin with respect to 1) anti­Pseudomonal activity, 2) aerosolization properties and 3) airway tolerance In a guinea pig airway reactivity model. One hour after dissolution of tV-colistin solution pH had risen to > 7.5, while Polymyxin E, pH remained atable. Incubation of tV-colistin with Pseudomonas eeruginosa demonstrated an apparent hydrolysis rete In culture media of about 3% par hour. 64 1111/ml Colistin was required to exact the same bactericidal effect as 0.5 11glml of Polymyxin E, after one hour of incubation with 1 o' bacterial cella per ml media. Polymyxin E, aerosolized more rapidly than tV-colistin doses In 1 Pari LC Plus jet nebulizer (4 ml dose In 8.3 ! 1.1 and 10.5 ! 1.5 minutes, respectively). Foaming and sputtering of Colistin may have lncreesed aerosolization times, an effect not observed with Polymyxin E, formulations. Under the same conditions, 4 ml of 0.9% saline eerosolized In 6.9+ 1.22 minutes. Other aerosol parameters (mass median aerodynamic diameters, respirable fractions) were similar for all three solutions. Guinea pig airway reactivity to acetylcholine challenge was measured following 20 minute exposures to either 20 mglml Polymyxin E, or Colistin, and no difference In airway reactivity was observed between exposure groups. These results suggest that Polymyxin E, could be used as 1 more effective and potentially safer aerosol than Colistin for treatment of Chronic pulmonary Infections In cystic fibrosis. Supported by PathoGenesis Corporation.

302 OPTIMAL METHODOLOGY FOR ANTIBIOTIC SUSCEPTIBILITY TESTING OF PSEUDOMONAS AERUGJNOSA. L Sajm;w1, J. Burns2, S. · Whittier', J. Krzcwinski2, R. Jones•. Dept. of Pediatrico and Pathology, Columbia Univ. N.Y.1.>, N.Y., Dept. of Pediatrics, Univ. Washington, Seattle, Wash.2

, Dept. of Pathology, Univ. oflowa, Iowa City,IO:

The optimal methodology for antimicrobial susceptibility testing of multiple drug resistant P. aerugiiJOSa, particularly mucoid and slower growing strains, has not been established. Accurate and reproducible detection of antibiotic resistance is critical for patient management, infection control and, potentially, to assess eligibility for lung transplant. We sought to determine the optimal methodology for antibiotic susceptibility testing by comparing several commercial systems. Agar dilution, the established reference method, was compared with microbroth dilution (PML Microbiologicals, Portland, Ore.) to determine if the fonncr technically demanding method could be replaced with a more convenient "gold standard". Three laboratories studied 98 strains: 48 P. aeruginosa isolates from individual CF patients (2S mucoid and 23 non-mucoid) and SO isolates from non-CF patients. Susceptibility to 12 antimicrobial agents was tested: amikacin, gentamicin, tobramycin, ciprofloxacin, pipcracillin, ticarcillin, pipcracillinltazobactam, ticarcillin/clavulanate, ceftazidimc, aztreonam, imipenem. and meropenem. Correlations between methods were excellent for all parameters examined. Comparison of 7,143 24-hour readings of. agar and microbroth dilutions read by the 3 laboratories yielded correlation coefficients ranging from 0.86 for ciprofloxacin to 0.96 for gentamicin. Correlation coefficients for 24 and 48 hour readings ranged from 0.91 for aztreonam using microbroth dilutions to 0.98 for gentamicin using agar dilutions. Serious discords in the categorization of susceptibility vs. resistance occurred rarely (1.1-2.1%) and were more likely to occur for CF strains than non-CF strains, 2.0% vs. 0.9% respectively. These studies confirmed that microbroth dilution could substitute as the gold standard for ongoing studies of SOO CF strains comparing antibiotic susceptibilities generated by E-test antibiotic impregnated strips, Kirby Bauer antibiotic disks, Vitek® and Microscan® microbroth dilution methodologies. These studies will generate clear recommendations to establish clinical microbiology laboratory standards for susceptibility testing of P. aeruglnosa strains isolated from CF patients. Funded by the CF Foundation

290 1997 Cystic Fibrosis Conference

303 305 BURKHOLDERIA CEPACIA EPIDEMIC STRAIN MARKERS

AMONG GERMAN CYSTIC FIBROSIS ISOLATES. A. Bauernrejnd1, S. SchwOrer1, R. Jungwirth1, C. Roller•, H. Bllrmeier, 1Max von Pettenkofer-lnstitut, MOnchen, FRG, 2Universitllts­

Kinderklinik, Erlangen, FRG.

Spread of Burkho/derla cepacia (B.ce.) among cystic fibrosis (CF)

patients is supposed to correlate with epidemic markers, namely the

cable like protein (cblA) or the B.ce. epidemic strain marker (BCESM)

described recently (Mahenthiralingam et al., J. Clin. Microbial., 1997).

We asked the question for the incidence of both markers in B.ce.

isolates of 62 patients from six German CF centres. Identification of

the B.ce. strains was verified by PCR with specific 16S rRNA primer

pairs. For detection of the cblA- and the BCESM-genes PCR with

specific primers was performed. Two cblA-positive strains (3%) and

25 BCESM-positive strains (40"/o) were identified. Only one of the

cblA-positive strains was as well BCESM-positive, the other one had a

different amino acid sequence (22 aa substitutions) in comparison with

the sequence of strain BC7 (Sajjan et al., J. Bacterial., 1995). Two

groups of patients (three resp. four) harbouring identical RFLP-types

of B.ce. were cblA-negative, while two out of three and one out offour

were BCESM-positive. We conclude that among our B.ce. strains the

cblA marker is rare while the BCESM is rather wide-spread. This may

indicate a so far low incidence of epidemic B. ce. spread among the

German population of CF patients.

304

The detection of genes encoding puUitlve transmissibility factors among epidemic and non .. pldemlc strains of Burlrholderl•

c.,.cl• In cystic fibrosis

FE eLOpE ME KAUFMANN, TL PITI

Epidemiological Typing Unit, Laboratory of HospitallnfC(;tion, Central Public Health Laboratory, 61 Colindale Avenue NW9 SHT

Bwlcholderia (Pseudomonas) cepacla is an important opponunistic pathogen in cystic fibrosis (CF) sufferers. Many patients with B.cepacla remain symptomless, but approximately 20% succumb to a severe often fatal necrotising pneumonia sometimes with septicaemia. The transmission of certain strains of B.cepacia amongst CF patients attending clinics and social gatherings has been repeatedly described but there has been a suggestion that all strains do not have the same propensity to spread. A study of B.cepacia from UK CF Centres showed that although there were over SO ribotype patterns identified among 178 patients, one profile, ribotype I, was widespread within and between centres. Other studies have demonstrated the presence of giant peritrichous flagella (cable pili) associated with these epidemic strains which has been associated with adherence to respiratory mucin. Funhermorc, a hybrid of two insenion sequence elements (I S 402 and IS/ 356), hu been reponed to be more prevalent among strains of B.cepacla tranamitted between patients than sporadic single cases.

We have detected genes encoding cable pili (cb/A) and the IS hybrid by PCR in a collection of6S isolates of the ribotype I epidemic strain and compared their fiequcncy against a collection of non-epidemic strains of various ribotypes. The control strains all lacked the cb/A gene and IS hybrid. In contrast all of the epidemic strains were cb/A positive and most of those teated so far have been positive for the IS hybrid. We conclude that the presence of cable pili and IS elements may be sood indicators of the potential ability of strains of B.cepacia to spread between CF patients.

THE MORBIDITY AND MORTALITY ASSOCIATED WITH TWO EPIDEMIC STRAINS OF BURKHOLDER/A CEPACIA WITH GENOMOVAR m STATUS IN CYSTIC FIBROSIS PATIENTS. CS Haworth ME Dodd, C Doherty. M Super. G Hambleton, P Vandamme, JRW Govan, AK Webb. The Adult Cystic Fibrosis Unil. Wythenshawe Hospilal. Manchester. UK

Molecular phylogenetic studies now suggest that organisms presently identified by conventional tests as Burkholderia cepacia represent several new Burkholderia species (or more correctly genomovars). Both epidemic strains or B Cepacta (strains I and 2) currently identified in lhe Manchester region have genomovar Ill status. Using other typing techniques, strain I has been classified as RAPD I, ribotype I and bacteriocin type Sl3/PO. Strain 2 has been classified as RAPD 13. ribotype 3 and bacteriocin type S3/P6. Bacteria within each strain ha\·e been shown to be identical by pulse-field gel electrophoresis. Two lransmission factors have been identified for B. cepacia; strains 1 and 2 both have the specific DNA transmission sequence. and strain I also has 1he cable pilus. We report the clinical outcome of two epidemic strains of B. cepacia with genomovar Ill stains in C).,.ic fibrosis palienl5 The following data was collected by a retrospective review of patient records. Group I, colonised with strain I. has 32 palicnts (20 alive and 12 dead) and group 2. colonised with strain 2. has 14 patients (8 alive and 6 dead). The FEY! % predicted a1 acquisition (median and range) or group I was jj j% ( 16.j-90.6) and of group 2 was 63.9% (28.3-101.6). There •s no significanl difference between them. Four patients have been removed from fun her analysis; in 1wo palienls from group I B. cepacia has been eradicated, which is unusual. and one from each group has received a transplant. There is no significant difference in I he median FEV I o/o predicted values at B. cepacia acquisition between those who are alive and those who have died within and between each group. There IS no Significant difference between the groups for the median time from B. cepacia acquisition to death: 2.8 years (range 0.2S-S.83) for group I and 3.7S years (range 0 3-7.6) for group 2. There is no significant difference between the groups for the median time from B. cepacia acquisition to the present time for those who are aJi,·e: S 00 years (range 2.83-7 9) for group I and S.08 years (range 2 0-8 66) for group 2. The number of deaths in each group did not differ significantly In conclusion. no sign;ficant differences in clinical oulcomc have been demonstrated between these two epidemic strains of B. cepacia with genomovar Ill status. This data provides additional confirmation of the morbidity and monality associated with genomovar Ill strains.

306 Biochemical and Genetic Characteristics of Epidemic Stnlns of Burkllolderla CqHICia Recovered from Patients with Cystic Fibrosis. E. Mahenthiralingam. D.A. Heruy, P. Vandamme•, M.E. Campbell, D.P. Speert. Dept. of Pediatrics, Univ. of British Columbia, Vancouver, Canada and l..aboratorium voor Microbiologic, Univ. of Gent, Belgium. •

Seven epidemic cystic fibrosis (CF) strains of Burlcholderia cepacia, types I, 2, 4, 6, 13, 17, and 40, were identified by random amplified polymorphic DNA (RAPD) typing. Types I, 2, 4, and 6 infected multiple CF patients attending clinic in Vancouver; type 2 B. cepacia encoded the cable pilus gene and is the major CF lineage, ETI2, responsible for epidemics in Canada and the United Kingdom; typeS 13, 17 and 40 were responsible for epidemics in Manchester, UK, Cleveland, OH, and New South Wales, Australia, respectively. Each of these epidemic strain typeS encoded the B. cepacia epidemic strain marker (BCESM), a DNA marker specific for transmissible CF strains which was identified by RAPD fingerprinting. All seven epidemic strain typeS were found to cluster within genomovar Ill of the B. cepacia complex. B. cepacia CF strains which to date were not associated with patienl-to-patient spread, or which were associated with transient patient colonization, did not possess the BCESM and were not within genornovar Ill. Examination of the epidemiological characteristics of RAPD typeS I, 2, 4, 6, and 40 demonstrated that these typeS were more likely to cause chronic colonization in either uncolonized patients or patients already infected with apparently nonepidernic strains. Biochemical analysis ofRAPD typeS I, 2, 4, 13, 17 and 40 demonstrated that all were positive for sucrose oxidation and lysine decarboxylation. B. cepacla type 6 (BCESM positive) and genomovar II strains were negative for sucrose oxidation and displayed variable reactions for lysine. Of I S9 BCESM negative isolates of unknown genomovar, 400/o were sucrose positive, 70% were lysine positive and 2S% were negative for both sucrose and lysine reactions. In summary. characterization of B. cepacia strains associated with ptltient-to-patient spread in CF demonstrated that they share considerable biochemical and genetic similarities.

307 NOSOCOMIAL TRANSMISSION DOES NOT ACCOUNT FOR A CHANGE IN PREVALENCE OF BURKHOLDER/A. CEI'A.C/A. INFECTION IN CYSTIC FIBROSIS PATIENTS ATTENDING AN AUSTRALIAN CLINIC. Robinson M'. Parsons SW1

, Paul M'. Torzillo P1, Hemming AL1

, Moriarty C1, Bye PTP1

•

Departments of Respiratory Medicine (I) and Microbiology (2), Royal Prince AI~ Hospital, Sydney, Australia.

Despite strict patient isolation procedures, an increased incidence of Buriholdtria upacia (Be) infection has been noted amongst patients with cystic fibrosis (CF) attending our hospital. Data from some North American and European centers have suggested the presence of a dominant strain accountin& for the majority of infections. This implies that the dominant strain is transmitted either nosocomially or by person­ro-person spread. Aim: To genetically type all Be isolates from sputum samples of patients with CF and determine if a dominant strain exists within this population. Methods: Be was grown from sputum samples using selective media. All Be thus isolated were examined using pulse field gel electrophoresis (PFGE) and examined for the presence of identical strains. Case records of individual patients were reviewed and associations between infected patients, in terms of temporal relationship of inpatient treatment, attendance 11 outpatient clinics and social contact were documented. Results: B. etpaeia was found to be transiently present in two patients in 1992. The following table documents the incidence and prevalence of Be in subsequent years.

1993 1994 1995 1996 1997 Total Clinic No. 96 108 Ill 119 131 B. eepacia +Ye 2 s 10 14 13 Prevalenee ( 'll>) 2.1 4.6 9.0 11.8 9.9 Newcasea 2 (2)0 s (3)0 4 (2)0 9 (3)0 0 Incidence ( 'J1,) 2.1 4.6 3.6 7.6 0

(n)• z Number of patoents who were B. ctpacw posouve pnor to theor first attendance at our institucion; 1' data only available to May. Be has been isolated in 20 patients. Three clusters of isolates involving 8 patients have been identified. Three siblings share a common strain which is unique to that family. These cases account for half of the 1996 new isolates from patients whose primary care has been 111 our institution alone. Two siblings and an unrelated third patient share a common strain that was acquired prior to transfer from another hospital. The two remaining patients with the third strain co-habitate. All other strains are unique to individual patients. Of the Be positive patients, three have died -all of who had severe lung disease and Be when lint seen Ill our clinic. There has been no significant deterioration in lung function in the group of patients who first acquired Be 111 our institution. Conclusloas: A dominant strain of Be does not exist in our CF population. For strains of Be present in two or more patients, close out of hospital contact was found in all but one case. This suggests that the spread of Be in these patients was person-to-person and occurred away from the hospital setting.

308 Comparison of culture and PCR drl«llon of B•rltiiDidlrlil t:tptldllln sputum of patients with cyallc flbrooll. P W Whitby', H .L. N. Dick', D. E. Tullis' and T. L. Stull'~- Departments of Pediatrica1 and Microbiology/Immunology', University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, 73104 and the Department of Medicine and Microbiology', The Wellesley Hospital, University ofToronto, Toronto, Canada M4Y 113.

Culture detection of Burltllo/derla eqxx:ia in sputum from patients with cystic fibrosis (CF) is problematic and may be partially due to the_recent demonstration thlll isolates currently identified as B. cqxx:ia consiSt of several

1peciea, the "B. ctpat:la complex." Growth of B. cepacia may require 72h or ~re, and species from other aenusea arow on selective media. requirina further testrng to establish an accurate identification. lmprovemenll in the detection of B. cepaciD would not rely on culture, would detect the organism in low quantities, and would be specific to B. cepaciD. Previously we had demonstrated the efficacy of polymerase chain reaction (PCR) in the epodemiologic analysis of B. cepDcia from a number of outbreaks. We investiaated the utility of PCR to detect B. cepDela directly from sputum samples. Followinaliquefaction of the sputa using N-ac~tyl lysine, DNA was isolated and analyzed in PCRs with three different p~mer paors. directed toward the ribosomal RNA loci. Two pnmer paors were putatively spectfic (or B. cepacla. The other pair, which universally amplifies a band from all bacteria. served as a control. Sputum samples were obtained from 104 adult and 110 child CF patients and analyzed independently by culture and by PCR to detect B. cepacla. The analyses were performed blinded with respect to each other. Results of the PCR with adult sputa demonstrated that primers directed to the 16S loci were more specific than those targetina the 16S-23S spacer reaion, and demonstrated a 9S% concordance with culture results. In addition, the 16S primer pair identified B. cepaciD in two patients whose sputa were culture negative. One of these p111ients is interrnittantly colonized while the other has not been culture positive. In contrast to the analysis of sputa from adult CF patients, PCR of specimens from children revealed multiple nonspecifiC bands, suggesting tha~ these primers may be less specifoc in the presence of mouth nora. The results of thts study indicate the utihty of PCR as a doagnostlc method for the identlficatoon of B. eepacla in the sputum ofCF patients. We anticipate thlll improvements on our taXonomic undentandina may allow the des ian of more specifoc primers for detection of B. upacla complex in sputum.

Poster Session Abstracts 291

309 MOLECULAR DETECTION OF BURKHOLDER/A CEPACIA FROM SPUTUM

Xu Jlru1, Jobg f Moorc1, Adrirnnr Sbaw1, J. Stuart Elborn2, A. Krvin Webb , Dennis J. Shalr .. Martin D. Curnn5

& Philip G. Murphy1 1 Molecular EpidemioloiY Research Unit Department of lla<teriology, Belfast City Hospital, l NOithem Ireland Adult Re1ional Cystic fibrosis Centre, Depanmenl of Respiratory Medicine, l.ncl B. Belfast City Hospi"'~ ' Brodbul)' CF Centre, Manchester 4 UWCM., Llandough Hospilal, Wales· 5 Nonhcm Ireland Tissue Typina Laboratory, Belfast City Hospital. Belfast.

Con_ventional techniques have misidentified other Gram negative orgamsms such as S/enolrophomonas mallophilia as B. cepacia, due lo colonial and phenotypic similarities. In addition, due to close phylogenetic relationships, separation of closely-related taxa is very difficult with conventional techniques but is of el<treme importance, as categorisation with B. cepacia infection has major significance in lenns of infeclion control, psychosocial issues an~ patient management. Therefore as it is important to detecl B. cepac1a efficrently, the arm ofthe study was to employ a sensitive PCR-based detection assay for both lhe detection and confirmation of B. cepacia from CF sputum. Various DNA exlraction protocols have been investigated with CF sputum in order lo optimise the recovery of chromosomal DNA from B. cepacia in sputum prior lo PCR amplification. Using a specific PCR method based on the amplification of a 209bp fragment of the 16S rRNA gene, all confirmed culture-positive isolates of B. cepacia, as well as all atypical B. cepacia colonies from the N.lreland Adult Regional Cystic Fibrosis Centre were confirmed. In addition, on el<Art1ination of sputum from CF patients, who were conventionally classified as culture-negative for B. cepacia by culture techniques, we demonstrated the presence of B. cepacia in the lungs of these patienls. Of lhrce centres examined, we detected 12/13 (92.3%) PCR-positivelculture-negative patients within the Belfast centre and at the Manchester Adult CF Centre, 13118 (72.2%) pat1ents were PCR-positivelculture-negative. At the CardifTCF Centre, where there is an extremely low incidence of B. cepacia, we were unabl~ to detect B. cepacia by PCR in 31/31 culture-negative sputa exammed. We postulate that prior to a rapid decline in pulmonary function of the CF lung due to B. cepacia, this organism may inilially exisl at very low numbers whrch are non-detectable using conventional bacteriological culture techniques. These studies probably demonslrate the presence of low numbers of B. cepacia in the CF lung. similar to the PCR detection of low numbers of Pseudomonas aeruginosa. This data therefore requires further investigations to ascertain its clinical significance in order 10 direct appropriate cross-infection control procedures, between patients who are PCR positive/culture-negative and those who are PCR posilive/culture­posilive for B. cepacia

310 OCCURRENCE OF BURKHOLDER/A CEPACIA IN THE ENVIRONMENT

John E.Moon:1, Brian Mcllhatton1, Dridre Gllpin1,

Judy Buchanan1, Adrienne Shaw', J. Stuart Elbornl, & Philip G. Murphy1 1 Molecular Epidemiology Research Unit N. Ireland Public Health Laboratol)', Belfast City Hospital, l N. Ireland Adult Regional Cystic Fibrosis Cenlre, Department of Respiratory Medicine, Level 8. Belfast City Hospital. Belfast.

Colonisation and subsequent infection with B. cepacia can lead to the rapid deterioration in lung function of the CF patient and therefore prevention of colonisation of the CF patient is very important in order to reduce carriage amongst CF patients. Consequently it is extremely important that sources and routes of transmission of this organism are fully elucidated, so that adequate cross· infection control measures are put in place to prevent environmental acquistion and person­to-person spread. The aim of this study was therefore to identify environmental reservoirs of B. cepacia, including natural niches of the pseudomonads such as food and water. Briefly, B. cepac/Q was isolated by initially preincubating the specimen [swab, food. water, device] in nutrient broth at 30°C for 24h to allow for recovery of any sublethally damaged organisms. Subsequently, I 0111 of broth was streaked onto B. eepDcia selective agar (MAST Laboratories Ltd., England) and incubated at 30°C for 48hrs and for an additional 3 days at ambient temperature. All presumptive positive colonies were con tinned using the API20NE system. A total of689 specimens were examined comprising of266 foodstuffs, 137 waters and 286 specimens from the CF clinical environment. Of these, 23 (3.3o/o) were positive for B. cepacia. where 14126 (53.8%) samples of raw unpasteurised milk were positive, as well as IllS (6.7%) sample of dried milk. In addition, 4/17 (23.So/o) air samples from in-patient wards [B. cepacia positive units) were positive, as well as 4/10 (40%) of sputum collection container surfaces. As all milk isolates were phenotypically different to the usual colonial morphology of clinical isolates. a specific PCR method was employed which was based on the amplification of a 209bp fragment of the 16S rRNA gene, whereby all milk isolates of B. cepacia were con tinned molecularly. This study demonstrates thai the practise of drinking unpasteurised milk by CF patients presents bacteriological risks associated with potential B. cepacia acquistion. In addition. the study shows that CF patients disseminate B. cepacia into their clinical environment. especially via air post

292 1997 Cystic Fibrosis Conference

physiotherapy and by hands. Overall this study confinns the need for good cross infection control procedures and continued segregation of colonised and non­colonised patients in order to minimise person-to person spread of this organism.

Supponed by the Wellcome Trust and Lilly Industries Ltd.

311 Molec:ular epidemiology of Pseudomonas aeruginosa and risk of c:ross-infedion in a cystic: fibrosis patients' holiday camp. K P Hw!Cejd•. C. Schmidt•, T.A. Wichclhaus•, B. Krackhardt•, H. G. Posselt+, J Bargon , J. Yahat", V. Schafer•, V. Brade•. •Department of Medical Microbiology, 1>epartment of Pediatrics, ' Department of Internal Medicine, University Clinic. FrankfurtJM, Germany, • Chaim Sheba Med. Center, Tel A\'i\', Israel.

There is C\'idence of cross-infection with P. atruginosa in CF patients both in the hospital environment and through social contact, but the means of transmission of this organism arc 110( well understood. We present an epidemiological study on P. a~ntginosa in a JrOIIP of 18 German CF patients (median age: 22,8yrs) during a three week holiday camp in Israel in January 1996. Sixteen patients showed prC\•ious chronic or transient colonisation with P. aeruginosa. Two patients were temporarily colonised \\ith S.aurcus before, but never acquired P. aeruglnosa. Sputum cultures at the beginning and one week after the camp were examined for P. a~ruglnosa. Suceptibility testing of the strains was performed by disc diffilsion according to the NCCLS crileria and the bacteriological changes 0\'CI' the holiday period were documented. Strains dissimilar in macroscopic appearance and/or antibiotic resistance patterns were separately processed. FiftySC\-en P. aeruginosa strains before entry and 5I strains one week after the camp \\'Cre obtained from 15 out of 16 prC\iously positi\'C patients. Spulum samples of the two formerly negative patients yielded no gro\\1h of P. aeruglnosa during the microbiological follow-up in 1996. Gcnotyping of the isolates was performed using pulsed-field-gel-electrophoresis (PFGE) after digestion of the genomic DNA with restriction endonuclease Spe I. Furthermore these isolates were investigated in comparison to 38 &imilarly processed sputum isolates obtained from 12 Israclian CF patients joining the camp for one week. Finally, genetic polymorphisms of DNA fragment patterns from all strains \\-ere studied for their 0\'Crall relatedness using a fingerprint analysing software system (CYBERTECH0

). No strict correlation between phellll(ypic appearance, antibiotic susceptibility patterns and genotype of the isolates was found. Most of the German patients \\-ere constantly colonised by one or two, fC\v by more indi\·idual genotypes during the four \\'CCI< screening period. Interestingly, 9 out of 12 lsraclian patients harboured one or two identical or closely related genotypes, possibly due to infection from a common source or as a result of preceding epidemic spread of certain strains. Additionally, isolates shO\\ing identical PFGE patterns could be demonstrated once in a male lsraclian patient and in a female German patient after the camp. suggcstins pmious cross-infection. Our study supports the occurrence of cross-infoction through social contact in CF patients. MotCO\'Cr, a marked difference in the epidemiology of P. aeruglnosa colonisation in the two groupt1 of CF patients from Germany and Israel was C\ident requiring further im'Cstigations.

312 CPPATIENJ'S DO NOT CONTAMINATE lliEIRBNVJRONMENT DtJRINO EXERCISE. .._ HernJcll, Linda Boykea, Bichant Ahre!ll. Dcpartmeab of IDianal Medlc:lne and PediaUtcl, UDiv. of !owl, Iowa City, lA, USA.

Ollce blcterlll ptllbopal, perllc:ularly mucoid P. Mrwg#tto.JQ (PA), '-no Clllblllbetlln tile 1111111 rl CF plticats (ptl), !bey an: rarely endlclletl. Pts who an: dii'Oalc:llly lnl'ec:fed by PA ud llleriCqllire S. 1111rn1 (SA), 111\'e a lipHk:andy ~bolter life expec:IIIICy lbaa Jill Infected by - « the other oflheac blctlria. 1'berefore, pmcntilla pll from IICIJ!Iirlnlacw Jlllhopal may be more clflot:llw lbaa IRIIIDIIUblaqucat lofoctioDI. lfmm-cr. we know little lbout the IIIUI'CC ofbcterla dlat IDI'ect the 1111111 rl CF pt1. Some -m.en think dlat CF pll an: 1D impartlnlaourcc al pltbopu ftlr other CP pll; odlen think that CIUII­

iJdlctloa II 1111 ...._ can11ct II iD1iJnue ud palcJapd. In addidoll, -cllaldiDs an: -.dlhat CP piiiJII'CIId Olplliau to other acepCiblc pta. In our halpltal. c:ardi8c IUINIIIJidon .wt quellioned wbcther It- life for CP pt1 to 1110-exercile equlpmeat. 1'berefore, we conduciCd tbl• study to detennlue wlledler CF pll ClOIItllllinaled the enW-olunent with vilblc orpllial wllea lbcy -a.tl Welllltlied 14 CP pt1 who- admitted ftlr ~of - pm__,. _.._, We cultated their banda bcCore ud after exac1te. 1bc pulse oxillleler and tile blntlla rltlle cxadle equlpmeat bclore ud after each pt -a.d. the air 36" from cacb pt'lllllllltb during maximal cxadle ud wblJe the pt - couablnl. and the pbyaic:ll thenipJIII' hands bcCore and after cacb pt excn:lacd. Qumti1dw .... c:ultanlwcnl allcalnecl durtDa RIUiiDe cllaic villll ud duriDc-'*'-. ()rpaitml.,._atll >to' CPU/ml-llllred. PA ud SA laolllel from the study ptl-~ by pulled field pi eleclrapboielil (PI'GE). ......_ ~ 10 lie idlllllc:lllt allllandllllllcbed, IIJbtJpcs of the - ..... It 1·3 handa dUIIInd, and dUrenat llnllallf >3 baadl cll&nd. T• ptl-llllle ud their medlu ... wu 25 ,..,._ On adllllllioa, the Jill carried: ~ PA (O'A; N • 2). mucoid PA (MPA; a • 5). O'A and MPA

(n • 3), CPA and SA (n • 2), and MPA and SA (n ~ 2). All environmental cullwes were negative for PA and SA with the following exceptions. Two air cultures from pts who were coughins each had I colony of MP A. Both pis carried MP A. One air cui lUre grew 1 colony of a mucoid P•udomona.r species 110(

identified in the pt's sputum. One pre-exercise hand culturc grew 82 colonies of SA but the pt did 110( cany SA. One physical therapist had > 300 colonies of SA on her hands after a pi exercised but the pi did not carry SA. Unrelated pts did not shasc strains. PFGE is beins done to determine if the 2 colonies of MPA obtained from air cultures matched those the pts carried. CF pts admitted for acute pulmonary exacerbations did 110( conWninate the environment with their PA or SA strains while exercising. Our data indicate that special infection control precautions arc not needed in physical therapy departments for CF pts who arc infected with PA or SA and that CF pts can usc common exercise equipment.

313 CEFTAZIDIME BID VS TID IN THE TREATMENT OF BACTERIAL RESPIRATORY EXACERBATIONS IN RUSSIAN CYSTIC FIBROSIS PATIENTS. V tvaqov'. L. Shabalova' C. Holmes2

, C. Hi113, P.

lllangovan3, S. O'Hara , S. Polikarpova•, N. Kapranov'

'Moscow Republican Hospital for Children, Moscow, Russia, 2Giaxo Wallcome R&D, Mlddlasex. UK.. 'southampton General Hospital, Southampton, UK, 4 Clinical Hospital N1 5. Moscow, Russia

Objectives: To compare the Clinical efficacy of 14 days of treatment with ceftazldime (CAZ) 1 SOmglkglday given tid with that of CAZ 1 50mglkglday given bid, In treating cystic fibrosis patients with acute respiratory exacerbations. The secondary objectives were to assess the bacteriological and safety profiles of the two treatments. Methods: In this open, single-centre study, 98 paediatric patients with an acute respiratory exacerbation of lhelr cystic fibrosis were enrolled. Patients were randomly allocated to 1 50mglkg/day CAZ treatment given as either 2 or 3 equal doses for 14 days. The signs and symptoms of rasplralory exacerbation, sputum bacteriology and lung function were assessed at entry to the study,~ 48 hrs posttreatment and 28-35 days posttreatment (follow-up). Adverse events were monitored throughout the study. Results:. In the clinically evaluable population, the proportion of patients cured/Improved at post-treatment was 58/63 (92%) in the CAZ bid group and 27132 (84%) In the tid group. AI the follow-up visit, 48198 (49%) patients were evaluable and of these, 23132 (72%) and 8/18 (44%) In the bid and tid groups respectively maintained their clinical cure. In the bacteriologically evaluable population, 197 pathogens were isolated from 88 patients of which 121/197 were eradicated by post-treatment. The eradication rate by patient was similar although equivalance could not be demonstrated. Fifty-nine pallents reported 75 adverse events during the study (33 during treatment and 42 post-treatment). Events considered by the Investigator lo be possibly or probably drug-related accounted for 23175 (31%) events which were mainly lower respiratory or gastrointestinal In nature. No patients were withdrawn from the study due to an adverse event. There were 3 serious adverse events: an exacerbation of respiratory symptoms requiring hospitalisation, pulmonary bleeding and surgery for osteomyelitis - all patients were In the bid group and all events were considered unrelated to the study drug. Conclusions: Ceftazldlme 1 50mglkglday given as two equal doses Is Clinically equivalent to lhe same dose given as the standard three equal doses, In the treatment of respiratory exacerbations of cystic fibrosis. Both treatments are well tolerated. Supported by GlaxoWcllcomc R.tD, Stockley Park. UK

314 CLONING AND SEQUENCING OF AN ANTIBIOTIC EFFLUX OPERON FROM BURKHOLDER/A CEPACIA. J.L. Burns, C. Goodall, J. BIIIT)'. R. Charron. Children's Hospilal and Medical Center, University of Washington, Seattle, WA 98105

Multiple antibiotic resistance is frequently found in CF isolates of B. cepacia. Potential resistance mechanisms include inactivating enzymes, allered antibiotic targets and decreased drug access. Resistance to the quinolones, trimcthoprim and chloramphenicol can be transferred from a resistant CF isolate, K61-3, to a susceptible laboratory strain by cloning a large fragment of DNA, suggesting that the resistances arc either genetically linked or the result of a common resistance mechanism, such as decreased drug access. The 5.6 kb fragment encoding this multiple resistance has been subcloned and sequenced. Five potential open reading frames have been identified, including three that are homologous with antibiotic efflux systems idcnlifJcd in P. atrugi1103a and other Gram-negative pathogens. opcM, encoding an outer membrane lipoprotein, has been previously reponed and is homolosous with several genes encoding P. aeruglttosa outer membrane proteins including oprJ, oprK and oprM. The name uo was

selected for "£epacia ~ffiux QPCron". ceoA is homologous with genes encoding several pcriplasmic link proteins. Its predicted gene product is -36 kDa. ceoB is homologous with genes encoding cytoplasmic membrane proteins of the resistance-nodulation-division (RND) family. Its predicted gene product is -130 kDa. S' of the start of ceoA is a partial potential open reading frame transcribed in the opposite direction from ceoABopcM which is homologous with the lysR group of transcriptional regulators. The finding of an efflux operon in B. cepacia composed of three structural proteins plus a potential regulator is similar to the mex opcrons reported in P. aeruginosa, but distinctly different from other organisms which contain contiguous genes for the cytoplasmic membrane protein and periplasmic link protein, without the contiguous outer membrane protein.

This study was supported by a grant from the CF Foundation.

315 CF MICROBIOLOGY IN YOUNG CHILDREN. J.~ Bums

1, D.

Yim1, S. McNamara', J. Emerson1, K. McCo/, P. Hiatt, B.W.

Ramsey1• 1Children's Hosp. & Med. Ctr., Univ. of Washington. Seattle, 20hio State Univ., Columbus, OH, )Baylor Univ., Houston TX

Examination of the microbiology of pulmonary colonization in young children with CF is difficult. Young patients do not routinely expectorate sputum and oropharyngeal (OP) culture is insensitive for predicting lower airway pathogens. Bronchoalveolar lavage (BAL) is required for accurate microbiology. Most studies to date using BAL culture are cross-sectional. This study enrolled children during the I st year of life and followed them longitudinally to age 3 yrs with annual BAL. Quantitative cultures were performed on selective media with microbroth dilution MlCs on all pathogens. 42 patients (pts) from 3 CF centers were enrolled; 28 have completed the study to date. Of 40 pts cultured at yr I BAL, 7 (18%) had P. aeruginosa (0 mucoid); 9 (23%) S. aureus; IS (38%) R influenzae; I (3%) B. cepacia; 3 (8%) S. maltophi/ia; 2 (5%) Klebsiella. 31 pts cultured at yr 2: 12 (39%) P. aeruginosa (2 mucoid); 10 (32%) S. aureus; 6 (19%) R injluenzae; 0 B. cepacia; 2 (6%) S. maltophilia; 0 Klebsiella. 25 pts cultured at yr 3: 8 (32%) P. aeruginosa (0 mucoid); 8 (32%) S. aureus; 6 (24%) R influenzae; 0 B. cepacia; 2 (8%) S. maltophi/ia; I (4%) Klebsiella. For the 28 pts who have completed the study, II never had P. aeruginosa, 5 had transient colonization, S had persistence, 7 were indeterminate. Southern analysis of P. aeruginosa i~la!es usin~ an exoA probe identified 3 patterns of lower tract colomzatton: perststence of a smgle genotype, replacement of one genotype by a second, and replacement of the second by a third genotype. Overall, 1/38 BAL isolates o~ P. aeruginosa was tobramycin resistant (MIC ~ 8 llglml); I was multiply resistant (susceptible only to a single class of agents). The numbers of pts colonized are higher than those reported in previous BAL studies and may reflect differences in fatient selection and culture technique. Of note is the relative absence o typical CF P. aeruginosa with ~uc~idy and antibiotic resistance. These data represent an important longttudmal study of BAL microbiology in young children with CF.

This study was supponed by a grant from the CF Foundation and the NIH.

316 P. AERUGINOSA COLONIZATION IN YOUNG CHilDREN WITH CYSTIC FIBROSIS. S. McNamara, B. Ramsey, J. Emenon, L. Ingham, D. Crist, CHMC, Seattle, WA; R. McConnell, P. Hiatt, Baylor, Houston, TX; C. Lawrence, K. McCoy, Columbus Children"s, Columbus, OH, USA.

Development of effective therapies for the treatment of early lung disease in the CF patient requires an undentanding of the aatunl history of P.urxgino111 infection in the lower airway. We are conducting a longitudinal study to define the association between P• colonization, aerology, and bost immune rnponse in tbe fint 3 yean of life among children with CF. Quarterly evaluation of subjecu includes assessment of pulmonary and nutritional status and collection of clinical specimens for bacterial and vinl cultures and serology. Infant pulmonary function testing is completed every 6 months. Bronchoalveolar lavage (BAL) fluid is obtained for bacterial and vinl cultures and inflammatory marker anal)l1tlat t, 2, and 3 yean of age.

Enrollment of 42 subjects ( 24 male, II female) at three CF centen (Seattle Children's, Columbus Children's, and Texas Children's) was completed in April, 1996. Subjects were enrolled by t5 months of age (mean 11.4 mo, SD

Poster Session Abstracts 293

3.3). All subjects are Caucasian; 3 of Hispanic origin. Of 33 subjects genotyped, 18 (55%) are homozygous AF508, and II(H'lb) are heterozygous AF508. All subjects are pancreatic insufficient with mild to moderate disease at enrollment based on Brasfield and Shwachman scores.

Preliminary analysis indicates a prevalence of P• in lower airway cultures of 18% (7/40) at year 1, 39% (t213t) at year 2, and 32% (1/25) at year 3. Findinp from simultaneously collected OP cultures demonstrate greater specificity than sensitivity for detecting lower airway P•. Serologic rnponse to exotoxin A has also shown limited diagnostic accuracy as a noninvasiw indicator of lower airway c:oloniz.ation. Examination of host inflammatory response reveals an endobronchial inflammatory response with elevated marken by year t; bow~er, there appean to be a blunted elevation of IL-10, an anti-inflammatory cytokme. BAL total WBC and percent neutrophils are higher in P• + infants. IL-111, IL·tra, and LPS show significant positive associations with density of lower airway p._ Clinical ch.tracteristics which may be associated with P• status include nutritional status (lower weight percentile) and pulmonary function, with year 1 and 2 data suggesting more air trapping among colonized infants. Completion of data collection and application of longitudinal analysit methods will allow further evaluation of these emerging trends and elucidation of facton associated with P• colonization in infants and young children with CF. (Supported by the CFF and the NIH)

317 MULTIPLE COMBINATION TESTING FOR ANTIBIOTIC SYNERGY IN MULTIRESISTANT PSEUQOMONAS AERUO!NOSA (PIA) AND IDJRKHQI PERlA CErACIA (BC) IN CYSTIC FIBROSIS (Cf). W. Ferril.l:l. MacDooa)d A.MR. Ma<:bmio. Univ. a( Ottawa, Ottawa, Ontario, Canada. ~: Combinatica llllibiolic thenpy hu been lbown to be beneficial in tho

IIWiapncnt of pulmonaly ....-bationo in CF. Wben PIA or BC ore multireoillant, ie. resistant to 2 or more 111tipiCIIdomonal agenla, oelectioa of 1ft ~ntibiolic combination il problematic. ~: To cle\'elop alqlrDducible. clinically n:lcvant labontory protocol for evaluatina ~ntibiotic combinaliono for treatment of resillant PIA or BC from CF paticota. l'dGiblldl: o.-Jopmont oC a multiplo oombinatioa - (MCT) procedure for 92 combination~ with veriJicatioa oC reproducibibty owr time, oorrelation with time kill curveo and witb anecdolal clinical validalioa. Syner&Y: defined as tho baclerieidal actioa of llllibiotic combination illisnificantly 1f011er than for each drua alone; 111tspism: dccrcuo in bactericidal activity witb tho addition oC another antimicrobial agent Antibiotica utilized included ceftazidime 32 jllml..; tobrarnycin I 00 and 200 jllmL, amikocin 32 jllml..; ootrimoxazolc I Oil jllmL, ciprolloxacin 2 jihnL, imipenem I 0 jihnL, IZilhromycin o.4 jllmL, piperacilliMazobactam 3214 f11mL. Ballll.l: s~ 1988, 39 PIA and 23 BC ilolalel from CF patients have been evalualcd using MCT. The method is reproducible: 4 of 4 ilolalel retested I to 2 yean later pw identical results to all combinalionolellcd. In 60% PIA and 67% BC epparent oynergistic combinationo were found; 41% PIA and 27% BC mtapistic: combinationo; and 0% PIA and 17% BC no cffectiw combination. Time lciU curveo uaina 1pparen1 ~Y~~Faistic: combinaliono IICCOfdina to MCT results -med I)'IIOfi!Y in I 00% of S6 combinationo 1148 boun and 77% 1184 boun for 4 BC i10late1 tcslcd with 14 combinaliona. Anecdotal clinical cue reports IIOicd clinical impnM:mcnt when I)'IIFaistic combinaliono were utilized in 9 o( I 0 patients where Ibis - reviewed . CgncJwions: CF multiple combinatioa - oC multiresistant PIA or BC u a buis for oelec:ting antibiotica for combination thenpy oC CF pulrnotwy exacerbationo appears to be reproducible and clinically relevant

318 IS no: ACQUISmON OF PSEUDOMONADS IN CYSTIC FIBROSIS PAmNTS INCREASED BY USE OF INHALED CORTICOSTEROIDS! UNEXPECTED RESULTS FROM A DOUBLE BLIND PLACEBO CONTROLLED STUDY Schmidt J, Davidson AGF Scear M, Wona LTK, Peacock D,Oravelle A. Menoa K, Cimolai N, and Specrt DP. Cystic Fibrosis Clinic and Departmenta of Pediatrics and Pathology, B.C.'a Childreu'a Hospital and University of British Columbia, VIIICOIIVI:f, Canada.

Pulmoowy discuc in Cystic Fibrosis (Cf) is characterized by 1 vi<:iouo cycle of inflammatioa, obstruction, infection and pulrnotwy damage. The use of inhaled corticosteroids ila potential means of inlcmlpting Ibis cycle by deacuina inflammation and minimising tissue destruction in tho CF lung. To assess tho effectiveness of inhaled lllcrOids in the treatment of CF airway di9CUC, we dcsij!llCd a double blind placebo conlrolled study wing inhaled tluticuone prq>ionate in pllienta apl 6 to 17 years. attending the CF clinic AI B.C.'a Children's Hospital. The proposed duration of the study was one year. with CF patients randomly usij!llCd to receive eilher placebo or SOO 1'8 bid Fluticasone via a metered dooe inhaler (MDI) and 111 aeroool spacer (Space Chamber no). Pulmonary function (FVC, FEV,, FEF lS· 75). oxygen saturation, sputwn or throat swab bacteriology, weight, height and clinical ststus was measured at the beginning of the study, and al three month intervals thereafter. After initiation of the study, but before enrollment was complete, it was noted lhat 6 of 17 patients became colonized with Pseudomonas aeruginou (PsA) for the farst time. M a resull, we suspended enrollment, tcrminalcd the study,and broke the code to determine which patients were on placebo and which oaoctive lherapy.

294 1997 Cystic Fibrosis Conference

Five of the 6 pllien .. newly colonioed with PsA were in the active drug trcabnent group, which had a total of9 patien .. enrolled. The other newly colonioed patient was in the plleebo group (group enrollment 8). This result was not statiatically signifiCallt. In addition, one patient in the active trestment group who was previously cultured negative for PIA or Burkbolderia Cepacia (B Cep) grew B. Cep for the tint time. There was no statistically •isnificant difference in pulmoowy function between the placebo ond active trestment lfOUP· Cultures of the MDlllld apacc:rs did not yield pseudomonas. PsA llrains were evaluated by RAPD. In liaht of these resul ... we retrospectively ..Wyzed the bacteriological mtus of the S6 CF patienb in our clinic (total clinic population 133 paticn .. ) who had previously been !rested with other aerosolioed !lleroids. Thirty-six of these patien .. were colonioed with PsA; ofwhoo! 27 had grown PsA before steroid therapy was started, and 9 patien .. became culture positive 3 months to 3 years after start of aeroeolioed steroids. AlthousJt the resul .. encountered during our clinical trial offluticasonc were sufticiently oonc:ernina to wmant diocontinuaticn of the propoaed clinical trial, the rate of acquisition ofPsA did not achieve statistical sisnificance. We sussest lhst future IIUdies involvins aerosolioed steroids in CF should include careful bacteriolosical monitorins.

319 ANTIGEN-SPECIFIC TH-l LYMPHOCYTE IMMUNE RESPONSE IN CYSTIC FIBROSIS PATIENTS WITH ALLERGIC BRONCHOPULMONARY ASPERGILLOSIS (ABPA) ~. L.K. Poulsen, C. Koch. Department of Pediatrics and Laboratory ofMedical Allergology, National University Hospital, Copenhagen, Denmark

A majority of patients with cystic fibrosis (CF) becomes colonized with Aspergillus fumigatus (A. fumigatus) in the lower respiratory tract with a 1-2 year prevalence of 40-57%. In these patients 1-11% develop ABPA. Early diagnosis of ABPA is important to prevent serious detonation of lung function, though difficult to establish because of similarities in the clinical and paraclinical findings in CF and ABPA. A role of antigen-specific TH-2 like T-lymphocytes in the pathogenesis has earlier been suggested. The aim of the present study was to evaluate the quantitative importance ofTH-2 cells using improved tecniques for measuring IL-4 and IL-5 secretion in unseparated mononuclear cell suspensions. Twenty CF patients heavily colonized with A. fumigatus were included. Half of the patients had ABPA. PBMC's were stimulated In vitro for 6 days by A. fumigatus antigen, and by Tetanus toxoid (TT) and medium serving as controls. Hereafter PMA and Ca-ionophore were added to increase cytokine production, and 24 h later supernatants were harvested and IL-4 and IL-5 (TH-2 markers) and IFN-y (TH-1 marker) were quantified by ELISA. In ABPA patients significantly higher IL-4 and IL-5 secretions were observed as compared to non­ASP A patients, i.e. 1.36 ± 0.76 vs 0.09 ± 0.01, (p<O.OI) and 36 ± 8.99 vs 2.91 ± 0.53, (p<O.OI) respectively. IFN-y secretion was similar in the two groups, as were TT and medium stimulated cytokine production. Conclusion: In CF patients heavily colonized with A. fumigatus a preferential and antigen specific TH-2 lymphocyte immune response is seen only in CF patients with ABPA as indicated by excessive IL-4 and IL-S secretion. Measurements of IL-4 and IL-5 may be useful in the diagnosis and as indicator of disease activity of ABP A.

Pulmonary

320* EFFECI'S OF INSPIRATORY FLOW RATE AND BRONCHODILATOR ON AEROSOL DEPOSmON IN CF D...LaubA. R. Jasbnaai, R. Dalby, P. Zeitlin. Departments of Environmental Health Sciences and Pediatrics, Jobns Hopkins Univenity, and Department of Pharmaceutical Sciences, Univenity of Maryland, Baltimore, MD, USA.

Inspiratory flow rate and the degree of airway obstruction are known detenniDants of aerosol deposition in human airways. The objective of

this study was to investigate the effects of altering inspiratory flow rate and pretreatment with a bronchodilator on aerosol deposition fraction and distribution within the lungs of patients with CF. On three study visits, five CF patients inhaled a saline aerosol containing Tc-99m DTP A that was generated by a Medicator nebulizer system (MMAD = 1.3 lUll) during 30 seconds of either continuous fast breathing (flow rate --43 Umin), slower breathing (flow rate -21 Umin), or slower breathing 1 hour after inhaling 2 puffs of albuterol generated by MDI (flow rate -16 Umin). Following aerosol inhalation, patients underwent gamma camera imaging of their lungs. Lung images were analyzed in terms of fraction of aerosol deposited in the lungs, expressed as a percent of inhaled fraction, and regional deposition, expressed as apical: basal zone ratio (an indicator of apex vs. basal deposition), skew (an indicator of overall distribution homogeneity), and inner: outer zone ratio (an indicator of large vs. smaller airway deposition). A Friedman analysis of variance showed that average {±sd) deposition fraction after rapid or slower breathing or slower breathing after bronchodilator pretreatment were similar with 47.2±12.3%, 58.4±18.0% and 57.4±14.8%, respectively. A similar analysis revealed that mean apical: basal zone ratio was unchanged on the three visits with 0.80±0.38, 0.70±0.25, and 0.89±0.60, respectively. Average skew values were also unchanged with 1.97±0.75, 1.66±0.63, and 1.74±0.74, respectively. After rapid and slower breathing, inner: outer zone ratio (1:0) were similar, with 2.86± 1.43 and 2.19±1.09, respectively. However, 1:0 ratio following slower breathing and bronchodilator pretreatment ( 1.71±0.60) was significantly reduced compared to faster breathing without bronchodilator (p=O.O 15). These results suggest that premedication with 2 inhalations of albuterol from an MDI followed by slow continuous breathing improves small airway distribution of aerosol generated by the Medicator delivery system in this small group of CF patients, compared to faster breathing and no bronchodilator pretreatment. Supported by the CF Foundation

321*

The aerosolization emclency of colistin Is nebulizer dependent. Dayjd .E, Gellert, Thomas.A. Standaert2, tThe Nemours Childrens Clinic, Orlando, Florida and 2 Cystic Fibrosis Research Center, Childrens Hospital and Medical Center, Seattle, Washington, USA.

Aerosolized colistin is an important antibiotic choice for CF patients infected with P. aerugjnosa. Many nebulizers cause the drug to foam, reducing its delivery; newer venturi-type jet nebulizers may eliminate or reduce this problem. We evaluated a variety of jet nebulizers to characterize their performance with this medication.

150 mg of colistimethate (Colymycin, Parke Davis), dissolved in 4 cc water, was placed in: Acorn II (Marques!), MistyNeb (Baxter), LC Plus and LC Star (Pari), Sidestream and Ventstream (MedicAid), and T-Updraft (Hudson). PulmoAide compressors were used. The mass median diameter (MMD, 1'), respirable fraction(% aerosol in 1-5 p. range), gravimetric output (ml/min), and nebulization time (minutes) was averaged for 4 nebulizers of each model. We calculated the respirable fraction of the colistin dose delivered. The quantity of active drug delivered is unknown using this gravimetric methodology.

MMD %1-SI' output neb time % deliv'd LC Star 2.6 70 0.11 30.4±7.9 55±1 Sidestream 2.5 66 0.23 12.9±1.7 52±3 Ventstream 3.0 64 0.22 14.7±1.5 50±3 MistyNeb 3.3 60 0.13 19.8±2.6 38±3 T-Updraft 4.3 48 0.11 27. 7±6.4 36±9 LC Plus 4.7 41 0.18 19.0±4.3 32±2

Relative to saline, colistin altered the performance of the 7 models. The colistin outpUt is low relative to other drug formulations assessed. No data was obtained from the Acorn II, since colistin foamed out of the nebulizer on all trials; the T­Updraft and Misty Neb did so inconsistently. Considering the nebulization time and the respirable fraction of colistin delivered, the Sidestream or Ventstream appear to be the optimal choice.

322* Evaluation of Aerosolized Tobramycin Delivery by 4 Major European Nebulizers. Thomas A Standaert', Bonnie Ramsey', Michael Vasiljev-K2 and Bruce Montgomerf. 1 CF Research Center, Childrens Hospital and Medical Center, and 2 PathoGensesis Corporation, Seattle, Washington, USA.

Mutilcenter US trials of the treatment of Cystic Fibrosis patients with preservative free tobramycin (Tobir") demonstrated improvement in pulmonary function and a decrease in sputum bacterial density. Anticipating future testing of this antibiotic formulation in Europe, we evaluated the performance of several commonly used European systems -the LC Plus and Star (Pari, Germany), Aiolos jet (Sweden), cps23 jet and LS260 ultrasonic (Syst'Am, France), and Sidestream and Ventstream (MedicAid, UK). Each was tested with the manufacturer's compressor; the reference standard was the LC Plus and DeVilbiss PulmoAide compressor, used in the recent Phase 3 trials.

Five cc of 60 mg/ml Tobi was aerosolized continuously, with no auxiliary inspired gas flow. The aerosol's mass median diameter (microns. Malvern laser). respirable fraction (RF, o/o in range of 1-5 microns), and gravimetric output (mUmin) were obtained in duplicate, on 4 nebulizers of each model. The respirable tobramycin delivery (mg) was calculated as (RF)[(Vol)(conc),....­(Vol)(conc),.,.J, with a maximum potential delivery of 300 mg. Stan'd Plus Star Aiolos Sidest'm Ventst'm cps23 L$260 95±8 97±6 154±3 170±4 152±9 140±6 52±7 130±2

All nebulizers perform as good or better than the standard, except for the cps23 jet (which improved to that of the standard with the use of a different compressor). Except for the Syst'Am jet, all nebulizers may deliver additional aerosol with increased inspiratory flow. In addition, the Aiolos and L$260 ultrasonic minimize wastage by 'triggering' aerosol delivery to the time of inspiration. Alternatively, poorly coordinated triggering could decrease delivery. Tobramycin delivery to the patients' airways may parallel these figures, but will depend also on multiple patient factors including inspiratory pattern and disease state.

323 DOPPLER MYOCARDIAL IMAGING DETECTS INFLAMMATION­INDUCED SUBCLINICAL RIGHT VENTRICULAR DYSFUNCTION IN CYSTIC FIBROSIS ~ A.A. lonescu, L.S. Nixon, A.O. Fraser, DJ. Shale, Department of Cardiology and Medicine, Univenlty of Wales College of Medicine, Univenity Hospital of Wales, Cardiff, UK CF4 4XN.

Right ventricular function il difficult to assess by conventional ec:hocardiography in patienll with chronic lung diseases. Doppler myocardial iJnagin& (DMI) il 1 new echocardiographic technique which measures regional cardiac conlnlction and relaxation. We hypothesised thll patienll with cystic fibrosis (CF) may have abnormal DMI indices of right ventricular (RV) function end that the C-reactive protein (CRP) level, measured by ELISA, might correlate with these indices. We lludied 13 pll. with CF (I males; mean age (SO) 23.7 (3.9) years; mean% predicted FEV 65.3(22.4)%; mean Shwachmann score 83.1; all were exacerbation-free and without Doppler si&ns of pulmonary hypertension) end 7 healthy non.CF subject~ controls. We measured (II end-expiration) systolic end diastolic velocities and velocity-time integrals of the RV free wall and of the tricuspid annulus. DMI parame!m of the RV were depressed in CF (Table; Mann-Whitney):

Parameter CF (mean[sd]) Controls (mean[ ad]) pvalue

D-VTI (em)+ 2.7[0.4) 3.7[0.9) 0.005

S-VTI (em)+ 2.2[0.3) 2.8[0.5) 0.007

B-Vel (cm/s)+ 12.0[1.6) 15.3[2.1) 0.004

o-VTI (em)++ 2.6(0.4) 3.1[0.5) 0.03

IVRT(ms)++ 57.8(13.7] <42.6[14.5) 0.03

+tricuspid annulus; ++-right ventricle; S-systolic; D-diastolic; VTI· velocity-time Integral; B-Vel-early diastolic velocity; IVRT-isovolumic relaxllion time.

In CF patienll the ntio of early and late diastolic velocities (E/A) of the RV free wall correlated with cin:uloting CRP (r-0.1, p-0.03). Doppler myocardial

Poster Session Abstracts 295

imaging shows a decrease of right ventricular systolic and diastolic function In patients with CF and chronic pulmonary lnfeclion. This auggesll sub-clinical ventricular dysfunction in the absence of pulmonary hypertension could be caused by persistent inflammation.

324 DEFINING A PUlMONARY EXACERBATION IN CYSTIC FIBROSIS M.Rosenfeld, J. Emerson. J. Williams-Warren, M. Pepe, B. Ramsey Cystic Fibrosis Center, University of Washington, Seanle, WA, USA

An acute change in pulmonary symptoms in CF subjects has been tenned a pulmonary exacerbation (PE). The frequency and/or severity of PEs has been a primary outcome measure in many clinical trials in Cf. Ye!, no stondardized definition ofa PE exists. The 1994 Cystic Fibrosis Foundation consensus conference on outcome measures for clinical trials in CF recommended that a definition of PE be established for universal use in clinical trials (I), We therefore aimed to identify the combination of signs and symptoms that best predicts the presence of a PE in patienll with moderate to severe pulmonary disease. Subjecll were patients enrolled in a multicenter trial of aerosolized tobrarnycin. Enrollment criteria included age:!: 6 years, FEV1 25% to 75% of predicted, and P. oel'llglnrua present in respiratory culture. At the initial screening visit and 2 subsequent visits, physician investigators were required to complete a PE questionnoire for each subject, indicating physical findings, changes in patient symptoms during the preceding 2 weeks. and impression of PE (presence/absence, and severity). Questionnaires were completed at screening for all 520 subjects enrolled at 69 centers. Subjects had a mean age of 21.2 years (range 6. I -63.1 ), and 54% were male. At the initial screening visit, 153 subjects (29"/o) were classified by the examining physician as having aPE (PE+). of which 133 (87%) were classified as mild, and 20(13%) as moderate in severity. The mean FEV1 (%predicted) ofPE+ subjects was 53% (SD 16) and of PE- subjects 49% (SO IS). The mean age was similar for the 2 groups. A wide range of signs and symptoms was more prevalent in the PE+ group than the PE- group. For example, increased sputum production was seen in 56%ofPE+ subjects and 2% ofPE- subjects (relative risk 56,95% Cl26, 121). Decreased exercise tolerance was seen in 35%ofPE+ subjects and 5,_,, ofPE­subjects (RR 38,95% Cll5, 98). Increased cough was seen in 61% ofPE+ subjects and 6% of PE- subjecll (RR 26, 95% Cl 15, 45 ). In conclusion, the prevalence of PE among subjects with moderate-to-severe lung disease was 29%. An array of signs and symptoms was significantly more prevalent among PE+ than PE· subjects in preliminary univariate models. Multivariate models will be presented assessing the relative importance of these signs and symptoms in discriminating the presence from the absence of a PE. Receiver operator characteristic (ROC) curves will be used to define 1 PE score with optimal sensitivity and specificity.

I. Ramsey B, Boat TF e1 al. Journal of Pediatric• 1994;124:177-92.

325 DIAGNOSTIC ACCURACY OF OROPHARYNGEAL CULTURES RELATIVE TO LOWER AIRWAY CULTURES IN INFANTS AND YOUNG CHILDREN WITH CYSTIC FIBROSIS

M. Rosenfeld!, J. Emerson', F. Accurso', D. Annstrong4, R. Castile', K. Grimwoocl',

P. Hian'. K. McCoy', S. McNamara', B. Ramsey1,J. Wagener'. 1 University of Washington, Seanle, WA, USA. 20hio State University, Columbus, OH, USA. 'Texas Children's Hospital, Houston, TX, USA. 4Royal Children's Hospital, Parkville, Victoria, Australia. 'University of Colorado, Denver, CO, USA.

As young children with cystic fibrosis (CF) do not expectorate sputum, the "gold sllndard" for obtaining lower respiratory secretions for culture from these patients is bronchoalveolar lavage (BAL). The invasive nature of this technique precludes its use on a routine basis. We conducted the largest study to date of the diagnostic accuracy of oropharyngeal cultures relative to BAL fluid cultures in infants and young children with CF, and the first to examine the effects ofage, bacterial density, and center. Subjects were children with CF <5 years old enrolled in studies involving BAL at the participating centers. Simulllneous BAL and oropharyngeal cultures were obtained, and BAL fluid was cultured quantitatively according to standard methods. Subjects in Seanle, Columbus and Houston were participating in a multicenter study. A total of 14 I subjects were enrolled: 40 in the multicenter study, 1S in Parkville, and 26 in Denver. Forty six subjects had I BAL. 49 subject~ had 2, 42 subjects had 3, and 4 subjects had 4 BALs. The mean age at first BAL was I I months (range Ito 44 mo). Defining a positive culture as growth at any density, the lower airway prevalence of Pseudomonas oel'llginrua (PA) lithe earliest BAL for each subject was 9%. The prevalence increased by oge. It was 0"4 for age S6 months, 16% for age 7-18 months, 23% for age 19-30 months, and 31% for age 31-52 months. For predicting any growth of PA from the lower oirway at the lint BAL perfonned in each subject, oropharyngeal cultures had a sensitivity of .46 (95% Cl .19, .75), specificity of .95 (.90, .98), positive predictive value of .SO (.21, .79), and negative predictive value of .95 (.89, .98). The diagnostic accuracy was similar if a positive culture was defined as :!:10' or :!:105 cfulml, and was also unaffected by center, age 11 BAL, and sender. The prevalence of H. injluenzae (HI) lithe first

296 1997 Cystic Fibrosis Conference

BAL for each subject was 35o/o, and the prevalence of S. alll'eus (SA) was 31%. The diagnostic accuracy of oropharyngeal cultures for HI was similar to that for PA. The accuracy for SA was slightly poorer. In conclusion, the specificity and negative predictive value of oropharyngeal cultures arc excellent, while the sensitivity and positive predictive value arc poor, confirming previously published results. Thus, a negative throat culture is highly predictive of a neaative lower airway culture, while a positive throat culture has poor predictive value. Supponed in pan by the CFF.

326 SHOULD PATENTS WITH N11AL1. Y NORMAL SPitOMETRY BE INCLUDED IN CLNCAL TRIALS EYALUATWG RATE OF DECLIIIE II FEY, OVER TIME? Ubllll, R.C. Ahr-. s-H Hen, M. Ternl, M. McCubbin, D. Ritchie, C. Lux, J. OM!. Depertment of Pldletrica, Unlv of lowll, Iowa Clty, lA; Children's Mercy Hosplbll, Ka- City, MO, USA.

Change In FEV, over lime Is commonly used n en outcome measure for CF cllnlcel trtels. Pllllenla Included In th- trtels should be expected to have e decline In FEV, In the ebeence of the therapy being evaluated. PB Devls et el (Pediatric R-erch 41:161, 1887) lhowed thlt higher lnitiel FEV, was -a.ted 1Mih • mont rapid rete of decline (-.41% predlyr when baellne FEV, < 40% predicted; but, -2.61, -2.111, end -3.30 % predlyr when baellne wea 40-5, 7G-88,11ld > 100% pred,l'lllp8dlvely). Thllsuggeltl thlt all pltlenll with beullne FEV, ~ 40% predicted dacllne rapidly. We subdivided patients with normellnltiel FEV, (> 10% predicted) Into 2 grou~:

1. Pltlenlll with completely normal spirometry. 2. PltlenliiiMihllbnonnelarneU llrwayftowl (abnormal FEF,..,. or scooping

of theft- volume loop). We postullted thlt Group 2 would have a more repld rete of decline In FEV, then Group 1. We grouped patients with CF followed It the University of Iowa ~ ID~performed In 1881 - Grou~ 1 & 2: n described ebove; Group 3: FEV, 6!>-79%; Group 4: 4~%; end Group 5: < 40% predk:ted. The rate of change In FEY, over the sublequent 3 yeera (111111-114) was celc:ulated for all ~with dltl SYIIHeble for this period. Rates of change In FEV, were as follows·

GROUP 1 2 3 4 5

n 37 16 14 11 5

tJ. FEY, 0.51 -3.311 -1.7 -2.1111 0.52

Std. Dev. 4.56 5.211 3.15 3.54 1.34

FEV, dec:lned llgnillceniJy more rapidly In Group 2 than In Group 1 (p-0.009 by t-tell). Our results conllrm end utend the llndlngs of DevJs et el by demollllrltlng 1 bell-ehllped ralltlonlhlp belwHn lnlllel FEV, end rete of decline. ~ 11111111 of decline - found In patlenls with completely normellnltiel apromelry (Group 1) ee well n thou with markedly abnormal FEV, (Group 5). Theee ftndlngs suggest thlt pltlenll with complltlly normal lnlllllllplrometry should not be Jnc:luded In c:Unlc:lll trials evaluating change In FEV, over the subMquent 3 yeara. Supported by the Cystic: Flbrolll Foundation R-arc:h end Development Program It the Unlvlnllty of lowe.

327 RELATIONSHIP OF PULMONARY STATUS BETWEEN INFANCY AND SCHOOL AGE YEARS IN PATIENTS WITH CYSTIC ftBROSIS. ~B. Tnn,J. Smldl. R. Tepper. DeplrtmeiU ot Pedletrk: l'll1moaoloiY and RadloloaY. JIIJiell WhiiCOIIIb Riley Holpltal fer Cblldrea, lndllm Ulliv. Medical Cealer, hdarwpnlla, IJidlaDa, USA.

Patledl with cystic: llbrolll (CP) are born with 11001181 lung fuiiCilon, howewr, they deftkJp proareatvc obatruc:dvc alrwly disease as )'011111

cblkten and evcaiUIIJy die ot rap1ratcry l'allure as yama IIWits. 1be objecllvc ot this study wu 10 ddermlne If a relltlolllhlp ex1111 between the raplnUJry IISIUIISielled by pulmDnlry fuDctloa and CXR durin8 Infancy and ot thaliSielled WrJDa IChool aae yean. we ran.pect~vcJy reviewed data trom 48 paliela (29 male, 19 female) with CF. An lnalysla wu performed on data plhered l'nlm one visit In Infancy and relaled 10 data Crom one visit WrJDa IChool •JO. Plllenll were clllllc:llly liable at both vlalt.l. Pulmoalry luDcdon wu ISieiiOd In the lllfada (rom parUal expiratory flow ~ume maneuwn (VIIIIXFRC), and In the IChool aae cblkten trom IIIII farclecl exJ*atay maneuwn. Stallsllc:al lnalyses were performed IIIIa& linear reareakJa. 1be faiJowlD& 81~ relationlhlps wac Couud: I) beUa' CXR IICXlRII cmJna lntulcy wac asaoclated with beUa' CXR IICXlRII IUiD& IChool aae )'an (pc:(l.OOI, r • 0.48), 2) beUa' CXR IICXJRII during IDlaacy and hlp 1w1a function cbiDg IChool aac

years [FEV1 (p = O.Ql, r = 0.34) and FVC (p = O.DI, r = 0.38)], aJXI 3) higher lung function during Infancy conelated with higher lung function OOrJn& school age years [VmaxFRC vs FEV1 (p = 0.02, r = 0.34); VmaxFRC vs FEFzs,, (p = 0.002, r = 0.44)). Our data suggest that better respJratocy status as assessed by pulmonary function and CXR during infancy correlate with better respiratay status during school age years. One could speculate that maximizing respiratay status In Infants with CF might result In better lung function during Infancy and during later life, thus slowing the progression of Irreversible airway obstruction In these patients.

328 DO WE NEED NEW REGRESSION EQUAllONS FOR LUNG FUNCTION IN CYSTIC FIBROSIS?: LESSONS FROM A STUDY OF INADEQUATELY NOURISHED CHI.DREN WITHOUT RESPIRATORY DISEASE. ~. A. Mehta, S. Ogston* and S. Mukhopadhyay Centre for Researoh Into HllllllfJ ClfNelopmert, Department of Child Health, "Department of Epidemiology, University of Dundee, Nlnewells Hospital and Medical School, Dundee 001 9SY, Scotland, UnHed Kingdom.

aackground In animal models, under-nutrition leads to profound and qualltltlve changes In the lung beyond a simple effect on organ size. This led us to challenge the assumption that correction for height is sufficient to normalise lung function for growth' In subjects with nutritional deficiency. We hypothesised that nutritional deficiency exerts an independent negative effect on lung function beyond such col1'8dlon for Hnear growth. and studied the effect of surrogate marltels of nutJttlonal stilus on lung function already normalised for height. M!!I!2!!J We assessed nutritional status (Weight, body mass Index, mid-upper arm circumference, subscapular end triceps sklnfold thicknesses) and measured lung function (FEV1• FVC, PEFR) In a nutritionally deficient c:ohOII of 3113 sc:hool-c:hlldren (M:F = 248:145) with no history or physlcel signs of respirstory disease. In a remote rural setting in India. .B!!l!l! Lung function, already normalised for sitting height and stature, declined with every measure of under-nutrition (p<0.01) In both sexes. The multiple regression of ln(lung function) vs ln(Weight) and ln(sltting height) reduced variability Signillclntly (p<0.001). In comparison to ln(lung function) against ln(sltting height or stature) alone. The Inclusion of additional variables did not, however, lead to any further improvement In variability (p>O.OS). Therefore, we added weight as an acldHional predldor to sitting height to celc:ullle a new set of reference prediction equations for FEV,, FVC and PEFR speclficelly sutted for under-nourished children. Conclusion Predlctlon of lung function In the under-nourished must ac:c:ount for nutritional differences If decline from pulmonary disease 1$ to be accurstely measured; this Is of particular relevance In respirstory diseases Ike cystic fibrosis (CF) that are assocllted with nutritional deficiency.' Further exploration of a nutrition-defined component to lung function decline In CF could leed to strstegles for Its corredlon: this could represent a new therapeutic Intervention for the disease. .!!!~'!!!!!!!! (1) Colel JE, l.8llhert Gl Lung function: -amen! ond application in medicine. Oxford. Blockw8115clentlllc, 11193. (2) Nlr Met II Thorax 1996;51:1023-7. Aliknowltdp!!'M!JI! Wa lhri t:r B. G. ~ (CM:Uta. hde). the tllllr of Mo)ine lliJIJnha ~end the......,. of-..16Qto. ~(\Mill Bengtr/. hla) tbr thei'h8t>. Thio~-~by1he-...Thllt, U.K., 1he~ ~. 1healllt, ,_end~~. U.K .. 1he8tlilh Metlcltlend Dentll Sll.dlnll' Tlllltend1he IMdaJ ,...._, COIIlC:I( U.K

329

ELEVATED INDUCER OF ANGIOGENESIS AND INCREASED MICROVASCULAR DENSITY SUGGEST A ROLE FOR NEOVASCULARIZATION IN CYSTIC FIBROSIS. S Crawford. V. Stcllnw:h, S. Mc:CoUey, 1. Jac:obitz, C. Backer, N. Boucle, Departments of Pathology, Microbiology-Immunology, Pediatrics, Cardiovucular-Thorac:ic Surgery, Northwestern University Medic:al Schoo~ Chic:ego, U., USA.

There is marked heterogeneity in the clinic:al presentstion end disease progreasion among CF patients (pta) who inherit identicel mutations in the CFTR gene auggestlng that genetic or physiologicel modifien play important rolea in disease severity. Since CF pts are prone to developing nua1 polyps and lung hemorrhage, one physiological process that may influence the manifestations of CF disease is angiogenesis, the induction of new capillaries from pre-existing veasels. Whether the cepillery vuculature remains quiescent or proliferates depends on the balance of inhibitory and inducing compoundl secreted by neighboring cells. Since one hallmark of angiogenesis is increased microvuc:ular density (MVD) within a tissue, MVD wu quantitated in specimens obtained from surgeries end autopsies of adults and children with CF end compared to non-CF

specimens. A statistically significant increase in MVD was observed in the lungs, nasal polyps, pancreas, and intestinal tracts of CF pts compared to parallel tissue of non-CF pts. Immunohistochemical examination of lung specimens for the expression of vascular endothelial growth factors (VEGF}, a potent inducer of angiogenesis, revealed that lung bronchiolar epithelium from CF pts stained more intensely than comparable non-CF lung tissue either with or without inflammation. Additionally, a striking increase in the level of VEGF was detected in the serum of CF pts (mean age 12.8:1:12.0 years) by EIA (n-22, mean-41S.Lt237.9 pg!ml) compared to age-matched non­CF pts (n-25, mean-31.1:1:14.1 pg!ml, p<O.OOOl). Data suggest that CF pts may have a higher propensity for angiogenesis due to high circulating levels of VEGF and that, in fact, vascular density is significantly increased in target organs.

330 CYSTIC FIBROSIS TRACHEAL FIBROBLASTS PRODUCE INCREASED PRO-INFLAMMATORY CYTOKINES IN VITRO. M.D. Infeld, T.L. Bonfield, R. Upshaw, R.W. walenga, M. Berger, P.S. Smith. Depts. of Pediatrics and Medicine, Case Western Reserve university, Willard Alan Bernbaum, M.D. Cystic Fibrosis Research Center.

The submucosa of the conducting airways in cystic fibrosis (CF) patients are exposed to increased mechanical stress as a consequence of mucus plugging and hyperinflation. We hypothesized that mechanical stimuli might participate in sustaining an inflamm~tory milieu in the CF airway and that a1rway interstitial cells may play an immunomodulatory role. Fibroblast cell lines from non-CF and CF tracheal necropsy specimens were isolated by explant outgrowth and characterized by cytoskeletal immunohistochemistry. supernatants from these cells grown in serum-free media in 3D collagen matrices at rest or exposed T,to cyclic JDechanical stretch in a FLEXERCELL apparatus were collected and assayed for interleukins 6 and 8 (IL-6 and IL-8) by ELISA. CF fibroblasts demonstrated equivalent basal secretion of IL-8 and two-fold increased secretion of IL-6 in culture. CF fibroblasts were stimulated to increase cytokine production by cyclic mechanical stretch in a dose dependent fashion, while nor~al fibroblasts only augmented cytokine product1on at the highest levels of stretch. Chronic JDechanical stress in the airway interstitium JDaY contribute to bronchiectasis, the final common pathway in CF lung disease.

Supported by NHLBI KOS-27890

331 IDENTIFICATION OF CYSTIC FIBROSIS IN PATIENTS

AFFECTED BY ALLERGIC BRONCHOPULMONARY ASPEROILLOSIS (ABPA)

A. P1do1n, L Enftnl, R. Levi. M.T. M1rnno, 0. Pizz.lmlgllo, A. Giunll CF C.,l« of Miiln, I.C.P. 20122 Milan, Italy

It' 1 Mil known that CF p81ientl are sUICIPiible to the d-'opment of /4BPA wtth 1 reported frequency of eboul 1~. More ,_,tly an lncrllled frequency of CFTR gene mut81oons h11 been reco-ed from p1li_,.. with ABPA. we how retrotpectlwly ev~lu81ed our CF populotion to lnwstigote If /4BPA diognoaio woo r .. aon for refarring pta to CF Center, 1clually to our CF Center (pldietrlc ond eduH unHa) patienll prnenting pulmonery diollll ore atudied to confirm CF doognoais. Since 1991 51

1ymptomotic CF pta wore doognoted (ogo .1-39 yra). Five potienll hod FICiived 1 prevtoua doognoa11 of ABPA: CF woa confirmed In ell. In !hill potianll (3 moln and 2 lomoln), dlognoala of ABPA wa1 midi 81 1 m•n ago of 20 yra 3 mo (rongo 8-27 yra), othorwou

Poster Session Abstracts 297

diagnosis of CF wos delayed. Mean age II CF dlognosla 23 y11 3 mo (ronge 13-34)(mean diognostic deloy 3yra). AI diagnoalt 515 prHented: Pleudomoneo ..ug/n011 lung Infection end bronchiectaaies ; 315 prHented chronic slnualtis ond noaol poiyposia, 2 males were azooapermic, one had thown recurrent episodes of ocute pancre81itis, none of them had liver disease. Pancreatic function Wll normol In ell. CF diagnoala was confirmed by moons of swoet chloride value end genetic anali1ys: 98 mmoi/L (M'508/T3381): 110 mmoi/L (M'5081R352Q); 55.5 mmoi/L (& F508/S1251N); 110 mmolll. (6F508/unknown); 55 mmolll. (& F508/unknown). I>J. pr11ent. ogH of potionll range from 17yra 8mo to 38 yra (m•n 1111 27 yra 7 mo). Conctualono: 11.8'!1. of 51 CF potlonta ev~luated for CF ralated symptom• had prevtoualy FICiived • correct diagnosis of ABPA. This lung dlseaaeled to ev~luation for the presence of CF, confirmed in oil. We think th81 ell patients effected by ABPA must ba lnveatigated lor CF In specialized Canters. Sweet chloride t11t could be borderline lhus en extensive genetic enaliayt (to Identify raro CFTR mutations) Ia • necesury slap to confirm dlagnoslt.

332 LACK OF CORRELATION BETWEEN CF SPUTUM SUSCEI"''mlLITY AND RESPONSE TO ANTIBIOTIC THERAPY. M.M. Saeed, M.S. Woo, S. Srinivasan, W.H. Mason, L.A. Ross, C.M. Bowman. Divisions of Pediatric Pulmooology and Infectious Diseases. Childrens Hospital Loa Angel01. USC School of Medicine. Los Angeles, CA. USA.

Are sputum susceptibilities necessary to determine antibiotic therapy In all CF pulmonary exacerbations? To help answer this queslion, we reviewed ~rds of 8 CF pts (CF-PR) with pan-resistant Pseudomonas atruglnosa (4& :4 9; mean aae 17.9±S.3 yn) and compared them to 8 CF pll (CF-S) with ausceptible organ­isms (4&:49; mean age 18.9 ± 2.4 yn). Data collecled included admission FEY, FEF,.,, Sp01, CF sputum cultures and susceptibilities and discharge FEY,, FEF,.,, Sp01• All pts were admilled for pulmonary exacerbations and were released after meeting the same discharge criteria (afebrile, no respiratory distress, baseline PFTs, etc). All patients received IV antibiotics, DNAse, and chest physiotherapy. Results were compared between groups using Studenl'l paired !-tests. Results reponed IS mean±sd. Significance WIS set IS p < 0.05.

Variables CF-PR CF..S p-YIIue

Age 17.9 ± S.4 yn 18.9 ± 2.4 yn 0.71

Hospital Days 15.6 ± 4.S 14.7 ± 4.3 0.68

FEV1 Admit 30 ± 11.7'1 32.6 ± 14.9~ 0.71

FEY! 0/C 47 ± 20~ 46 ± 15~ 0.93

FEF, Admit 14.S ± 7.S~ 13.7 ± 6.9~ 0.87

FEF,., 0/C 19.8 ± 8.3% 19.2 ± 9.2% 0.90

Sp02 Admit 90.7 ± 6.4~ 91.2 ± 4.2~ 0.8S

Sp02 D/C 9S.6 ± 1.8% 9S.6 ± 1.9'.\ 1.00

There were no significant differences In hospital days, pulmonary function teats, and oxygenation bciWeen pts with pan-resistant Pseudomonas and those with susceptible organisms. In this study, pll colonized with pan-resisliDt organisms had shon-term outcomes comparable to patients with susceptible organisms. Strict adherence 10 in vitro susceptibility testing may not be necessary for sucessful treatment of CF pulmonary exacerbation.

333 BRONCHIAL ARTERY EMBOLIZATION FOR THE TREATMENT OF HEMOPTYSIS IN PATIENTS WITH CYSTIC FmROSIS. GM Brinson, MA Mauro, MR Knowles. JR Yankaskas, PF Jaques,~- Cystic Fibrosis Pulmorwy ReseaJCh and Treatment Center and the Department of Radiology, Univenity of Nonh Carolina at Chapel Hill.

Hemoplysis is common in patients with cystic fibrosis (Cf). Bleeding may vary in severity, ranging from minor blood streaking of sputum, to expectoration of significant quantities of blood. Sub-acute or chronic mild hemoptysis (CH), while not acutely life tlueatening. may inhibit effective airway clearance. Major hemoptysis (MH), defined as bleeding greater than 240 ml/24 hr, represents a medical emergency. Bronchial anery embolization (BAE) is one or the treatment options for hemoptysis. At the Univenity of Nonh Carolina Hospitals we reviewed the insti!Uiional experience in the treatment of hemoptysis by BAE. From 1987 10 1997, 19 patients with CF (n•llM, Sf) underwent 37 BAE procedures during 30 hospitalizations for hemoptysis (n•l9 MH. n-11 CH). AI catheterization, the area judged likely 10 be bleeding (defined by the clinical data:

298 1997 Cystic Fibrosis Conference

history, chest radiograph. and any other investigations) was inspected. and all abnonnal arteries embolized. The median FEV I of the group was 30% predicted (range 13-84%). A high proportion of patients (68%) had severe lung disease (FEVl< 3S%) with a high incidence of multi-drug resistant bacteria (47%). Chest radiographs were reviewed for 26 of the 30 episodes; SO"Io of films were useful to reuospectively suggest a soun:e for the bleeding. One patient had a significant coagulopathy felt to be a contributory factor in the hemoptysis (PT•IS.4 on admission). Sixteen patients (n=27 presentations) were followed for a mean of 20 months after BAE. The overall efficacy of BAE for initial control of hemoptysis was 77% (n•23) after one session, 90% (11"'27) after two sessions, and 93% (n=28) after three sessions. Among patients who underwent BAE for recurrent hemoptysis on separate occasions, there was a high incidence of bleeding from non-bronchial systemic collateral vessels (n-12 of 16 presentations in 7 patients). One patient was demonsuated to have a patent large right bronchial artery (responsible for bleeding) despite having had that vessel embolized 10 years earlier with a metal coil. There were two deaths associated with massive hemoptysis despite BAE. Two patients had transienl neurologic dellcits during the BAE procedure, felt to be associaled with catheter manipulation near cerebral vessels. We conclude that BAE is a relatively safe and e!Jec:tive method of treatment for chronic mild hemoptysis as well as massive hemoptysis in CF patients. Patients with massive hemoptysis tend to have very severe CF lung disease, and a significanl number have multi-drug resistant bacterial infection. Patients with hemoptysis who have had previous embolizalion procedures should undergo rigorous angiographic inspection of all possible bleeding sites, including non­bronchial and previously cmbolized vessels. Supported bY CFF LS43

334 THE USE OF ANTI-INFLAMMATORY MEDICATIONS IN CYSTIC FIBROSIS: TRENDS AND PHYSICIAN ATTITUDES C M Oormann M.M. Sockrkler. Department of Pedlatrlca, Baylor COllege of Medldne, Houston, TX, USA M.W. Konltln. Department of Pediatrtc:l, C8le VVestem Reserve Univetlily School of Medicine, Clevel8nd, OH, USA

IDCrNiing evidence suggests thltt airway inftlrnrnation occurs early In the course of CF lung dlleiH and, • dlleiH advances, contributel to aignificlnt dutruction of lung tiaue. Thil observation his prompted intenae inlerelt in the potenlilll uae of anti-inftlrnmatory medicltiona to decreaae inftlmmation and preaerve pulmonary function in patients with CF. Long term uae of 01111 corticolterokla and Ibuprofen have been shown to be effective In slowing the progreulon of CF lung dllelle. Inhaled cortlcolterokll (ICS) have not been adequately ltudled thUI fir. Little il known nagardlng use trends and phyliclan attitudes toward these drugs. To examine thil illue, the diniCiorl of 111 US CF Centers MAl c:onlllct.d vii letter lnd liked to complete 1 two page survey. The survey laked t'tJr lnformltion regarding phylicilna' attitudes toward anti-infllmmatoriea, number of patients on therapy and number of phyllicllns prncriiMng therapy. 42 •Ul'VIIY• were retumed (38%). 53 non­reaponcllll'l MAl ~tlcted; 1110111 retuml IDS being IDIIyzld. The responding centers naported on <197 4 patient~ ( < 5 years • 961, 5-12 years • 1504, ~ 13 years • 2129, unknown • 380), 1037 (20.8 %) of whom IDS on routine antl-inftlmmatory drugs.

<6 5-12 ~13 TOTAL Oral Steroid• 10 48 120 176(3.5%) ICS 47 192 293 532 10.7% lbllprofen 11 187 131 329(6.6%)

RHponding Cenllrs rapraaented 139 phyalcilns. Of these, 53 (38%) pnaacribed long term uae of oralllerokla, 58 (42%) prncrlbed Inhaled sterokla, lnd 61 (44%) pnaacrlbed hlgh-dollllbuprolen t'tJr control of CF lung dileaae. The practitiot Mll'l surveyed naported flmililrlty and efllc8cy • the primary 1'811001 lor pr111crtblug oral COitloolterokll; concema over aide effects wens the major 11111011 for not pnaacrlblng. Regarding Inhaled cortlcotlterokla, the primary I'NIOIIS lor pi'IICrlblng -. flmUilrlty and llfety, with lick of llfllclcy being cited • the major 11111011 for not pmcrtblng. For Ibuprofen, etlk:lcy WM ranked hlghelt lrDODQ nanons for pnncribing with concem over llfety being the hlghelt ranked 01111011 for not pi'IIICrlbing. Further enelysil is undllrwly. As 1 group, lntl-inftlmmatory medlcltiona appear to be an under-utilized therapeutic modality in CF care. Thil II true in t1mt1 of numberl of patients on theae drvga • welt • numberl of Cll'l providers pretiCrlbing them. Additlonll ltudiel will be naqund 1o lddreu phylicllnl' concema reglrding the long term llfety and efllc8cy of antl-intlammltory druga in trutlng CF lung diiMM.

335 ANTI-INFLAMMATORY THERAPY FOR LUNG DISEASE IN CYme FIBROSIS: CURRENT PAEDIATRIC PRACI'ICE IN UK. 1M Balfour-Lynn' & C Dezateuxl. Respiratory Unit, Great Ormond Street Hoapltal for Children' and Unit of Epidemiology & Bioatatistica, lnltitute of Child Health', London, UK.

lutrodudloa: It lw been sugeated tiW IIUi-lnfllmmalory thmpy

may be an effective treatment for CF lung disease, but further evidence is required. While a recent survey' revealed 34% of CF adults were taking inhaled corticosteroids (ICS), similar data for children are lacking. This national survey of UK CF centres was undertaken to determine the current use of ICS as well as ibuprofen in paediatric CF lung disease. Methods: Postal survey to the Paediatric Departments of all CF centres (n=31) with over 50 patients (list provided by the CF Trust). Results: Replies were received from 30/31 (97%) centres treating almost 3500 patients under 18 years. In 70% of centres patients took regular oral steroids, and overall they were prescribed for 5% patients. Usage was generally low with median (range) 4% (1-23%) patients, but 5 centres had over 10% patients on oral steroids. The main indications were ABPA and asthma I wheezing; however 4 centres (13%) usually gave them for severe lung disease whilst 13 (43%) sometimes did. All centres had patients on regular inhaled steroids and overall they were prescribed for over 40% palients. Within the centres, usage was common (median 44%) although there was a wide range (10-90%). Most cenlres (87%) usually gave them for troublesome wheezing; 7 centres (23%) also usually gave them as anti-inflanunatory therapy whilst 13 (43%) sometimes did. The practice of prescribing for lung inflanunation started in the late 1980s and early 1990s in most centres. Only 7 (23 %) centres had ever prescribed ibuprofen for lung disease and 5 of these had only given it to a single patient. ConclusioJIS: The practice of treating CF lung inflammation in the UK varies widely between centres. ICS are used most frequently, with oral steroids reserved mainly for ABPA. The use of ibuprofen for lung disease is not routine in the UK. Randomised trials are needed to clarify the exact roles of these polent drugs in CF as they are nol without adverse effects, and in the case of ICS their widespread use will soon preclude such studies. I. Walter> S. ASJOC" of CF Adulu (UK) Survey 1994. Published by CF Trust 199,.

336* ASSESSING BREATHLESSNESS IN CWLDREN WITH CYSTIC FIBROSIS USING GOS 3-MINUTE STEP TEST. SO Randall', SA Prasad', IM Balfour-Lyoo'. Depts. of Physiotherapy' & Respiratory Medicine', Great Onnond Street Hospital for Children, London, UK.

Introducdon: Measures of perceived breathlessness and exertion are not readily understood by children and are somewhat subjective. We studied an objective score - Dyspnea Index (01), devised by Stanford Health Services USA and modified by University of Michigan Medical Center, but not yet validated. Methods: Subjects take a deep breath then count out loud to 15 (taking about 8 sees); the number of breaths to complete the counl is the 01. [1/ S4 patients with CF (mean age J2.S years) performed a standard 6-ntinute walk and GOS 3-ntinute step test (IS em single step with slepping rate of 30/ntin for 3 mins). Dl was compared with modified Borg score after exercise. {2/ A further 20 CF children (mean age 12.7 years) and 19 healthy schoolchildren (mean age 12.S years) underwent an incremental slep test (20/min for 2 ntins, 30/ntin for 2 mins, 40/min for 2 mins) using Dl and Borg score between increments. Results: [1/ Dl was sigrtificantly increased (p<0.0001) after both walk [mean (9S% Cl) 1.3 (1.1-1.4) vs 1.6 (1.4-1.8)] and step test [mean (95% Cl) 1.2 (1.1-1.3) vs 2.6 (2.2-2.9)], although step test made children significantly more breathless [mean difference (9S% CO 1.0 (0.8-1.3)]. Correlation of DI and Borg post-exercise was higher for the step test than the walk (r - O.S9 vs 0.19). [2} CF and normals had sintilar baseline scores for both Dl (mean 1.1 vs 1.0) and Borg (mean 0.4 vs 0.1), but after 6 mins of the incremental test, scores had increased significantly more for those with CF than normals [mean (9S% CI) difference 1.6 (1.0-2.2) va 0.6 (0.1-1.2) for 01, p<0.02; and 3.3 (2.5-4.1) vs 2.2 (1.5-3.0) for Borg, p<O.OS]. Both scores increased significantly over time, but the regression coefficients were significantly grealer for CF than normals [mean (9S% Cl) slope 0.28 (0.16-0.40) vs 0.10 (0.01~.16), p<0.02 for DI; and 0.56 (0.42~.7) vs 0.36 (0.24-0.47), p<0.03 for Borg). Regression coefficients for DI and Borg were better correlated in CF (r-0.52) than normals (r•O.IS). Condlllions: The DI has been validated as art objective measure of breathlessness. It is euy to explain and perform, and can be used by any child capable of counting fluently to IS, in any language.

337* TilE EFFECT OF INTRAVENOUS ANTIBI011C TREATMENT ON CHANGES IN SPIROMETRY AND SHliTil.E TEST PERFORMANCE IN CF. ~·. JM. Bradley", ES Wallace", V Hall", JS Elbom0 •

Regional Adult Cystic Fibrosis Cenue, Belfast City Hospital" and the Department of Sport and Exercise Sciences", University of Ulster, Jordanstown, Northern Ireland.

The shunle walk test is a standardised, incremental, externally paced 12 level walking test, that has been previously used to measure changes in exercise capacity in patients with chronic obstructive pullliOIW)' disease. During a pn:liminary study we investigated the use of the shuttle walk test as an assessment tool for exercise capacity in adults with CF. This led to the development of a modified 15 level shunle test in which patients wen: permitted to run. This modified shunle tes1 uses a greater range of speeds which accommodates the wide range of exercise capacities typically seen in a CF population. The objective of this prospective study was to assess the effect of a hospital based intravenous (IV) antibiotiC course on shuttle tes1 perfonnance in 10 male patients with CF, mean (SO) age 27(5), with an acute respiratory infection. In addition the n:lationship between shuttle tes1 perfonnance and spirometric measun:s of lung function was compared at the beginning (TI), and at the end (T2) of an IV course of antibiotics. The n:lationship between percentage improvement (T2·TI)xl00 in lung function and shuttle tes1 performanoc was also compared. Intravenous antibiotic treatment significamly improved spirometric measures of lung function and shuttle tes1 performance (p<O.OS). Then: was a mean percentage improvement of I 9"/o (FEV1), II% (FVC), 34°/o (FEF,,.,,..) and 21% (shunle test) following IV antibiotic treatment. There was a significant corn:lation between shuttle tes1 and FEV1 at the beginning (r-0.76, p<O.OS) and at the end of antibiotics (r-0. 70, p<O.OS). However there was no corn:lation berwecn treatment induced changes in shuttle tes1 performance and FEV, (r-().1). The shuttle tes1 is a sensitive measun: of exercise capacity in CF adults. Treatment induced changes in exercise capacity an: not solely dependent on changes in lung function.

338* A TWELVE MONTH COMPARISON OF STANDARD VERSUS MODIFIED CHEST PHYSIOTHERAPY IN TWENTY INFANTS WITH CYSTIC FIBROSIS. BM Button R. Heine, A. Catto-Smlth, A. Olinsky, PO Phelan, I Story. Departments of Physiotherapy, Gastroenterology and Thoracic Medicine, School ot Physiotherapy, University ot Melbourne, VIctoria, Australia.

Chest physiotherapy (CPT) II widely accepted as part of routine treatment In CF. Before newborn acreaning, CPT usually commenced attar the onset ot respiratory aymptoms. With the Introduction of screening In 1989, CPT has been Introduced around two months of age. we demonstrated In a study of twenty Infanta with CF using pH monitoring that there was a algnlflcant Increase In the number of reflux episodes during atandard physiotherapy with head down tilt (SPT) compared to modified without tilt (MPT) (1). The aim of this study was to compare the effectiveness ot MPT versua SPT. The twenty infants In the previous study ware randomise<! to dally SPT or MPT for the following twelve months. Both regimens ware carried out after Inhalation of normal saline. While asymptomatic, Infanta were treated once a day, with respiratory symptom• these were Increased appropriately. Parents were Instructed to complete a diary recording number ot treatments, presence ot Increased cough, upper respiratory tract Infections (URTI), wheeze and antibiotic use. Radiograph• were taken et diagnosis and twelve monthly thereafter. Twelve rnontha after entering the atudy, the diaries were analysed and the number ot days with Increased cough, URTI, wheeze and antibiotic use were calculated, together with hospital edmlssions and radiographic findings. Sixteen aubjacta completed the study (eight In each group). SPT veraua MPT results: annual cough days: 118 :1: 73.56 vs. 83 :1:119.86, p• 0.50; annual URn days: 70 :1: 32.78 vs. 37 :1: 24.91, p=0.04; annual wheeze days: 41 :t 32.46 vs.25 :1: 54.54, poo0.50; annual antibiotic days: 116 :t 88.56 vs. 67 :1: 58.62, p• 0.21; radiographic reports: 5 patients VI 4 with pulmonary changea; hospital admissions for respiratory problema: SPT • 2 patients had two admissions each; MPT • 2 patients had one admission each. We found no signifiCant differences between the parameters measured In the two groups except for URn days. We conclude that MPT should be considered as the most appropriate treatment regimen for Infanta In the first twelve months. Reference: 1. Button BM, at til (1997) Archival of 0/aease/n Childhood 78:148-150. Funded by: Physiotherapy Research Foundation and RCH Foundation.

Poster Session Abstracts 299

339* "FLUTTER VERSUS PEP"t A LONG-TERM COMPARATIVE TRIAL OF POSITIVE EXPIRATORY PRESSURE (PEP) VERSUS OSCILLATING POSITIVE EXPIRATORY PRESSURE (FLVlTER) PHYSIOTHERAPY TECHNIQUES. PM Mc!lwainc L.T.K. Wong. D.Peaoock. A.O.F.Oovidson. Deportment of Paediatrics, Uni>ersity of British Columbio and Department of Pbysiothenpy, BC Children'• Hoopital, VIIIOOII\'ef, B.C. Conada.

Oac:illating Positive Expiratory Pressure physiotherapy delivered by the Flutter device hu been recently recommended for cywtic libroaiii(CF) patiento. However, lhenl have been no long-tam controlled clinical triola comparina Flutter to other Physiothc:rapy techniques in CF. We have prcvioualy reported that phyoiothcnpy uaina the Astra·Meditec positive expiratory preuure muk (PEP) hu aignificant advantoges oompored to con-.tionol pooiUral droinoge ond ,...,....;on in the tre.olmenl of CF 1•

The~ of the lllldy reported here wu"'- the eft'ccta of physiothenpy .wn, the Flutter device a oompored to the Astra Mcxlitec PEP muk uoal ia our prcvioua lllldy. Forty paticnto between the ages of 7 ·17 )'elrl wilh Shwachman acon:a between 54-911 ottending the BC'a Childn:n'a Hospital cywtic fibrooia clinic ws-e enrolled in the lllldy. AU paticnto were performing PEP prior to the COIIIIIICIICCIIt of the lllldy. During the lllldy period. paticnto were randomly assigned to one of two llfOUpa, Group A wen: the control and continued performing pbysiotherapy using the PEP nwJc for 1 one year period while paticnto assigned to Group B performed oacillating PEP ...U., the Flutter device for the umc one year period. Clinic physiciana were blirxlod u 1o the group 10 which the patiento wen: ISiignod. Compliance with phyaiotherapy by both groups wu closely moniton:d throughout the lllldy. Clinical statua and pui!IIOIIIfY function(FVC, FEV1, FEF,..,) were meuurcd 11 the beginning of the lllldy and Ill regular intervals throughoul the lllldy. Five patients dropped out 6:om group B u they subjectively felt that the Flutter wu ineffective in clearina their aecrctiona. A fw1her 2 patiento in group B(Fiutter) wen: withdrown by clinic phyaiciana due 10 clinically aignificont detcriontion in pulmoowy function. 2 patients wen: dropped 6:om Group A (PEP)' due to DOD-canpliancc. The number ofbospitalizotions and use of antibiotics becouae of deteriorating respiratory statua wu aignifiCIIttly more ia the Flutter group (22 admissions compared 10 S in the PEP group). The FVC doc: lined significantly in the Flutter group as compared to the PEP srouP(P-0.01) and thercwuo greotcr downward tn:nd in FEV, and FEF,.., ollhoogh theae did not n:ach otatistical aignifiCIIICe. Our results indicote that Fluner ;. not u eft'ective as PEP in maintoining pulmonary function ia CF patientollld ;. more costly due to inc:rcued need for hospitalizations ond antibiotics. References I. Long-tam camparotiw trial of OOIIWIIIionol pooiUral drainage and percussion venua positive expiratory pressure ph)'liothenpy in the tre.olmenl of cystic tibrosia. P.M.Mcllwainc.ct II. Pcxliatric Pui.Suppll2,ablt 268.1995.

340* THROUGH RANGE COMPUTER GENERATED INSPIRATORY MUSCLE TRAINING IN CYSTIC FIBROSIS K ChaJhamu, A. Ionescu•, C. Davies•, 1 Baldwin .. , S Enright• .. , and 0 J Shale•. Departments of Respiratory Medicine• and Physiotherapy .. , Llandough Hospital, Cardiff and Salford University••• UK.

Systemic exercise is commonly pn:scribed as part of the management of CF patients. Although respiratory failun: is the fmal common pathway to death, the potential benefits ofstn:ngthcning the inspiratory muscles by specific training has not been extensively explored. Evidence fTom a meta-analysis of respiratory muscle training ( RMT ) in chronic lung disease' suggests that the equivocal results -.t are due to 1 paucity of well designed trials coupled with RMT methodologies which have not guaranteed an effective training load. We compared two RMT interventions; Thn:shold RMT with load set at JO"Ao of peak and computer generated through range inspiratory muscle training ( TIRE1 ) in which load was ftxed at 80".4 of individual capacity. I 8 adult CF patients were randomly allocated to the RMT groups. All patients completed 8 weeks of TIRE RMT, but 3 subjects did not complete Threshold training. TIRE subjects increased respiratory muscle suength ( RMS) from 118 t 31.S to 149 t 24.7 em water pressun: ( p < 0.04) and respiratory muscle endurance from 16812 t S071 to 23754 t 7344 pressure time units ( PTUs; p < O.D2 ). RMS was also increased through range when measured II 2S and SO% of tho original training template from 73.7 :t 19.7to 110.6 tIS em water pressure 1125% ( p < 0.001), and Sl.8 t 13.6 to 76.8 t 8.8 11 SO% ( p < 0.001 ). These changes resulted in incn:ased single bn:ath force generation with sustained maximum inspiratory pressure incn:asing from SB I t 193 to 852 t 292 PTUs ( p < 0.000 I ). In addition VC and MEF SO both incn:ased in TIRE subjects ( p <0.001, p < 0.01). Patients olso reported subjective improvements in chronic respiratory disease questionnaire parameters (mastery and emotion; p <0.01 ). In contrast, the Thn:shold trainers exhibited no chango in n:spiratory muscle function or any other measure. We conclude thll TIRE RMT set at 80".4 of peak is effective in improving respiratory muscle perfonnance throughout inspiratory range. These changes have a positive impact upon lung function and quality of life. I. Smith et al. Respiratory Muscle training in chronic airflow limitation: a

meta-analysis. Amer Rev ofResp Dis 1992;14S;S33-S39. 2. Chatham et al. Fixed load incn:men respiratory muscle training

physiotherapy 1996;82;7,422-426 .•

300 1997 Cystic Fibrosis Conference

341 343 COMPARITIVE EVALUATION OF TilE FLU1TER AND TilE CORNET IN IMPROVING TilE COHESIVENESS OF CYSTIC FIBROSIS SPUTUM. Bopnje pygupta. Sciicbi Nobmura, Ernst M. App• IIIII M.lcolm King. J>u1mcxwy Rescan:h Oroup, Uniwnity of Alberta, Edmooton, C1nada; •Dept ofPncwnology, LMU-Univcrsity, Munich, Germany.

Canpn:bensivc lrcalmcnt of cyllic fibrosis (CF) hms dioeuc COIISistl of,_..,.... directed at increasing the clearance of exceoo bronchial secretions, thus improving lwtg limctim IIIII oxygenation. The effectivity of the Flutter in improving the viococluticity of CF airwoy oec:reti""" bu alreody been established In vivo (Am J Reopir Crit Cere Med 1995; 15J:A737). The Comet, a device similor to the Flutter, usisto CF paticnla in mucus clearlnce. Our goal wu to evaluate the efficacy of two different devices, the Camet IIIII the Flutter,"" the cohesiveness (thread formation) of CF sputum. Equal volumes of ten different aliquota from pooled CF sputum were subjected to the following protocols: I) buclinc (no lrcatment); 2) airflow~ of 1.5 Us produced within the Flutter mounted at mid-position; 3) airllows of I.S Us produced within the Comet. For each protocol, the sputum cohesiveness (in mm, meon ± SE) wu measured by meons of a tilaoccmeter at bucline IIIII at 30 minutes. In comparil(lft to the bucline value (13.63 ± 1.33), the Cornet demonstrated a modcot decrease in cobcsivencss at 30 minutes (9.73 ± 1.14, p • 0.04). In conlrUt, the Flutter produced a sublltlntially pater decrease in cohesivenea ofCF sputum (13.63 ± 1.33 vo. 5.67 ± 1.25 at 30 minutes, p • 0.0004). A reduction in cobcsi- is related to a lower viococluticity, thus predicting enhanced clearance of mucus from the airways of CF patients. Although both devices improved the quality of the sputum, this In vitro study suggests that the Flutter is more efficacious than the Camet in reducing the cobeoivcness of CF sputum. This difference belwoen the devices moy be alimction of the &equency IIIII amplitude of oscillations they generate. Other aspects of theoe devices (such u variati""" in expiratory tlow rates IIIII 111gle of usage, and the reduced pressure requirement for activllina the Comet) moy modulate tbeoe In vitro elfccta"" mucus rheology. Thus, further clinical studies"" the Camet IIIII the Flutter ere necessary to fully uccrtain their eft"ccta "" CF patients.

342

THE EFFECfS OF ADDING PEEP TO HIGH FREQUENCY CHEST COMPRESSION ON OSCILLATED TIDAL VOLUME IN CHILDREN WITH C.F. C F Dolllllll. I. Tabak, P.C. Zuberbuhler, R.L. Jones. Departments of Pediatrics and Medicine, University of Alberta, Edmonton, AB, Cansda.

Children with C.F. have thick mucous obstructing their airways which requires physiotherapy for its clearance. High frequency chest compression (HFCC) increases mucous clearance in adult C.F. patients. However, by compressing the chest wall, HFCC decreases end-expiratory lung volume (EEL V), which further increases an already high airway resistance. Using PEEP with HFCC can increase EEL V back to FRC which, in turn, should decrease airway resistance and perhaps improve the eft'ectiveneu ofHFCC by increasing oscillated volume change at the mouth (Vosc). Nine pediatric CF patients (9-16 years) underwent HFCC alone (mean chest wall pulse pressure 8 ~o. frequency I 0 Hz) for 20 minutes, and on a separate day underwent HFCC +PEEP (mean 2 cmH20) for 20 minutes. Trapped air wu measured u the ditrerence between helium-dilution n.c and body box n.c, with measurements taken before and after the treatments. Wrth HFCC, EELV decreued to a mean of93% of bueline FRC. With HFCC +PEEP, EELV increased to 104% of baseline FRC (p<O.OS). Wrth HFCC, Vosc during spontaneous expiration avenaed 30.2 mL. With HFCC +PEEP, Vosc increased to 34.3 mL (p<O.OS). Despite the increased Vosc with HFCC +PEEP. there wu not a significant dift'erence in air trapping compared to HFCC alone. The use of PEEP with HFCC prevents the decrease in EEL V that OCCUI'I with HFCC alone. HFCC + PEEP also increases Vosc. In our study, thele did not result in decreased air tripping. However, cmly a amall amount ofHFCC and PEEP were used. A larger study with longer treatment periods may yield the decreased air trapping that we expect from the PEEP-induced increases in EEL V and Vosc.

Supported by American Biosystems Inc.

PHYSIO'IliERAPY: PATIENT PRAcnCE, KNOWLEDGE AND ADHERENCE MAY BE SUBOPTIMAL: llffi NEED FOR EVIDENCE BASED CLINJCAL GUIDELINES.

L.!l!u!!ro:. F Edenborough, D E Stablefonh, A Stracban Adult Cystic Fibrosis Unit. Birmingham Heartlan<k Hospital, Birmingham, Enaiana.UK

The I11118J of people with cystic fibrosis are often chronically colonised by bacteria from an early age. The principles of treatment include airway clearance tec:bniques (ACT) following bronchodilation, prophylaxis and early treatment of infection by antibiotic:s. Despite regular review and tuition, petieut practice, lalowlcd&c and adbcrence may be subofltimal. This was asseascd by self administered anonymous questionnaire. 118 (7~'Yo) of I ~8 patients responded. Acr were used alone or in a~mbination. The Active Cycle ofBreatbing Technique (ACBT) (32%), Autogenic DraiJUI&IC (AD) (26%), AD with Flutter Device (26%), postural draiJUI&IC with or without pen:ussion (23'Yo), AD with Positive Expiratory Pressure mask (11%). 8~% performed Acr at least once daily. However. duration was suboptimal in 74% when well and 44% when unwell. Frequency was IIUboptimal in 33% when well and 14% cUring an exac:crbation. Regular exercise was performed by 7S% of respondents. Type, duration and liequency wried according to individual capacity and preference. Drugs were ndlulised at suboptimal times in relation to Acr by 4% using bronchodilators, 6% DNasc and 7% antibiotics. 18% were nebulising DNasc with 10 inappropriate air compressor. Tbc results of this audit showed petieat (XliCiiQe, lalowlcd&c and adbcrence to be less than ideal. Several practical ~ to the aervicc have been implemented including the production of evidence based clinical guidelines distributed to staff, patients and carers, blc:ked up by regular review in the Adult Cystic Fibrosis Unit. Further assessment in twelve months time moy indicate their impact and complete the audit loop.

344 Use of Nasal lntennittent Positive Pressure Ventilation in Cystic Fibrosis Patients with Type II Respiratory Failure. Esmond GM. Mikelsons C. !kin NJ Empey DW. Adult Cystic Fibrosis Centre, London Chest Hospital, UK.

The use of Nasal Intermittent Positive Pressure Ventilation (NIPPY) has been demonstrated to correct type II respiratory failure in neuromuscular disease, chest wall disease and Chronic Obstructive Pulmonary Disease (COPD). The current indications for use in Cystic Fibrosis (CF) are uncertain and recommendations are largely confined to its use as a bridge to transplantation. We reviewed the use of NIPPV in 12 adult CF patients with type II respiratory failure. Indications for use were: 2 for bridge to successful transplantation, I during pregnsncy, I post thoracic surgery, I post extubation, I pneumonia and 6 for end stage disease to aid symptom relief. In the cases where NIPPV was used to support the final stages of pregnsncy, u a bridge to transplantation, post thoracic surgery, post extubation ventilatory support and in one of the end stage patients positive outcomes were obtained (increase in PaCb and decrease in PaCCb, symptom relief and improved quality of sleep). However in the I case of pneumonia and S cases of end stage disease poor outcomes were obtained (no change in PaCCb despite increased PaCb, poor symptom control and increased sputum retention). We conclude that NIPPV is most effeaive in short term ventilatory support for specific reasons, and decreasing PaCCb is a guide to its efficacy.

345 EXERCISE CAPACITY, VENTILATION AND SYMPTOMS AT PEAK ARM VERSUS PEAK LEG ERGOMETRY IN CYSTIC FIBROSIS. AJ Moorcroft. ME Dodd, and AK Webb. Adult Cystic Fibrosis Unit, Wythenshawe Hospita~ Manchester, UK.

There have been few studies of upper body exercise in CF and no comparisons with lower body exerc:ise. · We hypothesized that interference with respiratory muscle function would result in a disproportionate

limitation of IIpper body exercise with greater breathlessness. Fifty-three adults with CF perfonned both progressive cycle and ann ergometry tests to a symptom-limited maximum with measurements taken at peak exercise. The mean (SE) age of subjr.;•.J Nas 23.8 (0.8) years, BMI 20.9 (0.3) kglm1

and FEV1 61.7 (3.0) %predicted. Results expressed as Mean (SE) values at peak ann vs peak leg exercise were as follows: Oxygen uptake Jl.21 (.05) vs 1.73 (.07 )Umin'] and Heart rate [156 (2.4) vs 168 (1.8) bpm) were lower for arm ergometry averaging 70.8% and 92.9"/o of leg values respectively. These ratios arc consistent with previous estimates in nonnal subjects1

•

Ventilation [51.9 (2.2) vs 66.5 (2.8) Umin'] and blood lactate (5.8 (0.3) vs 6.9 (0.3) mmoi/L'] were similarly lower for arm exercise. The Ventilatory equivalent for carbon dioxide (VEIVC~) was raised [36.5 (0. 7) vs 31.5 (0.6)'] and along with a lower end-tidal pC01 [37.4 (0.6) vs 42.7 (0.8)mmhg'] suggest relative hyperventilation at peak arm exercise. This could not be explained by a difference in oxygen saturation [93.2 (0.7) vs 93.0 (0.7)%). Tidal volume at peak exercise was lower for ann ergometry (1.24 (.05) vs 1.68 (.07) L'] therefore subjects didn't fully increase it- the usual initial mechanism for raising minute ventilation. Instead a greater respiratory rate was seen [43.1 (1.3) vs 40.5 (1.3) Umin"]. Interference with respiratory mechanics presumably resulted in this breathing pattern being adopted to minimise respiratory effort. The Borg score [.or breathlessness was slightly lower for arm exercise [3.7 (0.3) vs 4.4 (0.3) ]. Subjects perceived muscular fatigue as the limit for both arm and leg ergometry with almost identical mean Borg scores for muscle fatigue [6.3 (0.3) vs 6.1 (0.3)). Conclusions; Ann exercise capacity is preserved relative to leg exercise capacity in CF. Breathing patterns arc altered however muscular fatigue still limits both fonns of exercise.

I. Franklin BF. Sports Medicine 198~:2;100- 119. 0 p<O.OOI 00p<0.05

346 COMPLIANCE WITH PHYSIOTHERAPY IN CYSTIC FIBROSIS ADULTS IN

r-=.~~!~i~ ~::~nt of Public Health & EpideMiology, university of li l"llinghu, Bl5 2TT, Englilnd

Introduction poor compl1ilnce with self-physiotherapy has been previously. reported, particulilrly among young adults and adolescents Wlth CF. However it is difficult for a patient to admit non­compliance to a health professional. An independent survey was used to detennine factors affecting compliance with self­physiotherapy, as well as the IIM!thods used by adults in practice. Method confidential postal questionnaire to 1870 adults with CF in 1994 known to the Association of Cystic Fibrosis Adults (UK). Response rate was 54". Participants were asked whether they did any physiotherapy at home, who helped the~~, and what ~~~ethods were used. Results 76" of adults reported doing any physiotherapy at home. 66" did their own physiotherapy• 36" had some help from family or friends, 3" had regular proressional help (NB over l<Xl" because some had 110re than one input). 8<l" of those attending specialist treatment centres did physiotherapy, compared with 62" attending non-specialist clinics, p<O.Ol. Other factors affecting physiotherapy compliance were age (8~ under 20, 73" 20-29, 6~ 30-39, 81X 40 plus, p<O.Ol) sex (72" 01ales, 8<l" females

1 p<O.Ol) and receiving advice ~rom a physiotherar:>ist

in the ast year (8l.X vs Sal', p<O.Ol). There was no soc1al class difference, but responders with higher educati'?nal level (A-Levels) were significantly l•ss likely to do phys1otherapy (50710 vs 6~, p-<1.02 after adjust01ent for severity). The proportion doing physiotherapy fell with sevuity (85" 11ild, sa 110derate, 68" severe p<O. 01 after adjusting for sex). Patients who reported dolng physiotherapy and who attende~ specialist centres were significantly less likel~ to recewe help from family and friends (46" vs 55", p-<1.04 , and require domiciliary physiotherapy 0" vs 7X, p-<1.04}. T ey were 110re likely to be using autogenic drainage (8" VI lll, p<O.Ol), conclusion COIIPliance with physiotherapy as IIM!asured by an independent survey is low, and affected by age, sex 1 educational status, severity of symptoms, attending speciallst clinics and receiving advice from a physiotherilpist. Patients attending specialist clinics were less likely to be dependent on others for their phys 1 otherapy,

347 THE EFYECTS OF LOW AND HIGH DOSE SALMETEROL ON THE LUNG FUNCTION OF HOSPITALIZED PATIENTS WITH CYSTIC FIBROSIS Nl Hordyjk.. CO Judy, PH Sammut. lL Colombo Dcpanment of Pediatric Pulmonology. Univenily of Nebrasksa Medical Cenler. Omaha. Nebraska. USA. ..

In a rccent investiption we found that hospitaliled patients \\ith cystic fibrosis who received 0.~ of 0.~% albuterol nebulizer solution TID significantly increased

Poster Session Abstracts 301

their pulmonacy function across the day but fell back to baseline overnight. To see if this fall could be prevented we initially compared the clinically recommended dosages of salmeterol mdi (2 puffs. 42meg BID) to albuterol mdi ( 2 puffs, I HO meg QID) in a placebo controlled 3-way mndom crossover. double-blind trial. Eighteen patients on three consecutive days of testing nx:cived either salmcterol. albutcrol. or placebo wilh cac:h of four chest physiOihempy ICSSions given a1 7 AM, II AM. 3PM. and 7PM. The mean% change in FEVI from prethcmpy at 7AM to post thempy at 7 AM was only ~-~% wilh 2 puffs of salmctcrol compared to 9 0% wuh 2 putTs or albuterol (p-ns) and -1.2% \\ith 2 puffs of placebo (p-0.1146). Howe\ -cr. the mean %change in FEVI wilh 2 putTs of salmcterol increased 10 12.1"/o as measured from prethempy at 7AM to prcthcmpy at JPM vs onl)· ~.4% "ith placebo (J>"(HXI2). After lhcral')· at JPM. the mean % change in FE VI from prclherapy at 7 AM was 11.4% \\ith 2 puffs of salmeterol compared to 9.4% \\ith 2 puffs of albuterol (P""ns) and 6.0% with placebo (p-0.16). This suggests lhat 2 puffs or salmclerol ia slower in onset but better dosed lhan 2 putTs of albuterol. The follol\ing morning after salmcterol tn:auncnl (2 putTs BID and lwelve hours aller lhe last dose). the FEVI dav baseline was still elevated 7.3,. compared to ~-7% aller treatment with 2 puffs of albuterol QJD ( .,..0.048), and U% after 2 puffs of placebo QID (p-0.039). The carryover effects salmcterol significantl)· correlated \\ith indices or milder lung disease. were nolably less for the FVC, FEVI. and FEF2,·?$ in lhc 8 paticnls \\ilh 508/(508or GS42X) genotypes vs. other genotypes (n-9)(p< 0.22, 0.11. 0. IJ). and were sustained through treatment with albuterol and placebo suggesting thai salmeterol had both bronchodilator and mucous clearance effCCIS. We then mndomly ~nrolled 10 of the original 18 patients bock into the same protocol and doubled the inhaler dosages from 2 to 4 puffs. The mean % change pre-to-post themP.V at 7 AM with salmeterol in lhese 10 patients increased from 11.2% with two puffs to 17.Wo with four puffs for the FVC (p-ns). 4.8 lo 22.7"1. for the FEVI (p-0.064). and -~.89 to 44.2% for the FEF2S-75 (JI"'(l.027). Also. the carryover effCCI incrcased from 7.0 to 7.0% for the FVC (p-ns). 5.48 to 14.7% for the FEY I (P""ns). and from S.8S to 46.~% for the FEF 2S-7$ (p-0.084). No side eiTCCIS were observed al low doses of drug and one patient experienced tremor with high doses albuterol and salmeterol. There were significantly greater improvements in lung function across the day and night \\ith high dose salmeterol which may be due to greater and/or more SUSiaincd bronchodilator effects, muc:ociliary effects, anli·inllammatocy effects, and/or effects on chloride chaMel function. ( Rnlnl from Gla~o-Wellcome Inc.)

Transplantation

348*

LONG TERM RESULTS OF LUNG TRANSPLANT AT! ON FOR CYSTIC FIBROSIS (CF). K..Qyj, •c Dyke,+ A

Khaghani, ~ Radley-Srnith, B Madden, 'N Banner, M Hodson, +MYacoub

Dept of Cystic Fibrosis, Royal Brompton Hospital, London SW3 6NP. •Harefield Hospital, Harefield, Uxbridge, Middlesex, UB9 6JH.

Between 1984 and 199S, over an II year period, 129 patients

underwent lung transplantation for end-stage cystic fibrosis (CF) at Harefield Hospital and Royal Brompton Hospital. This report

reviews the long-term outcome of these patients. There were 66 males and 63 females with mean age of24 yrs (range 7-4Syrs). All patients were severely incapacitated due to respiratory failure

with mean FEV1 of22% predicted, FVC 3S% predicted, Pa02 of

6.8 Kpa and PaC02 of6.46 Kpa on air. lOS patients (81.4%)

had heart-lung transplantation, 19 patients had double lung

transplantation either en bloc or bilateral sequential single lung

transplantation, 4 had combined heart/lung/liver transplantation

and one patient underwent bilateral lobe transplant from two live

donors. The actuarial survival for whole group was 67% at one

year, 47"A. at S years and 33% at 8 years. For lOS heart lung

transplant patients the survival was 72% at one year, SO% at S years and 35% at 8 years. 63 patients died and early monality was mainly due to infection, haemorrhage and multiple organ failure. Obliterative bronchiolitis (OB) was an additional cause

of late mortality. 3S patients developed OB. The cumulative probability of developing OB (70% confidence interval) was 17% at one year, 42% at 3 years and S 1% at S years. Lung

function improved post-operatively and FEV 1 at 1,S,8 years were

70%, 75%, 77%, and FVC at 1,5,8 years were 74%, 87%, 70%

respectively. Retransplantation procedure was performed in 20 patients.

302 1997 Cystic Fibrosis Conference

349* Panreslatant Bacterial Infection In Cystic Fibrosis Patients: Influence on Lung Transplant Outcome. BM Alii. PH Gilligan, IP Neuringer, KK Gott, J Rea, & JR Yankaskas. Division of Pulmonary & Critical Care Medicine, Oepts of Medicne, Microbiology, & Transplantation, University of North Carolina, Chapel Hill, USA.

The number of cystic fibrosis (CF) patients undergoing lung transplant continues to rise as long-term survival improves. One major contraindication to this potentially life· saving intervention is infection with multi-drug resistant bacteria. We undertook this retrospective study in 72 transplanted patients over 7 years to determine the influence of pen-resistant bacteria on transplant outcome. The in vitro antibiotic susceptibility pattem of respiratory tract bacteria obtained pre- and/or intraoperatively was used to categorize patients into Panresistant (n = 32) [Burkholderia cepacia n = 6 and Pseudomonas aeruginosa (n = 26)) or sensitive (n = 40) groups. Post-operative ventilator days, hospital length of stay, and antibiotic days were similar for both groups (p > 0.2). The Incidence of bacterial bronchitis (28% for the panresistant group and 33% for the sensitive group) and pneumonia (30% and 37%, respectively) did not differ between these groups (p >0.2) at 6 months. Likewise, one· year (76% and 74%, respectively) survival was similar for both groups (p > 0.2). As expected, panresistant B. cepacia patients had a lower 1-yr survival (50% vs. 80%, p < 0.05) and had a higher mortality attributable to cepacia (50"k vs. o•A., p < O.Q1) compared to pan-resistant P. aeruginosa patients. Our results indicate that CF patients infected with pan-resistant P. aeruginosa have similar transplant outcomes as patients with sensitive bacteria and should not be excluded from lung transplant based solely on this criterion. Patients Infected With panresistant B. cepacia seem to be at higher risk for poor transplant outcomes, but the number of such patients in our cohort was small. Further studies are required to determine if post-transplant survival is significantly lower in CF patients infected with B. cepacia.

350* EXERCISE PERFORMANCE IN CYSTIC FIBROSIS BEFORE AND AnER BILATERAL LUNG TRANSPLANTATION. PA Oalbam D.M. Syatrom, S.L Zorb, C. Wright, J.C. Wain, L.C. Glnns. Pulmoll8ly I Crllcal Care & Thoracic Surgery Unb and Lung Transplant Program, Massachusetts General Hospllal, Boston, MA, USA.

Bilateral lung trlnlplartatlon (BL T) Ia an accepted modality for the treatment of ~Qq patlenta wlh cyllllc fibrosis (CF) because I lfT1lrOvts lung function and quality of lie. A ayatematlc evaluation of exercise capacity In patlenta with CF following BL T hal not been deacrlled, however. To determine I reduced Oz delvery or uptake by nuc:1e are In part responsllle for limiting exerclee following BL T, lt'len pa11enta wlh CF (age • 27 ± 3 years) performed Incremental exercise telling before and alter (16 ± 5 months) BLT. Expired gaaea and Ve were meuurecl brelth·by-breath using a conmerclally available melabollc cart (MGC 2001, St. Paul, MN). Arterial blood gases, pH, and lactate were eempled each min during exercise, and cardiac output was meaaured by 11111-pasa radlorucllde ventrk:ulography (System 77, Baird Corp., Bedford, MA). Systemic Oa extraction ratio (OzER) was calculated as Caoz-Cvo/Cioz, where ~was derived by the Flck ~ (Caoz.CVOz • Vo-/0). FoAowlng BLT, FEV1 1118/ked1y lnlX'oved (0.90 :t 0.18 VI 2.12 :t 0.24 L, mean t SE, p • 0.002). VOzmu lfT1lrOved considerably, but remained well below predicted Valu81 (29 :t 4 VI 41 :t 3 'Yo pred, p • 0.03). Hemoglobin concentration W81 reduced after BLT (13.7:t 0.8 VI 11.6 :t 0.5 !jell, p • 0.05), Exercise was llmlled by pulmonary mechanics In Ill p1or to BL T (breathing reeerve < 11 Umln), but In only one (14 %) after BLT. The "log-log" lactlle Uveahold (L T) oc:amed In an patlenla pre- and post·BL T, and was abnormaRy early,though Improved following BLT (18 ± 1 Vl22 :t 1 %pred V02max. P • 0.006). Peak exercise OzER lncreaaed to near-nonnal after BL T (39 ± 8 va 58 t 8%, p • 0.02); hoWever, neither canlac: output (53 :t 6 VI 58± 7% pred), nor Oz delivery (2.14 ± 0.21 va 2.03 :1: 0.16 I.Jmln) Improved (p > 0.05 for eiCh). Deaple 1111ne:rt.e In peak exerclee RVEF Iller BL T (311 :1: 1 va 47 :t 3 %, p. 0.005), left ventrtc:ular ltrake wlume remained low (87 :t 11 vs. 68 :1: e ml, p .. 0.05). In c:onc:lualon, the data suggest that patients with CF, although Improved, Sill have significantly ln1l&lred exercise tolerance following BL T.

Decond~ionlng and anemia may be imponant contributing factors to the exercise lim~ following BL T In CF.

Supponed by the Wil Rogers lnstnute, AHA Grant-In-Aid, and the Monteiro Lung Transplant Fund.

351* Eurdse Perrormance aad Pulmonary Function In Adult BUateral Uvlng Lobar Lune Transplant(BLLLn Redplenll wltb Cystic Fibrosis. MA Finnerty, BJ Shapiro, RG Barbers, SM Farr, VA Starnes and KM Chao. Divisions of Pulmonary and Critical Care Medicine and Cardiolhoracic Surgery, University of Southern California, Los Angeles, CA, USA.

The shortage of cadaveric lung donors has led to the use of BLLL T for patients wilh end-stage cystic fibrosis. The replacement of whole lungs by two mature lobes may result in limitation of exercise ability and pulmonary function. The assessment oflhese parameters is described. Metboda: Patients who underwent BLLLT for cystic fibrosis and survived at least 30 days were evaluated using a modified submaximal Bruce exercise protocol and spirometty at post-op days 30, 90, 180, 365 and 7 40. Patients !hat exceeded submaximal Bruce stage 5 were evaluated using !he maximal Bruce exercise protocol. Exercise was scored in the following manner: the amount of work at each level of the submaxirnal and maximal Bruce protocol was estimated in METS. This allowed a predetennined comparison between !he two protocols and provided a framework as to show !he level of exercise as it translated to activities of daily living. Rnulll: I 3128(5 males, 8 females) patients (mean age 25± 5) undergoing BLLLT (between 1/93 and 8/96) and swviving > 30 days

. d . . al . perfonned exerctse an s~ trometrtc ev uatton.

MUS Ac!MI)' rvc n:vt FI:VIIFVC RF%Pr JAw•inll•t %Pr %Pr

p,.-op(-IJ) - - Jz-,9 20<7 51%9 604

Doy 41 (o•IJ) 2.5%2 WllkiJia 39±10 45•10 93• 6 70±23 2mph

Dtly90 (o-13) 6.4%3 Wllkinc 51•16 55•16 81±12 61%30 5mph

DtlyiiO (o-Il) 7.7•3 Joain1 61•17 64•17 17•11 71%34 lmph

Dtlyl65(.-U) IIZl Buketbotll 67•16 69±16 85•13 74<37 -DtiJ'7<10(-) 11%2 Buketbotll ~15 -16 77.t:l6 52%33 -.. Coaclualon: BLLL T rectptents demonstrate unproved pulmoruuy function and

exercise ability over a two year period of time. The high level ofactivity acltieved and !he near normal pulmoruuy function found in lhese patients further supports !he continued use of BLLLT as an alternative to cadaveric lung transplantation.

352* Cytoklne Expreaalon In a Mouse Airway Gran Model of

Obliterative Bronc:hlolltls ualna RT -PCR

I Neurin&er, S Walsh, D Fenstermacher, M Parsons, S Gabriel, and R Aris, Cystic Fibrosis/Pulmonary Research ~d Treatment ~nter, Dept. of Medicine, University of Nonh Carolina at Chapel Hill, and the Division of Nephrology, Dept. of Medicine, Duke University Medical Center Obliterative bronchiolitis (08) is the major cause of morbidity and monality in lung transplantation after the fust year. Fifty percent of cystic fibrosis patients who have undergone lung transplantation at UNC develop 08, a fonn of chronic rejection, and 08 has become the leading cause of death in CF patients post-transplant. The heterotopic mouse trachea allograft model reproduces human 08, and demonstrates a dense early CD4+ and CD8+ infiltrate followed by a sustained macrophageiCD4+ response. We used this model to study cytokine gene expression and the role of Th I, Th2, and cytotoxic T cells in the evolution of chronic rejection. Tracheal allografts (BALB/c to C57BU6) and controls were harvested from subcutaneous skin pockets at 2, 4, 6, and 10 week intervals. RNA was extracted from tissue using the guanidine/cesium chloride method, reverse transcribed, and PCR amplified using primers specific for U..-2, y-interferon, U..-4, U..-1 0, and granzyme B. and compared to the expression of Pectin. Bands for IL-2 and y-interferon were evident early at 2 week timepoints, and were expressed with greater intensity in allografts compared to isografts from week 2 through week 10. Granzyme 8 was also evident early, peaking at 2 weeks, with greater intensity in allografts compared to

isografts, but declining in all samples thereafter. ll.-4 and ll.-10 bands also appeared early at 2 weeks and with greater intensity in allografts compared to isografts, persisting through week 10. These studies suggest that Th l, Th2, and cytotoxic host responses, are playing a role in aitway rejection in a mouse modelof OB. Future studies are underway to examine the impact of the immune response upon aitway epithelium. Funded by ALA of NC

353* Conversion of Sandimmune to Neoral in Luna Tnnsplant Recipients with Cystic: Fibrosis. RE Dupuis, KK.Q2n. A W Smith, U Paradowsk~ TM Egan, RM Aris. Departments of Medicine. Surgery & Tnnsplantation, Schools of Pbannacy & Medicine, University of North Carolina II Chapel Hill. Chapel Hili,NC.USA.

Cyc:losporin(CYA) is poorly ab!orbed after administration of Sandimmune(SIM) in lung transplant recipients(L TX) with cystic: fibrosis(CF). As a result, many of these patients often require increased doses and frequent administrstion and/or use of cyc:losporin-sparing agents, suc:b as ketoc:onazole. The new formulation, Neorai(NEO) has been reported to improve cyc:losporin absorption over Sandimmune(SIM) in L TX with CF. This increase in absorption may result in higher blood levels and potentially a change in the safety pattern of cyclosporin. In addition, NEO may reduce the need for administration of agents, like ketoconazole. We reviewed the records of lung transplant recipients who were converted from SlM to NEO. Convcnion was initiated on a I: I mg with previous SlM dosage. NEO dose was adjusted to maintain similar CY A 1roUgh levels as with SlM. Data was c:ollec:ted 6 months prior to and ~ 6 months after conversion. Twenty L TX(8 M.12 F; age 30(8.6) years; weight SO(l0.4) kgs) with CF were converted to NEO, 7·S4 months post-transplant. Seventeen were on ketoconazole(KETO) at time of conversion. I 0 were discontinued during conversion. Time to stable NEO regimen was 4.4(2.9) weeks. CY A doses were baseline SlM 7.1(4), NEO I month 6.4(2.9), NEO 3 months 6.3(2.4). NEO 6 months 6.7(2.2)mg/kglday. CYA levels were baseline SlM 267(92). NEO I month 334(144). NEO 3 months 282(133), NEO 6 months 220(S4)ng/ml. Nine patients had an increase in NEO dose(719 had kctoconazole discontinued; mean increase of 29.2%), 6 a decrease in dose( mean decrease of 41.4%), S no change. All patients requiring a dosage reduction once NEO was started bad a baseline SlM dose of> 8mglkglday, Irrespective of KETO. Overall, Serum creatinine was no1 different, with 6S% L TX having no change, 20% an increase and IS% a dec:rease. There were no significant differences in episodes of acute rejection, blood pressure, potassium. magnesium levels and gastrointestinal or neurologic side effects. Overall, conversion to NEO wu well tolerated. L TX with CF on higher SIM doses(>Bmglkg/day) require a reduction in NEO dose. Most L TX with CF receiving NEO. along with KETO. will need an increase in NEO dose if KETO is discontinued. The use of KETO does affect NEO dosage and lhe cost-benefit of this agen~ along with NEO, should be evaluated.

G 1/N utrition/Endocri ne

354* THE COMBINED USE OF CYCLOOXYGENASE INHIBITORS AND PANCREATIC ENZYMES CAUSES INTESTINAL AND HEPATIC INJURY. R. Kimura J lloyd·Siill, V. Arango. Department of Pediatrics, Rush Medical Collage, Chicago ll •

A recent outbreak of fibrosing colonopalhy in patients with Cystic Fibrosis (CF) has been related lo lha use of high doses of pancreatic enzymea (PE). The use of high d011ea of a cyclooxygenase inhib~or. ibUprofen (60 mglkglday) in patienta with CF caused a decrease in tha r~~te of progreasion of lung injury. Because of thia encouraging study, an CF patients are now being treated with l:>uprolen. In our in~ial studies. intraduodenaJ infusion of high doses of PE ahowed minimal effects on tha intestine. When intestinal permeabilrty was increased, PE caused an increase In tha number of intestinal and hepatic lesions. Since cyclooxygenase inhibitora Increase intestinal panneabilily, we hypothesized that the combined use of cyclooxygenase inhibitora and PE will resutt in damage lo tha inteslina and the liver. The effect of indomethacin, a cyclooxygenase inhibitor, with and without PE on rat intestine and liver was investigated. For 10 daya. 4 groups of rats were infused via an Indwelling duodenal catheter with 1) normal aaline-NS; 2) PE (150,000 un~s/kg/d of lipase); 3) indomethacin-INDO (3 mglkg/day, equivalent to 15 m!¥((g_of ibuprofen); and 4) PE + INDO. Tha rats were aacrificed and the intesttna and

Poster Session Abstracts 303

liver were fixed and stained (H&E). Uaing a 'Swisa Roll lechnique' to examine the entire Gl tract, the number of ulcera were counted. Severe ulcers wera defined es ulceration extending past lhe muscularia mucoaa. The presence of inflammatory hepatic nodules were observed and quantified.

Sttxti Grow Cit) NS C3l PE 181 INDO (l!l INOO+PE (:i) I rats w~h severe Ulcera 00 Or'8 G'8 4-5 • f hepatjc nodyles 3+1 5 8+2 3 2319 131±58 Values are means±SEM. 'significantly different from PE or INDO alone Fisher exact Test pc0.05

These studies clearly indicate lor tha first time, tha aynergistic effects of a cyclooxygenase Inhibitor and PEon creating intestinal and hepatic injury. These studies support our hypolhesis that increasing Intestinal penneabilily predisposes the intestine to injury by pancreatic: enzy..-. Since many CF patients ere using high doses of ibuprofen with PE, these patients have a strong possibilrty of the development of aevere Intestinal injury resuhing In ulcers in the intesline and fibrosis in the liver.

355* ONTOGENY OF EXPRESSION OF THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR mRNA AND PROTEIN IN HUMAN FETAL LIVER. Peter Van Eyken, Valeer Desmet, Helen A. John and Andrew E Mulber11. Department of Pathology, Catholic: University, Leuven, Belgium and the Division of Pediatric: Gastroenterology and Nutrition, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania. ·

Virtually nothing is known about the regulation of ion transport in nonnal biliary epithelia or its pathobiology in cystic fibrosis (CF). The aim of this study is to identify the ontogeny of expression of the c~ fibrosis transmembrane conductance (CFTR), a cAMP-dependent Ct· channel within the fetal human liver. The expression of the CFfR within the human fetal liver has not been previously explored. An understanding of expression of CFTR within fetal liver may be important towards understanding the pathobiology of CF effects upon the liver. METHODS: Cryostat sections of fetal, neonatal, infantile. pediatric and adult human livers (ranging in age from 13 weeks of gestation to 45 years) were stained for the CFfR mRNA and protein using suitably developed nonisotopic in situ hybridization (NISH) probes and the poiyclonal antibody pAb3145 in an avidin-biotin complex technique (after blocking of the endogenous biotin) (Resta et al, J Clin Invest. 1995;96:646-652) RESULTS: In regards to CFfR protein expression, developing and mature hepatoc:ytes were negative. At 13 weeks of gestation, no positive staining was observed. From 16 weeks of gestation on, weak positivity was restricted to the tubular pans of the ductal plates. Tubular structures that were being incorporated into the mesenchyme of the portal tracts were more strongly positive. Interlobular and segmental bile ducts fully inco'1'orated into large portal tracts were even more strongly positive. The mtensity of the immunostaining for CFfR did not increase with gestational age. NISH experiments corroborated these findings in selected livers of selected gestatiOnal age. CONCLUSIONS: Immunoreactivity for CFfR is related to the maturation of bile ducts: no or weak expression in ductal plate cells was restricted to tubular structures; stronger staining of tubular structures that are being incorporated in the mesenchyme of the portal tract and still stronger staining in large bile ducts fully incorporated in the portal tracts was appreciated. CFfR expression and its relationship to the differentiation of biliary epithelia remams to be elucidated. Supported by DK02077 and a Pilot Project Grant from the Human Gene Therapy Center of the University of Pennsylvania School of Medicine and (AEM).

356*

HEPATO-BILIARY DISEASE IN CYsnC FIBROSIS: ENZYMATIC AND IDSfOPATIIOLOGIC RESPONSE TO URSODEOXYCHOLIC ACID TIIERAPY. Wong LIK'·',Davidson AGF1J,Jevon G1,Jameison D',Peacock D'·2,Gravelle A1,Schmidt J'. Cystic Fibrosis Clinic'. Departments of Pediatrics', Pathology' and Radiology', B.C. '1 Children's Hospital, Vancouver, Canada.

We have utilised a simple protocol of clinical, liver enzyme and ultrasound (US) monitoring in our pediatric Cystic Fibrosis (CF) clinic during the past S years to demonstrate a high prevalence of CF related liver disease'. Ursodeoxycholic acid (UDCA) bas been well shown to improve liver enzyme abnormalities in such patients'; but It is not known whether there is concomitant improvement in the liver histological changes, or whether these continue to progress. We now report the effect of UDCA therapy on histological as well u enzymatic: and US abnormalities in patients detected in our clinic to have CF related liver disease (Focal Biliary Cirrhosis, FBC).

304 1997 Cystic Fibrosis Conference

Method : CF patients identified with HBD and confirmed by liver biopsy 10 have histological evidence of FBC were offered UDCA therapy in the dosage range of 20-30 mglkgitlay. Efficacy of therapy was monitored by liver enzymes monthly and by US every 6 months. Histological response was assessed by percutaneous liver biopsy after at least 12 months of UDCA therapy. Liver histopathology was assessed and scored by one pathologist for the degree of fibrosis, bile duct proliferation, inflammation, inapissation of bile and steatosis. US was scored by the radiologist according to the method of Williams et at'. Radiologist and pathologist were "blinded" as 10 the clinical, enzymatic, or treatment status of the patients and as to the "scores" reponed by each other. UDCA Therapy : 22 patients received UDCA therapy. Liver enzymes (AST ,AL T, GGT) improved to normal range within 2 months in 18 patients. However 4 continued to have mildly elevated enzymes. 10 patients bad repeat biopsy after one year of UDCA therapy: histopathological score improved in 8, The non-responden were either non-adherent to UDCA therapy or receiving inadequate UDCA dosage ( < 20 mglkg/tlay). US was less sensitive to the changes over time. CONCLUSION : UDCA therapy favourably affected hisropathological as well as enzyme abnormalities in these CF patients. • see ac:companyina abltract 1 Colombo C ct al. HeparoiOIY 1996;23:1484-1490 'WUiiams SOl et al. J llepoto1199,:2BtH21

357* LONGITUDINAL CHARACTERIZATION OF GROWTH AND GROWTH TRACKING IN INFANTS AND TODDLERS WITH CYSTIC FIBROSIS IDENTIFIED BY NEWBORN SCREEN.

MK.Simll& , AA Drescher, FJ Acc:uno, NF Krebs. Departments of Pediatrics and Preventive Medicine and Biometrics, University of Colorado Health Sciences Center and The Children's Hoopital, Denver, CO, USA.

Growth failure, a common manifestation of cyatic fibrosis (CF), is related to pulmonary motbidity and mortality. Growth parameters have primarily been studied crou-aectionally precJudina accurate analysis of srowth velocity and trackins. We proapeetively studied srowth pattems of 152 children (77 female, 75 male) diallftOied with CF by newborn acreening in Colorado since 1982. Growth messurernents were obtained according to atandardizel! procedures at birth (weight only), 2, 6, 12, 24, and 36 moutha of aae. Data analyaia was performed through longitudinal mixed effect modeling in SAS Proc Mixed. Age and gender were used aa independent covariatea in models for weipt and length. NCHS z-acorea were used to facilitate comparisons to national norms. The best model for both weiaht and beipt were cubic fimctiona of age. Z.acores were modeled using a quadratic 1\mction of age. Mean weipt z..acore in CF did not reach the median z..acore for nonnab for either males or fomaloa, duoupout tho 36 montha. Mean birth weipt z acorea (WAZ) were -0.65 for males and -0.75 for females. Mean WAZ for males' (z • -0.90, p<O.OI) and females (z -1.04, p<O.OI) continued to decline until 12 montha of age. By 36 montha, the mean W AZ for males bad improved to -0.39, and the mean W AZ for females bad reached -0.65 (p<O.O I). Males' moan heipt for ago z..acore (HAZ) fell from -0.49 at two montha to -0.60 at 12 montha. Females' mean HAZ fell from -0.55 at 2 montha (p<O.OI) to -0.78 at 12 montha (p<O.Ot). After tbeae ages, mean HAZ improved. By 36 months mean HAZ for males waa -0.23, and for females waa -0.41(p<O.OI). Low weipt for age (<5• percentile) at 12 montbl waa correlated with low weight for age at 24 (r • 0.39, p • 0.0001) and 36 montha (r • 0.45, p • 0.0001). Similarly, low height for age (<S* percentile) at 12 moutbl waa correlated with low hei&ht for age at 24 (r- 0.41, p • 0.0001) and 36 DIOIIthJ (r • 0.32, p • 0.0001). Theae meuurea of tracking indicate a risk for low weight and height at 36 months if the infant bad low weiaht or beiaht at or before 12 montbl. 1bese data also 11tJUC11 tblt problema with growth in the lint year of life are not neceaaarily corrected by aae three.

358* Qateoporoela In cystic fibrosis: Role of cslclum IMI.tMiorDdon. .BM...Aila. 5. Dingman, G. Lester, and D. Ontjes, Depts ol Medicine ~opedlcs, lJnlvenslty of North carolina, Chapel HID, NCJ. USA.

The bone mineral den8lly (BMD) ollndlvkluals with cystic nbrosls CCFl tans prograsalvelv below nonnal with advancing age. MeChanlsrils for low BMD In CF Include starold admlnlafratlon, low leYela of sex aterolds, chronic Inflammatory diseaSe ph~ In~, and chronic rnaJabaOrptlon of calcium (C8j8001or vitamin D. The pu!p988 of this atudy waa to comp811t the fractional absorption oi45Ca and urinary excretion of ca In CF subjects and normal controls following a breakfast containing 625 mg or ca and 5 mlcroCI45CaC12. Eleveh young adults with CF with pallcreatlc Insufficiency (8 maleS; 6 f8ma181; average age 26.7 y11) were studied on two sepantte occaslona, either with and Without administration of panC18811C ~ (5 Pancreaae MT·16) wtlh the meal. Eleven healthy yocJIIO adults (7 males; 4 females; average age 26 y11) with normal BMD measurements served as controls.

The mean T -score for lumbar spine BMD for the CF subjects was ·1.47. Following baseline, lasting collections, limed serum and urine samples were obtained for 5 hours alter the meal. Fractional absorption of 45Ca was estimated by the method of Nordin and expressed as the fraction of Ingested 45Ca in the extracellular space at varying limes. Postprandial excretion of Ca In timed urine samples was expressed as the ratio (mg per mg) of Ca to creatinine. Mean values (SEM) are shown below.

Time alter Meal Controls

CF (-) Enzymes

CF (+)Enzymes

Fractional Absorption of Ca 1 hr 3 hrs 5 hrs .052 .101 .106

(.010) (.017) (.018) .038 .on .091

(.005) (.007) (.009) .045 .084 .099

(.008) (.016) (.018)

UdnaiY Ca/Creat, D-2 hrs 2-4 hrs

.142 .198 (.021) (.022) .075 .151 (.OtO) (.012) .069 .160

(.009) (.023 )

Without enzymes, CF subjects showed slglflcant (p < 0.05) reductions In the absorption and excretion of dietary calcium following a high calcium meal. Addition of enzymes partially compensated for this deficiency, but urinary calcium excretion remained low. Calcium malabsorption Is likely to be a factor In the development of osteoporosis In CF patients and calcium supplementation may be useful in building and maintaining bone strength.

359* CAROTENOID LEVELS ARE ALTERED IN CF PATIENTS WITH PANCREATIC SUFFICIENCY. C. Galaban, A. Chaffin, A. Leculre and J.P. Chazatette. CERM, Hopttat Ren•• Sabran (Hopltaux de Lyon), Bd E. Herrlot, Glena, 83408, Hytrea Cedex, France.

Patients with CF have been prevtousty shown to have low levels of plasma carotenolds (Cds) (1 ,2,3) wHh no close correlation wHh fat absorption percentage (2,3). In this study we Investigated the reasons for this apparent Cds dellclency In CF patients. Serum Cd levels were detennlned In CF patients wfth pancreallc sulllclency (CF· PS) (n-26; aged 2·39 years), CF patients with pancreatic Insufficiency (CF·PI) (n-68, aged 4·23 years) and non.CF controls (n-38; aged 2· 46 years). All CF·PI patients continued to take pancreatic enzyme supplements during the study. Serum levels of Cds (zeaxanthin+luteln, cryptoxanthin, tycopene, a-carotene, and b· carotene) rellnol and tocopherol were determined by high­performance liquid chromatography. Serum trypsinogen and lipase were determined by lmmunoenzymalic assey. As expected, Cd levels were significantly lower In CE-Pis aa comparod to Mn!mls (means :1:. standard deviations; unpaired t-tests): zeaxanthin+luteln n :1:. 86 nmol/1 versus 613 .:1: 308 nmol/1, P<0,001; cryptoxanthin 28 :1:. 22 nmoVI versus 242 :1:. 166 nmol/1, P<O,OO 1; lycopene 25 :1:. 39 nmol/1 vesus 341 .:1:231 nmol/1, 1)<0,001; a-carotene 28:1:.33 versus 85:1:.63, P<0,001; b-camtene 52 :1:. 38 versus 280 :1:. t83, p<O,OOt. Cds levels were significantly lower In CF-PI!I " comparod to CF-PSs: CF-PS zeaxanthln+luteln 415 .:1: 226 nrnoVl, p<0,001; cryptoxanthin 163 :1:. 146, P<O,OOt; lycopene 227 .:1: 155, p<0,001; a-carotene 56 :1:. 34, P<0,001; b-carotene 175:1:. 122, p<0,001. Most Interestingly, Cd levels were slgnlicantly lower In Cf-psa u compa!l!d to contmla: zeaxanthln+lullln p.0,007; cryptoxanthin p-0,05; lycopene p.0,03, a-carotene p-0,03; b-carotene p-0.01. Serum Cd levels In CF pallents poorly correlated to serum levels of pancreallc lipase (zeaxanthlll+luteln r-0,47, P<0,01; lycopene r-0,43, P<0.01) and to serum levels of tocopherol (zeaxanthin+ lutein r-0,30, p<0,01 ). These data Indicate that Cd deficiency In CF Is not only due to fat soluble compound anered digestion and absorption In these patients. The significant decrease ot Cds In CF-PS patients suggests an Increased turnover ancllor metabolism of these antioxidants that could be consumed during the oxidative burst occurfng In pulmonary Inflammation. Ref.: 1) Knopfle G. & a11975, Z. Klnderheilk 119: 279·91.

2) Congden SW & al1981, Arch. Dis. Child. 56: 708·14. 3) Homntc:k ON & al1993, J. Pedlalr. 122: 703·7.

360* CYSTIC FIBROSIS RELATED DIABETES (CFRD) IN CHILDREN: GENOTYPE, PHENOTYPE AND CLINICAL DETAILS. ltL Solomon, D.C. Wilaon, M. Corey, J. Zielenaki, L.-C. Taui, P.B. Pencharz, P.R. Durie, N. Sweezey. CF Clinic, Div. of GI/Nutrition, and Dept. of Paediatrica and Genetics, Reaearch Institute, Hoap. for Sick Children, Toronto, and University of Toronto, Ontario, Canada.

The importance of CFRD as a complication of CF ia being increaaingly recognised. In the largeat aeries of CFRD reported to

date, we previously studied the Toronto adult CF clinic (Pediatr Pulmonol 1996; Suppl. 13, 251 and 323). We found that 53 (45% females) of the 326 adults attending this clinic in the period of April 1991 to March 1996 had CFRD, a prevalence of 16'1>, and all were pancreatic insufficient (PI). Only patients with the more severe classes of CFTR gene mutations developed CFRD. CFRD wu a late complication of CF, diagnosed at a mean (range) age of 24 (12-42) years. In view of the high prevalence in adults, we aimed to evaluata prevalence of CFRD in children, and also genotype, phenotype and clinical status at and subsequent to the diagnosis of CFRD. Here, we report findings in the Toronto childhood CF population. From April 1994 to March 1996, 301 children (< 18 yr.) attended the CF clinic; 24 (8%) were pancreatic 1ufficient (PS), 8 (3%) were PS who later became PI; and 146 (49%) were girls. Four girls and no boYB had CFRD, a prevalence of 1.3 %. One additional girl with CF developed IDDM at age 4 yean. Mean (range) age at diagnosis of CFRD wu 14.2 (12.2· 15.6) yeare. All were PI, and none have died or developed diabetic microvascular complications. The genotypes are AF5081AF508 (n•2), AF50814016insT (n•ll and AF508/Ell04X (n•ll. Contrary to a recent report (Arch Die Child 1996; 75: 546), none of these 57 combined patients with CFRD bave the N1303K mutation; Nl303K waa present in 12 of the 627 (2%) children and adults genotyped at our clinics. No child had risk factors other than CF (PI) at the diagnosis of CFRD, such as oral steroid therapy or nocturnal enteral feedings. Although CFRD baa a lower prevalence in childhood than in adults, thia appeara to rise in adoleoence. It apparently occurs only in those children with severe CFTR gene mutations, all of which are asiiOCiated with the PI phenotype, consistent with our adult data. We recommend consideration of ocreening of all PI children for CFRD after 10 years of age. (Funded by NIH, Canadian CF Foundation, and MRC).

361* GLUCOSE TOLERANCE IN ADULTS WITH CYSTIC FIBROSIS: PHENOTYPE-GENOTYPE CORRELATIONS. DC Wjlsgn C. Stewart, A.K. Hanna. M. Corey. J. Zielens!ci, L . .C. Tsui, P.R. Durie, P.B. Penchan, E. Tullia. CF Clinic and Division of Endocrinology, Wellesley Hospilal, Toronto; Division of OutroenteroloJYINutrition and Departments of Paediatrics and Genetics, Research Institute, Hospilal for Sick Children, Toronto; and University of Toronto, Toronto, Onlario, Canada.

Endocrine pancreatic disease Ia not well characterized in adults with cystic fibrosis (CF), especially with respect to its relationship with CF genotype and exocrine pancreatic diseue. We hypotheoizo thai the occurrence of slucose intolerance in CF correlates with the severity of pancreatic disease (pancreatic sufficiency (PS) or insufficiency (PI)) which is in tum innuenced by genotype. From April 1996, we have prospectively screened 119 CF adults (aged 17·S2 yr.) who regularly anend the Toronto clinic. A further 36 patients have known CF related diabetes mellitus (CFRD). Genotype was defined iD a novel 1 class system, by composites of functional classes of CFTR gene mutations (Annu Rev Genet 199S; 29: 777). TheseS functional classes include both severe (1,11, and III) and mild (IV, V) mutations, a11ociated with the PI and PS phenotypes respectively. Screenina for alucose tolerance was done usina a modified WHO test: fastins plasma alucose, followed by a 7SI oral alucosc load, then a 2 hour plasma alucose. Patients were catesorized into normal (NOT: <7.8 mmoi/L fastinsalucose, <7.8 mmoi/L at 2 hr.), impaired (IGT: <7.8, 7.8-11.1 mmoi/L), and diabetic alucose tolerance (DGT: <7.8, >I 1.1 mmoi/L) and known or screened CFRD (fastinJ >7.8 mmoi/L). Results are Number(% of class):

Genotype Claas Known Screen: Screen: Screen: Screen: ~ (~>&nc. phenotype) CFRD CFRD DGT IGT NGT

11·1 (PI s 31%1 0 I 16%) fll9%1 7144%)

11·11 Pll 26 (33%) Ill% 8110%1 9ti2%1 34 44%

II · lii IPII 2 (17%) 0 2 (17%) 3125% sl4z<~>\

II -IV (PSl 0 0 0 0 fii00%1

li·V PSI 0 0 I (14% lti4%1 s 111'1>)

Severe - Severe IPII 3(15%) 1(5%) I (5%1 4 (20%1 11155%}

Mild • Other IPS I 0 0 0 I 16%1 16194'1>)

CFRD only, and DGT and JOT m&Jnly, occurred on psllents wtth the more severe classes of CF Jenotype, all or which are associated with the PI exocrine phenotype. We speculate that severe exocrine pancreatic disease is 1 prerequisite of CFRD. (Funded by NIH, CCFF and MRC).

Poster Session Abstracts 305

362* EFFECTIVENESS OF ADEKs• IN CYSTIC FIBROSIS AS A FAT SOLUBLE VITAMIN SUPPLEMENT, WITH SPECIAL ATTENTION TO VITAMIN K. DC. Wilson, M. Rashid, P.R. Durie, A. Tsang, D. Kalnins, M. Andrews•, M. Corey, J. Shin, E. Tullis, P.R. Pencharz. Divisions of Gastroenterology/Nutrition and 0 Haematology, Research Institute, Hospital for Sick Children, Toronto; CF Clinic, Wellesley Hospital, Toronto; and Dept. of Paediatrics and Medicine, Univenity ofToronto, Onlario, Canada.

Patients with CF risk developing fat soluble vitamin deficiency due to pancreatic insufficiency (PI) and/or CF-usociated liver disease (CFLD). Funher, indications for, and dosina requirements of, vitamin K (Vit K) supplementation in CF are controversial. Havins reported oub-c!inical Vit K deficiency in almost all children with PI (Pediatr Pulmonol1996; Suppl. 13: 313), we then prospectively evaluated the effectiveness of ADEKs• (Scandipharm Inc .. Birmingham, AL) as a Vit K, vilamin A (VitA) and vilamin E (Vit E) supplement. In 1S children and adults (40 males) with PI and 1 mean (SD, range) age or 15 (II, 0.6-46) yr, daily vitamin supplements containinJ VitA and Vit E. but not Vit K, were replaced by ADEKs, containing similar amounts of VitA and Vit E. Before (pre) and at least S months after (post) slarting ADEKs, plasma PIVKA·II (prothrombin induced in vitamin K absence) assay (ELISA; a sensitive indicator of vit K deficiency), prothrombin time (Pl) and vit A and vit E assays (HPLC) were performed. Seven (9%) patients had CFLD with cirrhosis. Results below are mean (SO, range); • p=0.004 by Student's paired t test; •• pzO.OOOI by Wilcoxon rank sign test.

Assay (Normal ran2e) No. Pre Post VitA (0.7·2.1 umoi/L) 64 1.3 104 0.4·2.Sl 1.2 10.4 0.5-3.21 Vit E 12·46 umoi/L) 64 24 II 6-53 2018 8·48).

PIVKA·II f< 2.1 ng/mLI 72 17127 2·1701 10f27 2·200) ••

The number ('I>) with subnonnal VitA was S (8%) both pre and post the change. Despite the drop in mean Vit E level, the number (%) with subnormal vit E rose insignificantly, from 7 (11%) to 8 (13%). By contrast, the number(%) with abnormal PIVKA-U fell significantly (pzO.OOI by McNemar's test) from 67 (93%) to 42 (58%). Four patients (2 with CFLD) had prolonged PT (> 13.S sec.) at both time periods. The median (range) PIVKA·II fell from 9.S (S.S-S5) to 2.2 (2.0-65) in the 7 patients with CFLD. We conclude that ADEKs is an effective supplement for vit A and vit E. and that it improves, but does noc resolve the ncar universal problem of vit K deficiency in Pl. We recommend further Vit K dosin& studies be performed in CF patients with Pl. (Funded by Scandiphann Inc., Birmingham, AL.)

363* THE DIAGNOSIS OF CYSTIC FIBROSIS RELATED DIABETES: A SELECTIVE APPROACH IN PERFORMING ORAL GLUCOSE TOLERANCE TEST BASED ON A COMBINATION OF CLINICAL AND BIOCHEMICAL CRITERIA ll..Ylm&. M Kemp•, 1 Hooper", and ME Hodson. Departments of Cystic Fibrosis and •Clinical Biochemistry, Royal Brompton Hospital, London, SW3 6NP UK.

The diiiJIIOiis of cystic fibrosis related diabetel (CFRD) iJ impol1ant u the development of this condition may be usociatcd with a deterioration in clinical status which can be reveraed with prompt treatment Fastina blood alucoae (BG), alycoaylated hliCIIIOg)obin (HbAlc), and the presence ofsymptoml of hyperglycaemia (SH) wben used alone, have been found to be insensitive in the diagnosis of CFRD. A 2 hour oralalucose tolerance teal (OGTT) is generally regarded u the 'gold atandard' diagnostic method but performing OGTfa in all adult palients is time and I"CSSOIl'CC consumina and ia inconvenient for the patienll. Aim: To evaluate a more selective approach in performing OGTfa based on the use of a combination of biochemical and clinical criteria in the diagnosia of CFRD. Method: Clinically \Yell adult CF patients, not known to be diabetic, attending this clinic for annual assessment had random BG and HbA I c measured. The presence of glycosuria and SH were determined. All these patienll were invited to have OG1Ta performed within a month to determine their glucose tolerance status. The results of their OGTTs were matched with results of their annualasscssmenl Results: Between Aua. 96 and April 97, 86 patients were recruited for the study. 73 OGTfa were carried out to date. 4~ and 16 pstients had normal and impaired glucose tolerance (GT) respectively. 12 had diabetic GT response. Of these 12 patienll, 2 (17"/o) had glycosuria at their annual assessment; 7 ('8%) had SH, 3 (25%) had abnormal fasting BG, 10 (83~.) had abnormal HbAic, 4 (33~.) had nndom BG above II mmol.ll. lf OGTTa were performed only in those patients who had abnormal HbAlc (> 6.1%) !!!l!.l2!: presence ofSH and /or a random BG over II mmol/1, only 25 of the 73 patients (34%) would have undergone OG1Ts of which II of the 12 patients with diabetic GT would have been identified (sensitivity: 92%). 14 patients without diabetel would have undergona OGTT (specificity: 77"/o). Conclusion: Pcrformina OGTfa only in those with one of morc of the above clinical or biochemical criteria would identifY the vast majority of patients with CFRD and relatively few pstients without diabetel would have undergone OG1Ts. Such approach will lead to a considerable amount of 11vin1 in lenni of time and resource for both the medicalstalT and patienll.

306 1997 Cystic Fibrosis Conference

364* DIABETIC RETINOPATHY IN PATIENTS WITH CYSTIC FIBROSIS RELATED DIABETES (CFRD). l!...Y!m&, A Landers•, B Mathalone•, K M Gyi and M E Hodson. Department of Cystic Fibrosis, Royal Brompton Hospital, Fulham Road, London SW3 6NP and *Department of Ophthalmology, Chelsea and Westminster Hospital, Fulham Road, London, UK.

Cystic fibrosis related diabetes (CFRD) is likely to become increasingly common as patients survive longer. Diabetic microvascular complications were considered to be very rare in the past. Only 10 cases of diabetic retinopathy (DR) had ever been reported in CFRD patients mostly in patients who had diabetes for more than I 0 years. Aim: To prospectively determine the prevalence of DR in adult patients who had CFRD for S years or more in a national CF centre. Method: Adult patients (age 16 or above) with CFRD for S years or more attending Royal Brompton Hospital were invited to take part in an eye examination. Their eyes were dilated with 1% Tropicamide eye drops. Examination of the fundi with 780, 900 lenses and slit-lamp were carried out by experienced ophthalmologists. Results: 31 of the 36 patients with CFRD for S years or more were examined to-date (median age: 26 yrs, range: 19-43 yrs; median duration of diabetes: 9 yrs, range: S-29 years). All patients were treated with insulin for their diabetes. S of the patients (16%) examined were found to have DR (duration ofCFRD: 23*, 12••, 12, 9, 6 yrs ). I patient• had proliferative retinopathy, 1 had maculopathy*• and 3 had background retinopathy. The prevalence of DR in patients with CFRD for 10 yrs or more was 23% ( 3 of 13 patients ). There was no difference in the levels ofHbAlc in patients with or without DR (median: 6.8% vs 7.3% respectively, p- O.SS). Condusion: The prevalence of diabetic retinopathy is likely to increase as patients with CFRD live longer. The high prevalence of diabetic retinopathy we found in patients with CFRD for S years or more emphasises the importance of regular screening for this potentially devastating complication in this group of patients. We thank Prof. D Geddes Cor allowing us to study his patients.

365*

Geaenllzed Atherosc:lerosls Ia an Adalt with CF and Diabetes MeiUtus. PM Schlesinger, DS Holsclaw, Jr. and 08 Fyfe. Depts of Pediatrics and *Pathology, MCP+ Hahnemann School of Medicine Alleghany University of the Health Sciences, Philadelphia, Pa.

In spite of progressive pulmonary disease, most CF patients now live into adulthood and many develop extrapulmonary complications. Cardiovascular involvement bas included right ventricular hypertrophy with pulmonary hypertension, hypoxemia and cor pulmonale. Indeed, CF patients have been felt to be "protectedn from atherosclerosis by their life-long malnutrition and bypocholesterolemia. The autopsy of this 41 year old white female with CF, pancreatic: insuft"~eiency and insulin-dependent diabetes since age 16 dem­onstrated severe and widespread atherosclerosis. Diagnosed at age 14 months with failure to thrive, steatorrhea and positive sweat tests, genotyping later revealed her to be homozygous GS42X. Complications included diabetic retinopathy, myopathy with generalized weakness, neuropathy with involvement of the long tracts of her legs, and gastrointestinal pseudoparesis. Hypertension and proteinuria progressed and at age 31 a renal biopsy showed nephrosclerosis. The hypertension and chronic nephrotic syndrome remained stable for the rest of her life. Cholesterol values averaged 200 mf'dl from the age of 22. Her pulmonary disease worsened through her third decade, leading to chronic respiratory failure, and oxnen dependency. Serial echo­cardiograms and EKGs were essentially normaL There were no symptoms of myocardial ischemia, infarction, or rhythm abnormalities. Her pulmonary disease progressed and she died of respiratory failure at age 41. At autopsy, the expected findinp were observed in the lunp, pancreas and intestines, llli was advanced diabetic nephrosclerosis. Tolally unexpected was a dramatic: degree of diffuse atherosclerosis. The coronary ttrteries were eggshell in consistency to palpation and revealed I 00% occlusion of the right circumflex ttrtery, 90 • I 00 % obllruction of the left anterior descending ttrtery and SO"~ obstruction of the circumflex artely. The entire abdominal aorta, including the renal and iliac bifUrcations bad extensM, palpable calcifiCation. There was mild left ventricular hypertrophy. This is the firSt well documented case of atherosclerosis in an adult CF patient Others may not have been

identified. since autopsy is frequently not performed in patients dying of pulmonary disease. We hypothesize that atherosclerosis may become more frequent as our patients live longer, maintain a high fat intake and develop other predisposing conditions such as diabetes.

366* LIPID METABOLISM IN ADULTS WITH CYSTIC FIBROSIS. C. Stewart D. C. Wilson, A.K. Hanna, M. Corey, P.R. Durie, P.B. Pencharz, E. Tullis. CF Clinic and the Divisions of Respiro1ogy and Endocrinology, Wellesley Hospital, and the Division of Gastroenterology and Nutrition and the Department of Paediatric&, Research Institute, Hospital for Sick Children, and the University of Toronto, Toronto, Ontario, Canada.

Lipid metabolism is poorly characterized in adults with cystic fibrosis (CF). We hypothesize that patients with pancreatic insufficiency (PI) will have lower plasma lipids than those with pancreatic sufficiency (PS), and that some patients with diabetic glucose tolerance (DGT) or frank diabetes (CFRD) or CF liver disease may have a secondary hyperlipidemia. Since April 1996, we have prospectively screened 112 patients (26 PS, 86 PI) at the Toronto adult CF clinic. Fifteen (13%) have DGT or CFRD and 3 (3%) have CF liver disease with cirrhosis. After a 14 hour fast, blood was drawn for plasma triglyceride (TG) and plasma cholesterol (choJ) assays. Our normal TG range is 0.5-2.4 mmoVL (45-220 mg/dL) and chol range is 3.4-5.2 mmoVL (130-200 mg!dL). Nine patients, (7 PS, 2 PI) have high chol and 10 patients (6 PS, 4 PI) have high TG. Only one with high chol and none with high TG have DGT, CFRD or CF liver disease. 42 patients (4 PS and 38 PI) have low chol and none have low TG. Mean (SO) chol was significantly (p< 0.0001) higher in PS than PI patients, at 4.5 (1.1) and 3.5 (0.6) mmoVL respectively. Mean (SO) TG was also significantly (p< 0.01) higher in PS than PI patients, at 1.7 (1.3) and 1.1 (0.6) mmoVL respectively. We conclude that plasma lipids are lower in PI than PS adults with CF, presumably due to lower exogenous lipid absorption due to maldigestion. Further, secondary hyperlipidemia& seen in conditions such as diabetes mellitus and chronic liver disease appear to be rare in adults with CF. This apparent contradiction may be because these chronic complications occur in CF in patients with Pl. (Funded by NIH, Canadian CF Foundation and MRC).

367 HIGH CYPIA2 EXPRESSION IN LIVER BIOPSIES FROM CYSTIC FmROSIS PATIENTS ~1 , A. LiDdblad2.3, Y. Terelius4

, M.Jnaelnwi-Sundberg', M.1erliDg4, M.

Wakelbmp1, B. S~ 1Diviaioo ofClUW:al Plwmacolosy, HuddiDge UniveBity Hospital, 2Divisioo of Pediatrica, Huddiaae University Hospital, 3~nt of Pediatrics, Slhlgrensb University Hospital, 4Astrs Arcus, Slklertllje, Department of Medical Biocbcmistry and Biophysics, Kuolilllka lllltitu~ Stockholm, Sweden

IDcressed metabolic elimiDation of some druga has been abown in CF and tbeophyUine bas been reported to be cleared at a greater rste.We investigated the level ofCYPIA2 inliverbiopsiet and clearance of theophylline (marker drug forCYPIA2 activity) in the ssme aubjects. Twentyfive CF patients (4-44 yrs) with liver diseue were exuniJied. 1bey all bad elevated levels of aminotransfersses for at least one year and/or pathological ullralowld of the liver. Two patients bad macronodular cinbosia and one bad aians ofbepatie failure. Fifteen patients were bomozyiOUI for the AFSOS mutation, 9 patients were betcrozygOUI for .t.FS08 and one patient bad two lllllalown mutations. The immuDOresctive content ofCYPIA2 was quantitated by Western blots in 4-2011111 tiuue samples. The amount ofCYPIA2 was expreaed in units relative to a atandard liver sample and allowing for microsomal protein content. Controls were obtained from the hospital liver bank. Theophylline clearsllc:e wu determined after an iv dose in 13 of the patients. lbe amount ofCYPIA2 in the liver biopsies from the patients wu nnu:h biaher compared to the controls. In CF, CYP I A2 bad a mean and median value oC 3.4 and 4.0 relative units, respectively (rsnae 0. 7-7). There was DO aipific:ant deviation from a normal distribution. The controls bad a mean and median vslue of 1.2 and 1.0 relative units, respectively (range 0.6-4.1 ). There was a highly signifiCIIIt deviation from a normal distribution (p<O.OOOI). Theophylline clearance in the patients did DOt differ from tbat of an aac matched reference aource. This study showed an unexpected and bigbJy increased ofCYPIA2 expression in liver tiuue

from CF patients. However, there wu DO increase in tbeophylline clearmce iD our patients and no correlation between CYPJA2 content and theophylline clearance. The increased content of CYP IA2 could possibly be due to the disturbance of liver function in CF. CYPJA2 is a highly inducible enzyme and one could conceive that the pathological process causes autoinduction. The increased expression ofCYPJA2 could explain the increase in theophylline clearance observed in oome studiea. However, this fmding is not consistent, probably because the total capacity for a specific drug metabolic pathway may be impaired by the general condition of the liver reducing total and microsomal protein contcnl

368 Measuring Taste Sensitivity in Cystic Fibrosis. CE Comas' AC Clucas 2, DCK Roberts' and RL Henry' ' Dept of Paediatrics, The University of Newcastle, Newcastle, NSW, Australia. 2 Dept of Nutrition & Dietetics, The University of Newcastle, Newcastle, NSW ,Australia.

Chemosensory dysfunction has been found to exert profound effects on body weight, dietary intake and food enjoyment in patients diagnosed with taste and smell disorders. Chronic sinusitis in cystic fibrosis (CF) is reportedly associated with anosmia or hyposmia. This has important clinical implications as CF is a disease that places extreme nutritional demands on patients, predisposing them to malnutrition. To optimise growth in CF a diet high in fat, sugar and salt is recommended. Disordered taste perception has been studied in CF with variable results but is associated with weight loss in other patient groups. The taste senses of sweet, salty and sour were investigated in 27 children with CF (5 -16 years) and 40 control children without CF (10- 14 years). Subjected were presented with nine solutions of increasing concentration for each taste. The levels of taste detection (D), the value at which they detected there was something in the solution; recognition (R), the value at which they correctly identified the solution taste; and threshold (1), the value at which they no longer tasted the solution as more concentrated, were recorded for each subject. Mean values were compared for the Jwo subjects groups. Significant differences were recorded for sweet D, R and T. Concentrations of sweet solution were approximately 60% greater for subjects with CF. Salty tastes were significantly greater in the CF subjects for D, and T by approximately 60% also. No significant differences were detected for sour forD and R butT was approximately 1 5% greater. Both subjects with CF and controls showed a high dropout rate for sour taste with only small numbers completing taste testing of all nine solutions. The clinical application is that recognition of taste impairments in children with CF should be utilised to enhance energy intake, weight gain and food enjoyment through feeding children with CF more sugar and more salt and the development of CF specific functional foods. This project war carried out In conjunction with final year students completing a Bachelo1' of Applied Science (Consumer Science) as part of course requi,.ements within the Faculty of Medicine and Health Science, The University of

Newcastle.

369 GASTROINTESTINAL MUCOSAL INFLAMMATION AND IMMUNE ACTIVATION IN CHILDREN WITH CYSTIC FIBROSIS Nicholas M Croft'·•, Rosalind Smyth~ Una O'Hea2

, Ken Humphreys', Tom 0 Marshall\ Anne Ferguson'. 101 Unit, Western General Hospital, Edinburgh, UK 'Respiratory Unit, Royal Liverpool Children's Hospital, Liverpool, UK 'Royal Hospital for Sick Children, Edinburgh, UK • Now Royal Hoopltal for Sick Chlldrea, Cluaow, UK

Pnevious IIUdiel vary u to whether children with CF have Intrinsic tmmune or Inflammatory lbnormalilieS ol the gastrointestinal mucooa. In response to the recently clescribed complication ol fibrosing colonopsthy, thought to be related to high dose enzyme ussge, we have studied children with cystic fibrosis using whole Jill lavage (WGL) with commercially available polyetbylene-glycol baled solutions. WOL has been shown to be a perfusion !lyltem which allows estimation of the secretion rate ol proteins and other subslanc:lCI Into the GJ traCt. Patleats and Methods: 23 pancreatic insufficient children with CF (12 taking high dose preparations, 11 low dose preparations) were lludied and compared with 12 controls undergoing whole pi lavage prior to colonoscopy or u a lrCIItment for severe constipation. The whole pt secretion rates (calculated u per k& per day) for blood (Hb), proteins suggestive of active mucosal inflammation (albumin. a I AT and !gO), cytokines (IL-l and IL-8), granulocyte elastase, secretory JgA (n•l7), and eosinophil cationic protein (ECP) (n-17) were

Poster Session Abstracts 307

compared using the Mann-Whitney·U telL U they had a proclucllve cough the children were lsked to expectorate any sputum during the lavage. Results: None of the children bad significant blood lou. The secretion rates of albumin (p<O.OOJ), a IAT (p-0.07), !gO (p<O.O~). ICCreiOry lgA (p<O.O~). lnterleuldn-1 (P<O.O~). interlcukin-1 (p<O.OOI), granulocyte elastase (p<O.O~) and ECP (p<O.O~) were higher In the children with cystic fibrosis compared with controls. There were no differences In the secretion rates between the children taking high or low dose preparations. In addition three children studied following surgery for librosing colonopatby, one studied one month prior to FC beina diagnoaed, plus one child with pancreatic insufficiency but prior to starting enzymes had abnormalities similar to the remaining CF children. One child with CF who Is pancreatic sufficient had normal parameters. Dl~a~uloll: Our data suggests that there is generalised Immune activation and minor lnllammalioa (1101 of the severity found in active Inflammatory bowel disease) In the OJ mucosa of pancreatic insufficient children with CF.

370

MAGNETIC RESONANCE CHOLANGIOGRAPHY IN CYSTIC FIBROSIS. I Durieu, 0 Pellet, C Gaillard, G Bellon, P Vital Durand. Departments of internal medicine. radiology. pediatry. Centre Hospit.alier Lyon-Sud. 6934S Pierre-B~nite. France.

Oblec!lye: to evaluate the prevalence and the nature or biliary tract abnormalities in adult cystic fibrosis (CF) population with a non invasive diagnostic procedure : the magnetic resonance cholangiography (MRC). Methods and patients: 16 CF adult patients (mean age: 27 years; 19-39) were prospectively included in the study whatever their hepatobiliary status. Among them 3 patients received acid ursodeoxycholic (AUDC) treatment because of abnormal hepatic tests: 2 or them have underwent cholecystectomy 3 and 10 years ago. Among the 13 other patients only 1 had abnormal biologic hepatic tests. All patients underwent abdominal ultrasonography and MRC with a 3 D TSE MR scanner (Philips 1,S T). The sequences were obtained with a multislab acquisition mode: 120 panitions with a partition thickness of 1.6 mm and followed by a MIP reconstruction. The MRC images were assessed for overall quality, and presence of dilatations, strictures or lithiasis of intrahepatic or extrahepatic biliary tree. By analogy with retro~;ra~e cholangiographic critena. we diagnosed cholangitis when assoctauon of strictures, dilatations and rigidity of intrahepatic biliary tree. ~ ultrasonography showed hepatomegaly in 3 patients, non foncuonnal gallbladder in 7. gallstones in S. It was considered completely normal for 6 patients. MRC images were obtained for IS patients. No anatomical abnormalities of the comon bile duct were diagnosed. One patient had a common bile duct lithiasis. Intrahepatic stones were observed in 2 patients. Complete criteria of cholangitis were present in 6 patients but S others presented also some abnormaliti~ of intrahepatic biliary tree : rigidity (n=2); strictures (n=l); dtl~~uons (n=2); ab~ormal distal visibility of biliary tree (n=S). Abnormal1ues were predommantly located on left hepatic lobe. Among the 6 patients with cholangitis, only 2 had abnormal hepatic tests, one of them being treated with AUOC Conclusion: among 16 adult CF patients, 11 presented abnormal MRC images with cholangitic aspects while only 4 patients had abnormal biological tests.

371 CORRELATION BETWEEN FECAL WEIGHT, FECAL FAT AND FECAL ENERGY IN PATIENTS WITH CYSTIC FIBROSIS L.fll.i5, D. Kalnins, M.Corey, P. Pencharz, P. Durie. The Research Institute and OepaJtment of ~:cs, The Hospital for Sick Children, and University of Toronto, Toronto,

Many patients with cystic fibrosis (Cf) and pancrcstic insufficiency (PI) continue to experience high fat and energy losses despite pancreatic enzyme therapy. Severs! s1Udies< 1.ZJ have suggested lhst stool energy losses or slool weight may be a better indicator of efficacy of enzyme lhcrapy than fecal fal. To study the correlation beiWeen (I) fal and energy losses in stool and (2) wet weight and losses of fat or energy in stool, we analyzed data from 3 of our previous studies (94 observations) involving 43 patients with CF and PI (mean age 21 yzs, range 7-42). All patients were experiencing gastrointeslinal symptoms, and/or weight Joss. Sevenly-two hour stool collections were analyzed for wet weight, fat and energy content Dietary intake wu weighed and enzyme dose recorded.

308 1997 Cystic Fibrosis Conference

Reaulll:

Energy intake (kcaVday) Fat inllke (gm/day) Lipase/units/gm fat/day Fecal weight (gm/day) Fecal fat(% intake) Fecal energy • % intake

• kcaVday

Mean:i:SD 2615±760

100±33 2475±1190 230±117 20±12 17±9

431±239

Ranae 1233-5020

40-201 477-5765 27-592 4-59 3-43

64-1158

There was a strong correlation between % fat malabsorption and % total energy malabsorption (....0.80, p..(),OOOI). Wet weight of stool was correlated with both % kcal malabsorbed (r-0.60, p-0.0001) and % fat malabsorbed (....0.43, ~.0001). Regreasion analysis ot% fat malabsorption against stool wet weight mdicated 1.6 kcal were malabsorbed per tpn of stool. This was not affected by age or weight of the patients. Fat malabsorpllon accounted for 41% (range 14-83) of fecal energy !oases.

Coneluoton: Fecal fat and fecal enerey are both effective methoda of evaluating malabsorption. Correlations with stool weight are not as strong, and may reflect the many extraneous facton that influence weight of stool ie: dietary composition, water content, bacterial flora etc.

I. Murphy etal Arch Dis Child 1991 2. Wootton et at J. Royal Soc. Med 1991

Supported by Canadian Cystic Fibrosis Foundation

372 SUBSTRATE Ufll.ISATION IN INFANTS WITH CYSTIC FIBROSIS. Shepherd RW, OJm..RM, Wainwrigllt C, Thompson M. 6iJa: Infanls with cyltic fibrosia display higher IIII«8Y expenditure and lower body fat stores than nonnal infants. In order to define further the medlanism for difl'erences in nutrition, the aim of this study was to determine whether infants < I year have the same substrate utililltion rate u nonna1 children. Mlllltlla: Restina IIII«8Y expenditure and respiratory quotient (RQ) were measured by indirect calorim«ry. Z.scores for heisJ!t and wtisJ!t were calculated uaina the anthro program. Results are IIIEl

±scm. Bm!bl: In infants aged between one and six months, the CF infants (n-24) had significantly higher RQ (0.9&lo0.2 vs 0.88%0.17, p<0.05) and lower z-scores for heigllt (-0.64±0.29 VI 0.56:1:0.15, p<O.OOI) and weisJ!t (-0.43±0.20 vs 0.29±0.18, p<O.OS) than nonna1 infants (n=28), and there was no difference in REEJk& (0.27±0.10 vs 0.26:1:0.10 MJik& n.s.). Betwealsix months and one year, there was no diffennoe between RQ (0.92±0.08 vs 0.89±0.02, n.s.), or z.scores for heigllt (-0.31±0.37 VI 0.12±0.32, n.s.) and weight (-0.49±0.40 vs 0.09±0.26, n.s.) but REEJk& (0.36:1:0.16 vs 0.25±0.01 MJ/Ics. p<0.05) was sipificantly increued. Cwlpiow. We postulate that the observed difFerences in RQ may be related to difFennt growth dynamics in cyltic fibrosis infants. When these infants are nutritionally compromised, as reflected in lower z -scores, carbohydrate is favoorecl u the metabolic aubstrate as fat stores may be low and fat malabiOiplion poorly controlled. As nutrition is improved and cstch up growth occurs, more fatty acids may be utiliaed, u reflected in nonnalilltion ofRQ.

373 MotUity ud 1eeretloa Ia tile upper patrolatestlaal tract Ia patleau wltb CJitlc ftbi'OIII (CF) K Hallbcrp J. Dalenblck0 , B. SlriUidvik. Departments of Pedialric:sllld Surpry0 , Faculty of Medicine, OiiCebcq Univenity, Ol!tebor&, Sweden.

In bealthy subjects, a number of pstroinleldul secretory producll YrrJ cyclicllly in auocialion wilh the lntadipslive motility pettern, the miJra1in1 mo10r complex (MMC). Altbou&h patrointeatiul aymptoms IIIII dysfunctions n commonly reported amona patients with CF very little is known about llllderlyin& palhophysioloaical mechanisms, eapecia!ly conc:cmiDa IIIOiility and motility related secretions. In the c:unentltlldy, 2 womea IDIIl men, ..... lS-46 yn with CF IIIII

lbeonul - test - IIIUdied. Two pstieata - homozyJOiel for 41'508, one wu ~late llld one hlld unknown mutati0111, beiDa the one patient without clinical ~ insufficiency. One patient hlld dilbeta IIIII one paliont wu operated on due to meconiumi!eus. All were in stable clinical condition without sips of infeetlon at the lime of the IIUdy. After u overni&ht futiJIIa CODitalll·flow, pslric perfullon technique wu utilized to ccntinUOUIIy measure pH lllld PC02 of pstric eftluen~ with aubaequent calculations of pstrie acid llld biclrbonale outputs, with

concomitant registration of interdigestive gasuoduodenal motility with open­tip manometry(l ). All patients exhibited MMC phase m activity, however differing from normal patterns by a simultaneous onset of contractions at all the 8 gastrolduodenal registration sites. Furthennore, phase m activity was preceded by a long periode of low motor activity, not fullfilling phase n criteria. Gastrin levels were norms! lllld did not alter with the current phase of the MMC. Gastric acid output varied as in healthy subjects with a substantial rise during gastric phase ill activity (14~±16.0 11moVmin vs phase I 81.9±8.0 11moVmin) while gastric bicarbonate output was low lllld without ordinary MMC related variations (5.9±0.8 lllld 6.2±0.7 11moVmin during both periods). All patients exhibited a constant and significant duodenogastric reflux as measured by the amount of bilirubin and duodenally infused [ 14q. labelled PEG in gastric effluent. Net gastric fluid secretion was less than a third (0.9±0.0~ mVmin) as compared with normal subjects (3.4±0.1 mVmin). Obvious differences in both gastroduodenal fasting motility lllld interdigestive motility related secretory patterns were detected in the srudied CF patients ss compared with healthy subjects. Further investigations of these phenomena might lead to important knowledge about the underlying pathophysiological mechanisms behind gastroduodeoal dysfunction among CF patients. I. The pHIPC02 method for continuous detennination of human gastric a:id and bicarbonate secretion. J. Dalenbllck, L. Fllndriks, L. Olbe & H. Sj<lvall. Scand JGastroenteroi199S;30:86t-871

374 Breast Cancer In Females with Cystic Flbroals. OS Holsclaw, Jr .. KS McCoy*, W Morgan•, OM Schlesinger, G Young* Dept. of Pediatrics, Allegheny University of the Health Sciences, Philadelphia, PA.; Ohio State University , Columbus, Ohio*; University of Arizona Health Sciences Center, Tucson, Az•.

Most reported cancers in CF have involved the gastrointestinal tract, (pancreas. hepatobiliary tree, distal ileum and proximal large bowel), have presented at a relatively younger age and with a rather aggressive clinical course. Surprisingly, In spite of its ductal and exocrine structure, there have been no reports of carcinoma of the breast. We report here the clinical features of three adult women with CF who developed breast carcinoma. l<.ULl, 35 year old white female. Homozygous delta F508 with moderate pulmonary disease and pancreatic insufficiency. A small breast mass biopsy revealed infiltrating ductal carcinoma with positive margins. Subsequent total left mastectomy with one positive node found. Right breast biopsy was negative except for fibrous mastopathy but elective prophylactic mastectomy performed. Chemotherapy was completed and well tolerated. Now doing well three years after diagnosis. ~ 50 year old white female with pancreatic insufficiency and severe pulmonary disease. Left breast biopsy at age 46 revealed invasive intraductal carcinoma followed by total mastectomy. Pathology report waa of free margins but with positive regional lymph nodes. Also noted was mammary dysplasia, atypical hyperplasia and sclerosing adenosis. She died at age 50 years from advanced pulmonary complications of CF but with no residual evidence of carcinoma on autopsy. ~ 40 year old white female with two prior fibroadenomas of the breast at ages 14 and 28 years. At age 37, another mass fell to be a fibroadenoma was removed but was found to be an Infiltrating ductal carcinoma. Following lumpectomy, estrogen receptors were absent, and DNA analysis by now cytometry revealed near diploid ONA content (DNA index 1.2). A partial course of chemotherapy was given. A recurrence necessitated a full mastectomy at age 38 followed by radiation therapy. 22 lymph nodes were negative. Metastasis to the chest wall and liver were noted by age 39 and she died of brain and spinal cord metastases at age 40. Both pulmonary and nutritional status was excellent.

All three cases had their onset between ages 35 and 46 years of age and were infiltrating ductal carcinomas. None had a family history of breast cancer. Heakh care providers Involved In the comprehensive management of patients with CF be aware that carcinoma of the breast does indeed occur. We urge the Inclusion of sequential breast screening for females with CF starting In their 30's as the age of survivorship steadily increases.

375 Histopathology of the Female Breast In Cystic Flbroals. OS Holsclaw Jr. FU Garcia*, Depta. of Pediatrics and Pathology*; MCP+ Hahnemann School of Medicine, Allegheny University of the Health Sciences, Philadelphia. PA

Although an exocrine gland and thus expected to participate In the ductei epithelial abnormalities characteristic of CF. the basic histologic structure of the post pubertal female breast has not been studied. The only report, a single caae by Ward (1972) concluded that there was complete lobular agenesis, which II contrary to our clinical obSifVatlons of normal breast developmen~ pregnancy, and successful lactation. Qu!gn: Breast tissue

from 15 patients wijh CF was examined. Twelve had died of CF complications and had autopsy material for review with one prior breast biopsy also available. Two lumpectomies for breast carcinoma from outside institutions were revi-ed. An addijionaJ living case had both a breast biopsy and bilateral mastectomies (for breast carcinoma and prophylaxis). ~: Ages ranged from 18 to 46 years (median 32) and all had normal sexual development. 14 of the 15 had pancreatic insufficiency. Genotypes done in six we111 varied with three homozygous for delta F508. Two patients had been pregnant and fiVe patients had insulin dependent diabetes. The breasts from 40% of the patients showed well developed lobular units, 27% demonstrated partial obliteration and 33% had near total atrophy. One of the latter group had complete normal development by breast biopsy three years prior to death. The common feature of all breasts was the development of. varying degrees of fibrosis. The fibrosis was an ongoing process with sonne areas showing well-established dense fibrosis with total obliteration of lobular units and other areas showing only early fibrosis which appeared to start in the perilobular stroma. Dilated ducts were present in the areas of dense fibrosis. The elongated shape as well as the tapered ends of these ducts seemed to indicate uneven compression by fibrosis throughout the duct system. The majority showed inspissated eosinophilic material which partially filled the ducts. Some chronic inflammation (lobulitis, ductitis) was noted. Other changes noted were epithelial hyperplasia without atypia, apocrine metaplasia, microscopic papilloma, sclerosing adenosis, diabetic fibrous mastopathy and eosinophilic mastitis. Three patients developed mammary carcinoma, ductal (NOS), grade I In two and high grade ductal in one. gonclusions: Patients with CF demonstrate either present or past normal breast development and do not evidence complete lobular agenesis as previously reported. There is ductal dilatation and inspissation and an active progressive fibrotic process which involves lobular un~s as well as the duct system and which can develop proliferative lesions and carcinoma.

376 EXTREME VARIABILITY OF RESPONSE TO ENZYME TREATMENT IN PATIENTS WITH CF. D Katpjna L. Ellis, M. Corey, P. Penchaa, P. Durie. Tbe Researcb Institute and Depanmelll of Pediauica, Tbe Hospilal For Sick Childreo and The Univenlty ofTonm~.Toromo.~

Enzyme therapy for CF patienta witb pancm~tic insufficiency (PI) promotea growtb and weight Jain. However, few dosinJ and efficacy atudiea bave been conducted. Caregivers and patienta often arbitrarily alter enzyme dole 10 allevtate aymptoms, witbout objective evaluation of tnaldigeation. OB !ECJWES and METHODS· We lllalyzed data from tbree of our previous 1wdiea wbicb included patients witb aymptoml of maldigeatioll and/or poor weight gain. A total of 94 observations in 43 patients witb PI (mean age • 21.2 yean. range 7.0-41.9) weno evaluated. Seventy-two boor otool collectiona.weno lllalyzed for wet weigb~ fal and enersy absorplioo. Dietary inlake wu wetgbed and enzyme dole recorded. Patients continued tbeir UIUal enzyme dole, perceived to be appropriate by caregivers (based 011 age-adjuated enzyme dolins Juidelinea). All patients were 011 enla'ic coated enzyme products. BESUI.TS· Pslienta' mean enzyme intake was 247S:tll91 Lipaae Units/g fallday (range 471-S16S), Tbe mean ,. fecal energy malabsorption wu 17±9 (range 3-43). The mean ,. fecal fal malabsorption wu 20:t.l2 (range 4-S9) wilb 18/94 at <10,. and 14/94 a1 >30-.. Enzyme dosage, fecal fat and 10181 energy malabsorpliOD did not correlate witb age. There wU DO IU8Jeltion in tbe eobort, or any subgroup, of a correlation between enzyme doae and fecal fal or total energy malabsorption (p >0.3 for all conelaliona). There were DO outliers or bigb influence points tbal may bsve obscuml a real relatiooship in tbe rest of tbe dala. CONCI!JSIQNS and RECOMMENJ)ATIONS· In spite of die very large ranse of values, tbere was DO relationship between enzyme dole and fal or energy losses, indicating tbat an cqulvalen~ albeit very variable level of control wu being achieved ala wide range or enzymes doaea. However, tbeae doael may not reflect actual enzyme requiremenu. In patienll witb auuested aymptoms of maldigestion, objective atudiea aucb u 72-bour fecal f8l arc recommended to delenDinc efficacy of enzyme tberapy. Symptoml tbat may iDclude frequent bowel movements or abdominal pain and au sbould not be uaed u aole indicaton of adequacy of enzyme therapy u tbey arc not oecesssrily related to inadequate enzyme dole. Otber potential c:auaea or sasuointestinal symptoms abould be oonsidered before enzyme dole Ia altered. Fwtber IUidiea are warranted to explore tbe mechanism or nutrient maldlgestion in CF.

Supported by tbe Canadian Cystic Fibrosla Foundation

377 PLASMA ZINC AND ALBUMIN IN CYSTIC FIBROSIS INFANTS IDENTIFIED TIIROUGH NEWBORN SCREENING: RELATIONSHIP TO ENZVME TIIERAPY

KullLJ:I£ Sontag MK, ACCIIno FJ, Hambidge M. Sectioru of Pulmonology and Nutrition, Dtportmmt of Ptdiatrlcl. Tire CAildrm '1 Hospital a11d the Univenlry of Colorado Health ScitiiCtl Center

Poster Session Abstracts 309

Infants with eyatic fibroaiJ and exocrine pancreatic insufficiency bsve relatively low absorption of dietary zinc and poor inteotinal conaervotion of endoaenoua zinc. This evidence of impaired zinc homeootuiJ indicates that these Infanta arc at enhanced risk of zinc deficiency. Pluma zinc concentrationa in CF infanll weno therefore examined in relation to pancreatic exocrine enzyme replacement therapy. Subjects were 4o4 CF infants leu than 4 months of aae who bad beea detected initially through the Colorado neonatal acreenina proaram. Resulta accordina to enzyme therapy status at time of blood collection were:

II 18 26

A&Umllil l.S 1.6

PI Alb C&fdll 3.56±0.89" 3.S2;t0.84

p•0.88

PI Zn Cu w/dl) 69.8± 3.2 82.2± 5.2

p• 0.048 •· mean± standard etror

Twenty-one (46%) infants bsve plasma zinc S 70 llllfdl. There wu a ai1nificant positive correlation between plasma zinc and albumin (r • 0.41, p • 0.001). It ia concluded that tbe mean plasma zinc eoncentralion of cyatic fibrooiJ infanta not receiving pancreatic enzymes ia significantly lower than tbe mean for cyatic fibrosis infants of comparable age who are receivina enzymes. ThiJ difference Ia not explained by the pooitive correlation between plaama zinc and albumin and IU8Jestl tbat nonnal zinc homeootasiJ may depend on adequate exocrine pancreatic enzyme activity.

This research wu aupported by Grant Number MOl RR000069, General Clinical Reaearcb Ceo ten Program, National Center for Research Re10urces, NIH

378 COMPARISON OF GROWTH STATUS IN CHILDREN WITH CF BETWEEN THE UNITED STATES AND CANADA. H...t..J.!Y, M.R. Koaorok, S.C. FitzSimmons, M. Corey and P.M. Farrell. Department• of Pediatrics and Biostatistics, University of Wisconsin-Madison, WI and US and Canadian CF Foundationa.

We compared growth statue and prevalence of growth failure aa identified by anthropometric indicators in children with CF in US and Canada (CA) based on analysis of height (ht) and weight (wt) data obtained from the 1992-94 CF Patient Registries (n•14730, US; n•2152, CA). Growth percentile• and% ideal wt/ht (IWH) were computed ba~~ed on NCHS growth referencea. The occurrence of growth failure was categorized by sex, age, meconium ileua (MI), time of CF diagnosia, FSOS status and pancreatic enzyme supplementation (ENZ). Our resulta show that the percentages of male (52%) and Caucasian (96%) are similar between US and CA. Mean age (yr) ia younger in US (9. 7 • 5.4) than CA patients (9.1 1 5.3), p<O.OO 1, whereas mean age of diagnosia (yr) ia older in US (1.9 • 3.6) than CA (1.5 • 2.6) patients, p<O.OOl. The incidence of Ml appears to be higher in US (22%) than CA patienta (18%). Overall, CA patients show significantly higher median hi/age (28"'o/oile) and wt/age (27"'%ile) but a similar% IWH (103%) compared to US patients (ht/age•21Mo/oile, wt/age•22""%ile & % IWH•103o/o). Consistently, the prevalence of ht/ age and wt/ age<5"'%ile Ia higher in US than CA whereas that of% IWH<85o/o is similar between US and CA, !!~.l!llmml!l.~!t.~I9)Y ............................................................... ..

%patients with: h1/age<5th96ile wt/age<9'%ile IWH<8S9(,

1m ~ 1m ~ 1m ~ all patients < 18 yr 22* 16 20* 16 7 7

by age: < I yr 38 35 40 32 16 11 1-3yr 20* 15 171 14 3 3 4-IOyr 171 10 121 9 3 2 11-14 yr 251 19 25* 17 6 5

................ ~~.::Ill yr. ....... ~!!' ...... ~0 .......... ~~· ...... ~~ ........ ~ 1.' ..... J.:, .. c-pco.ooot mw1 apc0.05, chi·-auate _.,....

The higher occurrence of ht/age and wt/age<5"'o/oile noted in US than CA patients persist after adjusting for gender, age, age of diagnosis, Ml, F508 status or ENZ. Within each country, growth failure is particularly prevalent in infanta and adolescents and in newly diagnosed patients (ht/age<S"'o/oile: US•33%, CA•32%). Additionally, patients not taking ENZ show lower occurrence of ht/age<S'"%ile (US•I2%, CA•9%) and wt/age<S'"o/oile (US•IO%, CA•7o/o) than patients taking ENZ (ht/age <5'"%ile: US•22%, CA•I7o/oand wt/age<5"'%ile: US•21%, CA•16%). (Supported by NIH granta DK34108 and RR03816)

379 Plasma Antioxidant Capacity In Children with Cystic Fibrosis

L.C Lands, C. Grenier, V.L. Grey, D. Drury

Division of Respiratory Medicine and Department of Biochemistry, Montreal Children's Hospital-McGill University

310 1997 Cystic Fibrosis Conference

Patients with Cystic Fibrosis (CF) are predisposed to oxidant stress as a result of chronic lung inflammation and malabsorption of antioxidants. Several studies have demonstrated a deficiency in plasma antioxidants, despite seemingly adequate levels of individual antioxidants. We report the use of a new, simplified assay to assess plasma antioxidant capacity, and compare these results with other blood antioxidants, nutritional status, and pulmonary function. Fourteen children (5 male), 5 of whom were hospitalized had blood drawn for plasma total antioxidant capacity (T AC), Vitamins A and E, cholesterol, uric acid, and selenium (8 patients). Thirteen also underwent spirometry and whole body plethysmography. Despite being well nourished (% Ideal Body Weight: 91.2 ± 11.8), 11114 (78%) patients had a low TAC. There was no difference in TAC between hospitalized and non-hospitalized patients. The T AC in our patients was significantly lower than that of II healthy adult volunteers. The proportion of patients with a low T AC is less than the proportion of patients with low concentrations of Vitamin A (29"/o), Vitamin E (7%) or uric acid (0"/o), but not less than the proportion with a low concentration of selenium (SO%). There was no correlation between the T AC and any of these concentrations. Furthermore, there was no correlation between the T AC and the % Ideal Body Weight or lung function (FEV1 or RVffLC). Plasma antioxidant capacity can now be simply and readily measured. The reason for the disproportionately low plasma antioxidant in CF patients is unclear. We are investigating whether it relates to altered glutathione stores.

Supported by the Canadian Cystic Fibrosis Foundation and Fonds de Ia Recherche en Sante du Quebec

380 LONG SEGMENT FUSIFORM STENOSIS AND COLONIC SHORTENING EQUIVALENT TO FIBROSING COLONOPATHY IN THE COLON OF ADOLESCENT PIGS EXPOSED TO SODIUM LAURYL SULPHATE AND POLYSORBATE 80, A CHRONIC TOXICrTY STUDY. L.M. Ball, C L Lannon, 0. van Valzen. Department of Pathology, Dalhousie Unlver~lty, Halifax, N.S., Canada. Btekprpynd: Long 1egmant fullform 1teno111 and bowel shortening Is Integral to the pathology of human Flbroalng Colonopethy. Induction of the fibrotic component In animal modele thu1 far requires co-exposure to Eudraglt L30055 and the dispersion aolvent containing Sodium Lauryl Sulphate ISLSI and Polyaorbate 80 (PS801, retained In the final enteric coating. The poaalblllty of a separate role for theae aurfectanta with known Irritative potential In long term expo1ure would 1eem to warrant further Investigation. Mllblull: Female adoleacent plg1, weight 70 kg, were exposed by three tlmea dally caecal gavage through tranlabdomlnal 1tenta, to ltandard strength Eudraglt dispersion 1olvent containing 11.6 mg/kg/day of SLS and 38.5 mg/kg/dly of PS80 with 100 (n•16) or 60 ln•8) mg/kg/day of Eudraglt L30055, to aolvent diluted 10 fold with 111ine In • 81 containing 50 mg/kg/day of Eudraglt L30056 or to stand11rd ltrangth dispee~lon 1olution In •11 1 only. Animals, with recording of animal welghtl, ware terminated at humane endpoint or at 90 day1 expo1ure. Full length colon specimens ware mea1ured for mean and least circumference, length end weight. Buulla: Long Hgment fusiform 1tanoaea, often multiple, and with reduction• to 3-6 em circumference (normal 10-181 were found In the po1tcaecal and d11cendlng colon of all anlmala expol8d to full atrength dlaperaion solvent, Independent of and without co-expo1ure to Eudreglt L30D66 and to a much lall8r extent in the colona of animal• expoeed to 11ilne diluted diiPBCiion aolvent. Significant bowel shortening, with coincldantli reduction In colon to bodyweight ratio, was found in 4016 of animal• and was similarly related to exposure to full strength disper1ion solvent. Cpne!y•IQM: The role of Sodium Lauryl Sulphate and Polysorbate 80 in the development of the pathology typical for Fibroling Coionopathy require• further ltudy. Theil agant1 with known bowel irritant characterlltlcl may play a crucial role In the pathogene~la of FC, and may ba Independently rHponlible for the ltanolld, 1hortaned aspects of the affected colon.

381 DEVELOPMENT OF PANCREATIC INSUFFICIENCY IN INFANI'S wmf CYSTIC PIBR.OSIS IDENTIFIED nntOUGH NEWBORN SCREEN; LONOinJDINAL RELATIONSHIP TO SERUM IMMUNOREAC'I1VJ! 11lYPSINOGEN

CO I !apnl MK Sontla, KB Hammoad, FJ Accuno. Department of PedialriCI,

The Children's Hospital and University of Colorado Health Sciences Center, Denver, CO.

Newborn sc=ning and early diagnosis allows the opportunity to evaluate the development of early pancreatic insufficiency (PI) in infants with cystic fibrosis (CF). Alma: I) To descn'be the development and incidence of PI in infants with CF during the fust 2 yean of life. 2) To detennine if there is a longitudinal relationship between serum inununoreactive trypsinogen (IR T) and the percentage of fat excretion in infanta with CF. Methods: We prospectively evaluated 62 infanta with cystic fibrosis identified through newborn screen, at 2, 6, 12, and 24 months. Infanta were admitted to the clinical research center for 72- hour fecal collections at 2, 6, 12, and 24 months. Pancreatic enzyme replacements were withheld 24 houn before admission and throughout the stool collection. Fecal fat was expressed as a percentage of fat malabsorption (grams of fecal fat I grams of fat intake x 100). PI was defmed as > IS% fat malabsorption in infanta < 6 months and > I 00/o in infants > 6 months. Serum IRT measurements were also obtained at 2 months, 12 months and 24 months. We analyzed the data using repeated measures, mixed effects modeling in SAS. Resulta:

2 months 6montbs 12 months 24 months lRT 276±13 196±9 108±13 44±17 "lo or Fat Ezcretlon 21±2 25±1 32±1 46±3 % Patlenta PI 63 74 93 92 Percent of fat excretion increased siBJiificantly with age (p - 0.0001). The incidence of pancreatic insufficiency was 63% at 2 months and increased to 93% by 12 months. IRT decreased significantly as a quadratic function of age (p<O.OOI). IRT sipficantly decreased as percent of fat excretion increased, but age was a better predictor of percent of fat excretion than was IRT. Four pancreatic sufficient (PS) infanta had elevated IRT levels (>100 nglml) at 12 months of age consistent with observations in older PS patients. However 300/o of PI infants also bad elevated IRT levels at 12 months. Thus IRT did not discriminate between PS and PI infanta. Conclusions: I) The incidence and degree of pancreatic insufficiency increased throughout infancy. 2) IRT decreased with age but did not predict pancreatic status.

382 Famotidinc (F AM) Phannacokinctics (PK) in Patients with Cystic Fibrosis (CF). L.MiW1. M. McCubbin, L. Lctzig, G. Keams. Departments of PhaDnacy Pmctice, Pediatrica & Pharmacology, Univ. of Missouri • Kansas City 111¥1 the CF Cclller, the Children's Mercy Hospital, Kansas City, MO, USA.

Famotidinc may have several advantages over other H, .. ntagonists 111¥1 is beiJ18 '-' in patienll with CF; however, the PK in this population are not known. This investigation is designed to char.lcteri2e the PK of famotidine in patients with CF. Methods: We conducted a PK study of F AM in 6 patients with CF (10.3 to 28.3 yr., mean weiglu (±SO) 43.0 ±11.7kg, 111¥13 male) who were given a single IV (0.50 ±0.1 S mglkg) and oral (UKJ ±0.30 mglkg) dose in a crossover design with a wash-oot period of <!: 2 days. Repeated blood samples were obtalncd over 12 hr after IV (11'"12) 111¥1 oral (n-9) FAM dosing for quantitation of plasma concenlllltion by high-performance liquid chromatography. Concenllation VI. time data were curve fit using a non-linear, WLS algorithm with weiglu • IN(calc). PK panunctcrs wen: calculated from the resultant polyexponellial ptiiiiiiCtcr estimates. Resu Ita: after .rLdll.liD&. PK parameters (mc:an ±SO) were: Cmax • 496.7 ±228.3 nglmL, tl/2~ • I.S3 ±0.29 hr, CL • 0.92 ±0.27 Uhr/kJ am Vdsa • 1.60 ±0.41 Llkg. PK parameters after ll.llli..dl:lsi were: Cmax • 131.82 ±64.25 nglmL, nag • o.S4 ±0.3S hr, Tmax • 2.62 :1:1.31 hr, tl/2 elimination • 1.81 ±O.SO hr, CL (corrected for F) • 0. 99 ±0.361Jhrfk& Vdss (conected for F) • 2.37 :1:1.00 Llkg 111¥1 F (bioavailability) • 0.63 ±0.31 (range 0.24 to 1.00). Significant differences were absent when elimination rate constant, CL 111¥1 Vdss WCRI

compared between the oral and IV dosing periods. Comparison of present data to those ~ly noported for infants 111¥1 children without CF (James LP, ct at. JC/In Pharmaco/1996;36:48-S4) n:veakd an approximate 34% increase in CL for patients with CF and a similar value for Vdss (i.e., 2.4 ±1.7 Ukg for mn­CF subjects). Conelullona: 1). M with other dnlgs which have both renal and non....,nal CL routes, the CL of FAM appears increased in patients with CF; 2). The om! bioavailability of F AM in patients with CF appears comparable to Rpotted values for adults without CF am 3) The CL of F AM in CF would appear to 111pport a Q 12 hr dOling interval to maintain concenlllltions above the I!C50 for the dmg.

Supported by the Children's Mercy Hospital Research Vision Con: Laboratoty Project Gnml and grant fiUOIHD31313~ for the Pediatric; Pharmacology Relealth Unit Netwodc from the National Institutes of Health 111¥1 Human Development

383 THE USE C. THE MooiFlED Tunu! Tl!sT (1' ARn AND 1liE PROLONOED PH PRall! mDYINlliEJ::l!AcNJslsarO~REn.ux(GER)INCHiuJRENWnH CYSTIC FlllltOSJS (CF). .!ennjfq Kpjghl M Hepdenog Rour!c Jr J Marc Majwe ~oCPcdiatrica, Duke UDiwnity Medical Center (DUMC). Durbam, NC, 27710,USA ~ rdlux (GER.) is., impoNDI cauoe oCmorbidity in dlildreD with

cy11ic fibrosis. The prevalence oC OER in CF is sufficiently high dill ai'CCCIIIIIUdy '"""''''"".dod pHmetry in tNefC'J 111:w CF palieDI. Ala> ol COIIOa1l ..., the reports of Bom:t'• ~in in adoleocaJt CF poti<DIS. Early cliagnoeis md-..., impo1tam. The sold llmdaid b diagDoeis is the prolcnpl pH probe test. Sboitcr, Jcaa costly pH lludia haw DOl been tbousbt to be uoeful ill previoua IIUdieo. A mrospective .mew - dooe o( all pH IIUdies clooe in inflllll md dlildreD II DUMC Ol.OIJ91-12131194 to determine the utility ol the Modified Tuttle Acid Reflux Teat (TARn in c:hiJdieD with CF who bod symptoms COIIIisteui with GER. The palialt- maaitorcd b cme bour Iller apple juice (acid) bol111. The TART wu deemed llmcnnal if there wae 2 cr illlli'C rcllux epilodca with esophageal pH <4 b llleut IS 80il<lllds -=b. cr uingle rcllux epioode with pH< 4 b- 10% oCthe k:ll time. Tbil- cnnporcd to lbe prolonged (18 boun) pH tco1. These were IICOI'Od primarily m the buil o(the rcllux iDdcx (%limo pH<4). 'J'bere wae IS prolonged pH IIUdies thai wae prcccdcd by the TART pcrfamcd in childrcu with CF. The -.itivity o(the TART k:11 wu 100% IIIII the specificity 28.6%. The poailiw mdncptiYe(X'cdiclive valueo wae 53.3% IIIII 100% lelpOCiively. Though theilUIDbcn 11e 1i11111, lbildala IUgscsll thll in cbildRn with CF, the TART may be m<R useful tban previously thou(!hl u a acreening teot fer GER . ID Ibis popuiA!ioo, all GER + poolic:ma were id<mified by 1he ICI'O<II. IIIII all TART - patients were in fiiCI GER - • The TART may be a coot clfcctive way to approecb Ibis diagnoois. Of the 8 patienll with pooitiw prolonged pH IIIUdiea, 5 were below third wci(!hl centile. Tbey all bid gatroolaDy blbeo pl....t with limultaoeoul fundoplicatioa. Ouc paticnl oolhe 25th IXUlile lbo bid p.stroot<my lUbe ODd fimdoplicatioa. Ouc potiall who wu well DOUrisbcd wu llealcd medically. Ouc patieul transfcmd elsewbere for -"'- opiniOD. The pooitiw pH ld in 1he aettina a( failure to thrive is impoi1anl to prevenl continued OER with the risk oC aspiratioa wbal eatenl feeding il accelc:ntcd.

Supported byCF Ccntcr,DUMC

384 ENERGY BALANCE IN PATIENTS WITH CF WHEN lliEY ARE STABLE AND DURJNG AN EXACERBATION OF RESPIRATORY SYMPTOMS. M McCloskey, Christine McCabe. Siobhan Pyper, A 0 B Redmond, J S Elbom. Adull and Paediatric Cystic Fibrosis Unita, Belfast City Hospilal and Royal Belfast Hospilal for Sick Children. Belfast. N. Ireland.

Resting Energy Expenditure (REE) is elevaled in patients wilh Cystic Fibrosis (CF) wben they are liable and there il a fwthcr increase during an exaccibation of respiralosy aymptoma. The aim of lhislludy wu to compare REE and Total Energy Expenditure (TEE) with Energy Imake (EI) in patienll when they are clinically liable with a respiratosy exaccibalion. Energy balance wu assessed in 10 patienll aged 14-40, wilh mean FEV1 53% (11.6) predicted. The energy balance was measured when 1hc patient wu liable and when the patient had an exacerbation of respiraiOsy aymploma requiring IV'a. During each analysis period 2 measure men II of energy expenditure were laken I 4 days apan. An exacerbation of respiratosy aymptoma wu defined u wonening aymptoms and > 10"/o decrease in FEVo requiring inlravenous antibiotic lreatmenl. REE wu measured by open circuil indirecl calorimetsy wilh a face mask and TEE wu measured by a 24 hour heart rate method. Energy lnlake wu measured by 3 day food diarica. Physical Activity Levels (PAL) wci'C delermincd by calculating TEEIREE. REE wu increased at the beginning of a respiratosy exaccibation 37.5 (H) kcallkg/day compared 10 the end oflhat analysis period 32 (4.4) kcallkglday, p-0.03. There was no difference between REE at the end of an exaccibation and REE during the stable period 32 (~) kcallkglday. TEE mcssuremcnll were not different between the twa analysis periods, 40.9 (6) kc:allkg/day during an exaccibation and 40 (7.4) kcallkg/day during the liable analysis period. There wu no difference beiWeCn 'ICE at the beginning and end of an elUICC!bation period. PAL wu lower at the beginning of an clCIIcerbation I. I (0.05) and increased following IV'S. 1.3 (0.2), P" 0.006. There wu no doffercnce in PAL between the end of IV's and the stable analysis period 1.2 (0.1). El during the exaccrbation wu ~5.6 kcallkg and 53.7 kcallkg during the period of stability. There wu a aignificant relationship belween TEE and El during the liable period (M.48, p-0.03) but no relationship between El and TEE during the exaceibauon period. TEE in patients with CF is not altered during a period of exaceibation of respiratosy aymploms when compared with a period of clinical stability. There is a significant decrease in physical activity al the beginning of an exaccibation but at the end of 2 weeks of antibiotics patients generally have PAL similar 10 when they are at home. Energy inlake docs nol change during an exaccibation compared to when stable. Supported by the CF Trust (UK).

Poster Session Abstracts 311

385 O.teoporosil In Adulll wllh Cystic Flbroola. ~lid. K. Herlyn, A. Lapey, E.J. Anderson, D. Schoenfeld, J.S. Finkelsteon. Depts. of Medicine and Ped•aarocs, Harvard Medical School IIIII Massachusetll General Hospital, Boston, MA. USA.

Prupo .. : To comprehensively atudy the prevalenc:o, prosreuion, and pathophysiology of osteoporosis in aduh patienll (pta) woth cyauc fibroaia (CF). Background: Pts w.ith CF have muhiple potential risk factorl for osteoporosis oncludong poor nutnllon, lean body mass, malabsorpllon of vnamin D, ledentary lofc style, glucocortJcood use, delayed puberty, and poa11bly others. Prior atudoea have found a high prevalence of osteoporosis among choldren woth CF but more limited dsta exists_ f'!" adults. No longitudinal study measuring the rate of progression of osteoporosos on CF has been done. M'tloob: Eighty adulu with CF will be atudoed at basehne and at two years with detailed medocal examinationa, chart reviews, nulritional &UeSSments, and pulmonary function testa. Bone density ia meuured in the lumbar opine by dual energy x-ray absorptiomeary (DXA) and in the forearm by songle photon absorpllomeary (SPA). Calcium, vitamin D, and parathyroid hormone levels are measured alons with biochemical marken of bone formation (osteocalcin (OC)] and resorption [N-telopeptide of type I collagen (Ntx)J. Rmlll.: Data from the baseline visit of the first 25 pil entered in thia protocol are reported: 14 men and I I women, age range 20.55 years (mean 32). All p11 are Caucasian and none were . laking glucocorticoids. All of the women had regular mensuual cycloa and all of the men had normal testes sizes and serum testosterone levels. All pts had normal serum calcium levels and no patient had markedly reduced vitamin D levels or elevated PTH levels. As a group, the pll with CF had significantly reduced trabecular bone density. 19125 pts (76%) had spinal AP DXA measuremcnll below the mean for their age (p<O.OI): lS/25 pll (60%) had spinal AP DXA bone density measuremenll ~I standard devoatoons (s.d.) below the age-adjusted mean. Thia prevalence of mild osteoporosis was significantly greaoer than the 16'1> expected for a normal IWilplo (p<O.OI). 7125 pts (28%) had AP DXA bone density measuremenll ~2s.d. below the age-adjusted mean. This prevalence of severe osteoporosis is also significantly greater than the rate «peeled for a normal population (p<O.O I). Similar resulll were seen tn SPA measurements of conical bone. A vesy interesting association between CFI'R genotype and bone density was observed: 819 p11 homozygoua for the AF508 allele had AP DXA bone density measuremcnll ~I a.d. below expected for age; however, only 6/16 pts 1101 AF508 homozygous had bone density measurements <:I s.d. below expected for age. This difference in osleopenia according to genotype is greater than expected (p<0.05). Additionally, markers of bone turnover were significantly abnormal: 13125 (52%) pts had NTx values >2 s.d. above control mean (8 men; S women) while only 3125 (12%) pts had OC values >2 a.d. above control mean (2 men; I woman). CoiiCiusiotu: lbesc preliminasy resulll demonstrate that ~ognoficant osteoporoSIS exosll among adulll with CF and that there may be an omponant assocoatoon between CFTR genotype and bone density among pts with CF. Ongoing bone resorption occurs in adults with CF and appears to be uncoupled from bone formation. Results of the full longitudinal study will better define the extent and pathophysiology of osteoporosis in pta with CF and perhaps lead to effective treatment mterventoons.

386 ORAL ERYTHROMYCIN EFFECT ON GASTRIC EMPTYING IN CYSTIC FIBROSIS PATIENTS. A....MJm&k t, F. Bonnio2

, M. Gerardint,

M. Le Bourgeou\ B. Bok1, J Navarro' 1. Pediatric Gastroenterology Hospital Robert Debrc, 2. Nuclear medccine dpt. Hospilal Bcaujon, Paril, Clichy -FRANCE.

Malnutrition in revere CF patients needs conlinuoua enteral nutrition by gastrostomy or nasogastric tube. Unfortunately this technique may be poorly toleralcd. In view of treating an eventual delayed gastric emptying """ evaluated the effect of erythromycin, given orally, u prokinetic agent. lA!imll 7 CF patients • Shwachman ICOre 4S ::1: S • Nocturnal enteral feeding for SO to 70 % of RDA - Exclusion criteria : prokinetics agents, antibiotics with clawlanic acid, crythranycin. ~ Scintigraphic dytwnic acquisitions of lOs-images were recorded during one hour after simultaneous ingestion of 10 MBq of Ill ln-DTPA Oi'lll18e juice and I 0 MBq of 99 mTc-sulfur colloid in egg while. The patient was placed in supine position. Treatment by Elhylsuecinale erythromycin (Smgt1cgfi'ID) was initiated immediatly afier a first scintigraphy (E-). A second one (E+) was performed 15 days later.

Results S(%) L %) E- E+ E E+

1 40 45 52 69 2 3S 61 79 90 3 Sl 62 78 83 .. 53 82 70 97 s 68 93 76 93 6 36 32 70 71 7 78 32 80 S9

~lll!iRitm NS aSS% %of aolid (S) and liqwd (L) evacuation at I hour. Subjectiw digestive sympiOmS (nausea, wmilin& abdominal discomfort) improved for all the patients. No scintigraphic gastrooesophageal retlux was detccled. Conclusion Such a short aerie& of patients only yieldJ to preliminary results IIC\'CrtbciCSI orally erythromycin may contribute to a better tolerance of liquid continuous enteral feeding.

312 1997 Cystic Fibrosis Conference

387 THE RELATIONSHIP OF BYPEROXALURIA TO THE SEVERITY OF FAT MALABSORPilON IN CYSTIC FIBROSIS PATIENTS. ~·. J. Markat, C. Fooyt, D. Homnick1

, L. Yelton\ K. Anderson•, L.Oiiwr, ATones1., 1Deplrtment ofPediatric:s, Michigan State University­Kalamazoo Center for Medical Studies; 2 Phannacia &. Upjohn Inc, Kalamazoo, MI, USA

Cyllic Fibrooio (CF) ....... wilb varyina clepeeo al"t".l malablorptioa wae otudied lo determine wbelbor byperaocaluria wu clirocdy rclaled lo lbe cJcarcc of ma1aboorptiocl. Pllienb wilb coadilioniiUCb u Crolm'1 m-oe. jejUIIOileal bypul, chronic poncreatilis, and '*-ia1 OYII"JP"OWih wbich readl ill mallblorption or maldigeslion ..-e known 1o be II ll"calcr rill< for calcium oxaiiiC IIODC fonnalioo. Tbe lbeory is lhll inleslinal calcium is p""-ially bound by ~ tatty ICidl. lbua aJiowins unbound oxa111e 1o be lboorbod inlo the bloodllrelm. ThiiiiUdy propoocalhll CF pllienls hi"" the IIIIIC enlcric

etiolo&Y Cor hypero>Wuria u - ill 1be 11bow conditions or mallblorplion llld llllldipmon. Ten CF lllbja:lllreceivinspon:RIIic enzyme oupplcmenlllion wae ncruilcd &an the MSUI1CCMS CF Clinic and Clllq)IAd lo lix narmal bealthy controb rea"Uilcd from lbe commllllity. Subjectl were liked 1o coDilluc wilb UIUal pancrcllic: enzyme oupplemc:nlalioa mel 1o bop a diet diary IIIII limit calcium (<1200 msfday) llld oxaiiiC (<IOOOJDWday)ilrooowodc,lltcrwbich time a 24 bow urillccollcoclion wu oblainedfor ....._ Cllciln, cilrlle IIIII cbemiltriel. ScNn cbemillriel were done illldddilion 1o 25-Hycmcy and I ,25-Diby&mly V"lllmin D. A 72 bow Cecal t".llllllylil wu done Cor cleF= rLr.llllilblarption Fecal !".! readlllbowed lhll 4 CF lllbjects bad ~mR mallblorption nnsinl from II-59%; 6 CF IUbjecta bad mild mallblorption nnsiiiB from 78-9S%; and normal bealthy cootrols bad Cecal flllblorption nnsilll from 93-98%. Tbe difl"ermce ill cleF= or ma11b1orption wu llalistically lipificant rcr lbe 11:\'a"e maJabqoben and lbe normal beaJ1hy oaNrola II p<O.OOOI. Tbe 11mn mallblorption PIP bad F""lcr urinary OOCIIIIecxaelionl(36.9mWm2Jday :t:I2.4),IXJIIIINRCI lo !be normal bealthy cootrols (20.3 mWm2/day :t:1.9) which wu llllilticaJIIy lianificant II p<0.05. Tbcdilfacncc in oxalate cxaelionl rLmild mallbabcn IIIII narma1 beaJ1hy cootrols wu DDlllalistically liplif"ICIIII. Tho calciiDI CIIIICIIIlioD- 1111'11111 ir all Mjecllll <4maJk8fday. ThiiiiUdy indiclla lhll alipillca nepiYe ccrrelllica .-betweeD oxalate ClfiCI"ecioo IIIII t".llblorption (r-. 73, p-.OOIS). Plllicnll wilb 11mn mallblorption 1111)' be II riaUorpalbolosical eft'ects or hypcroxaluria IIIII raal 110DC formllioa. Due lo Ibis limilcd 11111ple lize, lllnbcr IIUdico .,.. .-led in CF pllienll wilb _.., Cal malab8orplion.

~by BCIU Cmmmity ~Fund llld Michipn StiiC Univenity-KaiiiiiiZOO Cenlcr Cor Modical Studiea.

388 ANALYSIS OF PLASMA VITAMIN D CONCENTRATION IN CHILDREN WITH CF. G.C. Burdge, I. Doull, C.J. Bolles. Child Health, University of Southampton, Southampton, UK

VItamin (viti D playa a central role in the regulation of calcium homeostasis and bone mineralisation. Vit D is obtained either pre-formed from the diet or synthesised de novo in skin from sterol precursors. Previous studies have demonstrated low plasma vlt D in pancreatic insufficient CF patients, particularly In adolescent and post-adolescent subjects. However, these data preceded the introduction of enteric coated mlcrosphere IECMI pancreatic enzyme preparations. In the present study, we have determined the vit 0 status of CF patients whose dietary maintenance included vit 0 and ECM enzyme supplementation. Our long-term aim is to provide a logical basis for vit D supplementation in CF.

CF patients (aged 2.8-19.4 years (median 11.9), n•21) were recruited from our outpatient clinic between December and March, corresponding to the period of minimum UV irradiation and vit D photosynthesis. Sera were collected and 25-hydroxyvitamin D (2510HIDI concentrations determined by competitive binding assay.

Median serum 25(0H)O concentration in these CF patients was 13.8ng/ml (range 3.9-41.01. However, in 20/21 sublects serum 25(0HID concentration was leas than 50% of normal range 110-50ng/ml), and in 3 patients serum 25(0HID concentration was below 10ng/ml. In contrast to earlier studies, there was no correlation between serum 25(0HIO concentration and patient age.

These preliminary data from our on-going study indicate that contemporary nutritional management does not provide

sufficient vit 0 during winter months to support normal serum concentrations and suggest e requirement for further interventions.

389 BODY COMPOSmON,ANAEROBJC MUSCLE POWER, AND PULMONARY FUNCI'JON CHANGES Wrnf AGE IN BOYS AND GJRI1l HETEROzyGOUS (HET) VS. HOMOZYGOUS (HOM) FOR AF!!OS. Q.YL S!llliiJII, R. Hanoing, C.J. Blimkie, R. Calvert, B.V. Ayub, 0. Bor-Or. Childrea's Excn:i8c IJid Nutritioo Ccutre (CENC), McMaster Univenity, Homiltoo. ON, Canoda.

To compor<: body C<llllpOOitioo, IIIMI"Obic mWICic power, and pulmonary functioo changes wilb age between CF boys (M) IJid girb (F) HET vs HOM for AFS08, data were retroopectivcly eumined from 41 childmt over 3S4 visits lo CENC between 1985-1996. HOM (1M, 16F) llld HET (9M, IOF) childrcu riDged in age from 5-18y. Body mau (BM), beipt (liT), and% ideal body DIUII (%IBM) (NCHS) were clctermiDod, u weD u body fat (BF). % body fat (%BF). llld fat IRe mua (FFM) from 4 lite lkinfold tiiCUIIRIIIC8ll. Peak power (PP) and IDWI power (MP) wen: measured usinslbe Winpte IIIMI"Obic teot: a 30 lea cycle crgomclly. FVC and FEV, u% predictod were~ using spiromelry. 1llere wen: DO signifiCODt difl"ercuccs in body COOIJIOSiticm cbaages wi1b qe between HOM-M and HET -M. HET-F vs HOM· F, boweva, badpcaterpinl ill BM (losBM,..r• 1.12 + Josl02y vs logBM.o..• 1.43 + Jos0.83y, p-0.01) and FFM (sqrtFFM.,..,• 0.58 + IC)rt1.39y vs sqrtFFMIIOM. 1.36 + oqrtl.lly, p-0.002), tbou@b DO difl"ermcea for HT and %BF. There wu a F""lcr treDd, albeit NS, for %IBM 1o decline wilb age in HOM-F than HET -F (sqrt%1BM.,., • IO . .S9 -IC)IIO.I4y YO oqrt%1BM.o..- 11.04 - IC)rt0.43y, p-0.143). pp bad greater mcrea-wilb II!" in HET·M than HOM-M (oqrtPP .... • 17.83 + IC)rtl0.76y VI

oqrtPP..,..• 12.23 + oqrt9.14y, JR).043), but not for PPIFFM. indicating lbe difl"crcuoe wu due to greater miiiCie mau in HET -M. A similar pattern wu fOUDd for MP in M (NS). PP baciF""ter a.crca- wilb age in HET·F than HOM·F (logPP..,• 1.28 + Jos1.83y VI JosPP..,.. • 2.16 + Jos1.38y, p-0.003) evcu wbeu oomcted for FFM (Josi'PIFFM..,. • 0.04 + Jos0.97y YllogPPIFFM..,.. • 0.62 + log0.68y, JR>.OI3, 111galin81be difl"ermce m.y be due 1o more elftcient mWICie in HET -F. Similarly, MP pial"""" greater in HET·F VII HOM-F (JosMP..,• 0.93 + logl.79yva JosMPHON • 1.85 + Jos1.35y, p-0.003). HET-MYI HOM·M maintaiDod higherFVC (sqrtFVC...,. •5.09 + oqrtl.31yYIIC)rtFVC"""•6.48 + 1Qrt0.64y, p-0.033) and FEV, (oqrtf"EV1111T • 5.44 + oqrtl.l2y VI oqrtFEV,HOM• 7.59 + IC)rt0.27y, p-D.003) over time. No difl"erCDilel for FVC or FEV, change were foWICI between HOM-F and HET • F. In COIICiusioa, HET ·M VI HOM-M showed greater aboolute anaerobic power and pulmotwy limctioo with increuod age, but DO dilfcrcuces in body compositioo. HET • F VI HOM-F had pcater ~in BM, FFM, llld anaerobic power with age. ill spite of similar changes in pulmoowy fuactioo. Pbenotypic 6FS08 expression may be apporent for body c:ompooition,IIIMI"Obic muacle function, and pulmonary limctioo, IIIII illibly dilfcratt betwoeo tbe -·

390 A COMPARISON or ANTHROPOMETRIC STANDARDS FOR EVALUATION or Cr PATUNTS • L....ShiD. M. Corey, D. Kalnilll, P. Penclwz. Dcp1rtmen1 oC Gutroenterolo&Y and Nutrition, Hospital for Sick Children, University ai"Toronto, Toronto, ONT ARlO, CANADA.

Three anthropometric lltiDdardl for IIKIIins nutritional 11at111 In CF patiCDII were ClOIIIpancl: I) U.S. National Center Cor Heallb Statistics (NCHS) 2) Britilh Tanner-Wblteboule (T -W) mel 3) a new Brillsh QOIIIPOiite (UK90). Computerized venlonl or Ill 3 were used lo c:ompule belsht and weiabt pen:entiles and % Ideal weiJht for ap and beiJht (%1Wt), when: ideal weiabt is that weipt which corrapontls 1o the ame percentile for qe u the obsemd height percentile Cor ap. The CaDidlan CF RcJIIIIy data for 1994 (N•I860) were used 10 investiBIIC dl1rerellcel in the ca1cu11tet1 indices for indivldulls •sed 0 to 18 yean. We bypotbclized that us1ns the %1Wt would minimize the eft"ect of any l)'llelllllit: dl1!'eRDccl bclween the lllndartll.

The proporti01111bat Cell below lbe 51b and SOth IICI"Ct:lltilelwere u follows: NCBS T-W UKfO

Reltdlt Welot Helot Welot Helot Welallt ~-%lie MaiCI (N.,.-959) 15.8% 13.7% 12.7% 10.2% 11.4% 15.1% Femalea (N,-901) 12.8% 15.0% 9.8% 11.6% 9.0% 11.0% Total (N•I860) IU% 14.2% 11.3% 14.3% 10.2% 16.5% a• %lie Mala 74.2% 71.5% 67.7% 66.6% 75.2% 69.1% Femalea 70.9% 70.0% 64.0% 71.9% 71.9% 73.5% Total 72.6% 70.1% 65.9% 69.2% 73.6% 71.6%

NCHS ltllldaldl for beiJbt were JCDCI'IIIy hipcr, 10 that more CF paticnll were clulltled u IIUIIted than with T-W or UK90. Tbe same pattern wu 1eCD in weiabt data for J1111a, 10 i11at - were clulltled u underweiJhllllinJ NCHS. However the appolite paucm wu IIICll for fcmalel, lbat is, Brillsh weight llandardl were b!Jber, ~ more CF feataJes u andcrweilht than wilb the NCHS. Further pattern tlilc:repuclel were obaerwtlacroa dill"erent qe JIOUPI.

1besc discrepancies induced 1:0mplicatcd diffcmu:cs in patterns of ,.-a!Wl In females of all age groups, the average ,.-a!Wt using the NCHS standards were higher than with the T-W or UK90, although relative differences varied across age groups. In males, the average ,.-a!Wt generally agreed better among all 3 llandards. However, there were age-specific discrepancies which were different than those in females. We 1:0ncludc that the "o!Wt is DOC Cree of bias due to llandards U.lcd, cspccially in the 1:0mparison of males and Ccmalcs. (Supported by Canadian Cystic Fibrosis Foundation).

391 WATER-MISCIBLE TOCOFEROL IS NOT SUPERIOR TO FAT-SOLUBLE PREPARATION FOR VITAMIN E ABSORPTION IN CYSTIC FIBROSIS (CF). S.Soltani, E. Gronowitz, H. Andemon, B S!rJndyjk DeptJ of Pediatrics and Clinical Nutrition, Faculty of Medicine, G<ltebora University, Goteborg,Sweden.

Viwnin E deficiency is a major problem in CF ond may result in neuropathy, sastrointcstinal disturbances ond myopathy. We have had-­miscible a-rocoferol acetate cOIIllllefciably available since years but still haw had difticultie& to oblain normal levels if not &iving wry high dose& of the preparation. 1be dcwlopmcnt of more efficient pancreatic supplementation may also improve the absorption of the fat-soluble vitamins 10 make water dispersions leu importanL In order 10 compare the absorption of fat-soluble (F) and water-miscible (W) tocofcrol acetate (E-vimin •, Astra, Sweden), equivalent amounts '"""' siven 10 five CF patients and five age-and sex-matched healthy controls. aged 15-47 years. All patients had pancreatic Insufficiency and had been on pancreatic enzyme and vitamin supplementation for yean.The vitamin E supplementation wu not given 10 the patients one week before the tests. 1be preparations, 10 mg/ kg BW, WCR Jiven one week opart after an over-night fasting 10Jether with a light atandardizJod meal. The CF patients took the aame number of pancreatic enzymes to the two meals. Serum wu analyml for vitamin E before and t,2,3,4,6,8,10,24 and 28 h after the oral intake with a HPLC-melhod. Serum futinalevels did not differ between patients ond controla, mean :1: SO beina 16.4:1: 3.6 and 18.2 :1: 1.9 jlmolll, respectively. 1be highest concenuation in serum in controls were 41.9 jlmolll after F and 39.8 jlmolll (medians) after W preparations, correspondinJ values in CF patients being 26.8 and 22.8 jlmolll. 1be median difference between pcnk concenuation in serum after W or F Jnpsrations in healthy controls wu • 7.2 )Lmolll and in CF patients 0.6 jimoll1.(.,.0.06). 1be peak concenuation wu a:hicved mucb earlier ia CF than in controls after adminiouation of I', median time beins 6 and 10 h, n:spectively (.,.o.03) but no difference wu seen for the W preparation. AUC wu si1nificantly lower in CF than in controls for both solutions (p=0.02). Although the AUC was consistently lower for W than F preparations both in CF and controls, lheae differencca were not significant. Our data do not justify the use of the more expensive water-miscible preparation of a-rocoferol acetate in CF patients on modem pancreatic

enzyme supplementation.

392 Hyperoxalurlaand bypocalclurla In 24 bour urine collection~ from children wltb cystic fibrosis (CF). M A IumeC, D. Goldwater ,I.H. Ward2

, B.M. Phillips1• Departments of Child Hcalth1 and Clinical

Biochemistrl, Booth Hall Children's Hospital, Manchester, UK

Calcium oxalate renal stones have been described as a complication of CF and other malabsorptive states. As a preliminary assessment of factors relevant to stone formation in children with CF we collected a 24 hour urine sample from each of 26 children with CF. Clinical data was collected for each patient to give age, weight, weight for height, lipase dosage & FEY 1. Oxalate excretion (expressed in mmol/24h) varies with age: <13y, mean • 0.4038, a.d • 0.114 n •13, normal• <0.35; >13y male, mean • 0.6600, a.d. • 0.2760, n• 6, normal• <0.48; >13y female, mean -o.5186,a.d. • 0.2256, n •7, normai•<0.52. 15 of 26 samples yielded normal excretion of oxalate, II of 26 yielded high excretion. For glycolate mean • 0.232, a.d. • 0.156, normal• 24126, high •2/26. For, calcium mean • 0.056, a.d • 0.034, normal• 9124 low • I 5/24. [Normal ranges: Glycolate <0.34 mmol/24h Calclum 0.1 mmollkg/24h or 2.5-7.5 mmol/24h). Multiple regression analysis revealed that oxalate and glycolate excretions were dependent on each other (T • 4.61, p < 0.0025) but there were no significant interdependencies between excretion and clinical variables. Of the 10 patients with high levels of oxalate excretion 7 had low levels of

Poster Session Abstracts 313

calcium excretion and 3 had normal levels of calcium excretion. Of tho 9 patients with normal levels of calcium excretion 6 had normal levels of oxalate excretion. Two patients had hypercalciuria. This data is compatible with a high frequency ofhyperoxaluria in children with cystic fibrosis. The normal level of glycolate excretion argues against a metabolic cause for this. Calcium binding to unabsorbed fats instead of oxalate in the intestinal lumen could reduce calcium absorption and allow increased oxalate absorption. Altered ion transport or hormonal factors may also be involved. The incidence of renal calculi is much less than the incidence ofhyperoxaluria in this sample. One child has had 2 macroscopic stones in a total clinic population of 1 SO. The urine of children with CF may contain substances that inhibit the formation of renal stones. Future work will address this issue. Supported by tho Cystic Fibrosis Trust.

393 LEPTIN IN CYSTIC FIBROSIS PATIENTS

S. van Koningsbruggent. J. MUIIer-Berghaust, F. Ahrenst, W. Blum2, E. Schtinaut, D. Michalkl, E. Rietschell. 1 Department of Pediatrics, University Hospital of Cologne, Gennany 2 Lilly, Germany

Introduction: Cystic Fibrosis (CF) is a chronic disease which often leads to wasting due to pancreatic insufficiency and increased energy demands resulting from chronic pulmonary infection. Leptin is a recently described hormone which is exclusively synthesized and secreted by fat cells. Its physiological role is unclear at present. Its function may lie in the regulation of energy metabolism. Wo therefore sought to determine the clinical relevan<X! of leptin in patients with CF. ~ Leptin concentration was measured using a RIA. Values were adjusted for gender, pubertal development and body mass index (BMI) (kg/1112) and were expressed as standard deviation IIOOTC (SDS). In a subset of patients bioelecbic impedance was used to determine fat mass. Pulmonary function was assessed by FEVl, MEF2.5 and VC. All results are reported as median ::1: standard deviation. ~ 27 CF patients ( 14 f; 13 m) were examined. Patients• characteristics: age 21 ::1:8,6 years, BMI 19 :t 2,7 kg, height SDS • 0,44 ::1: 1,4, FEV 1 57 ::1: 28%, MEF 25 21::1: 29,5% and VC 83 :t 18%. Results; SDS for Jeptin was· 1,8 ::1: 2,0. 13 of 27 patients had SO-scores less than -2. Leptin correlated positively with percenlage body fat as determined by bioelecbic impedance (n=17, r=0,72, p< 0,001). There was no discernible influence of parameters of pulmonary function on leptinSDS. Cooclusion; Leptin is decreased to very low values (less than 2 SD below mean) in a substantial number of CF patients of different age. The positive correlation to the percentage of body fat indicaJes that the regulation of leptin is maintained. The high proportion of decreased values may be related to chronic nubitional deficiL Leptin might be of importance in the nubitional slatus of CF patients but the clinical significance still remains unclear.

394 PATHOLOGY REVIEW OF WHOLE SMALL AND LARGE BOWEL POSTMORTEM SPECIMENS IN A PEDIATRIC POPULATION NOT EXPOSED TO METHACRYLIC ACID CO-POLYMER ENTERIC COATING BEFORE AND AFTER THE EMERGENCE OF FIBROSINO COLONOPATHY. D.M. Hughes, P. Pianos!, p yan Yetzen. Department& of Pathology and Respiratory Medicine, IWK Grace Health Center, Halifax, N.S., Canada.

The appearance of the normal colon of the paediatric CF patient, other than In resection specimens for (acute) Intestinal disease, or through mucosal biopsies, Ia not known, Thus studies of the pathogenesis of Fibroalng Colonopathy (FC) are compromised by lack of appropriate comparison material. Wa carried out a retrospective atudy of postmortem colon and smell bowel samples of 14 paediatric CF patients In a population known never to be exposed to methacrylic acid copolymer (MACPI, possibly related to the Induction of FC. 6 patient& (3M/2F, age 6-1 g years) came to postmortem during the period FC emerged 11992-1996). 9 Patients (M3/F6, age 2-22 years) were available from the period before the introduction of MACP (1968-1982). Pancreatic enzyme auppletion history ranged from 1 to 19 years. None of the patients suffered from Crohn'a Disease, Colitla Ulcerosa, Volvulus, Invagination or neoplastic bowel disease. Systematic analysis lor the presence of amall mucosal ulcerations, eubmucosal fibrosis, aubmucoaal

314 1997 Cystic Fibrosis Conference

artery changes and submucosal myenteric plexus neuronal complex increase showed ulceration and submucosal fibrosis to be absent in both groups, whereas vascular changes and neuronal complex increase ware found in 4/5 casas during and 6/9 casas before the period in which FC has been described. From the findings we conclude that in the absence of MACP no submucosal changes typical of FC, irrespective of exposure duration occur. In addition vascular and neuronal complex changes, often discussed In relation to changes of FC may reflect changes common to all CF patients rather than FC specific pathology of relevance to Its pathogenesis.

395 STOOL ELASTASE AS A DIAGNOSTIC TEST FOR PANCREATIC FUNCTION IN CJI. ~ T. Leung, D. Cubiti, A. Reynolds.

Departmeaus of Respiralmy Paediatrics and Biochemistry, Great Ormond Slrllel Holpital for Chi1dlal, Loudon, United KiJI8dom,

Teall of J)fUICie8lic: fimctioo in cbildRn wilh CF arc either highly iiiVliSive inwlvin& duodcoaJ intubation and paucrealic stimulation, involve 3 day stool collections or lack aeasitivity because of tbc illflucna:s of faecal floral activity, pauage time and exogeDOIII enzyme administnltion as in the stool chymollypsin tell The development of an ELISA test for faecal pancreatic elastase I (FPE) iibows polelltia1 u a rapid and simple test of psnc:reatic function lhat is not 1nt1ueoca1 by exoaenous eDZ)'IIIel, requires a single small stool sample and shows biJh aeasitMty and spccific:lty wbcn COIIIpiii'CII with the gold standard tests for penc:reatic fim:tiOD. We IJI8iyled a '' Slool sample for FPE in 26 CF cbildren (age 2days - 16 yn) with a ranae of geuotypes and 10 pancreatic suflicient (PS) cbildreD. Specimens were IJI8iyled according to manufacturers instructions (Sc:bcbo Tech, Gmbh, BaluJhofslr 6, D-H·m Wettcoberg, Germany)

CJI STOOL ELAST AS!t utrlo fN >200utrlt!l n 0-~ '0-200 >200 12 12

J-+T I I I I

GS42XIwlk I I G"IDIRIUIX I I

AF50lllwlk s 7 I 2 2

NORMALS 10 10 .. ot 26 CF cbildreo, 23 bad 'VaY low e1asase levels (22 had zero actiVJty). 3 CF children with normal PPE ac:tMty were c:linically PS. 2 oflhis group bad the same paotype. The lint lllbple of -.ium from 2 DeOD&tes (AF~AF~) post 1U1J111Y for IIIIIIXIIIium ileullbowcd zero activity wilh no rise cmr the ocxt lhree weeks. We coaclude that Slool ~ is a simple, reliable, aeasilive and IIIJCCific test for exocrine penc:reatic f'unl:tloo in CF. PPE is not illfluenced by exoaeuous enzyme lldmini&lratioo and is stable at room temperature for a week making baDdlin& easy aud post.qe of samples fessible. The test comc:tly identified PS individulls with UJIUIU8! CF paotypes. The observation of zero activity in De\>bom IIIIIIXIIIium JpeCiJDenl bigbliJbla lhe .-ys' polcntial for IIIMbom screening in CF • a test that can be UlldcnakcD 011 day 1 of Ufe and completed in boun.

396 INCREASED RNA DEGRADAnON IN CYSTIC FIBROSIS (CF). ASSOCIATION WITH DISEASE SEVERITY AND NORMALIZAnON WITH ANTIOXIDANT SUPPLEMENTS. B.M. Wjn~b1 • A. Herl~. G. Khoachaorur', G. Sch6ch, D.H. Shmerllng1

, H. To~. 1Dept. of Pedlatrlcl, Unlv. of Zurich, Switzerland; 2Reaearch lnsl of Child Nutrition, Dortmund, Germany; 'Dept of Surgery, Unlv. of Graz, Auatria.

Whole body RNA degradation rate• are related to metabolic rate, perhaps due to the ef'fec:ta of reactive oxygen apecie1 (ROS) formed u a reault of oxygen conaumptlon. In CF, oxygen conaumptlon and oxidant 1tren are lncreaaed. To atudy the relatlonahlp between RNA degradation and diseaH uverlty (Shwachman acore) and the effect of correcting Impaired vitamin E and jkarot.ne atatus, urinary excretion of modified RNA catabolltea, N'-ttveonlnocarbonyladenoaine (fA) and N2,N2-dlmethylguanoalne (mlG) (from tRNA) and paeudourldlne ('I'; from rRNA & tRNA), was meuured by HPLC and atandard­ized for urinary creatinine, age and HX (z-acore) (A) before and .rter 20().o4()() IU/d vltemln E (n = 17) and (B) before and .rter additional 0.5 mg/kg/d P-carotene (n = 19). BIIYb: RNA cata-

bolite excretion correlated inversely with Shwachman score (e.g., m:lG. r = -0.60, P = 0.006). (A) Plasma a-tocopherol in­creased (9.96±4.12 vs 28.16±6.47 f.11T10VL) and t6A, mlG and 'I' decreased (2.13±1.63 vs 1.23± 0.97; 1.80±0.84 vs 1.23±0.63; 2.04±1.07 vs 1.53±0.98; all P < 0.01). (B) Plasma J3-carotene increased (0.08±0.06 vs 1.21± 1.03 llmoVL) and t6A, m/G and 'I' decreased (1.48±0.94 vs 0.40±1.12; 1.61±0.93 vs 0.78±0.99; 1.71±0.95 vs 0.90±0.98), as did plasma malondialdehyde, a marker of lipid peroxidation (1.01±0.37 vs 0.63±0.13 llmoVL) (all P < 0.001). Normalization of RNA degradation, along with nor­malization of antioxidant status and lipid peroxidation, suggests that ROS are involved in increased RNA catabolism in CF; association with disease status suggests that changes in RNA catabolism may be clinically relevant.

397 Patients with Cystic Flbrosls(CF) benefit from &-Carotene

Supplementation for 6 months S.Renner',C.Wojnarowski',Koller DY',P.Rust',I.Eimadfa2,1.Eichler' 1 University Children·s Hospital, Vienna, Austria 2 Institute of Nutrition, University of Vienna, Austria

Background: Patients with CF have significantly decreased plasma concentrations of nutrient antioxidant vitamins. especially of B­carotene (BC), due to fat malabsorption and chronic pulmonary inflammation. The aim of this doubleblind placebo-controlled study was to investigate the effect of oral B-carotene supplementation for 6 months on clinical and immunological parameters. Methods: 24 CF-patients (20 females) mean age 11.7 years (range

6.7-27.7 years) were randomised to recieve BC (1 mglkg/day­max.50 mg/day, n=13) or placebo (n=11) for 6 months. Multivitamin­. vitamin E- and pancreatic enzyme-supplementation remained unchanged. At monthly follow up visits clinical-,lung function- and infection-parameters were assessed. Total antioxidant capacity (TAC) was measured photometrically. The number of pulmonary exacerbations, requiring antibiotic treatment (in days) was evaluated 3 months prior to and during the study. Bul.!J!L Plasma concentration of BC increased significantly into the normal range within the first 4 weeks in the supplementation group (SG). Malondialdehyde plasma-concentration decreased significantly (p<0,05) after 6 weeks, TAC was improved by 12 % only after 12 weeks. After six months there was an increase of weight (n.s.) and length (p<0.05 ) in both groups, but no significant changes of lung function and lgG-serum concentrations. Antibiotic (AB) treatment­days decreased significantly during the six months in the SG (p<0.05) compared to the three months before, but increased in the placebo group. No adverse events were observed during the study period. Conclusion:Supplementation with BC 1 mg/kg/day normalised plasma concentrations in CF-patients within four weeks and resultet in a significant decrease of pulmonary exacerbations. Our data suggest, that CF-patients may benefit from B-carotene supplementation.

398 PANCREAS CHANGES IN CYSTIC FIBROSIS • Retrospective study of 52 patients eeen in a 7 year period.

J, FEIGELSON, C. ANAGNOSTOPOULOS, y, PECAU, M. PQUET 153, rue de Sauaaure - 75017 PARIS - FRANCE -and J, CARRERE, J,p, CHAZALETTE • H&pita1 Ren6a Sabran 83 - GlENS - FRANCE

Pathologists describing lesions of the pancreas have obar­ved lipomatous degeneration at the end of the disease sin­ce the early stages of the recognition of cr. Recent deve­lopment& in imaging along with molecular biological data have contributed additional element•. Aima of the •tudy was establishing a correlation between morphological findings on imaging and clinical and bioche­mical data. A particular attention was devoted to lipase­mill and genetical classification. 52 cr patients, mala• 28, female• 24, aged 2 to 53 years.

followed up during a 7 year period, were investigated, of these only 2 were pancreatically sufficient. Lipaaemia was assayed at monthly or bimonthly intervale and this was recently supplemented by tripeinogen assays. Ultrasound and C•T scan evaluations were done annually or at 18m in­terval. RESULTS 1 imaging findings led to the recognition of five ~ps 1 Groupl 1 pancreas normal (H : 3), 6 •· Group2 incomplete limopatoeie of the pancreas (H : 12), 21 •· Group3 1 complete lipomatosis (N : 24), 46 •· Group4 • ma­croscopic cysts (N 1 1), 2 •· GroupS 1 atrophic pancreas (H : 13), 2S •• Changes on imaging evolved slowly and were clinically si­lent. Some patients of groupsl and 2, changed into group J. The group4 patients condition remained unchanged. All groupS patients remained atrophic and devoid of lipomatous changes. Pancreatitis wee only observed in groupS patients and occured in 7 outof 13 patients 1 6 of them were hete­rozygous and one homozygous for the F 508 deletion. Atro­phy of the pancreas consecutive to clinical and biochemi­cal findings of pancreatitis was eaen in two patients. our study convinced u• that lipaeemia assays and pancrea­tic imaging should become part of the routine workup of CF patient•·

Lung Defense

399* IKB DEGRADATION IN AIRWAY EPITHELIAL CELL MEDIA TED INFLAMMATION. M A Fiedler, K. Wemke­Dollries, B.A. Chini and J .M. Stark. Division of Pulmonary Medicine, Children's Hospital Research Foundation, Cincinnati, Ohio.

Airway epithelial cells play a key role in inflammation by producing inflammatory mediators (IL-8) and adhesion molecules OCAM-1). A significant number of these mediators, which play a role in CF airway inflammation, arc regulated at the level of gene transcription by the nuclear factor kappa B (NF·KB). Activation ofNF·KB results in increased inflammatory mediator gene transcription. In the control cell, NF-KB is present in an inactive form in the cytoplasm, bound to its inhibitor,IKB. Upon activation of the cell with TNFu, NF-KB relocates to the nucleus, and initiates inflammatory gene transcription. The hypothesis of these studies states that NF-KB activation in airway epithelial cells is dependent on l~tB degradation, and that inhibition of JKB degradation would prevent NF·KB activation and inflammatory mediator production. Treatment of AS49 cells (an airway epithelial cell line) with TNFcx resulted in loss ofJKB from the cytoplasm within 10 minutes, as determined by Western blot analysis. Associated with the loss ofl~tB, was an increased presence of NF·KB in the nuclei of the cells. Deletional analysis of the S' flanking regions of the IL-8 and ICAM-1 genes demonstrated that NF-~tB translocation to the nucleus was critical in transcriptional activation of these inflammatory mediators. Furthermore, inhibition ofh:B degradation using the protease inhibitors L-1, chlor-3-(4-tosylamido)-7 amino-2 heptanon· hydrochlorid (TI.CK) or acetyl Leu-Leu-Norvalinal (ALLN) prevented NF-~tB activation in the cells treated with TNFcx. This data is consistent with the hypothesis that the activation ofNF·KB and transcriptional regulation of a number of inflammatory genes is dependent on l~tB degradation. Future studies aimed at reve~ing.l~tB degradation may offer a means oflimiting airway inflammation m CF. Supported by NIH KOB ID..034Sl-Ol and a CFF Grant FJEDLE97GO.

400* lntcrleukla .. and lnkrleukla·IO Procludioa by Cystic Flbrosl1 Naul Epitbdial Celli After Tumor Neci'Oii1 Fldor-<a and Rnpin~tol')' Syncytial Virv1 Stimulation. Hugh R, Bla<l< MD JUICI R, Yanlwkas. MD, Larry 0. Johnson. MD. and Terry L. Noah. MD. Departments of Pediatrics and Internal Medicine, University of North Carolina at Chapel Hill. Chapel Hill. NC, USA.

Poster Session Abstracts 315

Tbe marked influx of acutrophils iato the lungs or patients with cyllic fibrosis (CF) has led to speculation that CF respiratory epithelium conlributes 10 either overproduction of neutrophil attracting mediators or underproduction of anti·inflammalory mediaton. We therefore measured inlerlcukin-8 (ll.-8) and interlcukin·IO (IL-10) in supemalants of primary cultures of nasal epithelial cells established from young children with CF and in similar cells from non.CF controls. Short tcnn llimulalion with tumor necrosis factor-a (TNF-a) and infection with respiratory syncytial virus (RSV) both induced significanl increases in IL... However, there wu no difference in the production or n.. .. between CF and control cells at any timcpoint. The effect of transfection of CF cells wilh Ad~.CBCFTR, an adenovirus vector expressing CFTR, wu also dclennined. A chloride emux assay demonslrated an increase in forskolin stimulaled chloride transport consistent with expression of functional CFTR in Ad~.CBCFTR lrcated cells. X-gal staining of parallel cuiiUrcs treated with AcB.CMVLacZ at the same multiplicity of infection showed nearly 100% of cells were infected. The Ad~.CBCFTR treated cells had TNF-a stimulated IL .. production that was not different from untrealed or Ad~..CMVLacZ lrealed conlrols. Lastly, immortalized tracheobronchial epithelial cell lines wilh and without functioning CFrR were llimulaled wilh TNF-a. Again. supemalant IL-8 did not differ significantly between cell lines wilh and wilhout functioning CFTR. We conclude that release of IL .. by cullured nasal epilhelial cell• from patients wilh CF, after incubation wilh the non-specific stimuli TNF-a and RSV. appean 10 be unaltered by the absence of functioning CFTR. Stimulation of measurable IL-l 0 release was 1101 observed in primary cell cultures or immortalized cell lines under any condilion. HOWC\'CI', CF cell• transfccted wilh Ad~..CBCFTR released measurable amounts of IL-10, while parallel untransfccted and Ad~..CMV LacZ lransfccted a:ll1 did 1101. Further investigation of the relationship between CFTR and IL-10 expression is wananted.

Supported by grants from the Cystic Fibrosis Foundation.

401* Optimization of adenovirus vector backbone for protection against lysis by adenovirus-specific cytotoxic T lymphocytes. J.M. Kaplan, R.J. Gregory, D. Armentano, A. Scaria, L.A. Woodworth, S.E. Pennington and A.E. Smith. Genzyme Corporation, Framingham, MA, USA

Transience of expression following gene delivery by adenovirus (Ad)-based vectors represents an obstacle in the development of gene therapies for cystic fibrosis and other genetic disorders. Several factors may affect duration of transgene expression including the induction of cytotoxic T lymphocytes (CTI.s) with the potential to lyse Ad· transfected cells. Therefore, the relationship between Ad vector backbone and susceptibility to en. lysis was investigated. Target fibroblasts infected with El"E3~4+ Ad vectors were found to be resistant to lysis by Ad-specific CTI.s !!! ~ although the cells remained susceptible to lysis by CTLs directed against the immunogenic poal transgene product. Elements from both the E3 and E4 regions appear to be required for protection against Ad-specific CTI.s since target cells infected with E3.E4" or E3"E4+ vectors were readily lysed. Testing of E3+ vectors expressing individual E4 open reading frames (ORFs) indicated that targets infected with an E3~40RF4 vector were highly resistant to lysis by Ad-specific CTI.s while the combination of EJ and E40RF617 or E40RF6 provided intermediate or undetectable levels of protection against CTLs, respectively. E4+ vectors expressing the E3 gene product, gp19K, under the control of its endogenous promoter or a more potent CMV promoter, were also evaluated since gp19K is known to inhibit antigen presentation to CTI.s by trapping nascent MHC Class I molecules of certain haplotypcs within the endoplasmic reticulum. Target cells infected with the gpl9K.E4+ vectors, however, only displayed panial resistance to lysis by Ad-specific CTI.s suggesting that gp 19K alone could not account for the full anti-cytolytic effect observed with EJ~+ vectors and that other E3 protein(s), in combination with E4, may also be involved in protection against en. lysis.

316 1997 Cystic Fibrosis Conference

402* ABSENCE OF INTERFERON-GAMMA SHIFTS THE AdS· lacZ FROM A T HELPER TYPE 1 (Thl) TO A Th2-MEDIATED IMMUNE RESPONSE. F.W. van Ginkeii, D.W. PascuaJ4, E.J. Sorscher2, J.M. Wilson3, H. K1yonol,and J.R. McGhee I. 1-Department of Microbiology and Oral Biology and 2· The Cystic Fibrosis Research Center, University of Alabama at Birmingham at Birmingham, AL, USA. 3-Wistar Institute, University of Pennsylvania, Philadelphia, PA, USA. 4-Veterinary Molecular Biology, Montana State University, Bozeman, MT. USA.

Intracellular pathogens such as wildtype adenovirus type S (AdS), induce T helper type I (Thl)-mediated immune responses. Like wildtype AdS, the EIIE3/E2a deleted AdS vector displayed an attenuated Thl· mediated CD4+ T cell response to the vector, while an EIIE3 deleted replication-deficient AdS vector was characterized by a mixed Thlffh2 response. The immune response to the AdS expressed transgene, ~­galactosidase (~-gal), was Th !-dominated regardless of the vector used. Based on these observations the importance of IFN-y for the AdS vector and ~-gal-specific immune responses were determined following intratracheal administration of AdS-IacZ to IFN-y deficient and normal BALB/c mice. Cytokine analysis of immune CD4+ T ceJis stimulated with heat killed AdS or recombinant Jl-gal demonstrated an increase in Th2 cytokines in cultures derived from IFN-yl· mice, when compared to those derived from normal mice based on the frequency of cytokine secreting cells as determined by the ELISPOT assay and cytokine levels in the culture supernatant as determined by ELISA. The antibody profile to both AdS and Jl-gal demonstrated an associated increase of IgG I and decrease of IgG2a and IgG3 antibodies. The lgA antibody levels were not affected, while a slight decrease of IgG antibody levels was noted in especially the Th !-mediated jl-gal response induced by the E 1~3 deleted AdS-lacZ vector. Ongoing studies with the EIIE3/E2a vector w~ll further establish the importance of IFN-y for immune responses to ~h1s vec~o~. This study demonstrates the propensity of ~-y ~nockout m1~e to.ehclt Th2-type immune responses to AdS-IacZ and md1cates alterations m the observed IgG-subclass distribution obser~ed in. norm~! m1ce.. Th~s, blocking IFN-y should decrease cell med1ated 1mmumty. wh1ch ~Ill benefit AdS-mediated transgene expressiOn and decrease mflammauon. (supported by the Cystic Fibrosis Foundation pilot study 464, AI 65298, and AI 40288).

403 ADENOVIRUS MEDIATED TRANSFER OF FCyRIIA RECEP­TOR eDNA TO RESPIRATORY EPITHELIAL CELLS. f. ~ M-K Kim, S Worgall, J-G Park, A Schreiber, I Kovesdi, RG Crystal. The New York Hospital-Cornell Medical Center, New York, NY, University of Pennsylvania School of Medicine, Philadel· phia, PA, GenVec, Inc., Bethesda, MD, and CorBec Pharmaceuticals, Inc., Horsham, PA Colonization of the respiratory system by bacteria (Pseudomonas

aeruginosa, Staphylococcus aureus) in patients with cystic fibrosis may be associated with abnormal innate immune mechanisms. One form of innate immunity - internalization of bacteria by bronchial epi­thelial cells with subsequent sloughing of cells and removal of bacte­ria - plays a role in the maintenance of the sterile environment in the airways and is defective in cystic fibrosis. The present study investi­gates the feasibility of turning non-phagocytic pulmonary epithelial cells into phagocytic cells by using adenovirus (Ad) vector-mediated gene transfer of the phagocytic receptor (FcyRIIA) eDNA. Following infection with an Ad vector containing the FcyRIIA gene, A549 cells, a human respiratory epithelial cell line, showed binding (21444 :1: 1781 dpm; compared to 1499:1:212 of control) and phagocytosis (2921 :1: 465 dpm; compared to 41 5 :1: dpm of control; p<0.02, all comparisons) of !gO coated ' 1Cr-labeled sheep red blood cells. Fol­lowing infection with an Ad.FcyRIIA mutant, in which 2 of 3 cyto­plasmic tyrosines have been replaced with phenylalanine, we ob­served binding (16693 :1:939 dpm; compared to 1499:1:212 of con­trol) and not phagocytosis (415 :1:775 dpm; compared to 21444:1: 1781 of Ad.FcyRIIA infection). This observation suggests that In vi­tro Ad-mediated FcyRIIA receptor eDNA transfer to the pulmonary epithelial cells is feasible and F cy RIIA receptor conveys to these nor­mally non-phagocytic cells the ability to bind and phagocytize opso-

nized cells. This strategy has a potential for in vivo application to uti­lize nonphagocytic cells to clear bacterial infections and prevent colonization of airways by bacteria in patients with cystic fibrosis.

404 HIGH EXTRACELLULAR CHLORIDE CONCENTRATION PROMOTES APOPTOSIS AND LYSIS OF NEUTROPHILS FROM CYSTIC FIBROSIS PATIENTS AND CONTROL SUBJECTS. AM Jager1, J. Wu1, A. Lapey2, and M.W. Vermeulen I. 1 Pulmonary and Crtical C8re Unl and 2Pediatric Pulmonary Unk and Cystic Fibrosis Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.

Loss o1 cystic fl:llo8is tranemembrane regulator (CFTR) function causes detective ton transport by cystic fibrosis (CF} respiratory epithelial cells, lkely resulting In abnormaiMies In the Ionic COIJ1)0Sitlon of the airway surface lluld (AS F) covering these eels. ASF secreted by C F resplratOI)' epllheUa has been noted to have a higher chloride (CI·) concentration than ASF secreted by control epfthella. Respiratory epithelia recently have been demonstrated to secrete a jklefensln whose antl-rnlclobial activity Is Inactivated by the abnormally high salt content lound In CF ASF. Thls may force a greater reuance on recruited neutrophHs (PMN) to defend against lntectlon In the lungs ol CF patients. However, abnormal ASF Ionic composition also may Impair PMN function, further predisposing CF patients to pul1110f1111Y Infections. The IHespan ol recruited PMN Is limited. PMN are preprogrammed to undergo apoptosls, associated with a loss of functional ability. Stimuli promoting PMN apoptosls therefore may Impair their ability to cteer Infection. We therefore determined apoptosls, and subsequent lysis, of PMN Isolated from CF patients (n • 5) and heaHhy control subjects (n • 6), when cultured In media of Increasing Cl· concentration (108, 135, and 170 mM). PMN apoptosls was assessed by Wrtghi-Giernsa staining. Lysis was determined by the decrement In trypan blue-excluding cells. For PMN from CF patients, the percent ol PMN that were apoptotlc alter 18 hours In culture Increased lrom 48.5;~:7.0% In media wfth 108 mM Ct. to49.1;1:9.7% In 135 mM Ct. and to n.2;1:5.6%'1n 170 mM Ct. The percent of PMN that had lysed after 48 hours increased from 50.5;~:4.5% In 108 mM Cl· to 59.2;1:9.8% In 135 mM Cl·, and to 81.1;1:3.9%• In 170 mM Ct (•p < 0.05, 170 mM vs. 108 mM Ct, ANOVA, tor both apoptosls and lysis). The percent ol PMN lrom non-CF controls that were apoptotlc alter 16 hour& Increased trom 74.3±4.4% In 108 mM Cl·, to 79.0,t4.9% In 135 mM Cl·, and to 89.1;~:3.7%• In 170 mM Ct. The percent o1 PMN that had lysed alter 48 hours Increased lrom 87.3;1:3.9% In 108 mM Ct,to 78.7;~:3.5% In 135 mM Ct, and to 91.1.t.1.7% In 170 mM c1· (•p < 0.05, 170 mM vs. 108 mM Cl·, ANOVA lor both apoptosls and lyalll). PMN apoptosla thus was accelerated by high Ct concentration, causing lewer PMN to survive at 48 hour&, both lor PMN lrom CF patients and lrom controls. Thls acceleration of PMN apoptosis by high Ct concen­tration could contrbJie to impairment ol host defense In CF airways.

405 GENE TRANSFER TO ALVEOLAR MACROPHAGES . .S. ~ R. Singh, N. Topf, P. Leopold, R.J. Kaner, R.G. Crystal. The New York Hospital-Cornell Medical Center, New York, NY.

Alveolar macrophages play an important role in pulmonary host defense. In the context that genetic modification of Alveolar macrophages could be one strategy to strengthen pulmonary host defense in cystic fibrosis, murine Alveolar macrophages were infected In vitro with an adenovirus (Ad) vector coding for green fluorescent protein (AdGFP). Although higher doses of Ad vector were needed to efficiently transduce a significant number of cells compared to the A549 respiratory epithelial cell line, expression of GFP persisted In vitro for more than 2 wk and the viability did not differ from uninfected cells over that period. At an infection dose of 200 MOl 604/o of the cells expressed GFP 48 hr postinfection as evaluated by flow cytometry. In vitro infection of murine Alveolar macrophages with an Ad vector coding for murine granulocyte­macrophage colony-stimulating factor (Ad mGMCSF) led to GMCSF production (13 :1:39 ng/10' cells-24 hr) In vitro and to Alveolar macrophages proliferation (32l.t.34% in 4 days). To analyze if Ad modified Alveolar macrophages will survive In vivo, murine Alveolar macrophages were infected In vitro with Ad GFP (200 MOl) and 2x!O'cells were then transferred to lungs of mice via the intratracheal route. After 3 and IS days the lungs were lavaged and the recovered cells evaluated for GFP expression. At 3 days 2.0:0.6 % of the recovered cells were GFP positive compared

to 1.6±0.7% cells at day IS. Thus, gene transfer to Alveolar macrophages using Ad vectors is feasible and that the modified Alveolar macrophages can survive in vivo. This approach could have potential applications for developing strategies to increase pulmonary host defense.

406 A SERUM FACTOR INDUCES INTERLEUKIN-8 SECRETION FROM CF NASAL EPITHELIAL CELLS AND A CF NASAL EPITHELIAL CELL LINE. M L. Aitken, T.R. Hinds, M.V. Pier, T.R. Martin, C.L. Halbert, R.B. Goodman and S.J. Skerrett. Departments of Medicine and Pharmacology, University of Washington, and the Fred Hutchinson Cancer Research Center, Seattle, W A.

The role of epithelial cells in regulating CF airway inflammation is incompletely defined. We previously reported that serum induces IL-8 release from airway epithelial cells in a dose dependent manner with maximum effect at 20% serum and minimum at 0.1% serum (AJRCCM 1996 A854). IL-8 releasing activity was maintained after multiple freeze-thaws, heating to 6SOC for 30 min, and was partially reduced with exposure to pH extremes. We now report further characterization of this serum­derived IL-8 releasing factor (1L-8RF). CF airway epithelial cells were isolated as previously described (Human Gene Therapy: 1996: 7:1871) and a CF nasal epithelial cell line (CFI-16) were used as the target to assay IL-SRF. The cells were cultured in keratinocyte serum-free medium (KSFM) with epidermal growth factor and bovine pituitary extract until confluent. The monolayers were washed, counted, refed with KSFM without growth factors. Ion exchange chromatography on DEAE cellulose suggests that the protein is neutral or positively charged at pH 7.5. The molecular weight of IL-8RF was estimated by gel filtration to be 135kD using Sephacryl 200-HR. Taken together with SDS and Gradient gel electrophoresis suggests that the protein may be composed of more than one subunit. Identification of this serum-derived IL-8RF provides a mechanism whereby serum exudation can cause mflammation in the airway.

Supported by HL 50052 and GM37696

407 NITRIC OXIDE-DEPENDENT INHIBITION OF HETEROLOGOUS CFTR EXPRESSION. I..1i.l.l.in&. I. Y .. Haddad, Eric J. Sorscher, •seng H. Cheng and S. Matalon, Gregory Fleming James CP Research Center, Departments of Physiology & Biophysics, Pediatrics and Anesthesiology, UAB, Birmingham, AL, 35294 *Genzyme Corporation, Framingham, MA 01701-9322 .

In response to various inflammatory stimuli, mucosa-associated immune cells and epithelial cells produce nitric oxide ("NO). and reactive oxygen species. It has been shown that the production of "NO can adversely affect the function and expression of ion channels and the heterologous expression of reporter molecules. Since the CF lung is in the state of chronic inflammation, and various gene therapy approaches may further inc~ease airway inflammation, the goal of our studies was to charactenze the effect of "NO on heterologous CFfR expression in an in vitro model system. When LLCPK1 cells stably transduced with wtCFTR (LLCPKwtCFTR) were exposed to "NO produced by 3T3 cells stably transduced with the inducible nitnc oxide synthase, the production of "NO correlated with a significant decrease of CffR protein expression in LLCPKt cells. To provide more d1rect evidence that "NO inhibits heterologous CFTR expression, LLCPKwtCFTR cells were treated with a slow release "NO donor, diethylenetriamine (DETA). In the presence of 12S~tM DETA CFTR expression was reduced in LLCPKwtCFTR cells by 64.08± 5.24% (mean±S.E.M.; n=4). Freshly isolated human red blood cells (i.e., "NO scavengers) abrogated the inhibitory effects of DET ~ on CFTR protein expression up to JmM DETA concentration, indicating that the effects of DETA were due to "NO generation. The "NO-dependent inhibition of CFTR protein expression was not a general cytotoxic effect, since in the presence of 12511M DET A there was no significant increase in LDH release from LLCPKwtCFTR cells (control: 3.50±0.08%, J2S~tM DETA:

Poster Session Abstracts 317

3.77±0.33%; mean±S.E.M.; n=6, percent release of total cellular LDH). These findin~ indicate that "NO can inhibit heterologous CFTR protein expression in vitro. To investigate whether "NO­dependent inhibition of heterologous CFTR expression exists in vivo and to delineate the mechanism of inhibition might aid the design of more efficient gene therapy protocols.

408

ACTIVATION OF p38-.ot IN MACROPHAGES BY SODIUM CHLORIDE. G S Kerby, E. D. Chan, and D. W. H. Riches, Dept. of Pediatrics, National Jewish Medical and Research Center, Denver, CO, USA.

Airway disease in cystic fibrosis (CF) is dominated by inflammation and elevation of proinflarnmatory cytokines including interleukin-8 (IL-8) and tumor necrosis factor • a (TNFa). Recent studies of airway surface fluid (ASF) composition in CF have demonstrated an elevation of sodium and chloride compared to normal or disease controls. Hyperosmolarity has been shown to induce a stress signal in the ceU causing the release of proinflarnmatory mediators. Increased osmolarity and tonicity lead to macrophage signal transduction via the mitogen­activated protein kinases (MAPKs). The p38_. member of the MAPK family has been shown to be activated in osmotically shocked cells, and when this pathway is blocked by a specific p38- inhibitor, IL-8 synthesis is inhibited. We hypothesize that exposure of macrophages to elevated sodium chloride (NaCJ) concentrations will activate the p38-pathway. Mouse bone marrow derived macrophages were exposed to described conditions and lysed. p38- was immunoprecipitated, and an in vitro kinase assay was performed using A TF-2 as a substrate in the presence of [32P) y-ATP. The reaction mixture was separated by 50S­PAGE and transferred onto a nitrocellulose membrane. Phosphorylated proteins were visualized by autoradiography and quantified by phosphoimaging. Our results demonstrate an increased signal via the p38- pathway after exposure to increasing concentrations (0 • 200 mM) of NaCl for 10 minutes, with the activity at 200 mM NaCI appearing equal to 1 nglml of TNFtt. We conclude that the p38_,.. pathway is activated in macrophages exposed to increasing concentrations of NaCI. Thus, the p38- pathway may play a role in mediating proinflarnmatory cytokine production in the CF airway.

This abstract is supported by grants from the Cystic Fibrosis Foundation, NIH SCOR HL 56556 ond ROJ HL 55549, and National Jewish Medical and Research Center CJC.

409 ANALYSIS OF THE PULMONARY INFLAMMATORY PHENOTYPE IN THE CF MUTANT MOUSE. G MorrjsM J. Dorin, D.Porteous, K. Crook. MRC Human Genetics Unit, Western General Hospital. Edinburgh.

85% of deaths In adult CF patients are caused by lung disease foUowlng a sustained inflammatory response to a P"rsistent pulmonary Infection. However, even before there is any evidence of lung Inflammation due to Infection, the profile of pro-Inflammatory eytoklnes In the CF lung differs from that of the nonnal lung. This raises the question of whether bacterial pathogens cause the excessive local inflammation or if they simply aggravate an underlying defect In the host defence system resulting from the CFrR mutation. The aim of this study was to attempt to answer this by analysing the pulmonary inflammatory phenotyP" of the Edinburgh CF mutant mouse (cftr"""'"l under specific pathogen free (SPF) and conventional housing conditions and also foUowlng an Inflammatory stimulus. Bronchoalveolar lavage fluid was assayed for relevant Jnllammatory markers Including the cytokines TNF-a and IL-1P and the chemoattractants KC and CP-10. Immunohistochemistry was used to study the expression of the ceU adhesion molecules JCAM-1 and E­selectin. TNF-a levels were found to be significantly different between +I+ mice and cftr•'""" mice In the conventional housing conditions in the absence of any detectable pulmonary infection. Conversely, levels of TNF-« were not significantly different In SPF housed mice or foUowing Intratracheal instillation of LPS. These results lead us to propose that the pulmonary inflammatory cells of the cftr"',.." mouse function normaUy when given a direct inflammatory stimulus and that another aspect of the host defence system. such as a defensin molecule, Is not functioning adequately to destroy low level non­specific bacteria. A human airway defensln molecule, hPd 1, has

318 1997 Cystic Fibrosis Conference

recently been described whose bactericidal properties are inactivated by high salt concentrations as found in the airway surface fluid of CF patients. We have identified a homologue to h~dl which is expressed in murine airways and whose expression is upregulated in response to LPS. Results from the examination of the expression pattern of the molecule, as well as from functional analyses to determine if its bactericidal potential in the airways differs between +I+ and cftr-'HCU mice will also be presented. This work is supported by Glaxo Wellcome.

410 ABNORMAL INFLAMMATORY RESPONSE TO INFECTION IN CYSTIC FIBROSIS M. Mub!ebach-Sooner. T. L. Noah, M. W. Leigh. Dept. of Pediatrics, The University of North Carolina, Chapel Hill, NC, USA

Infection and inflammation are c:haracteristic:s of pulmonary disease in cystic: fibrosis (CF). Several reports showed early and severe pulmonary inflammation in infants with CF and increased inflammation compared to infected control patients. Infection in CF may differ from normals in terms of specific: pathogens and numbers of bacteria. To further evaluate the relationship between infection and inflammation in CF, we studied bronchoalveolar lavage fluids (BALF) of infants and children who needed bronchoscopy for clinical indications. Children were excluded from the study if they had received anti-inflammatory medications within the two months preceding bronchoscopy. Total cells were counted in an aliquot of the native lavage fluid and differential cell count was determined on 200 cells after staining with Wright stain. Quantitative bacterial cultures were performed in the hospital microbiology lab. Immunoreactive interleukin 8 (IL-8) was measured in the cell free supernatant with a c:ommen:ially available ELISA kit. 53 CF and S I non-CF patients were included of which 28 and 25, respectively, had ~ SO,OOO c:fu bacterial pathogens per ml BALF. Mean numbers of polymorphonuclear neutrophils (PMN) and IL-8 levels were significantly higher in the CF than non-CF patients (p<O.OOI). Calculation of linear regression for inflammatory markers as a function of bacterial colony counts yielded significantly greater slopes for CF compared to non-CF BALF. Patients with P. aen~gfntna tended to be older but did not have more severe inflammation than CF patients without this pathogen. As P. aen~ginosa is rarely seen in non-CF patients a comparison cannot be made for this organism. H. injluenzae was isolated in 12 CF and II control patients and was the preclomin10t pathogen in S and 7 children, respectively. Comparing patients infected with H. injluenzae showed that the CF group had higher PMN and IL-8 values than the non-CF group. Relating these markers to bacterial counts showed a trend for more inflammation per colony forming unit of H. lnjluenzae in the CF group. We conclude that the inflammatory respo1110 is increased in CF even after controlling for specific bacterial pathogen and for bacterial quantity.

This study was supported by the James Aycock Endowment Fund.

411 INCREASE IN FREE IL-8 CONCENTRATION AND THE CHEMOTACTIC RESPONSE OF NEUTROPHILS TO DNASE TREATED CYSTIC FIBROSIS SPUTUM. .l!..fiW. J.O. Warner•, J.K. Shute, University Medicine and *Department of Child Heahh, University of Southampton, Southampton, UK.

The viscosity of sputum from patients with cystic fibrosis (Cf) is largely due to the high concentration of DNA (3·1 S mlfg) derived from necrosing neutrophils(PMN). The major PMN c:hemoattrac:tant in CF sputum is interleukin-8 (IL-8), a basic peptide which has an atrmity for polyanionic molecules including heparin. In this study we investigated the binding of IL-8 to DNA and the in vitro effect of DNase on levels of free IL-8 in CF sputum. We also measured the chemotactic response of PMN to CF sputum, comparing DNase with non-DNase treated samples. Human plac:ental DNA (S mlfml) was incubated overnight at 37"C with hr IL-8 (SO 11s'ml). The mixture was separated by heparin affinity chromatography and IL-8 detected by immunoblot analysis. Sputa from CF patients (n•l 0) were treated with DNase I (I 00 jiglml) or buffer control for 4 hat 20"C,IDd centrifuged at 20,000 g for 20 min. The soluble phase was assayed for free and total IL-8 by spec:ifK: ELISA. This phase was then assayed for neutrophil chemotaxis using modified Boyden chambers and the results were expressed as migrated PMN per high power field (hpf). Immunoblot analysis revealed that IL-8 was bound by DNA, which prevented the IL-8 from binding to a heparin affinity column. In untreated

sputum, free IL-8 (1.44 (0.24·6.04) ng/g) accounts for only 2.94% of the total ( 48.88 (0.034-392.839) nglg). DNase treatment resulted in a significant (p=0.002) increase in free IL-8 concentration (I 0.1 (0.27· 16.68) nglg) with no significant effect on totalll-8 (41.04 (0.012-383.763) nglg). There was a significant increase (p = 0.05) in the PMN chemotactic response of DNase treated CF sputum (33.5 (9.2·52.2) PMN per hpf) compared with untreated samples (19.6 (7.8-44.8) PMN per hpf). These results may indicate a potential risk of increased inflammation during rh DNase therapy in CF, and we are currently investigating the effect of DNase in vivo.

412 Paeudomonaa aarug/noae-lnducad cytoklne production by ra1plratory epithelial calli 11 a calcium-dependent procau. A J Ratner, E. DiMango, B. Bryan, and A. Prince. Columbia University, New York, NY.

The respiratory epithelial cell Is an active participant In defense against P. aeruglnosa pulmonary Infection, producing the prolnflammatory cytokine IL-8 following ligation of the glycolipid receptor aslaloGM1 by adherent P. aeruginosa. We explored the role of calcium In this signal transduction pathway, as well as the potential for pharmacological manipulation of this process. P. aerug/nosa PA01 Induction of epithelial IL·8 expression by 1HAEo· cells, an SV40-immortalized human airway epithelial cell line, was found to be calcium-dependent. IL-8 production was completely suppressed In the presence of 1 mM TMB-8, a chelator of lntracallular Ca2•. Incubation of the cells with 1511M thapslgargin, which stimulates release of calcium from the endoplasmic reticulum by blocking the residant Ca2•.ATPaea, resulted In an amplification of the Pseudomonas-Induced IL·8 response. Verapamll, which blocks Import of extracellular calcium Ions, minimally inhibited IL·8 expression, whereas chelation of extracellular Ca2• with EGTA, which may cause release of Intracellular stores, potentiated the IL·8 response Induced by Pseudomonas. The generation of Intracellular calcium flux In response to PA exposure was confirmed by single-cell Imaging using epithelial cella loaded with lura-2/AM, a ratlomatrlc fluorescent dye responsive to changes In calcium ion concentration. Addition of PA01 to the epithelial cells triggered a rapid Increase In Intracellular calcium detected within 30 seconds of bacterial contact and maintained for several minutes before return to basal conditions. Theae results demonstrate that Ca2• Is Important In the Inflammatory response to Pseudomonas and suggest new targets lor possible therapeutic Intervention In modulating the exaggerated response to P. aeruglnosa Infection which typifies CF airway disease. Supported by CFF and NIH.

413 CMT -3, A CHEMICALLY MODIFIED TETRACYCLINE, INHIBITS ELASTASE ACTIVITY AND EXTRACELLULAR MATRIX (ECM) LYTIC ACTIVITY IN CYSTIC FIBROSIS (CF) SPUTUM. CL..Ban. M DeLemos, L Restrepo, EJ Roemer, and SR Simon. Depts. of Pediatrics and Biochemistry, State Univ. of New York at Stony Brook, Stony Brook, NY, USA .

Human neutrophil elastase (HNE) activity is present in great abundance In CF sputum, and is thought to play a key role in mediating lung damage in CF. CMT-3 is devoid of anti­microbial activity, but can Inhibit HNE and other proteases in vitro. In view of the potential Involvement of HNE in the pathogenesis of CF lung disease, we studied the effect of CMT-3 on HNE activity and ECM lytle activity in CF sputum. HNE activity was measured by chromogenic cleavage of MeO­Suc-Aia-Pro-Val-pNa. Lysis of ECM was assessed by incubating CF sputum with interstitial ECM laid down by a rat muscle cell line, R22. CMT-3 inhibited HNE activity in CF sputum in a dose-dependent fashion. At 100 11M CMT-3, sputum HNE activity was inhibited by 50% and 20% In the presence of 150 11M and 600 11M substrate respectively, suggesting that CMT-3 acts competitively. CMT-3 also protected ECM from degradation by CF sputum. Degradation

of both H' and s• labeled proteins was inhibited, suggesting that CMT-3 protects against lysis of both collagen and proteoglycans. In summary, our results demonstrate that CMT-3 is an inhibitor of CF sputum HNE activity and total proteolytic activity. The concentrations of CMT-3 used in this study are potentially achievable by oral administration. Based on our results, further efforts should be made to elucidate the mechanism of CMT-3's inhibitory effects and to evaluate the potential therapeutic role of CMT-3 in CF lung disease.

Supported by NIDR-D£10985, CF Foundation, and Collagenex Corp.

414 THE NUMBER, COMPOSITION AND FUNCTION OF INFLAMMATORY CELLS IN BRONCHOALVEOLAR LAVAGE FOLLOWING INTRATRACHEAL INFECTION WITH MUCOID PSEUDOMONAS AERUGINOSA (PA) DIFFERS IN RESISTANT AND SUSCEPTIBLE MICE. K. Sapru. P. Stotland, M.M. Stevenson. Center for the Study of Host Resistance, Montreal General Hospital Research Institute, Montreal, QC,CANADA. .

Patients with cystic fibrosis show a remarkable heterogeneity In the severity of PA-induced chronic bronchopulmonary disease. In the present study, we Investigated some of the possible mechanisms underlying this difference. Following Intratracheal Inoculation of bacteria impregnated agar beads, susceptible C51BU6 (B/6) mice showed sigmficantly higher cell numben, primarily polymorphonuclear cells, In bronchoalveolar lavage fluid suggesting an Inflammatory response In the lungs 7 days post Infection (p.l.) when compared to reststant BAUVc mice. However, the propon10n of macrophages was significantly higher in the BALIVc mice suggesting that these cells may play an imponant role In defense against chronic PA Infection. Determination of In vitro production of nitric oxide (NO) and tumor necrosis factor (TNF·a) by bronchoalveolar macrophages following stimulation with LPS or heat-killed PA was carried out at I week intervals for 4 weeks p.l. Both strains showed a decreasing trend in the production of NO with progression of infection. However, macrophages from resistant BALIVc mice showed a significantly higher response to LPS and PA at 7 days p.l. suggesting that, In these hosts, bronchoalveolar macrophages are Immunologically activated and may have a protective role in specific Immunity to PA Infection. Macrophages from susceptible IV6 mice showed higher NO production compared to BALB/c mice at all time points with high spontaneous production of NO at 7 days and again at 21 days p.l .. Thus, these hosiS appear to develop chronic inflammation which may play a role in disease progression. Allhouj!h TNF·a levels were stgnilicantly higher following stimulation m both strnin!z macrophages from BALIVc mice showed 3-fold higher levels ofTNr-a at 7 days p.i. compared to IV6 mice which showed 4·fold higher levels at 28 days p.l. These findings suggest that high TN F-a levels early In the course of the disease may be protective while high levels later on may contribute to disease

r.rogression. Taken together, these observations suggest that differenca n bronchoalveolar macrophage production of NO and TNF·a may

contribute to the heterogeneity In severity of bronchopulmonary disease among patients with cystic fibrosis. (Supported by the CCFF).

415 PRIMARY AND IMMORTALIZED CF AIRWAY EPITHELIAL CELLS EXPRESS A SELECTIN LIGAND. P.J. Park 1, B. Leeflang2

, J.F.G.VIiegenthart 2, A.D.

Rhim 1, IF, Scanlin 1 and M.C. Glick 1

• 1Departrnent of Pediatrics, University of Pennsylvania School of Medicine and Cystic Fibrosis Center, The Children's Hospital of Philadelphia, Philadelphia. PA, USA 2Departrnent of Bio-Organic Chemistry, University of Utrecht, Utrecht, The Netherlands

Specific ligands are expressed on cells for a family of selectins which are involved in multiple functions including the recruitment of leucocytes in the inflammatory proces.s (I). The selectin reacts in a calctum dependent way wttli tts hgand through the ammo tenninal carbohydrate recognition domain showing homology of C-type lectins. The main characteristic of selectin ligands IS the presence of fucosyl residues linked al,3 to an antennary N-acetyl glucosamine on the oligosaccharide of a glycoprotein which may or may not be sialylated . Immortalized CF airway cells (CFff43) and primary cells from CF tracheal ttssue were shown to contatn these spectfic fucosyl residues on membrane glyc?peptides. Almond al,J/4fucostdao;e releao;ed 27% of the [·'H]fucose residues from the membrane glvcoi?Cptides of CFit43 cells.

Poster Session Abstracts 319

These findings were verified 6y high resolution 1H-NMR spectroscopy which also gave information on the sialylation of the membrane glycopeptides. In other studies, an initial biochemical comparison of the sialylation of glycopeptides revealed less sialylation of glyco_lleptides from CFff43 cells when compared with those of non CF airway epithelial cells or CF cells corrected Wtt~ wt Crz-R. !hus the CF phenotype of alte_red glycosylation as revtewed m (2) ts expressed on a1rway eptthehal celfs. In addition, a ligand .for a sel~IJn ~s present on the CF airway cells. The fact that the tmrnortahzed atrway cells as well as the primary airway cells and skin fibroblasts (3) express this ligand suggests that ai.Jfucosyltransferase i~ expressed C!Jnstituttvely in CF rather than. transtently ~ an mllamrnatton tnduced response. It is posstble that the tnflammatory cascade which begms during the neonatal period in the CF .lung is initiated by this m~hanism and therefo"? may be allevtated by carbohydrate-based selectin antagorusts. Supported by CFF Student Traineeships (P.J.P. and A.D.R)

l Lowe, 1. B. and Ward, P.A. (1997) 1. Clin. Invest. 5, 822·826. 2 Lazatin,1. 0. et al. (1994) Glycosylation and Disease I, 263·270. 3 Wang, Y. M. et al. (1990) Clin. Cbim. Acta 188, 193-210.

416

DIFFERENTIAL EXPRESSION OF ADHESION MOLECULES AND CHEMOKINES BY CF AND NON-CF AIRWAY EPITHELIAL CELLS. Lisa M. Schwiebert, Robert P. Schleimer*, John Engelhardt+, and Sherry Sterbinsky*. Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL, USA, *Department of Medicine, Johns Hopkins University, Baltimore, MD, USA, and +Institute for Human Gene Therapy, University of Pennsylvania, Philadelphia. PA, USA

A hallmark of airway inflammation observed in cystic fibrosis (CF) is a massive neutrophil influx into the lungs. Airway epithelial cells express a variety of pro-inflammatory mediators that trigger the migration of leukocytes, including neutrophils and eosinophils, into the airway. The obj~tive of this study is to examine the expression of pro-inflammatory mediators that facilitate the recruitment and transmigration of neutrophils, including ICAM-1 and IL-8, and cosinophils, such as VCAM-1 and RANTES, in CF and non-CF primary airway epithelial cells (n=2 pairs), cell lines (n=4 pairs), and CFIR-corrected cell lines (n=l pair). ICAM-1 and VCAM-1 protein expression was monitored via flow cytometry while IL-8 and RANTES protein expression was measured via ELISA; mRNA expression of each of these molecules was determined through Northern blot analysis. Preliminary data demonstrate that basal protein and mRNA expression ofiCAM-1 are increased 3-4 fold in CF airway epithelia, as compared to non-CF epithelia. Furthermore, these data show that the protein and mRNA expression of IL-8 are increased by 2.5-7.5 fold in CF airway epithelia stimulated with TNFa when compared with similarly treated non-CF controls. Additional findings demonstrate that CF airway epithelial cells, when stimulated with TNFa, express 2-4 fold less VCAM-1 mRNA and protein and 21-28 fold less RANTES mRNA and protein than their non-CF controls. Together, these data suggest that CF airway epithelium is biased in its expression of pro-inflammatory mediators that facilitate the migration of neutrophils into the lung. (Supported by the CFF)

417

PHARMACOKINETICS OF IBUPROFEN SUSPENSION IN YOUNG CHILDREN WITH CYSTIC FIBROSIS. C. Scott. G. Reisch-Bogan, R. Kustra, B. Johnson. 1. Dong, P. Smith. Schools of Phannacy and Medicine, The UniveiSity of North Carolina at Chapel Hill, Chapel Hill, NC.

Antiinflammatory therapy with ibuprofen slows the progression of lung disease in children with cystic fibrosis (CF). Initiation of chronic, high dose ibuprofen therapy requires therapeutic drug monitoring to verify that adequate peak plasma concentrations of SO - I 00 j.ig/ml are attained. The objectives of this study were to evaluate ibuprofen suspension phannacokinetics and determine optimal blood sampling times that reflect peak concentrations in young children \\ith CF. Subjects were admitted to the clinical research unit after an overnight fast and given one 20 mg/kg oral dose of ibuprofen suspension. Blood samples were

320 1997 Cystic Fibrosis Conference

obtained at 0, IS, 30, 45, 60, 120, 240 and 360 minutes. Samples were promptly centrifuged and separated; the plasma was stored at • 70"C until analyzed by high performance liquid chromatography. Peak concentration (C-), time of peak concentration (T ...,J, area under the concentration-time curve from time zero to infinity (AUC), and tcnninal half-life (T,n) were estimated from concentrations obtained using noncompartmental analysis (WinNONLIN." v 1.1). Apparent clearance (CLIF) and apparent volume of distribution (Vd,IF) were calculated and normalized to body weight. C,_ and AUC were normalized for dose and body weight ( mglkg) to compare with literature values published for ibuprofen tablets. Fourteen patients (mean age 5.2 years; range 3.1-8.5 years) were studied and received a mean dose of 20. I mglkg. Results (mean± sd) arc as follows: c_ 84.1 ~ 23.2 ~glml: c_t(mg/kg) 4.2 ± I 1: T_ 40 8 ± 23.4 min: AUC 9.8 ± 3.1 mg•minlml: AUC/(mg/kg) O.S ± O.IS; T112 84 ± 30 min; CU(F•kg) 2.2 ± O.S mV(min•kg); and Vd,/(F•kg) 280 ± 130 mllkg. AUC/(mglkg), T,12, CU(F•kg) and Vd,/(F•kg) are similar to literature values reported for ibuprofen tablets in older patients. T,.., is shorter in comparison with tablets (40.8 vs. 66.0 and 93.8 min). C_t(mglkg) is the same for suspension and tablets, which is unexpected. Based on these data, ibuprofen suspension peak concentrations occur earlier, necessitating blood sampling at 30 and 45 minutes, in addition to the usual 60 nunute blood sampling for tablets.

Supported in parts by the University of North Carolina General Clinical Research Center (NIH RR00046), the Pharmacy Foundation of North Carolina and the Boomer Esiason Heroes Foundation.

418 INCREASE IN FREE IL-8 CONCENTRATION AND THE CHEMOTACTIC RESPONSE OF NEUTROPHILS TO GELSOLIN TREATED CYSTIC FIBROSIS SPUTUM. ~. B Perks, J.O. Warner• University Medicine and •Child Health, University of Southampton, Southampton, UK.

The abnormally high viscosity of sputum from patients with cystic fibrosis (CF) is attributed to the release of cellular components from necrosing neutrophils (PMN) in the airways. One component, actin, has been shown to contribute to this viscosity by forming long protease-resistant filaments which are highly viscoelastic. It has been suggested that gelsolin, a protein that severs actin filaments, may have therapeutic potential as a mucolytic agent in CF patients. IL-8, the major PMN chemoattractant in CF sputum, is a basic peptide which has an affinity for polyanionic molecules including heparin. This in vitro study investigates the binding of IL-8 to actin and the possible effects of gelsolin on free IL-8 concentration and PMN chemotactic potential of CF sputum. Actin (0.5 mglml) was incubated overnight at 37°C with hr IL-8 (50 J.lg/ml). The mixture was separated by heparin affinity chromatography and IL-8 detected by immunoblot analysis. Sputa from CF patients (n=IO) were treated with gelsolin (250 nM) or buffer control for 4h at 20"C and centrifuged at 20,000 g for 20 min. The soluble phase was assayed for free IL-8 by specifiC ELISA and PMN chemotaxis using modified Boyden chambers. Results of the chemotaxis assays were expressed as migrated PMN per high power foeld (hpf). lmmunoblot analysis revealed that IL-8 was bound by actin, which prevented the IL-8 from binding to a heparin affinity column. Gelsolin treatment resulted in a significant (p=0.004) increase in free IL-8 concentration (4.79 (0.446-11.167) nglg) compared with controls (1.23 (0.094-3.984) nglg). There was a signifiCant increase (p=0.003) in the PMN chemotactic reponse to gelsolin treated CF sputum (165 (59-283) PMN per hpf) compared with controls (79.59 (27-127) PMN per hpf). These results suggest a potential proinflammatory effect of gelsolin in the airways, prompting further investigation prior to its therapeutic application.

419 IXAGGUATID INFlAMMATORY RISPONSI IN CP AIRWAY IPl'l1DUAL au.s. A. 3tppkp R. Breya-, A. c-mco, R. Dwonld, o. Kin& K. TCIIii,K.snp.m. C...b'l..unl~ Vlllderbilt Uaiwnity, Nuhville, TN, USA.

Pulmoury jnf!emmetim in CF ocoun -'Y llld may Jll**lc iDfectim. D..-8 (a pro­iaflammatcry cytokine produced by RIJiiratoly epithelium) is found at hiab lewis in the IIIIIP of inflllll with CF. Treatmenl of CF paticntl with the C)'I:IOOXJII"JIIX (COX) inbibikr ibujlrciD liiiM the docline in luag 1llDcticn. Tberefcxe, we hypotbesized that CF bronchW epithelial celll haw 111 eugenled lL-11 fC1!10111C to iDfllmmalcry llimuli becoule Cbe lllljor COX product oCbrondliol epithelium, POE,, is pro-iaflammatcry. We lludied: primlry lllllmll bumlll brondliol epitbe!W celll (NHBE); lrllllfOI'IIIed lllllmll 1m-. brmdUI epidleli.ll C.U. (BBAS); 2 dilliirent lwmlll CF airway epiCbeliol celllineo (2CF and m3); llld m3 celll triDifcnned to olio expreu wild-type CFTR (C38). Cells _.. culllnd to cm6Jin:le in llllltiple well plllel. plaoed CIIIIIUID·he media for 24 bn,

then incubated with increasing concentrations ofTNFu (0.01 to 300 ng/ml) or IL-l~ (0.1 to 1000 pglml) for 24 hrs. Supernatant IL-8 concentrations were measured, the total number of ccUs in the well counted, and 24 hr IL-8 production expressed as ng IL-8/million cells. With either TNFu or IL-lp, IL-8 production in the CF cells (2CF and IB3) was significantly greater (p <.05 when comparing the slopes of the dose response curves) than in normal cells. At the maximwn dose given. IL-8 production was increased 86- to 138-fold ov..- baseline in the CF cells and 15- to 26-fold in the nonnal primary or transfonned cclls. The TNFu response in the "corrected'" CF cell line (C38) was similar to that in the normal cells. In C38 cells, TNFu at moderate doses (I to 30 nglml) increased IL-8 production 2· to 6-fold and the maximwn response was a 43-fold increase in IL-8. To detcnnine the role of COX products in modulating the response to inflammatoJY stimuli, we treated IB3 or BEAS cells with 40 11M indomethacin for 24 hrs prior to a 24 hr incubation with increasing concentrations of TNFu. COX inhibition decreased TNFu stimulated IL-8 production by 30% to 70'/o in IB3 oells but increased production by greater than 200% in BEAS cells. Since these data suggested that a COX product was pro­inflammatory in CF cells and sinoe POE, is the major COX product of bronchial epithelium, we exposed normal and CF cells to I 0 .. to I o·• M POE, and measured 24 hr IL-8 production. IL-8 production was unaffected by POE, in IB3 and BEAS cells and increased only slightly in 2CF cells. However, incubating CF cells with 10'7 to 10'' M POE, for 2 hrs prior to adding 30 nglml TNFu increased TNF u stimulated IL-8 production (10"' M • 2.25 fold increase in IL-8 production over TNFu alone; 10~ M = 6.8 fold: to·• M • 8.6 fold). In contrast, pre-incubation with POE, had minimal effect on the TNFa response in BEAS cells. In cooclusion, CF bronchial epithelial cells have an exaggerated IL-8 response to cytolcine stimulation compared to nonnal cells (either primarY or transfonned). CF cells which also express wild type CFTR do not show this exaggerated response. COX inhibition returns this response clo!ICI' to normal. Prelimirwy data suggest that POE, priming is required for full indu.:tion of the IL-8 response to TNFu in CF epithelial cells. Support: CF Foundation.

420 LONG TERM STABILITY OF AIRWAY INFLAMMATION IN INFANTS AND YOUNG CHILDREN W1Tit CYSTIC FIBROSIS

Wascpcr JS, Sontag MK, Accurso FJ. Deportment of Pediatrics, University of Colorado School Health Sciences Center, Denver, CO, USA.

Neutrophil-dominated airway inflammation cbaractcrizet the lung disease of older cystic fibrosis (CF) patients and baa been identified in some infants with CF. To detennine longitudinal patterns of airway inflammation we studied 24 infants with CF identifiCd throuah newborn screening for up to S years. Repeat bronchoscopies were performed after time periods varying from 2 to 36 months. BALF cellularity and leveb of IL-8 (a pro-inflammatory cytokine), elutue activity, and elastue· alpha 1-antiproteaJe (NEAP) were meuured. BALF total neutrophil count, IL-8. elastue activity, and NEAP levels in individual patients varied greatly. Percent neutropbila, however, varied little over time. Ten of II infants with less than 20% neutrophils during their flllt year of life remained in this range for up to 5 yean. Of the infants with greater than 20% neutrophils in their BALF, 8 continued to have an increased percent over time, suggesting persistence of their airway inflammation. These fmdinas suggest that the majority of CF infants with little airway inflammation during the flllt year of life will remain atablc for several years. However, CF infants with increased airway inflammation during the fust year of life may not be effectively treated with currmt routine care and new therapeutic options, pouibly directed toward reducing inflammation or the stimulus for inflammation need to be ltudied.

Supported by a Jllllll from the Cyatic Fibrosis Foundation and the NIH Pediatric GCRC propam.

421 MYELOPEROXIDASE IN BRONCHOAL VEOLAR LAVAGE FLUID OBTAINED FROM INFANTS AND YOUNG CHILDREN WITH CYSTIC FIBROSIS

Wa1cpc;r JS Kettle A. Depar1ment of Pediatrica, Univeraity of Colorado School of Medicine and tbe Free It.dic:al Raearc:h Laboratory, Univmity of Canterbury, Chrisldmrcb, New Zcalllld.

Tiuuc injury Clll occur followinfl expoaure to elevated leveb of oxysen ftee radicab. Myeloperoxidue (MPO), a neutrophil-derived enzyme which can generate tbe potent oxidizina •sent hypoc:blorou~ acid, is increased in bnmchoalveolar lavaae t1uid (BALF) from older cyatic fibroais (CF) patients and correlatea with their depe of luna em-e. We meuured MPO leveb in infants with CF detected by newborn acreeninl to - if this potential cauae for airway injury cxista early in life. Thirty-two BALF aamptea from 21 CF infanta (IIICBil age 22.3, ranse 2-60 IDOIIIba) prospectively aclected u part of 1 longitudinal atudy of airway inl1ammation were wed. MPO wu meuured apectropbotometrically and COIIIpaJed with ltUidard concentrationl. Addili9Dal meuurea of airway

inflammation included BALF c:eUularity IJid levels of IL-8, elastase activity, IJid elas1ase/1l-antiproteue complexes (NEAP). MPO was present ill CF infants with greater than 10% neutropbils ill their BALF. MPO weakly COrTelated with neutrophil count, IL-8 IJid elastase activity, but not with NEAP. MPO bad 1 strong linear correlation with percent neutropbils ill the BALF. These firulings iDdicate tbat MPO ia preaent ill the linn)'l of CF infaats who have inCJeued ainny inflammation. The "'lationsbip to percent neutropbils auagests that the presence of u increued atimulua for neutrophilia ill more important than the absolute Dumber of neutropbils ill the airway. SiDc:e oxygen free ndicals COD produce tissue damage, these fmdinga auageat that therapy may DCCd to be din:ctecl at decreasing the llimulus for neutrophilic inflammalioo IJid ~ing antioxidant defenses.

Supported by 1 grant from tbC Cystic Fibrosia Foundation IJid the NIH Pediatric GCRC prognm.

422 BRONCHIAL CYTOKINE EXPRESSION IN CYSTIC FIBROSIS CHILDREN WITH STABLE LUNG DISEASE AND WITH AKUTE PULMONARY EXACERBATION Claudia W91narowski. T. Frischer. E. Hofbaue,., C. Muller, I. Eichler, OY. Koller, W. MosgOIIer, and R. Zie!Che*. University Hospital, Dep. of Pediatrics, and "Oep. of Pulmonary Medicine, A-1090 Vienna, Austria.

we have studied the expression of Inflammatory cytokines (IL-8, I potent neutrophil chemoattractant. and TGF-11. an Important cytokine for inducing collagen production) in bronchial mucosa of cyst1c fibroSis (CF) patients with stable pulmonary disease and with acute pulmonary exacerbation. Exacerbations were defined by Increased cough and sputum production, fever with Of' without new pulmonary Infiltrates, deterioration of oxygen saturation and > 1 0% decline of lung function (LF) and weight loss. Fiberoptie bronchoscopies were performed under general anesthesia (intravenous midazolam 0.1-0.1Srng/kg, propofol 3-7rng/kg, and nalbufinehydrochlonde 0.2mglkg) with the Olympus BFP30 bronchoscope. The endoscope was Introduced through 1 laryngeal mask. Mucosal biopsies were taken of subearinae of the right lower and middle lobe using an Olympus19C forceps. Analyses of expression of the IL-8 and TGF-1! gene were performed by qualnative and quantitative RT-PC~ with a competHor gene diluted In aeries from 10 attomole to _10 attomole. Aliquots were elec:trophoretically aeparated on a Nu-S1eve GTG/agarose gel, visualized by ethidium bromide staining and the number of mRNA molecules was determined by calculation. Twelve patients with CF ( 5 girts, 7 boys) with an age between &-17years (mean age 10.11 years) were enrolled. Five patients had an acute pulmonary exacerbation. Increased IL-8 levels were deteded In patie~ts with ·~ without pulmonary exacerbation (range between 10 • 8x1 0 moleculesljdmRNA). TGF-~ was expressed In all patients with chronic stable disease (1o'.ex104 molaculestjdmRNA) but was absent in patients with acute exacerbation. The elevated expression of IL-8 and TGF-~ in patients with stable lung condition was lndepende.nt of the severity of their pulmonary manifestations (as assessed by clinical evaluatoon, LF, and HR-CT). Conclusion: Even In patients wfth mild pulmonary disease and stable LF a cytokine that has been demonstrated to be Involved In remodeling of lung tissue (TGF-11) Ia upregulated In the bronchial mucosa.

423 PULMONARY CLEARANCE OF MUCOID PSEUDOMONAS A.ERUGINOSA. AND INFLAMMATION IN NORMAL AND IL·IO KNOCKOUT MICE. H.....Yil1, M. Hanesl, C. E. Chrispl, J. C. Boucherl, V. Deretic.l IDepl: of Microbiology and Immunology and 3Unit for Laboratory Ammal Medicine, Univ. of Michigan Medical School, Ann Arbor, MI. and lLaboratory Animal Resources, Univ. of Texas Health Science Cenler at San Antonio, San Antonio, TX, USA.

Chronic endobronchiolitis compounded by infections wi.th Pseudomonas aeruginosa is the major cause of morbidity and mortality in patients with cystic fibrosis. Conversion to the muc:oi~. exopolysaccharide alginate-overproducing phenotype in P. aeruginosa JS believed to facilitate the establishment of chronic mfection. In order to initiate investigations of the role of mucoidy in P. aeruginosa persistence and analyze inflammatory processes associated with P. aerugino~a infections in CF, a mouse model of repeated respiratory exposure to th1s pathogen was established. We first compared an isogenic pair of nonmucoid (mucA.•) and mucoid (mucA22) P. aeruginosa and found that the mucoid strain was cleared several-fold Jess efficiently than its parental nonmucoid strain during the initial stages of the exposure

Poster Session Abstracts 321

regimen. The microscopic pathology findings and proinflammatory cytokine levels were similar in mice exposed to nonmucoid and mucoid P. aeruginosa throughout the infection. While a single exposure to P. aeruginosa aerosols caused only mild histopathological changes, repeated exposure to the aerosols resulted in significant lung pathology in CS7BU6J mice. The peak of histopathological changes and inflammation was characterized by subacute lymphohistiocytic bronchopneumonia and persistent elevation of tissue-associated TNF-a and MIP-2. We also tested lung histopathology and proinflammatory c:ytokines in transgenic IL-10 deficient mice (IL-IOT). On initial challenge, significant mortality was seen in IL-l OT mice not observed in normal animals. While no histopathological differences in the lungs could be observed between C57BU6J and surviving IL-IOT mice upon single exposure, increased pathology was observed in IL-IOT mice at the end of the repeated exposure regimen. These results are consistent with the proposals that anti-inflammatory c:ytokines may play 1 role in P. aeruginosa-induced tissue damage, and that the reported reduced IL-10 levels in the lungs of CF patients may be of significance for the respiratory sequelae in this disease.

424 ENHANCED ADENOVIRAL GENE TRANSFER AND EXPRES­SION IN MACROPHAGES AND DENDRITIC CELLS USING A VECTOR WITH GENETICALLY MODIFIED FIBER PROTEIN WITH POLYLYSINE REPEATS R J Kaner, E. Stolze, T.J. Wickham, I. Kovesdi, R. Granstein, R.G. Crystal. The New York Hospital-Cornell Med­ical Center, NY, NY and GenVec, Rockville, MD

Enhancing host defense may be a useful strategy to reduce the frequency or severity of lung infections in cystic fibrosis (CF). Cells of monocytic lineage are important participants in host defense, either in the phagocyto­sis of organisms and elicitation of an inflammatory response (macrophages) or in antigen presentation (dendritic cells). We modified the genetic pro­gram of human alveolar macrophages to produce 1 potent activator of anti­microbial activity, interferon-y (up to ISOO pg/10' cells·24hr) for 16 days by in vitro infection with an adenovirus vector containing the human interferon-y gene [AdiFNy, multiplicity of infection (MOJ)a200). To en· hance the effectiveness of gene transfer to macrophages and dendritic cells (DC), we tested the hypothesis that genetically modil)'ing the fiber protein by addition of poly lysine sequences that could interact with negatively charged binding sites on the cell membrane would enhance viral entry and thereby increase transgene expression. Mouse skin DC line XS52 and pri· mary human blood mononuclear cells were infected with a MOl of SO Of'

200 with a null vector (AdNull); Adpgal, a 1st generation Ad vector con­taining the p-galactosidase gene; or AdZ.FpK7, 1 2nd generation Ad vector with fiber sequences modified with the addition ofpolylysines. After 72 br the cells were fixed and stained with Xgal. All cells infected with AdNull were negative. The percentage of positively expressing human monocytic cells (MOI•200) was S%1% Adpgal; and 100%0% AdZ.FpK7 (p<0.05). AdZ.FpK7-infected monocytes were also unifonnly positive at an MOl of SO. At MOJaS, there were no positively expressing AdPgal-infected mono­cytes, while ll%3 %of AdZ.FpK7-infected cells were positive; 30:1:4% of mouse skin DC were positive 30:1: 4o/o, MOI-200) vs I 00:1:0 % exp..,ssion by AdZ.FpK7-infected DC at MOJaSO. Thus, more efficient transgene expression in macrophages and DC can be obtained with an Ad vector which expresses additional poly lysine residues on its fiber protein. Aug­mentation oflocal host defense by Ad vectors targeted to macrophages and DC may provide another approach to reducing infections in CF.

425 INTERSTRAIN DIFFERENCES IN MURINE SUBMUCOSAL GLANDS. E.A Cowley, E. Zysman-Colman, M. King and D H Ejdelman. Meakins-Christie Laboratories, Montreal Chest Institute Research Centre, McGill University, Quebec, Canada.

One of the limitations of murine CF models is that the airway anatomy of mice is incompletely understood. Although mucous glands are said to be largely absent in mice, we have been able to harvest mucus from the proximal trachea of mice, particularly those ofCS7BI/6N background. To identifY the aource of this mucua we systematically examined the location and relative abundance of

322 1997 Cystic Fibrosis Conference

submucosal glands in three inbred strains of mice: ClH-HeJ, All and CS7BV6N. Tracheae were formalin fixed, paraffin embedded and S j.!m thiclc cross-sections stained with Masson's trichrome. In CS1BV6N mice, large submucosal glands were consistently detected (rr=S). Glands were always located in the most proximal portion of the trachea, immediately below the larynx. Glandular acinar cells stained positively for PAS. Glandular tissue appeared to be organized into bilateral glands, with two gland ducts opening into opposite sides of the trachea at the same level. In contrast to CS7BI/6N tracheae, submucosal glands were rarely seen in A/I (n•S) and ClH-HeJ (n-4) mice and when present, tended to be considerably amaller than those in CS7BI/6N. A. a basis for further study, sagittal semi-serial sections were examined from three C57BI/6N mice and estimate& of the total glandular volume and length calculated to be 78 ± 6 111 and 0.98 ± 0.04 mm respectively. This work indicates that tracheal mucous glands are present and available for study, at least in some inbred mouse strains. Interstrain differences in submucosal gland volume in mice may contribute to differences in host defence capacity. (Supported by the Canadian Cystic Fibrosis Foundation and the J. Costello Memorial Fund)

426 RHEOLOGY OF MOUSE TRACHEAL MUCUS. ~ Kina. E.A. Cowley, E. Sudo, D.H. Eidelman. Pulmonary Research Group, Univ. of Albena, Edmonton, Canada; *Meakins-Christie Labs, McGill University, Montreal, Canada

One of the limitation of murine models of airway disease is the difficulty in collecting sufficient mucus or airway surface fluid for conventional analysis. We have found that it is possible to harvest tracheal mucus from laboratory mice in quantities sufficient for rheological analysis by means of judicious choice of sampling site, prolonged collection times, and further miniaturization of the magnetic rheometer technique for rheological testing. Mucus was obtained from adult male mice after xylazinelpentobarbital anesthesia by performing a tracheostomy and inserting a small (PEIO) catheter retrograde towards the larynx. The catheter was left in place for periods of I or 1.5 hours, and then withdrawn and placed immediately in a test tube containing paraffin oil to minimize evaporation loss. Adherent mucus was scraped off the catheter and transferred to an analytical container inserted into a magneticmicrorheometer. By using steel balls of 70-90 JIM size, we were able to reduce the sample volume requirement down tom. 300 nL. Where necessary, tracheal samples from up to four matched mice were combined to achieve samples sufficiently large to analyze. Eleven samples of control tracheal mucus (0.51±.27 mg) were collected from 23 C57BU6 mice. In addition, four samples of mucus were obtained from five mice receiving methacholine (5J1L of .01 or .1 mglmL) by irrigation external to the larynx. The control mucus was rheologically similar to control tracheal mucus from dogs and ferrets (rigidity modulus log G* at I rad/s = 2.38±.28 dyn/cm2), and the response to meth­acholine was also comparable (mean o log G* = 0.49, p=.025). The volume of mucus collected per hour per mouse was 0.23±.16 mg. while after methacholine, the mucus collection rate increased to 2.6±1.5 mg (p=.025). This study demonstrates that collection and analysis of tracheal mucus from laboratory mice is feasible. These findings thus provide background support for studies involving animal models of mucociliary dysfunction. Supported by Canadian CF Foundalion.

427 CLINICAL USE OF IBUPROFEN FOR CYSTIC FIBROSIS (CF) LUNG DISEASE: DATA FROM THE 1996 CF FOUNDATION NATIONAL PATIENT REGISTRY. M W Kl!llltln 1 and S.C. FitzSimmonr. 'Department of Pediatrics, Case Western Reserve Univenity School of Medicine, Cleveland, OH, USA, and 2(;ystic Fibrosis Foundation, Bethetcla, MD, USA.

Long-tenn, hiah-dose ibuprofen OBU) therapy has been shown to slow the progreuion of lung di- in CF patients ~ 5 years of age with FEV, ~ 60 % pndided in a four-year clinical trial (NEJM 1995; 332:848-54), and is being ldvocated u a new thenlpy for CF patients with ti*C clinical clwlcteristics. The objective of this report is to characterize the number and type of patients

receiving IBU therapy during a one-year period ( 1996), and to determine its safety profile in these patients. Data were obtained from the 1996 CFF National Patient Registry. Of 19,069 patients seen in outpatient clinics at 113 accredited CF Care Centers in the U.S., 8, 793 (46.1 %) met the clinical criteria for consideration of IBU therapy (age~ 5 yrs; FEY, ~ 60% pred). 1,354 (7.1%) patients at 81 (72%) CF Centers were prescribed IBU therapy. Regarding age, use was greatest among those 5-12 years (737 patients, 11.8%), followed by those 13-17 years (262 patients, 7.7%) and those ~ 18 years (282 patients, 4.4%). Interestingly, 73 patients < 5 years of age (2.5%) received IBU therapy. Regarding severity of lung disease, use was greatest among patients with FEY, > 60% pred (9.8 %) compared to those with FEY, 40-60% pred (5.8%) and FEY, < 40% pred (3.9%). A dosage of20-JO mg/kg BID was reported in 86.2% of IBU patients. The incidence of peptic ulcer disease, gastrointestinal bleed (requiring hospitalization), and renal failure (requiring dialysis) was determined. Ulcer disease was reported in 5 (0.37%) IBU patients vs. 43 (0.24%) non·IBU patients, G.l. bleed in 6 (0.44%) IBU patients vs. 31 (0.17%) non-IBU patients, and renal failure in I (0.07%) IBU patient vs. 28 (0.16%) non-IBU patients. Differences in the incidence of these adverse effects between IBU and non-IBU patients were not statistically significant. These data suggest that long-term, high-dose ibuprofen therapy is being used in the target group identified by the results of the four-year clinical trial. Although overall usc was low in 1996, usc has doubled since 1995. We speculate that low-use of ibuprofen is based on the complexity of obtaining a phannacokinetic study to initiate therapy, and on concerns regarding safety. These data may alleviate the fear in initiating therapy based on safety concerns; the annual incidence of ulcer disease, G.l. bleeding, or renal failure was not significantly increased in patients receiving ibuprofen. However, continued monitoring for these and other adverse effects is recommended to further determine the safety of long-term, high-dose ibuprofen therapy in CF.

SYNTHEnc PAOTEGAINS CAN ELIMINATE P. AERUGINOSA FROM THE MUCOSAL SURFACE OF CULTURED AIRWAY EPITHELIA. J. J. Smlth1.~. B. McCray, P. S. Thome, D. J. Loury*, J. C. Flddes*, 8rid M. J. vvelshl. Depts. of Pediatrics, Preventive Medicine, and Internal Medicine, IHoward Hughes Medcal Institute, University of Iowa College of Medclne, Iowa City, lA 52242 and *lntraBiotlcs Phatmaceullcals, Inc., Sunnyvale, CA 94086.

We recently suggested that bacterial Infections may develop In CF =because an lnc188!18 In the NaCI concentration Inhibits the actl of antlbactellal factors In CF airway surface fluid. Thus, a

lherapeutlc approach to CF lnfec:llons Is to deliver to the airway surface an exogenous bactericidal agent that Is not Inhibited by high concentrallons of salt. Protegrtns 8/8 a family of naturallv· occurilng antimicrobial peptides that relaln their bactericidal activity ra the presence of physiologic concentrations of sail In addition, In vitro studies lndk:ate that synthetic congeners ol the native protegrfns have bactertcldal activity against CF"pathogens, such as muookfand non-mucoid P. aeruglnoss, and methlciiUn-reslstant S. sureus. We therefore hvPotheslz8d that protegrfns may be useful for reducing the densltv of "P. BBnJdnoss on the muooaal surface of the airway. To test this hypothesfi, we Inoculated the air-covered, mucosal surface of rultured Calu-3 airway eolthella, a oel line derived fran a pulmonary adenocan:lnoma, wllh a laboratory strain of P. seruglnosa (10'·10' CFU of PA01S). After Incubation lor 2 hrs, 60111 of saline solution containing synthetic congeners of protegrin was added to the mucosal surface. Mel Incubation for an additional hour, the bacteria were recovered and plated onto agar for colony count. Under control conditions (exposure to saline alone), the number of bacteria recovered was greater than the Initial Inoculum. Thus, P. seruglnoss readily multlpled on the surface of Calu-3 airway epithelia. In contrast, PIOiearfn caused a dose-dependent decrease In the number of bacteria reCovered. At hlgler concentrations of protegrfn (200 l'ltml) bacteria were rarely recovered. These results suggest that an In-vHro model of the airway epithelium may be valuable for testing new treatments for airway disease. In particular, protegln's sustained bactericidal act!VItY In the presence of Nacl may prove useful for the development of new treatments for airway Infections In CF.

429. Oven ............ olhun..n Det--... B..Qmpp. M.

Frye, T. Waper,J. Baraon.

Med.Kiinik II. Uni•cnity of Frankfurt, Franlcfurt. Germany

The f..Uiy o( tbe defensins is defined by small (3-4 kDII) cationic, cyslein

rich pcplideo which contribute to., unspecific host defense. All memben of

this JroWing family share a eencral h:f1iary slni<IW'C composed a lhree II

shc:cts stabilized by lhree inuamolccular disulfide bonds. lbe number as well

as the spacing of the six cysteins is well conserved in all deC ensios. In vitro

synthesized defensins exhibit antibacterial activity, bowever,the activity is

raluced by a factor a 1000 compared to defensios isolated from human

serum. This loss of activity is most probably due to inconect folding of the

peptide.While in-vitro synthesilled pcptides have to achieve their conect

tertiary slrUCIUR post synthesis, eucaryotic expression systems take advantage

of the cellular machinery to synthesize and fold !he peptide oorrectly.

Therefor, we chose eucaryotic cell lines to overe•presa defeosios. Human

defensin (liD·5 and hBO.l) w= cloned into pCDNA3 and transformed into

CHO aod HT -29 cell lines. Supemalallt a transformed and control cells were

assessed for their aotihaclerial activity in a haclcrial assay. Supernatant a cell

lines transformed with IID-5 and bBD-1 exhibiled strong activity towards

~richia coli, Puudomonos aeruginosa and Staphy/ococnu aurew .lbese

data are most promising Mid could indicale that other euca-yotic C\prellsion

systems such as yeast or barulovirus have potential to express highly active

defmsins.

Funded by DLR

EXPRESSION AND ACTIVITY OF HUMAN B· DEFENSIN·1 (hBD·1). P.B McCray. Jr, S. Travis, P. Singh, B.D. Conway, W-G Forssmann, C. Lawyer, A.D. Anderson, B.L. Davidson, M.J. Welsh. IPF, Hannover, Germany, Southern Illinois University, Springfield, IL and University of Iowa College of Medicine, Iowa City, lA, USA.

Defensins are small cationic peptides with broad spectrum antimicrobial activity, and are therefore candidates for the bacterial killing factor(s) in airway surface fluid (ASF). We cloned the lull length coding sequence for hBD-1 from human airway epithelia and found the hBD-1 amino acid sequence shares homology with the slgnal!propiece and mature peptides of other B·defensins, Including bovine TAP and LAP. hBD-1 was expressed as a single N400 nt transcript in airway epithelia by Northern blot and by ribonuclease protection analysis hBD·1 transcripts were more abundant In the conducting airways than the gas exchange regions of human lung. In situ hybridization localized hBD-1 mANA expression primarily to conducting airway and submucosal gland epithelia. We tested the antibacterial activity of hBD-1 using a sensitive luminescence assay. Native hBD-1 peptide isolated from blood exhibited antimicrobial activity against E. coli, but was less potent than HNP-2, cecropin P1, or protegrin. Synthetic hBD-1 peptide was active against a recombinant E. coli expressing luminescence genes from Photorhabdus luminescens. An hBD-1 concentration of N30 11glml caused a 50% reduction in luminescence. An adenoviral vector was prepared to express the hBD-1 eDNA (Ad5/hBD·1 ). When Hela cells were Infected with Ad5/hBD-1, the hBD-1 mANA was abundantly expressed and antimicrobial activity was seen in a bacterial agar overlay. Human B-defensin-1 is an antimicrobial peptide expressed In airway epithelia and may contribute to the microbicidal activity of airway surface fluid and play a role In the mucosal defenses of the lung. (Supported by CFF and Children's Miracle Network).

A BURKHOWER.IA CEPAC/A MUI' ANT WITH ALTERED MEMBRANB PERMEABn.ITY CAUSING INCREASED SUSCEPI'IBn.ITY TO CATIONIC PEPTIDES. B R, McKay, D.P. Specrt, Depanmeol orMiCIOblology and lmmuDology, Univenity of British Columbil, VIIICOIMr, B.C., Canada.

Poster Session Abstracts 323

Blll'kho/du/11 t:tpiiCIII II a lung pathogen that II bccomlDJ or ln=uinJ lmponancc In pallents with c:ystlc flbrosi1. It II unclear bow B. t:tptJCia esllbUshel infcctlon in these patients and why IODIC patients show 110 l)'lllpCoiDI or eli- and olhen dm:lop a fulmlnatlna aecrotizlna piiCWJIOIIia. Wo are lnlerested In clari1Yina tho pathoaenic mechanism of B. t:ffJIIC/11, h weU. B. t:tptJCitl 11 a pathogen In chronic granulomatoul diseue patienll. Neuttophill from tholo pallents lack tho ability to undergo an oxidative bunt for dcstroyinalnlemallz.cd microbcl. Thcso ncutrophils are dependent on nonoxidative klllina. 1 ~or component beiDa tho cationic pcptides. B. t:tpaclll Is resistant to killing by tholo neutrophila and Is also resistant, '" vlfrotl, to high conoentratiolll or cationic peptides. Understanding how B. t:tpacla resiSII killing by cationic peptides may give us I clue about tho pathogenesis of B. t:tpacltl. A mutant or B. t:tpacla A TCC 17616 canying the transposon TnJ-7J/ inserted In Its genome (mutant 2607) wu isolated In a preliminary screen with the cationic antibiotic polymyxin. 2607 had I lowered MIC orsl'g/ml compmd to the parent (63 l'g/ml). Rcduocd MICI were also found for the cationic peptides human neutrophil defensin-1 (from >250 l'g/ml to 3.9 l'g/ml), and CP-26, 1 modillcd cecropin-mclittln hybrid (from >2SO l'g/ml to 3ll'g/ml). The MICJ for other families or antibiotics were not changed IIIS&esting this mutation specifically affedl resistance to cationic peptides. We have also shown that the outer membrane of this mutant has alleral pcnneablllty. Tho hydrophobic probe I·N-phcnylnaphthylamlne (NPN) can not pcnneabilize tho outer mcmbrano or all wild-type B. t:eptJCIII ICSied even In tho presence or cxtrcmcly high conoenuatio111 of polymyxin (up to 200 J'g/ml). NPN wu llble to pcnetrale the outer membrane of our mutant ltrain at 1 concentration or 1.6 l'g/ml polymyxin. A llllall clone canyinJ 1. 7 kb of chromosome acljacent to tho transposon bas been constructed. We arc screenina1 c:osmid library or B. t:ef'IICitJ ATCC 17616 using a probe from this clone In an attempt to ldcntiJY 1 c:osmid that will complement the mutant phenotype. Fwthcr aenetic characterization and phenotypic analysis of this mutant may lead to understandina lhe inlrilllic resistance and pathogenesis or B. t:tf111Citl.

COMPONENTS OF AIRWAY SURFACE FLUID HAVE SYNERGISTIC ANTIMICROBIAL ACTIVITY. f....SiJW1 and MJ Welsh. Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City.lowa USA

The use of synergistic antibiotic combinations can provide important benefits in the treatment of infections. These include increased killing of susceptible organisms, killing of microbes resistant to one agent when used alone, enhanced activity against polymicrobial infections, and decreased emergence of resistant strains. These effects would also be of benefit at mucosal barriers, such as the airway surface. The fluid lining the airway surface contains several antimicrobial components produced by different cell types including airway epithelial cells, submucosal glands, and perhaps. alveolar and immune cells. We hypothesize that in combination these substances may have synergistic antimicrobial effects. To evaluate this we measured bacterial killing using E. coli expressing luminescence genes. We used two established methods for testing antibiotic combinations. The checkerboard method differentiates synergistic, additive, and antagonistic interactions between antimicrobials. This technique quantifies the degree of synergy by the fractional inhibitory concentration (FIC) index. An FIC index of S 0.5 defines synergy and additivity is defined by an FIC index of 1. Lysosyme (produced by submucosal glands) tested in combination with ASF (derived from primary airway epithelial cultures) had a significant synergistic effect (FIC index of 0.2). The combination of lysosyme and lactofenin, a product of the sub-mucosal glands. also showed a synergistic effect (FIC index of 0.33). We confumed this finding using the kinetic method of synergy testing. Studies currently underway will elUIIlline the effect of combining other components of the airway surface fluid and products of the submucosal glands as well as exogenously adminislered antibiotics. We are also examining how changes in the ionic environment might alter these antimicrobial interactions. These studies suggest that native antimicrobial substances found at the airway surface enhance the effect of each other, and that this synergy maybe an imponant component of the mucosal host defense.

ANTIBACI'ERIAL ACTIVITY OF RESPIRATORY EPITHELIUM IS GENUS SPECIFIC AND INOCULUM DEPENDENT, Ymi!b. L. Cohn, M. Kaplan, A. Mings. Deparunent of Molecular Microbiology & Immunology, University of Missouri-Columbia, Columbia, MO, USA.

To gain insight into the general applicability of the observation that when low numbers of P. t~erugirwsa arc inoculaled on to the apical surface of

324 1997 Cystic Fibrosis Conference

polarized, respiratory epithelial monolayers growing at an air-liquid interface are not derccted on the cell surface 18 hours later, we sllldied 2 CF BAL i!olates: H. influenvu and Staphylococcus aurtus an compared them to P. turuginosa PAK. CF respiratory epithelial cells (183) and the corrected derivative (C38) were grown to confluence on collagetKoated filters (Transwells•, Costar) and placed at air-liquid interface for 7 days. Chromosomally marked H. l'lfluerrzt~~ and S. aur~us were added in graded inoculae to the apical surface: after 18 hours incubation, the apical surface was rinsed and quantitatively cultured, as was the basolateral compartment, while cell associated bacteria were released by tteatment with 0.1% Triton X-100. Data representative of 3 or more replicates with C38 cells is: H. injluenvu inoculum of 10"-10' resulted in no recoverable bacteria from apical cell-associated, or the basal comparttnent: 101-!Ql cfu inoculum gave !()6 cfu on apical surface, 10' cfu cell-associated and none basally; a !Ql-!()l cfu inoculum produced I 0' cfu on the apex, I 0" cfu associated with cells and I ()I cfu in the basal compartment. With 183 cells similar results were obtained: with an apical inoculum of 10"-101 cfu, similar to C38 no organism& were recovered from any compartment. An inoculum of 101-!Ql cfu resulted in 10" cfu on the apical surface, 10' cfu were cell-associated and 10' cfu in the basal compartment: an inoculum of 1Ql-10' cfu produced 10' cfu on the apical surface, 10" cfu cell associated and 10' cfu in the basal compartment. Similar dats were found with P. turuglnma PAK except for a greater tendency of an inoculum of 101-!Ql cfu to invade and b'anscytose IB3 cells, in comparison to C38 cells. Staphylococcus aurt!us was cleared from the apical surface of both cell lines at lnoculae up to 10' cfu. We conclude that H. l'lfi/Unvu, S. aurrus, and P. turuglnosa when placed on the apical surface of respiratory epithelia, may not be recoverable, ( < J()l cfu) may b'anscytose (102-10' cfu) or become tightly associated with the cells(> 1Ql cfu).

434.

CYSTIC FIBROSIS, LUNG INFECTIONS, AND HUMAN TRACHEAL ANTIMICROBIAL PEPTIDES. Y.....KQ, M. Delannoy, and P. Pedenen. Department of Biological Chemistry, School of Medicine, Johns Hopkins Univenity, Baltimore, MD, USA

Cystic fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the protein CFI'R (cystic fibrosis transmembrane conductance regulator). 1be most common mutation, the deletion of phenylalanine at position 508, results in infections by Pseudomonas aeruglnosa and other microorganisms in the lunp of CF patients. In order to undentand how the lunp of healthy people, unlike those of CF patients, are protected against lethal bacterial infections, we employed b'anlmission electron microscopy (TEM) on vertically as well as horizontally cut thin sections of several thousand human tracheal epithelial cells infected with bacteria to visualize their interaction. These studies revealed that P. aeruginosa do not multiply when planted onto cells from healthy humans, whereas the bacteria grow profusely on cells from C.F508 CF patients. Our results revealed also, and for the fint time, that these infectious bacteria gain entrance into CF cells. In addition, multiple bacteria tend to surround or target particulas CF epithelial cells at any one time rather than making a concerted atladc on the whole population of CF cells. Significantly, binding, multiplication, and entry of P. aeruglnosa into CF cells were visually observed at a concentration (104 mM) ofNaCI, considered to be in the near physioloaical range for airway surface fluid.

Attempts are beina made to identify antimicrobial facton that are secreted extra-cellularly or expressed in normal epithelial cells by use of mass spectral and molecular biological techniques. Mass spectral analyses show that a 5 kDa 1!pCCies is not present in the extra-allulas fluids from CF cells. In contrast, a 1.3 kDa and a 5 kDa species are present in normal tracheal cells. Furtherrnon:, human tracheal epithelial cells express an -4 kDa peptide (hT AP), which is known in its bovine form to exhibit bactericidal action against P. aerugtnosa. This suggests the presence of a first-line self defense mechanism in normal tracheal epithelial cells to protect against bacterial infection and the absence of this mechanism in CF cells, thus becoming wlnerable to bacterial atlllck. [Supported by grants from the Cystic Fibrosis Foundation and NIH (NIDDK))

435" .._....... ~ .__... l'nlllll (rBI'IaJ) ..

a.:teriddlllbl Hllw .... ,... ·-· ...................... ,.._ Cywdc l'l1lrosll ....._, L LAMBI!R.T, A. BREW!N aad P. SCANNON, XOMA Capalioa, Babley, CA. USA.

PmioUI IIIDdiel with r8PI21 baYe clemoJIItnded blctericldal activity apinlt pul-lleptiw aad cerlliD gnan1JOiitive organilma, both with aad without

supplemental antibiotics. Pseudomonas aer~~ginosa (Pa) isolates from Cystic fibrosis (Cf) patients characteristically exhibit reduced in vitro susceptibility to most antibiotics. In addition, the chronic nature of the infection RqlliRS continuous anbbiotic therapy, eventually resulting in Pa strains resistant to most anti-Pseudomonas drugs. We were interested in determining the in vitro bactericidal activity of rBPI21 against mucoid and non-mucoid clinical isolates of Pa alone, and in combination with standard antibiotics. Minimal inhibitory concentrations (MIC) were determined for 30 Pa, 3 Burkholderia cepacia (Be) and 3 Stenotrophomonas maltophilia (Sm) clinical isolates from two CF study centers. Studies were conducted using custom Dade MicroScan<BI panels containing only dried antibiotics, that when rehydrated with selected broths with and without varying concentrations of rBP121 , provided a sufficient range for evaluation of rBPI:,!antibiotic interactions. Kill curves using increasing concentrations of rBP121 were determined for representative strains. Both cation­supplemented (CSMHB) and non-supplemented (MHB) Mueller-Hinton broth (no modification ofNaCl concentrations) at pH 6.8 or 7.3 were used in the study. Lo~ phase inocula, quantified by plate counts, were adjusted between 1-SxiO CFU/mL. rBP121 alone exhibited antimicrobial activity against Pa in MHB at pH 6.8 (rBP121 MIC50 >8, range S2-32~<g/mL). Additionally, 2 ~<g/mL rBPI21 in combination with antibiotics reduced these MICs for all Pa strains by at least a 4-fold concentration. Conditions were identified where rBPI21 exhibited even more potent activity in CSMHB at pH 6.8 and 7.4 against Pa, Be and Sm. Kill cmves indicate that rBPI21 alone is rapidly bactericidal for sensitive strains. These results suggest that rBPI,1, in contrast to 11-defensin-1, is bactericidal for a IWll1bcr of multi-drug· resistant CF pulmonary pathollCIIS. alone and in combination with other antibiotics when assayed at physiological salt concentrations. Therapeutic intervention with rBP121 , a potent antibacterial and antioi:ndotoxin agent, may be beneficial in CF patients receiving antimicrobial therapy for multi-drug RSistant Pseudomonas infections.

Psychosocial/Behavioral/ Education

436*

PSYCHOSOCIAL RESPONSES OF ADOLESCENT RECIPIENTS WHO RECEIVED DOUBLE WBAR OR CADAVERIC LUNG TRANSPLANTATION. C L, Duqt, M.V. Hom, M.S. Woo, C.M. Bowman, V .A. Starnes. Divisions of Pediatric Pulrnonology and Cardiothoracic Surgery. Childrens Hospital Los Angeles. USC School of Medicine. Los Angeles, CA. USA.

What psychosocial issues do adoleacent Cystic Fibrosis patients experience after undergoing lung transplantation? To determine the common themes and emotional responses of these patients, we conducted in-depth interviews with 6 adoleacent Cystic Fibrosis lung transplant recipients. There were 3 male and 3 female subjects. Mean age at time of transplant was 1S.2 ± 2.7 years (range 12-18 years). The mean time interval since transplant was 25.4 ± 13.4 months. 5 patients received living donor double lobar transplantation and I patient received cadaveric whole lungs. Mean current age at time of interview was 17.3 ± 2.2 years. Initial findinas provide ipCCific insights into the adolescent lung transplant recipient. Major themes identified by patient self-report include: strong desire to set and attain meaningful long-range goals (specifically college and employment), the need to control as many aspecta of their lives as possible while dealing with parental ovcrprotectlveneu, and the adjustment of managing a new lifestyle. Common emotional responses Include: ongoing fear/anxiety of lung rejection and uncertainty of the future; impatience with disruptions of daily routines caused by post-transplant medical management and its effect on the attainment of set goals; and frustration with continued dcpendcnce on transplant team membera and parents. In general, patients reported a positive outlook on life, with greater emphasis placed on sought-after goals as well as interpersonal relationships. This study demonstrates that adolescent Cystic Fibrosis transplant recipients develop long-term goals and plans for Independence. By Identifying and anticipating the emotional nceda of this population, health care providen can assist patients in Improving the quality of their lives from a physiological u well as a psychological viewpoint.

437

SUPPORT NETWORKS AND THEIR RELATIONSHIP TO SOCIAL SKILLS IN ADOLESCENTS WITH CYSTIC FIBROSIS.

E.L. Jones, A L oymnor. s. Barry, D. Harris, s. LOWIIfY. Dept. of Psych. Indiana University, Bloomington, IN, USA

A large body of research indicates that social support plays an Important role In mediating the relationship between stress and psychological functioning (Quittner, Glueckaut & Jackson, 1990). However, studies examining the function of social support for adolescents is limited, in part, because of the absence of reliable measures (Eiser, 1994). Preliminary evidence suggests that adolescents with a chronic illness may benefrt from increased support, but to date, few studies have systematically evaluated either the structure or function of social support for this population (Varni, Katz, Colegrove, & Dolgin, 1994). Adolescents with cystic fibrosis (CF), for example, may have smaller social networlts and may seek support from different sources (e.g., heaHh care professionals) than adolescents without CF. In the current study, the structure (e.g., size, composition) and perceived quality of the social networi<S of adolescents coping with CF were assessed using a dialogue measure, "My Family and Friends" (Reid & Landesman, 1989). Forty-four adolescents with CF ages 13 to 18 were recruited from two major medical centers, and 44 adolescents without CF matched on age and gender, also participated. Information on network ' composition (e.g., who Is sought) and quamy of support (e.g., helpfulness) across different situations (e.g., needing medical information, sharing good news) was assessed for each adolescent. A measure of social skills was also completed. Results Indicated that the support networlts of adolescents with CF were smaller than their peers. Mothers were named as the most frequent providers of support (93%), followed by friends (91%), and then fathers (82%). Although health care professionals were named less frequently (24%), they were rated as more helpful than parents and friends by adolescents in the CF group. Mothers of adolescents with CF were perceived as more heipfulthan fathers for CF-reiated problems and for situations requiring emotional support. No relationships were found in tha CF group between social support characteristics and levels of social skill. Additional analyses comparing the social networlts and social skills of the two groups will be presented.

438

Supported by NIH GranlfiU9 HL4706o4 awarded to Dr. Alexandra L. Quittner

WAYS OF COPING IN ADULTS WITH CYSTIC FIBROSIS: THE DEVELOPMENT OF A CYSTIC FIBROSIS COPING SCALE. J...AbJlgn!, M. Dodd••, S. Gilling•, A.K. Webb••. •Department of Psychology, University of Central Lancashire, Preston, PRJ 2TQ, England. ••Manchester Adult Cystic Fibrosis Unit, Wythenshawe Hospital, Manchester M23 9LT, England.

How individuals cope with aspects of their disease has the potential to influence their ~elf management and the course of their CF. The Manchester Cystic Fibrosis Coping Scale was developed to assess the ways in which people with CF cope with a variety of CF concerns. The initial work involved the identification of patient concerns based upon the perceptions of CF patients themselves. A list of 23 CF concerns were recorded. For each concern the patient was asked what they did to ease the worry. From this interview dsta, a Jist of the 24 most common coping strategies was compiled. These formed a comprehensive set of items as to how people with CF act, feel or think about aspects of their disease. Subsequently, patients rated, on a three point scale, the extent to which they worried about their CF in general, and how much (if at all) they worried about each of the specific concerns. For each of the items that concerned them they were asked to select the statements from the list of coping strategies that they recognised as using themselves. These data were factor analysed using a Varimax rotation. The two factor structure was the most meaningful. Factor one was termed hopefulness and consisted of twelve items. This coping style reflected a optimistic, detennined and positive approach to CF. The second factor consisted of ten items and was named resignation. This style of coping consisted of an avoidant, passive and helpless way of dealing with CF concerns. Two of the original 24 items were omitted since they loaded equally on both factors. No significant correlations were observed between scores on either the hopefulneu or resignation scales and age or disease severity as measured by FEVa% predicted. However, males and females differed regarding their style of coping with specific CF concerns.

439 FINANCIAL CHALLENGES FACING ADULTS WITH CYSTIC FIBROSIS. p P El!errodt. BJ. Shea, J. Leon&. B.L. NyberJ, C.B. Hegl, J. Wilson, N.P.D. Brager and H.R. Rabin. Aduh Cyotic Pibrosil Clinic, Unlvmity of Cslgary Medical Clinic, foothills Hospital Medical Centte, CalCifY, Alberta, Canada

Poster Session Abstracts 325

Introduction: Significant financial cballengea face aduhs whh cystic libroals u they atrl~~e to meet both health and psyclqsocial needa. Alma: To ldenrlfy Jnccme aources and health c:osts of aduha with cyslic fibroaia atlendinJ the Southern Alberta Adult CP Clinic and to derme financial llrltegieL Method: A demognaphic: analysia was c:ooducced of 70 CP adults c:onslstina of 36 males (age ranac 18-44, median aac 27) and :W femaleo (age range 18-46, median aae 26). Results: 32 (46") CP clienll hs~~e .a high achool education or lesa c:ontributlnato 18 (26") belna Identified u the worki~l poor and 4 (6%) as social welfare mclpients. This croup may Jack adequste rUWICaal tes<liii"CCI to obcaln medical/drug coverage, meet nutritional mquin:menll, purchase physiotherapy equipment and/or fund COlli of luna transplanaarioo. 46 (66") of the 70 clients are worlcina, 10 (14") hsvlna moderate disease. Merely 27 (39") hs~~e access to prlwte dru& coverage through employment. Only 2 (3)" recci~~e work related Lon& Term Dissbility Insurance while the majority of Jncapocltated adulll access aovemment disability pensims [13 (19")) which provide auhst.onaially lea remun~rion. 27 (39%) H~~e with •IIJ>OUSC or algnincant other while 26 (37%) aolcly fund ~·r Uvina expe ...... 8 (I I") hs~~e returned to li~~e with their porems ror rUWICaaJ and psyclqsocial aupport, being too iU to manace independcnrly. 60 (86%) of the adult population do DOl hsw children. 10 (14") have become poren11 through available options which mquin: them to hs~~e auflicient health and ranancial reoourcea. 38 (~") hs~~e been hospitalized within the last 5 years, with 10 (14") requirinl ongomg funds for home oxyaen and 18 (26") for nutritional supplemen11. 30(43") hs~~e purchased physiotherapy equipmeDI. To pre~~ent or shorten bospit.olizarion within the ~ 5 yw-s, 13 (19%) accessed fundine for home IV therapy and 5 (7") hsve reccaved ga.m~ment f~d home care aervices. 3 (4") are lung transplant mclpienll and 3 (4") are walt-lasted for transplant, mqulrlnaaubst.ornial family and rmancial ~itmenll .. <_:onclusloaa: Considerable financial plannin1 Ia mquired by both claen11 and cluuc starr ID ensure that adequs te financial resources are aw ilable. Stratestes: The Southem Albcna Adult CP Clinic baa addreooed thcoe rmancial ~alle..'ges by: hsvlng the clinic social worker provide Information ora rmancial options mclu~ social ~~~~~~ and reproductivefporentin& choices; addin& a clinic ~rmacast ID moru'?' drug coverage and adherence; oiTerlna evcnin1 clinica for che~ who are w~lcinl ~-anendin& achool; holdina transplant plannin& c:onferences ror ~~~nil and thea~ ramal ... which Include a discuasiora of rmancial Implications; prov~na lnfonna~on 1o transltionina clienta reprdina medicalfdru& coveraae: submissions to IIOCa&l and prlwte agenciea in auppon of client needs; prudent 111e of home IV therapy and homecare aervlcea to pre~~ent or ahorten bospit.olizaalon; and management of provindal&overrunent fundina for newer !hera plea (e.J. Pulmozyme).

440

Pn!gnancy In Women with Cystic Fibrosis: An Emlc Penpeetlve. B. Ge!dmaket Medical College of VA, VA Commonwealth Univ., Richmond, VA, USA.

The purpose of this research was to generate a descriptive theory of the pregnancy experience from the insider perspective of pregnant women with CF. The specific aim of this qualitative, exploratory study was to describe and anal~ the following: the development of knowledge about sexuality; the practice of birth control methods; the informant's feelings about the pregnancy; the centrality of disease throughout the pregnancy; the infonnant's thoughts about becoming a mother; and the informant's perception of use of social support systems. The intent of inquiry precluded use of quantitative methods; a qualitative method allowed for probing of tacit or intuitive knowledge of the respondents. The design and method of reporting was case study. The study's sample consisted of the entire population of pregnant CF patients in a CF Center in a state in the mid­Atlantic region of the USA. Consent was obtained for audio-taped interviews. Data collection included the following per subject: 3 in-depth interviews (conducted during the second and third trimester of pregnancy and 30 days after delivery), participant observation, and document analysis (to detennine severity of illness). IRB approval was obtained. Grounded theory served IS the basis for data analysis. Data were analyzed IS

interviews occurred; transcripts were analyzed for themes individually and in comparison with other interviews. Special attention was paid to the specific context of situation and experience. Similarities and differences across experiences were examined. Internal validity of data was maintained through member checking, prolonged field engagement, and triangulation of data. Preliminary data analysis of second trimester interviews indicated 8 consistent themes associated with participants' views of how they perceived their sexuality and pregnancy. The themes were: need for infonnation; dating; future-death; being pregnant; baby concerns; CF concerns; support systems; and future-work/career. These were contextually embedded in respondents' views of their uncertain illness trajectory and death. Findings from this study may be useful to clinicians in developing counseling and treatment programs for sexually active women with CF.

326 1997 Cystic Fibrosis Conference

441 FACTORS AFFECTING EMPLOYMENT STATUS OF ADULTS WIT!i CF IN

THE UNITED KINGDOM s walters, Department of Public Health & Epidemiology, university of Birmingham, B15 2TT, England

Introduction Adults with CF may experience difficulties entering the employment market and keeping jobs. Revealing CF at interview J!laces adult:s with CF at disadvantage. This study looks at factors affecting employment status among adults with CF. Method confidential postal questionnaire to 1870 adults with CF in 1994 known to the Association of cystic Fibrosis Adults (UK). Response rate was 54ll0. Results 73ll0 of responders had been in full-time employment at some time in their life. The proportion rose with age, and showed no significant sex difference. 46ll0 of responders were currently in employment. The reasons given for not working varied with age an~ sex, but overall, 49!10 of those not employed gave ill health as a reason, with 34ll0 being students (71" in the under 20 age group). The mean number of hours worked was 34 per week, rising with age. The mean length of time in the current iob was 56 months, men significantly longer than -.ten (6~ vs 48 months), non-manual social class longer than manual (59 vs 49 months) and those whose employer knew about their CF longer than those whose employer did not know (59 vs 35), all p values <0.01. The proportion whose employer knew about their CF was 85!11, and this was significantly higher illllong -.ten (90 vs 80!111 p<0.01). 58!11 had less than two weeks' sick leave in the prev1ous year. Employment rates fell at all ages for both sexes between 1990 and 1994. Multiple logistic regression revealed the following factors to be associated with current emplo)llllent status: ever emplo')'ed full time, disease severity, possession of A'Level qualifications, possession of higher educational qualifications. Factors not affecting current employment status were: a9e, sex, revealing CF at interview and possession of 0 Level/GCSE ~ualifications. Model chi-s~uare ~~~cl~~~0C:,001, correct pred ction of employment status 3!11.

A relatively high proportion of CF adults are able to work full tillle at some time during their lives, and almost half were currently working, the majority of these full-time. Men, those in non-manual iobs and those whose employers knew about their CF were in theh current job for longer. Men are more likely to conceal their CF from their employer. A history of ever being employed full time, disease severity and possession of qualifications are associated with current employment status. CF adults should be encouraged to obtain higher qualifications since this enhances tfleir employability.

442 MCARE-PARTNERS" OF ADULT PATIENTS WITH CYSTIC FIBROSIS: ROLES AND RESPONSIBILITIES. I..H&ln:m, J. Lande!, S. Patton, D. Holsclaw •. M. Kaplan, D. Klein •. P. Lomas •. Department of Clinical and Health Psychology, • Department of Pediatrics, Allegheny University of the Health Sciences, Philadelphia, P A. USA.

This study examines several variables which define the tasks and characteristics of people who assist adult patients with CF with their treatment regimen ("care· partners) and the impact of this help on treatment adherence. Currently 21 partners (8 male, 13 female, M A&e • 41.8 years, .512 • 11.7) have completed the study. Partners are distnbuted among 3 categories: spouses ( 48%), significant others (28%); & parents (2Wo), have a mean education of 14.6 yean ® • 2.2); and non· parental partllm have known the patient an average of 12.2 yean (.512 • 7.1). Partners completed the Cystic Fibrosis Panner Responsibility Form (CFPR), a measure of partner involvement with 12 CF regimen tasks; the Caregiver Strain Index (CSI), a 14 item measure of caregiver burden adapted for CF (alpha • . 94 ); and the Profile of Mood States (POMS). PatienlS completed the Cystic Fibrosis Compliance Questionnaire (CFCQ), a self-report measure or adherence to specific regimen behaviors. Lung functioning was used as a measure of CF severity <M FVC"/o predicted • 45.3, m • 32.6; M FEVI% predicted • 39.1, SQ • 20.8, indicating moderately severe lung involvement). Care-partners were responsible for the range of 12 tasks relaled to CF treatment (number of tasks responsible for M• 5.0, SQ • 3.2). The most common responsibilities included actively participating in or reminding patients about dietary modifications (Active M • 68o/o, SQ • .5; Remind M • 59'Yo, .512 • .5) and lung clearance (Active M • 34o/o, st! • .5; Remind M • 55%, st! • .5), as well as providing emotional support <M • 73o/o, st! • .4). Although the partner's relationship to the patient was not significantly related to the overall number of tasks, spouses <M- 6.6, m- 3.0) tended to engage in more active tasks than the other care-partners <M- 4.2, m ·3.1; ~:c2. ">- 3.1.1! < .o7>. Importantly, higher levels of partner responsibility were not related to inmased cmocional diJireu or III8in in the care-partners (l!'s > .05). Next, we examined the role that care-partner uaiataJK:c plays in patient adberence. Significant findings were limited to lllCJlltive IIIIOCiation between partner responsibility for dietary modifications and patients' diet adherence {[ • ·.38, 11 < .05). This counterintuitive flndins may be becaule patients who have had a history of poor dietary adherence may elicit smater helping behavior from partners. Also, those patients who adhere to dietary changes may not need or solicit help from their partners. The role of care­partncn needl to be further lllle5Sed in order to optimaUy utilize this potentially important resoun:e for patienl care.

443 PAIN IN CF PATIENTS: A CONTRACTUAL APPROACH M Bgbbjps D. Chastain, N. Eksterowicz, D. Wilmoth, University of Virginia Medical Center, Charlottesville. VA. USA

Although the phenomenon is poorly reported in the literature, many processes may result in pain in CF patients. In the absence of specific maikets, pain may be ignored by the health care team. Scarrins caused by recum:nt infections. treatment for previous pneumothorax. or surgery can result in chest pain. Abdominal pain can cxx:ur as the result of biliary problems, bloating. ileus, and constipation and may be compounded by the incorrect use of pancreatic enzymes. Arthropathy, as wen as fractures due to low bone density, cause join! pain. Headache can be caused by hypoxemia, hypen:apoia. or sinus infection. Uorcported and undertreated pain can affect CF patienlS in many ways. Nom:ompliance with treatment regimens is perhaps the most obvious. Undertreatment of pain may lead to wbat is perceived as "drug seeking' behavior, resulting in the labeling of patients as narcotic abusers by bealth care providers unfamiliar with pain management. Unfamiliarity with drug dosages and conversions may lead to undertreatment. Narcotics may be misused as substitutes for other treatment modalities, for example in the treatment of anxiety. The individual experience of pain is inlluenccd by psychological and social factors. As CF patienlS transition to adulthood the issues of IJIUICCIJSiomed independence as well as issues of mortality may inftuence percepti0111 of pain. Many have not achieved esc­appropriate communication or coping skiDs. They may feel "out of control". Some feel victimized and entitled to receive wbatever care they demand. Situational depression may compound pain We utilize a multidisciplinary approach to manase the manifestations of pain in our patients. Our team consists of the medical director, nurses, and a psychologist and nurse from our institution's Aalte Pain Service. Our approach bas been to develop patienJ care contracts. These contracts contain I) common soals. 2) expected patient behaviors, 3) care plans for both the inpatient and outpatient setting. and 4) consequences for DOI!almpliance with terms of the contract. The patient participates in the development of the contract; allcr signing by all parties, one copy is given to the patienl and one copy is kept in the hospital chart. The benefits that we have observed as the result of contracting include 1) reduction in narcotic dosase. 2) replacement with IIIOJ'C appropriate medication or treatment modalities, and 3) IIIOJ'C active participation and increased compliance of patients with all aspects of their treatment plan. The challenges that remain are education of pb}'licians and nurses to pain syndromes and treatment in CF patients and the developtnent of appropriate consequences for patients who break the tenns of their contracts.

444* TRANSmON: THE MELBOURNE EXPERIENCE. JA Glazner, L Shea, N Collins, SM Sawyer. Department of Thoracic Medicine and Centre for Adolescent Health, Royal Children's Hospital, Melbourne, Victoria, Australia.

Approximately 400 children and edolescenta attend the CF clinic at the Royal Children's Hospital and 200 adulta attend the Alfred Hospital. The transfer of young adulta with CF between the two centres commenced 12 years ago: during 1997 15 young adults wl!! be transferred. The major psychosoclal!Huea we have Identified In this population Include; fear of the unknown and of change; the codependency that develops between the young person, his or her family end the paediatric care team; the reality of Increasing morbidity and mortality at this time; the changing role of parenta and their acce11 to Information; and the change from a structured environment to an adult oriented model of self care. Clear communication and early preparation underpin the transition process. The strategies we have designed to facllitata transition In this population Include; ( 1) Bimonthly meetings between allied health professionals from the edult and paediatric CF unlta. (2) The organisation of Information meetings for the young adults wtth CF who are transferring and their familial. These meetings are facilitated by adulta who are currenUy treated at the adult centre and are attended by members of both the adult and paediatric CF taama. A tour of the adult unit Ia part of this event. (3) The development of a lnlnsfar document which lncludea Information from the medical consultant, physiotherapist, dleutlan, CF co-ordinator and nursing staff. (4) Development of an education package (The Health Care Maze) designed to asalat young people In the transition of their health care from the paedlatrlc to adult facilities. (5) Transition Alert Letters, designed for thirteen, ftfleen and 118Venteen year olds, are a low key Intervention which aim to provide a reminder to the young person wtth CF, hls or her family and the Department of Thoraclc Medicine to consider certain developmentally approprlata lasuea. This programme Ia a joint Initiative of the Department of Thoraclc Medicine, Centre for Adoleacant Health (Adoleacant Unit) and Special School, Royal Children's Hospital and the Department of Relplratory Medicine, Allred Hospital. Good planning, good communication and goodwill, together with appropriate funding, are the e1ementa vital to ensure the optimal transition between paediatric and adult aervlcea.

445 ADOLESCENCE AND CF. ~ Department of Clinical Psychology, School of Psychology, Univ. ofBinningham, Birmingham, ENGLAND.

Adolc:sccuce is a c:rucial developmental stage. Major developmental goals central to normal maturation iDCiudc the achievcmcnt of independence from puents and the establishment of a sense of identity. The objective of this study was to examine adjustment amongst adolescents with Cystic Fibrosis (Cf). It . was hypothesised that poor self-amccpt. poor parental perception of adjustment and external locus of control would n:late to parental 0\'Crproleetion. Secondly. that internal locus of control would have a posltn'C impact on self-concept and puental perception of adjustment. It was expected that these relationships 'WOUld not be effected by objcctivc illdices of disease le\'Crity or the age and gender of the participants. All adolescents with CF. aged 13 10 16 and their puents attending a routine out-patient appointment at The Birmingham Cbildn:n's Hospital between May 1996 and Janu&JY 1997 when: asked if they would like 10 participate. The nature of lbe study was explained. Of tbe '3 families approached, 40 families participated. Families wen: seen either in their homes, 11 clinic: or on lbe hospital ward. The adolescents completed three qucstioonaires in the form of an interview t~ith the resc:an:ber; Locus of Control Scale for Children (Nowicki .t Slridland, 1973), Offer Self-Image Questionnaire (Offer, Ostrov .t Howard, 1982), and the Parental Bonding Instrwnenl (Parker, Tupling .t BI'OWII, 1979). A short interview 111'81 also completed which included a question on personal perception of illness severity. Parents wen: asked to complete a demographic questionnaire and the Personal A4justmenl and Role SkiUs Scale ill (Stein .t Jessop. 1990). Physical status measurea wen: collected from the clinic visit closest 10 the interview date and from medical n:cordl. This information included; FVC, FE VI, height and weight, age II diagnosis, number of hospital admissions and number of intravcnoua antibiotics in previous yean, details of any complications, and physician rating of prognosis and le\'Crity. Preliminary analysis illdicates then: to be a n:lationship between adolescents perception of fathers' parental style and the following measures; locus of control. aelf-image. pan:ntal perceptions of poycbosocial adjustment. Fwthcr analysis is being undertaken, the results of which will be discussed.

446 AN AUDIT OF THE SUPPORT NEEDS OF THE PARENTAL CARERS OF ADOLESCENTS WITH CYSTIC FIBROSIS AT THE TIME OF TRANSITION FROM PAEDIATRIC TO ADULT CARE ~ The Adult Cystic Fibrosil Centre, Llandough Hospital, Penarth, South Glamorgan CF64 2XX UK

The aim of this study was to examine the support needs of pan:ntal carers of adolesc:ents with cystic fibrosis 11 the time of transition from paediatric 10 adult can: with a view to devising standards of can: to enable a smoother transitional period for both the patient and the parental carer. Twenty-one pan:nll of ldolesc:enll aged 16-20 yean attending an adult cystic fibrosis centre cornpriled tho 1tudy population. The study was designed as an audit and data was collected by postal questionnaire. A n:sponse nrc of 15% (16:21) was echieved. The n:sults indicated that a number of key luuet which presented u difficulties for parenll during tho transitional stage. These issues appeared 10 be related to pan:ntal can:rs perceivina decn:asin& involvement In decision mal<ing regarding tn:abnent n:gimes and comin& 10 terml with a change in their role as the adolescent starts to take control of hislher lllness. These changes had pn:sented u difficulties for 43. 7"~ of the n:spondenll during the transition period. Respondenll also Indicated concern that then: wu a lack of information regarding their son'lidaughter's treabnent and '0% of respondents stated that they did not have sufficient Information regarding state social benefit righll. Of the respondents 62.6% felt lhll access to counselling would have been bcnefacial, 75% would have liked more contact with the cystic fibrosis team and 73% would have liked to have had the opponunity to meet other pan:ntal caren. Although the promotion of Independence and patient self can: an: accepted aims of adult cystic fibrosis units, responses would appear to suggest thll It was the promotion of these concepts which gave rise 10 these particular difficulties for parents during the transition stage. The conclusion of the study iJ that there iJ a aignificant unmet need regarding the support requin:d by the parental caren of adolescents attending the adult cystic fibrosis centre. This datasupporU attempts, such as transition clinics, pre-transfer visits to adult units and a wider Input of health professionals 10 address these identified deficits.

Poster Session Abstracts 327

447 A TWO YEAR FOLLOW-UP STUDY OF COMPLIANCE AND HEALTH STATUS IN CHILDREN WITH CYSTIC FIBROSIS SJL.!d!!l:1, M. Bryon

1, S.L. Madgc1

, A. Prasad1• I. Department of Child Health, Royal Free Hospital, London, UK. 2. CF Unit, Great Onnond Street Hospital for Children, London, UK.

Non-adherence to treatment is a perennial problem in children and adults wit~ CF. Studies ha~e suggested that 20-70o/o may be non-adherent to vanc;>us aspects of the1r treatment and physiotherapy regimes. No long tenn studieS have. shown how non-adherent children fare when compan:d to their more compliant peers. Alms: To compare decline in health status of self confessed poor adherers to those children with good compliance to treatment. To compare decline in health status in children who have received psychological intervention for their non-adherence to those who have not. To compare two different methods of assessing compliance. Methods: Fifty c:hildn:n aged 8.2,- 16.? years (mean 12.8) were assessed by means ofa self­completed quesllonnane, three areas of their treatment regimen were cove'7d by direct and i~dircct q~estioning: physiotherapy, enzyme taking and .d1~tary supplementation. The1r health status was measured using FEV h

C~mpm-Nonn~ (~N) score and body mas1 index (BMI). 10 of these ch1ldn:n were 1dent1 tied by their parents as non-adherent to tn:atment and subsequently seen by a psychologist. Two years later the same questionnaire and health status parameters were rccorded. An additional visual analogue scale of adherence was completed. The children were also asked what they felt were the three worst aspects of having CF. Rnulta: FEV1 decline over the two years for the group as a whole was -4.4% (0.3 7), e1<pressed as mean (SE). Then: was no statistical difference in the lung function decline when !"~.group seen by the psychologist was compared to the n:st nor when those m.111ally assessed as always complying were compared to the remainder. L1kew1se, then: were no differences in CN score or BMI, although there appeared to be a tn:nd towards an improvement in BMI in the psychologist treated group ~d a change in weight centiles of +12 as compared to -8 (p<O.O I). The Visual analogue scale showed compliance of 83% 88% and 6~o/~ for physic;>, enzymes and diet respectively. Indirect questioni~g yielded s1gmficantly d1ffen:nt results to direct questioning. Physiotherapy still rated as the w~rst part of having CF (~4%), however, only 26% of these rated it as the pr1me reason as compan:d to 71% the first time around. Conclusion: A longer study time or m~re se?sit!ve outcome measures may be necessary to show that poor compliance IS hnked to a more rapid decline in health status. However, the improvement in weight and BMI of the children treated by the psychologist lends strong support for early intervention in those suspected of poor compliance.

448 USING OBJECTIVE DATA TO MONITOR AND INCREASE USE OF CHEST PHYSIOTHERAPY (CPT) IN PATIENT EDUCATION. S. D'Angelo, T. McClendon, J. Kanga. Department of Pediatrics, Univ. of Kentucky Medical Center, Lexington, KY, USA.

Adherence to the medical regimen remains a pervasive concern In treatment for cystic fibrosis. Previous research documents that treatment elements that are most Intrusive Into daily life, and which do not consistently provide Immediate benefits, are those most likely to be omitted. Thus, chest physiotherapy is the treatment component In cystic fibrosis most often neglected. This project used objective data unobtrusively gathered from the alternating pressure vest to both monitor adherence and improve patient education. Eighty­five patients, ranging in age from 3 to 35 years participated. This paper reports on 77 patients who completed chest physiotherapy using the updated American Biosystems alternating pressure vest. Patients were prescribed chest physiotherapy by the pediatric pulmonologist, and education was competed by the CF team respiratory therapist. Readings of therapy administered were collected and reported by home health agencies. Adherence was measured as the percent of prescribed therapy completed. At baseline, the average percent of therapy rendered was 56.86%. 32.4% of patients were rated as having good adherence (greater than 70% prescribed therapy completed), 14.3% had moderate adherence (50 - 69%), and 53.2% had unsatisfactory adherence (less than 50% completed). Patients also completed subjective ratings of health status and satisfaction with method of chest therapy.

I

328 1997 Cystic Fibrosis Conference

This data was then used as a cornerstone of patient education to problem solve overcoming barriers to therapy and ways to increase therapy usage. We are now monitoring the effects of our interventions to determine whether objective monitoring can be used as a successful tool in patient education.

449 Tbe Impact of cystic llbraola oo llbiiDI relallollllll ... foster. C L., Bryon., M., and Biser, C.

Department of Psychology, University of Exeter, Exeter, EX4 4HR, Enaland Department of Psycholosical Medicine, Hospital for Sick Children, Great Ormond Street, London, WC IN 3JH, EnJIIIId

Most research investigatins the nature of siblins relationships where one child has a chronic condition suggests that the siblins relationship is compromised due to the physical and emotional demands of havin1 a sick child in the family. Parents may be unable to spend equal amounts of time with their children which could lead to reaentment 111d therefore more aures•ion and disagreements expressed in the oiblinJ relationship. However, in contrut, more recent work suuests that healthy siblin1s may express more empathy and greater appreciation of life thiJI siblinss of well children. This study aims to investiaate the nature of sibling relationships of children and adolescents with CF taking into account positive and nesative aspects of the sibling relationship. 44 well siblinas (mean ase 13 years) of children and adolescents with CF 111d 4S patients (mean ase 13 years) with CF participated in this study. 139 healthy children and adolescents formed the comparison group. All participants completed self-report questionnaires deoigned to assess the nature of their siblins relationships. Well siblings also rated how they felt about the disease and its impact on the family. The results indicate thatsiblins relationships of these children did not differ significantly from the comparison lfOUP· However, certain factors did relate to the nature of the siblins relationship and these include well siblinp' perceptions of the disease and its impact on the family system. Children reportins high levels of auression and disagreements in their siblins relationship were also more likely to report ncsative perceptions of treatment by others. Children reporting positive relationships with their siblings in the form of companionship and tcachins were more likely to feel sad about their siblinp' CF. Children who reported beins able to talk to others about their worries reported more positive encounters with their siblings than children who felt they could not talk to others. These results SUJiest that although as a group these siblinss do not appear to be experiencina poorer relationships thiJI their well peers, certain factors may well contribute to the nature of the relationship and this is likely to be related to the illness. Implications of these findings will be discussed.

Aclaoowledtemeoa Claire Foster is supported by the Economic and Social Research Council.

450 PSYCHOLOGICAL ASSESSMENT FOR TRANSPLANTATION: IS IT POSSIBLE TO WENTIPY SOME OUTCOME PREDICTORS1 B....tlllbili. A. GiuDta. c.ro eli ~o per Ia Fibroli Ciltic:a della Resicme Lombudia, ltliluti Cliaici di ~. Milano, Italy.

Patitnta and their fimiJiel filc:i"'the option of trlnlplantation have to 10 throuih a -.fbi, painfW and clemanditla experience. They have to face thai ia hardly poaible thai their J.hb can improve and 111 impendina death or. lnboopital and home treatment ia maicina them busier and busier; independenc:e.IICidemifljob life and ldf'-eonocpt proareuively decreue while iaolation, far, lid< of control iDc:rese. The ptyllholocial ~t for lrlrtsplantatio .... be aimed to identifY the atrenaJ11a, rilk &cion and psydlopatholosy • when the cue • in the patient• and family tllltllben _.... to lim/her. This --=nt is alto addreuedto iclentilY copilla pattam able to 111ppor1 and .,...,.-. adaptatioa to tnnsplantation. By - of cue iliuttndioaa, it will be llbowed how iDtbrmation emerpd ttom the ptyllholociala-..nt .,. coanected with JIOII-trllllplaiUion aclhennee to treatment, Ulldentandina oflrlrtsplantatio ._ and ability to IWWIIIIthe role of a healtby, incnuiaaiY iDdepeadiDt .,..._. Durina the paychoeocial__. our pllientl unclerao a Wechaler intellipnce ICIJe and Jlf'O.iec:tM lelts itt addition to clinical talks for I complete penonality eYIIualion. TID now I pelieala (3M, SF; II' ruae 12 • 32 ,_..; 6Juna ti'UIIplaDtation and 2 ~ tnnlpllnlationl) have been u.n.pillllecl 1111011& thole followed up at Millll CF c-. Other 2 patienta wwe evaluated and .,. waitina for tranap1antation 4 patienta more .,. underaoina the --=nt proceu. Only cme patient (lillie, 12 ,_..old, u- tnnlpliJIIation) llbowed an intellec:tull ftmctionina below the ...... popu1atioD tnelll (l.Q. Complete Sc:lle 89). No lli8n ofJIIYChopatboloa, u clulillecl by DSM·IV- found iD patienta. Patientt who lhowecl, .a. tnnlplantadon, poor aclhennee to-_.. thole with poor family 111ppor1, psycboloaical aymptoma iD pareat1 and undereltitnltion of the

emotional cost of transplantation considered more like a mechanical manor than a very stressful experience which need a great deal of motivation 111d resources. On the one hand, we ogree that patients with emotional or cognitive problems CIMOt be excluded from tnnspl111tation programs which CIJI save their lives; on the other hand our experience tells us that people with poor family adaptation need not only psycbological111d social support but also substituting intervemions. Clinical sketches concerning tfiJispllllted patients will demonstrate this concept in practice.

451 MEASUREMENT OF SOCIAL WORKER BURNOUT IN CF CENTERS. S. Wise, Social Work Services Departmen~ All Children's Hospital, St. Petersburg, FL, USA.

This study assesses the level of bomout in social workers at CF Centers based on the desiJD of a 1990 study by Coady, Kent, 111d Davis. Demographic characteristics of the lOS CF social workers surveyed by Coady et al. (1990) were compared to the demopaphic characteristics of the 87 CF social workers who participated in this study, a response rate of79%. This study looked at the frequency and percentages of the demopaphic variables of the participants which included age, gender, marital stmus. race, educational leve~ employment status, and job responsibilities 111d compared them with the Coady et al, (1990) responses. The occupational charactcristi<:s of CF social workers in this study were correlated with their burnout scores measured by the Mulach Burnout Inventory (MBI; Maslach & Jackson, 1996). The instrument was specifically designed for hum1111 service providers 111d contains three subscales to measure three components of burnout: emotional exhaustion, depersonalization 111d lack of personal accomplishment. The mCIJI scores 111d standard deviations in each of the subscales were compared to the scores obtained by Coady et al. (1990) 111d to the MBI normative scores. The fmdings indicate thai social workers in the 1990 111d the 1997 groups have lower burnout potential than the MBI groups which could be due to the well orpnized team approach of CF Centers. A one-way -lysis of variiJICC (111ova) was performed on each job variable. The mCIJis of the different variables were found using the Duncan Multiple Range Test. Four of the six job variables including hours per week providins services to CF patients; hours per week providing services to patients with chronic illness; perc:eived supervisory support; 111d perceived team support were sipificantly related to burnout (p<O.OS). A primary sipifiCIJit fmding indicated that respondents reporting support by their team had low burnout scores on the three subscales: emotional exhaustion (F[I,84]•12.SSI,p-.001); depersonalization (F[I,84)•13.024,p-.001); and personal accomplishment (F[1,84]=4.338,p=.040). The consistency between the fmdings of this study 111d the Coady et al. (1990) study clearly supports the relationship between supervisory and interdisciplinary team support and lower rates of burnout

Nursing Issues

452 GENDER DIFFERENCES IN EXERCISE CAPACITY, PULMONARY FUNCTION II WEIGHT AFTER EXERCISE TRAINING IN ADULTS.

~ AC HackDey. General Clinical Research Center, Sch. of Nursing, Dept. ofExen:iae & Sporta Science, University of North Carolina at Chapel Hill, USA.

Adults with Cystic Fibrosis (CF) usc various forms of respiratory therapy 111d exercise at home to maintain exerciae capacity 111d prevent respiratory deterioration. Thoup average ase of survival in CF is now 30 years, males on averqe ~urvive lonaer than females. Rosenfeld et al ( 199S) found relative risk of death wu 1.3S greater in females. Preliminary results have augestcd f'Ktors such u exercise capacity 111d nutrition may contribute to sender differences in Ions-term survival. This is a secondary data &Dalysia of aender differences after a nursins exercise trainins intervention. The intervention was done to maintain exerciae capacity 111d pulmonary flmction durin& the extended isolation for a aene lrlllsfer cliDical trial. The purpose of this -lysis is to compare ch111ges in exerciae capacity, pulmonary function tests (PFT) 111d weight between men and women before and after two-weeks of daily exercise trainins. Twelve adults (X • 29 yrs) were admitted to the GCRC 111d received pre and post testins with llalldard pulmonary function tests and a submaxirnal exerciae llmltcat uaina a modified Balke treadmill protocol, a constant 3 mph speed with four incremental pades of 0%, 2.S%, S'Yo, & 1.S%. Exerciae C8p8City wu measured by heart rate (HR) 111d eatimated VO, response~. An individual daily exen:iae reairnen wu preacribed based on the

submaximal stress test. Excn:i.se training ranged from 7-18 days (mean • 12). Paired t-tests showed a significant increase overall in mean estimated V02 (190 ml) in the post stress test, 11 • .03. When compared by gender, V02 increased 19.4% inS males and only 4.S% in 7 females. There were minimal changes in pre-to-post PFT's; however, males had small increases and females bad slight decreases. Overal~ subjects had a significant increase in body weight at the end of two weeks, 11 • .03; however, males gained 1.8 kg and females gained only O.S kg. In conclusion, results suggest that males maintained or bnproved exercise capacity, pulmonary function, and weight aftet a short monitored exercise 1raining during hospitalization; females had minimal increases in exercise capacity and weight and no improvement in pulmonary function. (Supported by M. Knowles, NIH RR00046.)

453 HOSPITAL SELF ADMINISTRATION OF INTRA VENOUS ANTIBIOTIC THERAPY IN CYSTIC FIBROSIS ~ I Nicholson, D McArthur, AP Greening. Scottish Adult Cystic Fibrosis Service, Western General NHS Trust, Edinburgh, Scotland.

Patients with cystic fibrosis have, in most cases a lifetime of experience in caring for their disease, and often have a greater knowledge of the treatments than many of the personnel caring for them. Once admitted to hospital, control over much of the treatment is removed from the patient, including administration of medicines. The aims of this study were a) to ascertain whether in-patients were receiving their intravenous (i.v.) drugs on time, b) to determine the amount of nursing time spent on preparing i.v. drugs, c) to allow patients the opportunity to self administer i.v. drugs in hospital if desired. ~ 7 patients were asked by means of a questionnaire about the timing of their i.v. drugs during their in-patient stay. Nurses were asked to time themselves preparing and administering patients' i.v. drugs. R.w.!lll In a TID regime 417 respondents reported their night time dose as being > 2 hours late. The morning and afternoon doses were > I hour late in 417 cases respectively. S/7 patients expressed 1 desire for control over their own i. v. drugs when in hospital. Nurses spent 16-50 minutes each time preparing and giving drugs. As a result of these findings, 1 pilot study has now commenced where selected in-patients have total control over the storage, preparation and administration of their i. v. drugs. Locked cupboards have been installed in the patient's rooms and drug kardexes are completed by the patients. Spot checks are carried out by nursing staff to ensure the correct balance of drugs. Early results show an increase in patient satisfaction, 1

saving in nursing time and less disruption to patient's sleep.

454 A NURSING AUDIT OF A NOVEL DRUG IN CYSTIC FIBROSIS (CF) HOME INTRAVENOUS THERAPY FOR PATIENTS COLONISED WITH STAPHYLOCOCCUS AUREUS M Pogson, D Hill•, S Mukhopadhyay. . Departments of Child Health and Microbiology•, Ninewells Hospt!BI and Medical School, Dundee, DOl 9SY, U.K.

Home intravenous antibiotic therapy has been used in the Ninewells Cystic Fibrosis (CF) clinic under supervision of the CF nurse ~ialist since July 1992. Our policy is that all patients who are chrorucally colonised with Pseudomonas aeruginosa (PA) and/or Staph aureus (SA) are treated electively with 2 weeks' intravenous (IV) antibiotics once every 3 months. PA and SA are the two most commonly occurring pathogens cultured from the respiratory tract in patients with CF and are associated with the progressive decline in respiratory function in these patients. The introduction of IV teicoplanin has allowed us to treat SA colonisation at home. Prior to the introduction of this drug, vancomycin, flucloxacillin, fucidin were used. These had many disadvantages including frequency of administration, side effects

Poster Session Abstracts 329

and difficulty in administration at home. In order that our patients could have their IV therapy at home and with minimum disruption we opted to use teicoplanin. This is given as 3 doses 12 hourly followed by a once daily dose for 14 days. It may be given along with an anti· pseudomonal drug if necessary. We have now been using teicoplainin since October 1992. We have compared the change in pulmonary function across patient courses of anti-pseudomonal alone with anti­pseudomonal plus teicoplain in doses ranging from I 0-2S mglkg!dose. Pulmonary function was measured before and after each 14 day course of antibiotics. Sputum was sent for culture and sensitivity pre and post therapy. Serum teicoplanin levels (trough and I hour peak) were also measured on day 7 of therapy. The initial results suggest greater clinical benefit with teicoplanin IS mglkg!dose in comparison to higher and lower dosage schedules. W<! wish to aclcnowledge tlw sup{KI't rec.lved from MIJI"/0#1 M<!Jft/1 Dow (now Hoechst Mar/011 R011n11l) for tlw a~ s'*.

455 LMNG DONOR LUNG TRANSPLANTATION, PREPARATION OF

FAMILY AND WORK-UP OF DONORS AT THE LOCAL CF CENTER J Swartz RN CPN. Vermont CF Center, S.Burlington, VT. USA

Patients and families who are considering a living donOr lung transplant have to cope with deterioratrng Clrnical statui, travel a long distance to a transplant center, and leave behind their familiar aupport systems. Because of Vermont's isolation from the nearest living donor lung transplant centers, we have developed a plan to do the preliminary screening of donors al our institution. We begon the procesa wotn a discussion with the patient and the immediate family to define their values and goals and to outline their available options which may inClude no transplant, continuing on a cadaveric waotrng list woth little hope ol a transplant, and living donor lung transplantation. When the patient and family have made their decision, they then invite. by letter or phone, their extended family and friends to 1 Family Meeting where the chosen options are explained with the help of the CF Center physician and nurse coordinator. For that subset of patients who choose living donor transplant, we describe the risks and benefits of this option, the donor workup, selection of donors, aurgical procedure, and posl-op care and recovery. Donor confidentiality is stressed and additional roles are discussed tnat include fund raising, setting up 1 tax exempt account, travel and lodging arrangements, and child care. Donor. are not asked to volunteer at this meeting but are encouraged to go home for a period of thoughtful contemplation and then to call us if they choose to be 1 donor. Blood typing is then arranged and those who are a match are asked to come for a donor screening package which includes chest xray, spirometry, blood and urine labs, sweat test, and EKG. A psychological evaluation is performed to determine the quality of information and Clarity of commitment. In order to provide confidentiality and freedom from coercion, these tests are offered as 1 package and the resuHs are revealed only to the donor and provided to the primary physician with their consent. The most su~able donor candidates are then chosen by the transplant center to accompany tne patient for final screening and surgery. In the highly charged context of living donor trenaplant, thos process provides an optimum level of Informed consent while maintaining the patient's autonomy.

456 MANAGEMENT OF TOTALLY IMPlANTABLE VENOUS ACCESS DEVICES (11VAD'1) IN PATIENTS WITH CYSTIC FWROSIS (CF); DESCRIP110N OF TWO CASES WITH TIV AD RELATED SUPERIOR VENA CAVA (SVC) SI'ENOSIS. P Tansey, R Coulden, R Appleton, D Biltoa. Adult Cystic Fibrosis Centre, Papworth Hospital, Cambridge, UK.

Totally implantable venous access devices provide Ill aa:ept.t>le alternative to repeated inscrtioa of venous lines in CP patients. However, lily intravenous line has the capacity to invoke an Inflammatory reactioa in the vein wall with subsequent thrombosis. We describe two c:uea where suboptimal line position and delayed interventloa have resulted in SVC Stenosis. Case 1; A 23 year old female with 1 llV AD in situ for 6 years was referred to our centre for continuing CP care. On initial assessment, Oushing the llV AD was exttemely difficult She gave a history of shoulder paiD 12 months previously during IV thetapy with 111bsequent decreasing efficiency of the line. Venography suggested SVC obsttudioa and cr imaging with 30 reconstruclloa revealed a light stenosis of the SVC lithe line tip with extensive llrge collaterals. Venoplasty bas been attempted without success because of inability to pass 1 guide wire through the

330 1997 Cystic Fibrosis Conference

stenosis. Case 2; A 2.5 yeu old female with 1 TIV AD in situ for 7 yws was refened for cmtinuing care. At referral a replacementTIV AD was requested by the patient because of sluggisll Oow. The TIV AD was removed llld 1 new line and port placed. 48 bours following replacement she developed clinical signs of SVC obstruction. Further questlonlag revealed 1 history of intermittent facial swelling on couahln& over the preceding 9 montha. cr angiography revealed an SVC stenosis and evidence of recent c:Jot. Thrombolyals with subsequent heparlnlsatlon was administered to attempt to improve SVC drainage. Although thrombus was resolved the patient required veuoplasty which maintained drainage for leas lhiD 7 days. Bccauae of poor lung func:tion making surgery impossible, llld ongoing c:Jinical signs and symptoms of SVC obslructlon a stent was inserted with good c:linlcal raolution. Theae lwO casca Ulustrate the severe consequences of oomplicatlons of aTIV AD. Euly warning signs of reduc:tion In Oow through the device should prompt thorough lnvestlgatlon. In addition Jines would be placed with the tip in the right atrium to avoid problems of SVC stenosis.

Epidemiology

457 AN UNUSUALLY LOW CYSTIC FIBROSIS PRBV ALENCE RATE IN A MAINLY CAUCASIAN POPULATION. H Cardoso, G. Luzardo, A. Mimbacas, B. Crispino, R. Poggio and I. .AznMez. Divisidn Cito~a. Inst. Invest. Bioi. "Oemente Estable". Montevideo. URUGUAY.

Different from others Latin-American countries, Uruguay bas only a total population of 3 millions people and approximately balf of them are located in its capital city, Montevideo. As un additional difference, DO

Amerindian poups c:an be observed living isolatcd in the country. According to gCIIdical studies (data from 21 blood groups, protein­enzyme and HLA loci), Montevideo is a cosmopolitan population and exhibit a higb degree of Caucasian component (87~ or higher) with about 6-11~ Afric:an admixture and 1-2~ of Amerindian one. The IDiin part of our C1uc•si•n population came from Spain, Italy ud Frmce. Tbcy emigrated to our country in the last 4 centuries. We estimated the prevalence of Cystic: Fibrosis (CF) in 1 sample of~ bellthy iDdividuals from Montevideo. 1bis one was obtained as a strldficd sample bued on private ud public Health Services according to aoc:ial ud ecooomical levels. In a PCR lllllysis we looked for AF508 mutatioa carriers. Only 4 iDdividuals were observed as bclerocygotes for this mutatioll (Ill~). Tbca, the probability to fiDel ooe bomocigous iDdividual for the .6.F508 mutation in our population would be 1/6~. In another band, we bave previously observed 1

frcquellcy of21.1~ of .6.F508/.6.F508 in a sampleofCF patients (40). Tbesc two data, permit us to estimate the prcvaleoce of CF (all mutations) for Montevicleo in I/131S8 individuals with a stllldanl error of 2.667xl0". 1bis ooe is probably due to the uncxpec:ted low number of .6.F508 carriers ud the size of the CF patient sample. A theoretical c:alculalioll bucd 011 edlllic: information would suggest a CF prevalence of 114000. Tbesc results would suggest tbat the observed differeoce bctwccn Soutlt-Europcan and our population are probably explained not only for geaetic admixture but also for otber factors such as founder effect. (Supported by Roche International Ltd,, Uruguay)

458 ELEVEN MUTATIONS ACCOUNT FOR MORE THAN II% OF THE CYSTIC FIBROSIS CHROMOSOMES IN SAGUENAY LAC-SAINT-JEAN (QUEBEC, CANADA). Marc 0t Braeulear (1), C.clle Marl (2), Claudine Verllngue (2), Christian Allard (1), Jean-Pierre Leblanc (1), Fernand Simard (1 ), Gervais Aubin (1 ),Claude Ferec (2) (1) Laboratolre de Recherche sur Ia Fibrose Kystlque, Univ. du Quebec a Chlcoutlml, Chicoutlmi, Canada & Cllnlque de Fibrose Kyatlque, Complexe Hoapitaller de Ia Sagamle, Chlcoutlml, Canada (2) Etabllaaement de Transfusion Sanguine de Bretagne Occidentale, Brest, France

Over the past few years, we have conducted a systematic study of 224 cystic fibrosis (CF) chromosomes in Saguenay Lac-Saint-Jean, a geographically isolated region of northeastern Quebec which has a high CF incidence (11936 live births) and carrier rate (1/15 Inhabitants). We identified 11 mutations accounting for 99.1% of the CF chromosomes. Three mutations, AF508, 621+1G->T, and A455E, accounted for 93.7% of the CF chromosomes. Three other mutations were found more than once; these are the Y1092X (3 chromosomes), 1148T (2 chromosomes) and 711+1G·>T (2 chromosomes) mutations. Five mutations were found only once. At present, two chromosomes remain uncharacterized. Our results indicate that denaturing gradient gel electrophoresis (DGGE) is a powerful method in identifying CF mutations. They have also considerable implications for genetic counselling and molecular characterization of doubtful patients. They make carrier screening technically feasible in this population.

Supported by grants from the Canadian Cystic Fibrosis Foundation, the 'Fondation de I'Universite du Quebec a Chicoutimi', the 'Association Francaise de Lutte contra Ia Mucoviscidose', and the CRI4U007B.

459 FOCUS ON SINUSAL POLYPOSIS IN ADULTS CYSTIC FIBROSIS PATIENTS •• Dl Cicco M., Coocaatlal D.•, Gervaolal N.•• C. Pizzamiglio

II Otorhinolaryngology Institute, University of Milan , Italy • lotitute of Pediatric• - CF Centre University of Milan, Italy •• Dept. of General Medicine • CF Adult Centre - ICP Milan, Italy

Cystic Fibrosis (CF) is characterized by abnormal transeptbelial sodium and chloride transport secondary to mutation of gene coding for the CF transmembrane conductance regulator (CFTR). Upper respiratory tract involvement is a common finding in patients with cynic fibrosis (CF) and an important aspect in the management of this pathology is the treatment of nasal and paranasal sinus disease. In Ibis study we reviewed the clinical experience with adult CF patients with nasal and paranasal sinus disease. We evaluated the long-term results achieved on 96 adults CF patients: in this group the 18% had nasal Polyposis , 89% had chronic sinusitis (X-ray confirmed but with no symptoms) , 20% had symptomatic pansinusitis (headache, sinus pain and pressure, nasal obstruction, congestion and infection (usually with Pseudomonas orJaoism). Tbe imaging was performed always with TC scan of the sinuses in the nasal polyposis group and in the symptomatic siuusitis. The routine use of the endoscope provide the early diagnosis of pathological changes of the lateral nasal wall. The medical approach to nasal polyposis was based on long term steroids (Beclomethasone Dipropionate) and nasal washes with aallne solutions. The best results of this medical therapy were obtenned in the early polyposis with polyps of small dimension ('%). The indication for nasal surgery is mainly on the basis of the history, ENT endoscopy and CT ocan of the sinuses.The surgical approach include polypectomy, etbmoidectomy, antrostomy (5%) or endoscopic surgery (B'Yo). In our series this last tecoique Is a succesful approach to treatment of both sinusitis and nasal polyps in cystic fibrosi1 : it reduces recurrence requiring further suraery for at least 2 years.

460 lllak of Bealp aad MaUpaut Dlleue ID ObUpte CF lleterozyJota. A.B. I.pwenfell, P. Maisonneuve, B. Palys, MH ScMni, B. Redemann, B AJaimo.Orolle. Department of Community and Preventive Medicine, NY Medical Colleae, Valha1la, NY.

1be rlalt oC diaeltive tumors has previously been shown to be elevated in CP patients. In addition, in ~everal other autoeomal recessive disordera, hcterozyaotea have an elevated r1a1t of cancer. Allll: to determine the r1a1t of malipant and non-maHanant dilease

in obligate a heterozygotes. Metbod: We conducted a case­control study of the health status of 1112 parents of persons with CF obtained from an active database (N • 1888) maintained by the International Association of CF Adults. Friends (N•688) of the CF membership constituted the control group. We used logistic regression analysis to compare the frequency of various diseases In cases and controls. For cancer data, we also performed a cohort analysis by comparing the number of observed cancers in CF parents with the expected cases In the background population. Results: The mean age ~SD of CF heterozygotes (53.4!,12.0 yrs) was nearly identical to controls ((54.6!.12.3 yrs). For the case­control study, there was no overall increased risk of cancer (OR • 1.0, 95% CI • 0.7-1.4) The risk of digestive tract cancer was similar in cases and controls: OR • 0.8 (95% CI• 0.4-1.7). For non-malignant disorders, only sinus disease (OR • 1.7, 95% • 1.4-2.3) and allergy (OR • 3.2, 95% CI•l.S-5.6) were reported more frequently in cases than controls. When compared to the background population, the overall risk of cancer In a beterozygotes was somewhat reduced: 91 cancers observed, 112 cancers expected (SIR•0.8, 95% CI • 0.8-1.0). Subgroup analysis revealed that this deficit was caused by a reduction in smoking­related tumors. Conclusions: This study suggests that CF carriers are not at increased risk for any type of cancer. With the exception of sinus disease and allergies, a carriers do not have an increased risk of non-malignant disorders. (Supported by a grant from Solvay Pharmaceuticals, Marietta, GA.)

461 LATE DIAGNOSIS OF CYSTIC FIBROSIS (CF) IN ITALY: A MULTICENTER STUDY. L Romano. C. Vanni, G.Pizzamiglio' R.Padoan', M.Antonelli•, S.Bertasi•, S.Bellodi, A. Villani", G.Mastella" Cystic Fibrosis Centers of Genoa, Milan', Rome• and Verona•,JtaJy

Diagnosis of CF is usually suspected because of symptoms (either typical or uncommon). CF is characterized by wide variations !n presentation mode and age. In the a1m to assess whether ~~a~os1s .m adulthood is related to late symptoms' appearance or to nusd1agnos1s, providing information in order to !mprove diagnostic su~picion and procedures, personal history and chmcal status at d1agnos1s of all CF patients diagnosed ~ 16 yr of age at 4 large Italian CF Centers in the last 7 yr were collected. Data refer to 123 patients (57 females, 66 males; mean age at diagnosis 23,7 yr, range 16-39). Genetic analysis is in course. Lung involvement was present in 97,5% of cases; mean age at respiratory manifestations' onset was 8,7 yr (range 0-36; 90th centile 18 yr). Asthma (14.6%), tuberculosis (13%), sarcoi~osis (6,5%) and generic recurrent bronchitis (51%) and bronch1ect~1s (25%) were the previous misdiagnosis. Only 471117 (40% )of the. ~!Ients had pancreatic insufficiency (PI); mean age at mald1gesuon chmcal onset w~ 3,5 ~r (range 0-34 yr; 50th centile 1 yr, 90th cenllle 7,4 y~). Age at d1agnos1s was not earlier in patients with PI. PI had been prev1ously d1agnosed as coeliac disease or "maldigestion" in 4,1% and 3,3% of cases, respectively. At diagnosis, FEV 1 ranged from 15 .to 143'11>pred (ayerage 65'11>pred). FEY 1 at diagnosis correlated ne1ther w1th age at resp1ratory symptoms appearance nor with age at diagnosis. The weight/weight for height ratio ranged from 70 to 160% (average 98%) and was ~ot related to either age at maldigestion appearance or FEY I at d1agnos1s. Sputum culture at diagnosis was positive in 89,4% of cases; S.aureus (60%), P.aeruginosa (50%) and P.aeruginosa mucoid .v.ariant (5,7%) we~ the commonest isolales. Sweat lest was always poslllve, excepted 6 patients who had a chloride concentration of the sweat in the border-line range (CI 40-60 mEq/1). Only 22 patients (18%) had had a sweat test previously. In 16122 patients (73%) the previous sweat test was positive, but its result had not been recognised. In conclusion, diagnosis of CF in adolescents and adults is usually a delayed diagnosis with respect to symptoms' appearance. Personal history of patients shows th~t misdiagnosis is very frequent; CF is often not taken into account m differential diagnosis, even in the presence of both respiratory and intestinal symptoms. Furthermore, the significance of the sweat test is undervalued. Our data show that, in Italy, the awareness of CF of primary physicians and specialists should be promoted.

Poster Session Abstracts 331

Other CF Care Issues

462 MEASURING QUALITY OF LIFE IN OIILDREN WITII C'VSTJC FIBROSIS: mE CYsTIC

1 FIBROSIS QU~IONNAIRES \CFQ). IL.Jfmly1, C. Groukopf', P.

Aussage , S. de Fonlbnme , J-M. Goehrs and lhe French CFQoL SIUdy Group I AII.COS, fiiiiCO Z l'rodulll ROCHE. f..,..

Cystic Fibrosis (CF) ldvenely affecll palienta' qualily of life from Infancy tJu:ough adult age. Heallh-Related Quality of Life (HRQL) measures applicable to tholdren and ldulll would be most beneficial for lnvestipt011 and clinicians to uscsa lhe multidimensional impoc:t of disease and natment ovenime. llsuco about child HRQL evaluation are now well documented. Should we consider lhe Child Heallh Status from a 10eietal penpeclive, usessina behavior and performance in relation to expec:talions for a pei'IOII of a aiven qe, or should we llllempt to concepiUalize lhe HRQL domain from lhe child viewpoint? Who should be asked, lhe dlild himself despite Vll)'ina cognitive capacilies and possible biues, a proxy responding on his behalf or bolh? And how to reconcile differing judgmeniS? We developped in parallel two questionnaires for evalulllina CF palienllaged 1-13, one exhaustive parent venion conc:enlrltina on behavior, performance and ll)'lllptoms, and a shorter dlild venion focusing on subjective peruplions and emotions. Items were aeneraled from analysis of 22 in!erviews conducted wilh 6-13 y- old CF patients and parenll. Two preliminll)' venions were completed by 14 I pain of palieniS and parenll enrolled in 24 French sites. Questionnaires were analyzed usiJoa descriplive statistica and fac:torial analysis. The 'parenl' queslionnaire was reduced to 46 items exploring 7 QoL dimensions: 'Physical functioning', 'Psychitlinlelpersonal relalions', 'Energy/fllliguelmood', 'Eat ina disiUrbances', 'Body bnage', 'School performance' and 'Treallllenl burden'. Two separate modules assess intensity of ll)'lllptoms and aeneral heallh pen:eplion. Internal consistency, construct validity, convergent and discriminant validity (muhilrlit analysis) have been previously reported, results on reproducibility and responsiveneu as well as comparison between CF patieniJ and genenl population scores will be discussed. The 29-item child questionnaire wu fielded into a JongiiUdinal study. Comprehension and ~bility are excellent. Intermediate analysis on 56 children shows significant correlalions across children scales, excep1 'Body Image'. Agreement between parent and dlild reporta are particularly good for Physical functioning, Eatins disiUrbantes, School anendance, Psychic/Emolional and Body image, but poor on FatigueiEnersY, Treatment burden and Genenl beallh pen:eption. Test-retest among 25 atable young palienll shows aood reproducibility (ICC-o.9S). Final results on factorial strutture, responsiveneu and clinieal validity will be reported. A third CFQ venion is concurrently developed for assessment of ldolesceniS. This comprehensive set of questiOMaires will pro~ide measures of heallh status and subjective quality of life of CF pediatric: patoents trom lhe age of B through !heir life span. Crou-cullural adaptations of the CFQ are developed in several European countries.

463 DEVELOPMENT AND IMPLEMENTATION OF A CYSTIC FIBROSIS CLIMCAL PATHWAY AND TRACKING TOOL. S Rushin&. D. Stokes, S. Cllllpbell, S. Erickson, B. Smith, P. Throop, P. C1111pbell. Dcparll11ellt of Pediatrics, Vanderbilt Children 'a Hospital, Nashville, TN, USA.

Sua:css in managed c:IIIC and excellent clinical practice requires the health system to identify and reduce practice variation and to continuously assess care quality and patient/family satisfaction. We have previously developed a pathway for plCWnOilia in cystic fibrosis (Cf) patients with input from all members of the CF team (Pediatric Pulmooology 1996; 135: 78-9). Out of this process admission and discharge criteria were defmed. A "tracking tool" was then designed to allow us to identifY variation from the pathway. We were able to identifY soun:es of variation with respect to admission criteria, use of ancillary services, and duration of hospitalization. We prospectively identified three measurements (body weight c:hange, oxygen saturation, and spirometry) as objective outcome measures of expected improvement along with an anonymous patienllfamily satisfaction questionnaire. Of 258 CF patients at Vanderbilt from 7/1/96 - 3/1/97,47 CF patients were entered into the pathway for pneumonia. Fmy four patients (94,-.) were admitted to the hospital and three patients (6%) were managed at borne for IV antibiotics without hospitalization. All patients met admission criteria and over 95% were discharged the day they met discharge criteria. All of the evaluable patieniS and families reported readiness for discharge as well as satisfaction with receiving appropriate discharge instruction. Comparable to 1 similar period the previous year, we found 1 substantial reduction in cost of respinlay care, laboratory, and pharmacy. The average c:ost of treatment for a CF pulmooary exacerbation decreased from $21,133 to $11,868 (exc:luding home care costs). Cost rcdlltlion was allributed, in part, to reduced lc:ngth of stay (LOS), once a day aminoglycoside dosing and elimination

I

332 1997 Cystic Fibrosis Conference

ofunnccesSII)' laboratory tests. Average LOS dccrcascd from 13 to 8 days. We conclude that the implemc:ntation of the cystic fibrosis pathway was associated with a reduced hospital LOS and dccreascd costs. The objective measures of care quality and patient/family satisfaction are continuously being followed to dctaminc the effect of these changes. We plan to continue to rcfme the tracking tool, to reduce variation and to document that quality of care is maintained or improved.

464

Cognitive Dysfunetioo in Adults with CF. A. Matt Maddrey. C.M. Cullum, C. Prestidge. Department of Psychiatry, Univ. of Texas Southwestern Medical Center, Dallas, Texas, USA.

The lifespan of patients with CF has historically been quite limited, and, therefore, very little is known about the neuropsychological functioning of individuals with this illness as they develop into adults. Disorders associated with episodic, or chronic hypoxia are known to result in neuropsychological abnonnalities, with particular evidence of memory disturbance (e.g., COPD, sleep apnea). Because cystic fibrosis is a disease which affects the pulmonary system, hypoxia is one outcome of this disease process. In the present study, a computerized screening battery (MicroCog) of neuropsychological tests was administered to a series of 31 CF patients over the age of 17 years. MicroCog includes an assessment of several cognitive domains, including aspects of attention, reasoning/abstraction skills, visuospatial functioning, and memory. Results revealed that 68% of the total sample (Ne31) demonstrated significant impairment in at least one or more MicroCog subtests. In particular, deficits were found on MicroCog measures that involved working memory and/or visuospatial components, when the subjects' scores were compared to existing norms for individuals of the same age and educational background. These findings are among the first to explore cognitive functioning in adults with CF, and suggest that a significant proportion of these patients show evidence of subtle neuropsychological impairment.