Abstracts of Workshops and Posters

Folia Microbiol. 35, 1-110 (1990)

Fifth Congress of Czechoslovak Immunologists

Plzed July 5-9, I988

A B S T R A C T S OF W O R K S H O P S A N D POS TERS

The 5th Congress of Czechoslovak Immunologists was organized by the Czecho- slovak Immunological Society at the Czechoslovak Academy of Sciences and by the Czechoslovak Medical Society of J. E. Purkyn6 (Section for Clinical Immunology and Allergology). The symposium and introductory lectures will be published separately as Proceedings; the Organizing Committee of the Congress asked the Editorial Board of Folia Microbioiogica to publish the abstracts of workshops and posters. A certain selection has been done and the reader will find these abstracts (total 345), together with an authors' index on p. 103.

Abbreviations of institutions occurring in the addresses of authors of the individual congress abstracts

AK AlvIN

BTA CHTF CSAV

DFN FaF

FDL FDNsP

FgU FN

FNsP FP FVL

IHE IKEM ILF

Agrocombinate Academy of Medical Sciences Biotechnoiogical Agrolaboratory Faculty of Chemical Technology Czechoslovak Academy of Sciences Pediatric Faculty Hospital Faculty of pharmacology Faculty of Pediatrics Pediatric Faculty Hospital and Poficlinic Institute of Physiology Faculty Hospital Faculty Hospital and Policlinic Faculty Policlinic Faculty of General Medicine Institute of Hygiene and Epidemiology Institute of Clinical and Experimental Medicine Postgradual Medical and Pharmaceutical Institute (Prague)

2 5th IMMUNOLOGICAL CONGRESS Vol. 35

IMV JEP JZD KHS KO'NZ LD LF LFH MBU MLU M ~ Z NRL NsP OHS OKB OKH OKI OKIA OMRF OUNZ PAN PF P~F RL ROZS SKNV SOKL SUKL SV~T SVU TRN I~EBE OEE OFF

OEO OEVM fJFGHZ

OI-ncr UJEP UK

OKEO fJKO O'MCH UMG O'NBR f.~z UOCHB UP

Institute of Medical Research (of) Jan Evangelista Purkyn~ Agricultural Cooperative Regional Hygiene Centre Regional Institute of National Health Postgraduate Medical and Pharmaceutical Institute (Bratislava) Faculty of Medicine Faculty of Medicine and Hygiene Institute of Microbiology Martin Luther University Municipal Institute of National Health National Reference Laboratory Hospital and Policlinic District Hygiene Centre Department of Clinical Biochemistry Department of Clinical Hematology Department of Clinical Immunology (Prague/Brno) Department of Clinical Immunology and AUergology (Plzefi) Oklahoma Medical Research Foundation DistriCt Institute of National Health Polish Academy of Sciences Faculty of Science (Bratislava) Faculty of Sdence (Prague) Reference Laboratory Resuscitation Department of Emergency Service Regional National Committee of Central Bohemia State Institute for Drug Control (Prague) State Institute for Drug Control (Bratislava) Slovak Technical University State Veterinary Institute (of) Tuberculosis and Respiratory Diseases Institute of Experimental Biology and Ecology Institute of Experimental Endocrinology Institute of Experimental Pharmacology Institute of Experimental Medicine Institute of Experimental Oncology Institute of Experimental Veterinary Medicine Institute of Physiology and Genetics of Farm Animals Institute of Physiology of Farm Animals Institute of Hematology and Blood Transfusion Purkyn6 University Charles University (Prague/Hradec Krfilov~/Plzefi) or Comenius University (Bratislava) Institute of Clinical and Experimental Oncology Institute of Clinical Oncology Institute of Macromolecular Chemistry Institute of Molecular Genetics Institute of Nuclear Biology and Radiochemistry Institute of National Health Institute of Organic Chemistry and Biochemistry Palack~ University

1990 ABBREVIATIONS 3

UPJg UPMD r',SOL U"l" I. CRCH VJ VDL VgCHT VgV VOCHR VUE V0FB vT_rrm VUHEM VUKEO VUL VOLB V67..H VOP V6"PL VURCH v~s v'fyla~

VUVL W Z WOLURCH 7;l~lP ZUNZ

~af~rik University Institute for the Care of Mother and Child Institute of Sera and Vaccine Umverslty of Texas h:stitute of Tuberculosis and Respiratory Diseases ~filitary Medical Research and Postgraduate Institute School of Chemical Technology School of Veterinary Medicine Research Institute of Rheumatic Diseases (Prague) Research Institute of Endocrinology Research Institute of Pharmacology and Biochemistry Research Institute of Human Bioclimatology Military Institute of Hygiene, Epidemiology and Microbiology Research Institute of Clinical and Experimental Oncology Research Institute of Drugs Research Institute of Medical Bionics Research Institute of Forestry Research Institute of Pediatrics Research Institute of Preventive Medicine Research Institute of Rheumatic Diseases (Pier,any) Research Institute of Stomatology Research Institute of Tuberculosis and Respiratory Diseases Research Institute of Veterinary Medicine Scientific and Research Base Woiker's Therapeutic Institute of Respiratory Diseases Complex medical-immunological unit Enterprise Institute of National Health


M~dt~ctorial and Polylunc~ml Clmracfer of Natm,al IImunity

~TEFANOVI(~ J. Department of Microbiology and Immunology, LF UK, Bratislava

The immune response can be conveniently divided into nonspecific (natural) and specific (acquired) mechanisms. The mechanisms of natural immunity are inborn, developmentally older, they react immediately after contacting the microorganisms, do not undergo ananmestic modification, do not possess memory, involve a wide variety of components that are able to inactivate and degrade non-self molecules. These mechanisms are involved in all phases of processing the antigen signal including the effector action on the target structures. Individual factors mutually cooperate and influence even the origin and function of specific immunity. Their structure and function in the maintenance of integrity, individuality and health of the individual is evaluated.

Pbalo~ot~ lqesOon Va/ues/n Cl~lrren wf~ Recurrent/a/ea/om

Clinic of Children's Infectious Diseases, FDNsP, Brno; Department of Pathological Physiology, LF UIEP, Brno

A total of 300 children (176 boys, 124 girls), aged 2 - 1 5 years, were examined. Comparison of four groups of children with recurrent non-streptococcal angina, scarlatina, inflammation of upper and/or lower airways revealed lower values of leukocytes, granulocytes and phagocytotic leukocytes in children suffering from recurrent scarlatina. In addition, in Isoprinosine-treated children, a significant increase of phagocytotic ieukocytes was observed 2 - 3 weeks after the beginning of therapy.

Stimulatory F__~ed el Strepto~ccus faecsUs on Phago~os/s

MArCDEL L., TALA~ArCrOVA M., TREmCHAVSk'Y I., TRAXrtclCEg J. Department of Gnotohiology, MBU C~dA V, Nov~ Hr~dek

A gradual age-dependent increase of peripheral blood leukocyte phagocytotic activity in conventional pigs (after delivery: 0.148 x 109/L of active phagocytes, i = 0.12; one-year-old piglets: 5.010 x 109/L, i = 0.62) was observed; on the other hand, phagocytosis in germ-free piglets remained low within the first year of life (one-year-old: 0.309 x 109/L, i = 0.15). Colonization of germ-free piglets with S. faecalis increased the number of phagocytotic cells (PMN leukocytes) up to 1.795 x 109/L (i = 0.18) within 24 h. Intraperitoneal administration of formalin-treated S. faecalis increased the number of active phagocytes in germ-free piglets to 7.865 x 109/L and the phagocytotic index to 0.74 within 24 h. A similar stimulation of phagocytosis could not be obtained after colonizing germ-free piglets with either E. coil or a mixture of anaerobes. Intraperitoneal administration of formalin-inactivated S. faecalis increased the phagocytotic index even in conventional pigs without increasing the total PMN leukocyte number. In addition, similar treatment increased the phagocytotic index also in cyclophosphamide-treated piglets. Thus, S. faecalis may be considered a natural stimulator of phagocytosis, its effect being based on stimulation of both the number and the activity of PMN leukocytes.

~1~ /a l fon at Gu/nea-P/g ~ e Func6on Us/n K S. aweus Vaec/ne

KOTULOVA D., FEgF_~CIK M., ~;TEFA~OVIC J. Department of Microbiology and Immunology, LF UK ; Institute of Immunology UK, Bratislava

The effect of Stava (SS), stock vaccine (ST) and autovaccine from S. aureus (SA) on guinea-pig macrophages (MA) was tested. The vaccines were administered subcutaneously in volumes of 0.1,0.2 and 0.3 mL, on day 12, 8 and 1 before examination. All three vaccines induced increased peritoneal MA (pMA) phagocytotic activity (PI) ; however, the percentage of phagocytotic cells (PA) has not changed

1990 ABSTRACTS $

after SS treatment and after treatment with SA and ST while PA was significantly decreased. Candidacidal activity (CA) decreased significantly in all three experimental groups, INT reductase activity and superoxide generation were found to be similar as in the controls. A significant decrease of myeloperoxid- ase and lysozyme was recorded after SS, SA and ST treatment which may be the cause of decreased CA and pMA. SS and ST induced a significant increase of INT in alveolar macrophages (aMA); superoxide formation was higher in all three groups. The most pronounced changes were obtained after SS treatment: increased PI and decreased PA and CA. Thus, various staphylococcal vaccines, used routinely, may interfere with various macrophage functions.

Influence ot Various Therspeutic lmmunoglobdn Preparations on the ~ m s ot Natursl lmmnni~

FERENC~K M., SLOBODNtKOVA L., KOTULOVA D., STARgL~ Z., ~TEFANOVI(~ J., STAC'H"~ A. Institute of Immunology, UK ; Department of Microbiology and Immunology LF UK, Bratislava ; Imuna, ~ari~sk6 Micharany

To increase the objectivity of evaluation of efficiency and safety criteria, the ability of various intravenous (Sandoglobulin, Sandoz AG; Gamma-Venin, Venimun, Behring; IVEGA and IGAMA, Imuna) and intramuscular (NORGA, IGAM, Imuna) immunoglobulin preparations to stimulate metabolic activation (INT reduction), superoxide production, release of myeioperoxidase, the phagocytotic activity (opsonization of killed bacteria), phagocytotic index and candidacidal capacity of human peripheral granulocytes, as well as the ability to activate complement in human sera (CHs0), were compared. Data obtained in these tests were scored by points: 0 - most effective, 3 - least effective, or preparation with the highest anticomplementary activity. Based on this score, the preparations examined can be classified in the following way: Sandoglobulin - 3, IGAMA - 8, IVEGA and Gamma-Venin - 9, Ven imun- 11, NORGA and I G A M - 16.

Actirily of Polymorphonndear Leukocytes in the Presence of Defined Immune Complexes (DIC)

~ALXOWCOVA M., ~I~_~ANOWC J., BucHwo~ D. Department o[ Microbiology and Immunology, LF UK, Bratislava

The effect of DIC on phagocytotic and candidacidal activity of human polymorphonuclear leukocytes (PMNL) and on their ability to undergo metabolic activation was studied. DIC (HSA-anti-HSA) were prepared in an HSA : anti-HSA ratio of 1 : 1 and in a 5-, 10-, 20- and 40-fold excess. Preincubation of PMNL with DIC for 45 min resulted in a significant decrease of the PMNL ability to ingest and kill C. albicans. Examination of the respiratory burst revealed that incubation of cells with DIC in the absence of zymosan increased the metabolic burst of PMNL. On the contrary, in the presence of zymosan the values were significantly lower.

The Influence of Antibiotics on the Metabolk Actirity of Phagocytes

~OLTISOVA D., Lor, nJ J., PmLICn J. Institute of Biophysics, C'vdA V, Brno ; FNsP Brno

The immunomodulatory effect of 12 antibiotics at different doses (including therapeutic) on the phagocytotic activity of human granulocytes was studied. Phagocytosis was determined using the luminol-dependent chemiluminescence. Therapeutic concentrations of antibiotics exhibited either stimulatory (clindamycin, oxacillin) or inhibitory (vibramycin, rifampicin, addamycin, erythromycin) or no effects (linkomycin, ampicillin, gentamicin, rolitetracyclin, chloramphenicol, septrin) on the metabolic burst of phagocytes. The immunomodulatory effect depends on antibiotic concentration, type of stimulant used (rye starch, opsonized E. coli) and is more expressed with antibiotics possessing the ability to penetrate inside the potymorphonuclears.


The Effect ot ASatoxlns on Reactlte Oxy~,~'.,~ Products in Polymmphonnclear Leukocytes

13VRAC"KOVA Z., BEROENDI L., RATEr OVA A. Department of Chemistry end Biochemistry, LF UK, Bratislava

The effect of afla:ora ~ BI (AFB~), a hepatotoxic product of A. flavus, on the generation of the superoxide ion radic Jl, J ydrogen peroxide (H202) and INT reduction by human polymorphonuclear leukocytes (PMN) was : tudied during phagocytosis of zymosan (20 particles per PMN). AFBI was added to the mixture ~37 ~ at concentration of 4.2 x 10 -s - 4.2 x 10-" per 3 x 106 PMN. AFB~, added to the mixture prior to zymosan phagocytosis, increased formation of superoxide and H202. In both experimental setups, AFB~ di ~ not influence significantly the INT reduction. Increased generation of the superoxide ion radica, and b ,drogen peroxide may under certain conditions alter the structure and/or the activity of some bio #gic dly important substances.

Alveolar I~ crophages in Immunocompromised Patients

SzR~ I., ~;v~mcovA I., TmaZA L., FI~ER F. VUTRN, t "ague ; Department of TRN ILF, Prague

The properties of human alveolar macrophage (AM) of patients suffering from lung carcinoma or patients subjected to a long-term therapy with corticoids were examined. Using monoclonal antibodies and indirect imm'unofluorescence method, we determined certain AM surface antigens: transferrin receptor, CD14 antigen, DR and DQ antigens. The phagocytotic activity was determined using synthetic methacrylate particles. No differences in DR antigen expression could be found among individual patients. As regards CD 14 and DQ antigens, we observed a high degree of variability of occurrence; in the case of the transferrin receptor, a significantly higher expression was recorded in patients with a disseminated lung process (79 _+ 12 %) as compared with local lung tumours or lymphadenopathies of various aetiology (39 -+ 17 %). No statistically significant differences in the phagocytor activity of AM could be demonstrated in the individual groups.

$igJg~cance of Alveolsr Macroph~e Fc Receptors in Lung smi Ikonchlsl Diseases

PALISA V., HOUDEK J., SEDLA~K D., KRIOAR V., STAJNEB A. Clinic of Infectious Diseases, Clinic of TRN, K(JNZ-FN, Pizeit

The weaker expression and function of alveolar macrophages (AM) Fc receptors in comparison with other, e.g. peritoneal MA, has been repeatedly discussed. We studied alveolar MA, obtained by bronchoalveolar lavage from 25 patients suffering from various lung diseases (bronchitis, pneumonia, bronchiectasia, fibrosis, sarcoidosis and the farmer's lung). Phagocytic activity of lung MA was examined using cadmium microcrystal particles coated with albumin (HSA) or y-globulin (NORGA). No differences in phagocytosis of either particle types were observed which does not suggest a decreased MA Fc receptor function in the above mentioned patients.

Are L j ~ t e s Pbsgoo~o~ ?

~.A~OXaCovA M., TOTH J. Department of Microbiology and Immunology, LF UK, Bratislava

During studies on the phagocytotic capacity of PMN leukocytes, phagocytosis of C. albicans by lymphocytes was accidentally observed. Two types of phagocytotic cells (5 - 7 ttm and 8-10 pro) with an almost round pycnotic nucleus and highly reduced cytoplasm were found in preparations stained according to Wright. In addition, cells with engulfed C. albicans, classified on the basis of morphological criteria as large granular lymphocytes, were also observed. Further experiments, using isolated T and

1990 ABSTRACYS 7

B lymphocytes revealed that approximately 0.4 % of T iymphocytes were able to phagocytose C. albicans.

Components ot Natural lmmma'ty in Tttchomonissis

D ~ P., Go~aogovA A., BUCHVALD D., V ~ M., JANC~KA A., ~ o M. Institute of Immunology, UK Bratislava; Institute of Parasitology, LFUK Bratislava, Clinic of Dermatovenerology, FN Bratislava ; 1st Clinic of Gynaecology and Obstetrics, FN Bra~lava

An in vitro interaction of Tnchomonas vaginalis with various factors of natural human immunity was studied. Differences in the susceptibility of the parasite to the cytotoxic effect of complement depended on the strain used, duration of in vitro cultivation and growth phase. Using a micro-INT test and light microscopy, interaction of T. vaginalis with human peripheral blood PMN leukocytes was also studied. Viable T. vaginalis was not phagocytosed even when opsonized with sublytic concentrations of serum. Killed cells of the parasite induced a metabolic burst of leukocytes, on the other hand, living protozoa did not activate the metabolism of phagocytes.

V~dn~ ot Natunll l m m ~ t ~ PatluDetelT in BrondJoJlveolJr Lavage

BORANSK'I' J., KOCAN I., BLAnOVA A. Department of Microbiology and Immunology, LF UK, Martin

Various parameters of natural immunity were studied in the bronchoalveolar lavage of rabbits who had received a single dose of a living culture of Pseudomonas aeruginosa (2 mL~ 109 bacteria per mL) intrapulmonally. The results, obtained at time zero, and after 0.25, 3, 7, 14 and 21 d following the infection were compared in the serum and bronchoalveolar lavage. The iavage possessed an antibactedai activity on days 7 and 14. Significantly increased lysozyme levels were found in the lavage on day 14 and in the serum on days 3, 7, 14 and 21. Total hemolytic complement (CH~) could not be demonstrated in the lavage. On the other hand, the following complement changes were recorded in the serum: a significant decrease on the 6th day, followed by an increase on the 14th and 21st days. In addition, an increased number of alveolar macrophages was found in the lavage on the 3rd, 7th and 14th day. Furthermore, antibody titres (determined by passive hemagglutination), spectrum of lipoproteins (HDL, VLDL LDL, Tg) and indicators of the acute phase of inflammation (PMNL, FW, T) were also determined.

Role 0t Fibronectian in the Nanspecific Immune Syste~

Pn0c'HA M., VOTROBA T., RAKOSNIK P. Department of Immunology, VUHEM, Prague

Fibronectin is a plasma protein playing a significant role in the functional ability of the macrophage-histiocyte system. Changes in fibronectin concentrations occur in a number of pathological conditions including sepsis. The authors isolated fibronectin using gelatin-Sepharose. Antibody to fibronectin was used for fibronectin determination in a group of 50 surgical patients (before and after surgery) and in a control group. The influence of operation on the fibronectin level was determined and fibronectin changes were found to be related to the occurrence of postoperative infectious complications.

Determination ot Adhesln K88 in a Strain Conslracted by Means ot Recombinant DNA

HOLODA E., MIKULA I. Department of Microbiology, Immunology and Veter/nary Hygiene, V~V, Ko~ice

A gene, encoding the K88 antigen, was isolated from field strains; the gone was subsequently incorporated into the pBR 322 vector. Recombinant DNA was transformed into E. coil strains HB 101

8 5th IMMUNOLOGICAL CONGRESS VoI. 35

and C 600. Expression of K88 antigen in various cultivation media was quantitatively tested using passive hemagglutination. The results revealed differences in K88 antigen expression in the field strains and in host strains carrying recombinant DNA.

Acthqty ot N o ~ Immr~ty Factors ~ Some Eaterobscteris

SIE(}FRIED L., PUZOVA H., JAN~OOVA V., I)UROVlCOVA J., HI_AuCovA J., HOCMANovA M. Department of Hygiene, Epidemiology and Microbiology, LF UPJS, Ko~ice ; OHS Michalovce

Phagocytosis and serum bactericidal activity against E. coli and klebsiella strains was tested in healthy individuals. The bactericidal activity was examined in 100 strains. In addition, phagocytosis was tested also in serum-resistant strains. Phagocytosis was determined by the fluorescence and plate method in 20 a-hemolytic E. coli and 20 ldebsiella strains. A 5 % and 100 % serum was used for opsonization of strains. Most of the strains examined were found to be serum-resistant. Despite the fact that the 100 % serum increased PMN leukocyte bactericidal activity, complete killing was not obtained with any of the strains used.

CIr. Follow-up Study is ,~ted Smrgicsl Stress $itmtJom

PALISA V., KRIOAR V., STAJNER A., KLECKA J., PODZIMEK A. Clinic of Infectious Diseases and Clinic of Surgery, KUNZ-FN, Plzefi

The authors followed 36 patients, divided into 3 groups according to the severity of the surgical stress. The first group included less severe operations (hernia, plastic surgery, retention of testes), the second group comprised patients with medium stress (cholecystectomy, explorative laparotomy), the third group included severe operations (resection of large intestine, stomach and lungs). CRP was measured before the operation and during subsequent five clays at 24-h intervals. It was found that in patients who underwent comparable operation, the CRP level was always higher in old patients compared with the young ones. It thus follows that tissues of elderly individuals are more fragile and that the operation procedure produces higher damage. Thus, CRP may be considered to be a criterion of surgical stress.

$igq~omce oI CRP Detenni~tion in lYdtetentiM Diagnosis ot Meningitis

~;TRUNcovA V., SEDLA~K D., PALISA V., STAJNER A. Clinic of Infectious Diseases, KUNZ-FN, P/zeh

In order to facilitate the diagnosis of the meningeal syndrome, determination of serum CRP levels was used. Common clinical examination, i.e. determination of body temperature, FW, differential leukocyte count and examination of cerebrospinal fluid was performed in 43 patients suffering from bacterial meningitis and in 19 patients with the aseptic meningitis syndrome. It was found that CRP determination may distinguish the aseptic meningitis from the bacterial one with high sensitivity (97 %) and specificity. CRP determination was proved to be the most sensitive test of all the laboratory determinations used.

Bindlag of Monoclomd AntlTmclics and the C3 Fragment to Complement Receptors

ZIDKOVA J. Department of Immunology, MBU CSA V, Prague

Complement receptors of CR1 type on the surface of human erythrocytes and receptors CR1 and CR3 on the monocyte cell line U-937 were studied. Monoclonal antibodies E11 (CR1), Leul5 and/or MN41 (CR3) and fragments of the C3 complement component were used for detection. The C3 component of human complement has two alleles (F and S). Presumably, these two alleles possess different biological activity. Therefore, the polymorphism of C3 was studied using electrophoresis in agaxose. Common C3 phenotypes (C3F, C3FS, C3S) and their fragments (C3b, iC3h) were isolated and their binding constants

1990 ABSTRACTS 9

determined. The affinity constants of C3b were of the order of 107 L/mol, while the iC3b constants were by one order lower - 106 L]mol. The F allotype had a higher affinity constant. The control monocional antibody El 1 had an affinity constant of 1.5 x l0 s L/mol with a maximum of 950 CRI per erythrocyte. The cell line U-937 possessed a higher affinity constant for CR3 after stimulation with phorbol ester, the number of CR3 being unchanged.

Activation of the Classical sod Alternative Complement Psthwsys by R.Strsins o! Grnm.Neptive Baaerl~

DL~nAr V., gI~CXOVA J., ~ I. Department of Immunology, MBU C"SA V, Prague ; UPMD, Prague

Most R-strains of gram-negative bacteria are killed in the presence of antibody by both the classical and alternative complement activation pathways. In the absence of antibodies, i.e. in precolostral newborn piglets, the ability of complement activation by various strains is different. Studies of 53 R-strains of Escherichia and Salmonella with defined surface structure revealed that the bacteridical effect of piglet serum can be completely blocked by specific antiserum against porcine Clq in 27 strains only. Removal of factor D of the alternative complement pathway by filtration of the piglet serum in a Sephadex G-75 column, resulted in the loss of the bactericidal activity in 13 strains. The extracellular phosphomannan of H. capsulata inhibited bactericidal activity in 15 strains. These results indicate that the mechanism of the bactericidal reaction exhibits great qualitative and quantitative differences in individual R-strains.

Effects of Physksl snd Chemical Fsctors o n the Activ/ff of Com#emeat

MLrRGA~OVA I., ~TE~ANOWt J., STAR~L~ Z., ABSOLONOVA O., BUCHVALO D. Institute of Immunology, UK Bratislava ; Department of Microbiology and Immunology, LF UK, Bratislava

Activity of complement (C) and its components (C3, C4) and of indicators of human serum proteolytic systems were studied with respect to pH of the medium and the presence of organic solvents (CHCla, Et20). Activity of C was measured by determination of CHso units (according to Mayer), the alternative pathway by radial hemolysis in agarose, C3 and C4 components, a~-AT and ~2-M by radial immunodiffusion. It was found that the activity of C depended on pH and temperature and was inhibited by organic solvents. Lower pH, decreased also the C activity, the changes being irreversible. At low pH values, an instant C inactivation occurred; a temperature decrease had a mild inhibitory effect. The C3 and C4 levels were changed at low pH within 5 minutes, at pH 3.5 the components were undetectable. Proteinase inhibitors could be detected in serum even at low pH values.

Polymorphism of Fsctor B of the Complement System: AJ/otype Freqnem'y/n the $/ovsk Polm/affoa

STANEKOVA D., STAR~IA Z. Department of Microbiology and Immunology, LF UK, Bratislava

The complement system is characterized by a high polymorphism of the individual components, regulators and receptors. From the medical point of view, of particular importance is the polymorphism of three complement components (factor B, C4, C2), whose genes are present on the 6th chromosome in the region of the HLA system, and determine its complotype. The aim of the present study was to establish the polymorphism of factor B in a selected Slovak population using a high-voltage electrophoresis in agarose with subsequent immunofixation. A total of 112 healthy, genetically unrelated individuals were examined. The presence of two main alleles F (fast) and S (slow) and two side alleles F1 and S1 could be demonstrated at the following frequencies: BfS = 0.799, BfF -- 0.151, BfF1 = 0.0044, BfS1 = 0.0178. Thus genealogical studies confirmed the autosomal codominant character of heredity of factor B alleles.


/ahem Clq ~ n ~ Associated wlth Systemic Lupus Ery~emstedes I,, Members ot Two G y p s y

F m a a ~

STAt~IA Z., 2rr~AN D., MozoLovA D., Buc M., DLUHOLUCKY S., TOMXCOvA E., Nw,~ M., TOTH J., ~;TEFANOW~: J. Department of Microbiology and Immunology, LF UK, Bratislava ; VORCH Pier,any; 1st Pediatric Clinic Brat/s/ava ; K ~ t Z Bansk~ Bystrica ; O ~ Z Rimavsk~ Sobota

An inborn C|q deficit was found in lO members of two families of Gypsy origin. In family A. two brothers out of 4 children (8 and 10-year-old) had a homozygous defect; in family B, one of 3 sisters (7-year-old). The complement deficit was associated in all cases with SLE. In addition, high CIC and Cl-inhibitor levels were found in all homozygotes, accompanied by an extremely decreased NK-cell activity, presence of the LE-cell phenomenon and ANF. As regards clinical symptoms, the butterfly-like exanthema, arthritis, vasculitis, photosensitivity and acral dystrophic or even sclerotic changes were found. This inborn defect was manifested in the homozygotic form due to kindred marriages between the first-generation cousins - heterozygotes of Clq deficiency. Further transfer of the defect to descendants is of autosomal recessive character.

The S/ga/ftcam~ lind Fncffoa ot Aml/ffen-Presentiag Ce//s hi the /mmue Reactions

V~TWOCA V., I~UHOVA B. Department of Immunology, MBO CSA V, Prague

Antigen-presenting cells play the main role in the induction of the immune response as they determine the character and level of the immune response. A number of cell subtypes are involved in antigen presentation (macrophages, B-lymphocytes, epithelial cells, fibroblasts); however, not all antigens are presented by these cells. The optimal course of presentation is influenced by a number of factors - expression of membrane markers, endocytosis, enzyme equipment, antigen processing and its presentation to immunocompetent cells.

An~eu Recogn~n by T L p ~ ? t e 5

REJNEK J., TUC'XOVA L., KOSTKA J. Department of Immunology, MI]~.I (~SA V, Prague

The T-lymphocyte receptor for antigen (TcR) was identified in T-cell clones possessing specific molecules for a given clone on the cell membrane. The development of gene analysis techniques permitted isolation of 15 and a genes encoding chains of this molecule, in which rearrangement of segments occurs similarly to genes for Ig chains. The originally isolated a gene could not be demonstrated in most T lymphocytes and was therefore renamed y gene. The TcRct gene was isolated later. The genetically restricted TcR, present on most T cells, is a heterodimer composed of a and l~ chains (40-50 kg/mol) which is in the membrane joined with the CD3 structure that is responsible for mediation of the activation signal induced by binding of the MHC product-antigen complex. Recently, the Y gene product was demonstrated in a low percentage of thymocytes and T lymphocytes possessing CD3, lacking, however, the TcRct, I~ heterodimer. This product was isolated together with a further polypeptide chain called 8, which is a product of gene X. It seems that the TcR3', 8 heterodimer does not undergo genetic restriction, it occurs in multiple forms differing in molar mass of the 3' chain and appears in early ontogeny of thymocytes prior to TcRa, 15; its function is unknown at present.

1990 ABSTRACTS 11

Sa'~n Dff/erenc~/n the Re~onse to Ant/gen Adm/n/sa~t/on in

ROSSMANN P., RIBovA B., RlnA I., V~x'I~.A V. Department or Immunology, MBU OSA V, Prague

The liver of mouse strains A/J and C57BL/10seSn was examined after primary and secondary antigen administration (100 ~g ARS-BGG i.p., the second dose after 14 days) using fight and immunofluorescent microscopy 5 rain, 1 h, 24 h, 7 and 14 d after the primary and secondary immunization. The primary antigen administration resulted in a sporadic occurrence of bovine and murine Ig granules in the Kupffer cells (1, 24 h), or the results were negative. Secondary immunization yielded a significant intrasinusoidai accumulation of granular aggregates of both murine and bovine IgG reaching a peak after 1 h; the aggregates disappeared after 7-14 days. The response was significantly weaker and more variable in strain B 10. Microscopic changes found in the B10 strain were restricted to hypertrophy of Kupffer cells, whereas in some A/J mice voluminous globular masses of precipitate with focal necrosis of the parenchyme occurred within 24 h. Thus, immunohistochemical changes after repeated antigen administration are stronger in A/J than in B10 mice and are accompanied by damage of the fiver tissue.

Polyelonal Aetivntion of B LymphotTtes by B ~ t e r i a 1 1 2 p o p o ~

HOFMAN J. Department of Immunology, MBU t~SA V, Prague

Polycionai activation of B lymphocytes results in secretion of lgM antibody against a number of antigens. Lipopolysaccharideoinduced secretion occurs already within 24-48 h after activation. Serum antibody titres as well as the number of plaque-forming cells against antigens tested are higher than in specific antigen-induced immune response. However, the polycional activation does not induce im- munoiogicai memory for the specific antigen. Mice immunized by two antigen doses bring about a secondary IgG response within 20 days. However, when lipopolysaccharide is given as the first, and specific antigen as the second dose, lgM antibody response increases in the primary reaction; however, the IgG response remains unchanged. The polyclonal activator is unable to differentiate B lymphocytes into the typical IgG secondary reaction, not even in the presence of T helper factors, injected during immunization.

Peripheral Molecules involved in Cell Recognition of SpontDeous L~/totozktty (NK, lO

Pc~sp1$~ M., KC~RYC'm J., CA~A M., BEZOU~;IC~ K., FI~;~OVA A., NovXx M., TABORSK~' O. Department or Immunology, MB(7 (~SA V, Prague; Institute of l~crobiology, University of P/sa; Department of Biochemistry, P~F UK Prague; Institute of V/ro/ogy, SA V, Bratislava

Certain iectins, released from the cell surface by EDTA and Triton X-100 were characterized. Molecules of a lectin fraction, found in NK cells and iymphocytes after isolation with EDTA and separation by high-power gel filtration, have molar mass of 65 kg/moi. Only the fraction present in a chromatographic profile obtained from NK cells possessed biological activity. Amino acid composition of both peripheral lectins differs in the acid to basic amino acids ratio. The isoelectdc point for the NK-specific lectin is 5.8, for the lymphocyte lectin 6.7. Both lectius differ also in the binding constant for the D-galactose terminal ligand (NK-specific lectin has a 100 times higher affinity). In addition, two other molecules (M 180 and 85 kg/mol) were isolated on immobilized mannan. The 180 kg/mol molecule corresponds to a previously isolated lectin from liver and is related to the recognition by NK cells.


Molec~es of PerlpbemI Fc Receptors Imvelved h. A a l ~ m ~ . ~ n t CenMsrO/totoxicity (ADCC) o t L ~

KUBRYCHT J., POSP|gIL M., BEZOUgKA K., TABOnSK'i' O. Department of Immunology, MB(J ~$A V, Prague; Depm'tment of Biochemistry, I~F UK, Prague

Porcine lymphocytes were treated with EDTA (10 retool/L, 0 ~ 10 rain), transferred to normal medium and their ADCC activity was determined. EDTA-treated cells lost their cytotoxic activity which could be restored by addition of molecules, eluted with EDTA from lymphocytes in a medium with normalized Mg 2+ and Ca2+concentrations. Fcy and Fc~t receptors were demonstrated in the fraction of molecules liberated by EDTA; such molecules were also isolated from various populations of porcine peripheral blood leuk,'r Isolated Fe? and Fctt receptors restored (similarly as the whole protein fraction) the cytotoxic activity of human and pig lymphocytes treated with EDTA (0.2 mg/L). Experiments with the most active preparation revealed the ability of molecules to influence the activity of untreated lymphocytes still at a concentration of 10 ng/mL. The molecules of this preparation bound selectively to the cell fraction, from which the preparation was isolated.

Cylo~ox/e Mom~k~md AJt ibod~ ~ Lymphocyte Ant/seas/n Cstt/e

SIMON M., DUgINSK~ R. OFHZ SA V, Koffice, Laboratory Ivanka pri Dunaji

Murine monoclonal antibodies (MAb) recognizing polymorphous antigenic determinants of bovine lymphocytes were obtained after fusion of SO2/0 cells and spleen cells obtained from BALB/c mice immunized with bovine lymphocytes. Five MAb of IgG isotype exhibited cytotoxic activity in bovine lymphocytes. Two MAb had a high association with BoLA antigens of class Iw8 or W 12. In other MAb, no clear correlation with BoLA antigens could be demonstrated.

Distribution of Immuaoglobulin. and MHC Antigen.Beming Cells in Bronchial and Lymphoid Tissues o t ~

FIRL~L~ V., DVOOAK P., M ~ R P. OFGHZ CSA V, Lib~chov

The presence of cells bearing the main Ig classes and class II (Ia) MHC antigens was studied in cryostat sections of the lung tissue and in the bronchoalveolar lavage (BAL) fluid obtained from healthy pigs and from pigs suffering from massive bronchopneumonia of unknown etiology. The above structures were detected by indirect immunofluorescence and immunoenzymically. Monoclonal antibodies against IgM, IgG and IgA porcine Ig isotypes were employed. Class II antigens of the SLA complex were detected by interspecies-reacting monocional antibodies against human HLA. As regards BAL of healthy pigs, approximately 70 % of Ia- and IgM-positive cells (mostly macrophages) and 10-15 % lymphocytes as well as PMN leukocytes were found. Up to 40 % of lymphocytes could be found in diseased animals.

Polyclom~! and ~ d c Stimulation of B Ceils by Bscterial Antigens

PROKE~OVA L., JOHN C. Institute of Medical Microbiology and Immunology, FVL UK, Prague

Bacteria contain in their surface layer (or release into the medium) a number of substances that may stimulate lymphocytes either specifically, following the interaction of an antigenic determinant with the receptor for antigen on the lymphocyte surface, or they can act as polyclonal activators. Polyclonal activation embraces a much larger lymphocyte population than antigenic stimulation can do, being limited to immunocompetent cells of certain specificity only. Substances of bacterial origin are mostly polyclonal

1990 ABSTRAC-q'S 13

activators of B cells. However, stimulation of B cells by various bacterial activators, has different requirements as to the cooperation with T cells. The exact mechanism of polyclonal activation is as yet unknown ; further studies of this phenomenon are needed for better understanding of the lymphocyte activation mechanism. Polycional activation is obviously involved in the defense of the organism. Bacterial polycIonal activators are used for the examination of reactivity of various lymphocyte subpopnlations.

Formation and Propert/~ at IgA and Its Subclasses.

TLASKALOVA H., ZIKAN J. Department of Immunology, MBU CSA V, Prague

The mucous membrane surfaces, where the first and most abundant contact between antigens of the external and the internal environment takes place, are protected by a complex of nonspecific and specific factors. One of the most important defense mechanisms is the production of IgA. Its daily output exceeds the production of all immunoglobulins of other isotypes. The major source of dimeric IgA-synthesizing cells are Peyer's patches that supply all secretory organs with these cells. The dimeric IgA is transported actively from secretory organs across the epithelial cells into secretions, or, in rodents, across hepatocytes into bile. The receptor for polymeric immunoglobulins and its proteolytic cleavage product, i.e. the secretory component that remains covalently bound to the dimeric IgA, play the key role in the above mechanism. Human IgA consists of two subclasses - IgA1 and IgA2, differing in the hinge region, saccharide components, disulfide bond arrangement and further properties such as susceptibility to bacterial proteinases, catabolism and distribution in secretions and tissues.

Interesting Conseqaenees o~ the Mathematical Model of the/Immune Res~ase

JtLEK M., PRmRYLOVA D., WANmWSKI J. Department of Immunology, MBf_J C~SA V, Prague; Institute of Biocybernetics and Biomedical Engineering, PAN, Warsaw

An earlier mathematical model of the immune response to normal antigenic stimulation was adapted to describe the origin of autotolerance and autoimmunity. Various prerequisites, leading to the origin of autotolerance, result in a number of further interesting consequences. The model of autoimmunlty establishment makes it possible to simulate the effects of plasmapheresis (removal of antibodies from the system) and immunosuppression (represented by a low intensity of cell division). The simulated plasmapheresis results in a stimulation of the system and in a rapid return to the original state (mostly due to stimulation of memory cells) ; immunosuppression leads to gradual decrease of the antibody level.

~bo d /e s a ~ t Pord~/gG AJ/o~s

ZaKAN J., DVOltAKOVA D., TRAVNi~XK J., Hum'? J., DVOraK P. Department of Immunology, MBU C~SA V, Prague ; (JFGHZ C"SA V, Libg'ehov

Immunization of miniature pigs (stock Lib6chov and Nov~ Hrfidek) with pig IgG yielded precipitating and nonprecipitating antibodies against IgG. Family studies of interaction of these antibodies with IgM confirmed that antibodies react with IgG ailotypes. All these antibodies interact with the Fcy-fragment (mostly with the C,3 domain). The nonprecipitating antibodies interact with IgG molecules of wide charge heterogeneity, while precipitating antibodies precipitate negatively charged IgG subclasses only. Comparison of these antibodies with sera possessing known allotypic IgH specificity revealed that nonprecipitating antibodies react with the antigen of IGH1-A2 or IGH2-B1 specificity. Precipitating antibodies detected the aliotype IGH3-C1 and a new IgH specificity. Nonprecipitating antibodies cross-react with this new IgH specificity. Further experiments provided characteristics of murine monoclonal antibody against porcine IgG allotypes.


Development ot Secretory lgA and Secretory Antibo~es in the S~iva oi

VANCIKOVA Z., ]~kDNIKovA-LoDINOVA R., TLASKALOVA H., MANCAL P., BARTEK J. MBU C'SA V, UPMD, USOL, Prague ; OKEO, Brae

Using the ELISA assay, the onset of slgA,~sIgM and secretory antibodies against E. coil was followed in the saliva of infants from birth up to the age of 6 months. Both the breast- and bottle-fed infants were examined; a part of infants was artiticially colonized with a non-pathogenic E. coil strain 083. The secretory immunoglobulin levels were highly variable. Artificial colonization with E. coli induced in bottle-fed infants an increase of secretory immunoglobulin levels while in other groups no increase was recorded.

61amses/a the M~rota/m/ar Appamtw of Msrhe Ly~cytew &m~ the/~mme Re~pome

Bn.EJ M., VgrWCKA V., ~tMA P. Department of Immunology, MBU CSA V, Prague

Murine peritoneal macrophages and lymphocytes have a characteristic morphological arrangement of the microtubular system. During in vitro cultivation in an antigen-free medium, 10-20 % of macrophages and lymphocytes form uropods with an axial bundle of microtubules. The proportion of cells with uropods increased significantly following immunization or during incubation in an antigen-containing medium. In addition, in thymus and spleen cells, the binding of radio-labelled monoclonal antibody against tubulin increases 2 to 3 times. Obviously, activation of immunocytes with antigen is accompanied by changes in the amount of polymerized tubulin and by reorganization of the microtubular system.

hmnm~nn~zlblaabn by Tsrgeted Drags. !. In Vivo Phsrmscololi~ Activity with Simsltueomly Deoeased Nonspecitic Togl~ty o! the Bo~d Cytomac Drug

i :~ovA B., S~RORm.~ J., U~mc~ K., ROSSMAm~ P., VL~rWCKA V., K o ~ x J. Department of Immunology, MBU CSA V; UMCH CSA V, Prague

Targeted drugs are synthetic substances with prolonged action, enhanced accumulation in the target tissue and removed undesirable side toxicity. The efficiency of a drug composed of daunomycin (DNM), antioThy 1.2 and Ia antibodies and a soluble copolymer (on the basis of N-(2-hydroxypropyl)meth- acrylamide) was studied. The composition of side sequences is decisive for controlled release of the drug. Undergradable sequences render the preparation ineffective. Drugs with degradable sequences decreased the primary reaction against SRBC in mice by 80 % similarly to free DNM. The~inding that DNM has a eytotoxicity decreased to 1 -2 % (tested by decreased number of haematopoietic precursors in the bone marrow) is of high importance. Morphological analysis confirmed the pharmacological activity of the polymeric drug with degradable sequences and absence of undersirable nonspeeific effect of DNM on liver Kupffer cells and heart muscle.

l m m w n o ~ t J O n by Targeted Drags. 11. MeebB~m ot Action

MBU CSAV, ~ : B R CSAV,/KEM, ~V/C/-/CSAV, ~ a ~ e

The targeted drug DNM-HPMA-anti Thy 1.2 antibody decreases the in vitro antibody response against SRBC already 10 rain after contact with splenocytes. In order to obtain a significant effect, a 3-h incubation is required. Such a time period corresponds to a maximum accumulation of 12SI-DNM in target organs. Free lzSI-DNM is rapidly eliminated (half-time 25-30 rain) from the blood, lungs, liver, heart, spleen, kidney and the bone marrow. The targeted I~I-DNM persists in the blood substantially longer and at higher levels. In the spleen, liver and thymus, accumulation reaches its peak after 2 h. When rabbit y globulin is used instead of specific antibodies, accumulation is significant particularly in the liver while in

1990 ABSTRACTS 15

the spleen and thymus it is 2 - 4 times lower. After administration of both the degradable and undergradable targeted drug, 125I-DNM is present in the blood in the form of high-molar-mass complexes 4 h after administration which suggests that the degradable products are eliminated by urinary secretion. Neither free ~25I-DNM nor ~25I could be detected in the blood.

Different Elfects of 5.FU on lmmsm)competent Cells of bbred Mome Strsim A/$ D d Bslb/c and a lgG.Deflcient Straim BIO

Department of Immunology, MB(J CSA V, Prague; OMRF, Oklahoma City

The effect of 5-FU on the antibody response to SRBC was studied in strains A/J, Balb/c and in an IgG-deficient strain B10. Administration of 120 mg/kg 5-FU 24 h before antigen injection inhibited IgM PFC in strains A/J and Balb/c, in the B 10 strain the number of IgM PFC remained unchanged. In order to suppress the IgM response, 5-FU must be given 12-72 h after antigen injection. Administration of gradually decreasing 5-FU doses showed that the lower susceptibility of IgM PFC in the B10 strain is caused by faster elimination of metabolites which supports faster restoration of the IgM reaction (1 - 2 days earlier). The IgG isotype is completely inhibited in the primary reaction in all strains. A single 5-FU dose, given 24 h after SRBC, influences the secondary response even after several weeks. The number of IgM PFC is higher in the secondary reaction, the IgG isotype is permanently suppressed. The results indicate that the IgG isotype is more susceptible to 5-FU treatment, its suppression is long-lasting which is obviously due to limited formation of memory cells during the primary reaction. The susceptibility of immunocompetent cells differs significantly in various genotypes.

Differences in the Ptoteo~k Activity of Peritoneal Exudste Celb in Vm~bus Mouse Inbred Strsim

L~pr4~z C., LANO~R J., RInOvA B., VgrWCKA V. Institute of Biochemistry MLU, Halle ( GDR ) ; MB(J CSA V, Prague

The proteolytic activity was determined at optimum pH 9.5 in a suspension of noustimulated peritoneal exudate cells (35-42 % macrophages, 50-65 % lymphocytes, 0.5-8 % mast ce!ls). Using azocasein substrates and 14C-labelled B. megaterium cytoplasmic protein it was established that at given pH the strains of mice used (CBA/J, C3H/He, A/J, B10, (AJ x B10)F1, B6D2FI, B10.LP, C3H, CBA), differed in the overall proteolytic activity. Addition of soybean and lima bean trypsin inhibitors produced an almost 100 % inhibition which suggests the proteolytic activity of serine type. Treatment with EDTA (10 mmoi/L) or o-phenanthrolin (1 retool/L) decreased the overall proteolytic activity by 30-40 % in all strains. This indicates the involvement of divalent ions. The proteolytic activity with a pH optimum at 9.5 is further inhibited by PMSF, Dip-F, chymostatin, DTE and TPCK. Leupeptin, antipain, pepstatin, E-64 and phosphoramidon ~ e ineffe&ive.

Modulation of Ecto.5'.nndeotidsse n d ATPsse Systems dm~g the ~ B d Mftogenic Cell Reaction

gozAzovA H., govA~O H., govARO F. Department of Onotobiology, MBU CSA V, Nov~ Ht~dek

The early phase of cell stimulation both with the alloantigen or mitogenic lectin is associated with changes of surface cell structures. Ecto-5'-nucleotidase (5'N), Ca2+-ATPasc and Na+/K+-ATPase are integral proteins, the activity of which changes duringthe above events. Spleen lymphocytes and brain cells isolated from genetically different inbred mouse strains A/0 la and C3H/Cbi/Bom were tested using enzyme assays. A significant inhibition of 5'N activity was recorded during the allogeneic reaction of spleen lymphocytes or brain cells and also during the reaction of these cells with ConA. On the other hand, the activity of Ca2+-ATPase and Na+/K+-ATPase was highly stimulated by allogeneic or mitogenic (ConA, PHA) stimulation of brain cells or lymphocytes.


The Effect of fmmoaomodalators on the Antibody Respome in M~ce

SvoaonovA M., ~)0LA K., NOUZA K. (.~EM CSA V, Prague

Cyclophosphamide administration 2 - 3 days prior to antigen injection significantly decreased formation of IgM and IgG hemagglutinating antibodies on the 5th and 11th day after immunization with SRBC. Cyclophosphamide, when administered 10-21 days prior to immunization, had a weak inhibitory effect. Splenectomy produced a slight immunosuppression in normal mice but it significantly potentiated the effect of cyclophosphamide. The monoclonal anti-Thy-l.2 antibody, administered 28 days before immunization, produced a pronounced IgG depression on the 5th day after the antigenic stimulus; maximum depression of IgM and IgG antibodies was obtained on the 5th and 11 th day when anti-Thy-l.2 was given on days -3 , - 2 and -1 . Double-stranded RNA showed both positive and negative immunomodulatory effect in nonirradiated and 3 C,y-irradiated mice, the effect being dependent on the time of administration. The Corynebacteriam parvum vaccine did not influence antibody formation against SRBC but it stimulated the IgM-IgG switch after immunization with lipopolysaccharide. Positive immunomodulating ~ubstances stimulated antibody formation also in individuals immunosuppressed by whole-body irradiation or by monoclonal anti-Thy-1.2 antibody.

The Major liistocompatibilJty Complex

IVA~KOVA E. IKEM, Prague

Considerable progress, contributing to a more precise definition of the major histocompatibility complex (MHC) has been achieved by introducing a wide spectrum of methods. Serological methods are employed for the determination of antigens of HLA-A, B and C (class I), DR, DQ, DP (class 1I) and the so called "I-like" (class IV - an analogy of Oa, T la of marine H-2) loci. Special procedures are used for the definition of class III of C2 and C4 component, Bf factor of the properdin system and 21 -hydroxylase. Further methods, i.e. biochemical, cytotoxic T-lymphocyte clones, Southern blot and DNA sequence, determine detailed structures of MHC subgroup antigens and, in addition, associations between MHC and certain diseases. Changes in HLA antigen expression are related to the clinical course of tumour diseases and rejection reactions in transplantations. Match in MHC antigens improves survival of the graft regardless of immunosuppression used. Involvement of MHC in the immunological reactivity of the individual and its general importance is well illustrated by the results of basic and applied research. MHC is involved in the formation of lymphocyte subpopulations and complement, in the origin of antibodies, survival after infectious diseases and effects of therapy used.

Ditlerentilltion Aat~ens of Haman Leukoeytes

Ho~.Jgi V. L?e/G t~AV, Prague

Considerable progress in recognition and identification of surface antigens of various human leukocyte types has been achieved by the wide use of monoclonal antibodies. So fax, at least 100 various types have been identified and probably many more remain unknown. Introduction of the "CD nomenclature" removed the terminological chaos existing in the field. In most molecules, the complete amino acid sequence was described and in a number of them even their function was determined (receptors for antigen, adhesive molecules, receptors for lymphokines and growth factors, Fc receptors). The employ- ment of these molecules as specific markers for identification of various subpopulations of normal or malignant transformed lymphocytes is of practical importance. For that purpose the following antigens are used : CD3, CD2, CD4, CD8, CD45R, CDw29 (T lymphocytes and their subpopulations), CD19, CD20, CD21, CD40, CD5 (B lymphocytes and their subpopulatious), a number of myeloid cell antigens,

1990 ABSTRACTS 17

CD9, CD10, CD33, CD34 (diagnostically important markers of some leukemias), CD30, CD25, 4F2, TR (important "activation antigens").

Natural HLA AntibodYes

MAJs~ A. OttKT, Prague

Natural HLA antibodies were determined in the sera of healthy males and females using the method of Tongio et al., including examination of monocytes. Out of 643 serum samples, 10.4 % sera were positive; further detailed examination showed the reaction to be either weakly monocytic or anti-HI.A-DR. The type of specificity of antibodies was determined in the remaining 21 sera (= 3 %). HLA-A and B cytotoxins were found in 16 sera; cytotoxins, however, could not be detected by cytotoxic tests using peripheral lymphocytes. Nine sera were obtained from males, in whom previous immunization could be excluded; antibodies found in these individuals can thus be considered to be natural antibodies.

Determination of HLA.A,B Antigem on Erythrocytcs

MMs~ A. UI-IKT, Prague

The problem of the presence of HLA-A,B antigens on erythrocytes is still open. We performed examination of HLA-A,B antigens on erythrocyles of healthy individuals and patients with various blood diseases by a direct antiglobulin test on a plate using HLA-polyspecific and monospecific antisera. The presence of HLA antigens could be reliably proven with some polyspecific sera only and not in all the erythrocytes used. Positive reactions were rare with 1 - 3 polyspecific sera. Positive reactions with a larger number of polyspecific sera were exceptional. In some cases, HLA-A or B antigen could be determined even by the corresponding rnonospecific serum. It follows that the HLA antigens are present on erythrocytes of some individuals but mostly in a weak form.

Ontogeuy of CIJm 11 Ttansplantmtiou Antigens

TP~alCHA VSk~ I. Department of Gnotobiology, MBU CSA V, Novy I-lrddek

Class If transplantation antigens are membrane proteins playing an important role in antigen recogntion and origin of the immune response. During ontogeny of the human immune system, they appear at three periods : in the 4th week of gestation in the yolk sac, in the 6th week in liver hematopoietic progenitors, and in the 8th week in periarteriolar regions of the nonlymphatic spleen and in thymus epitheiial cells. The first HLA-DR (DP, DQ)oproducing cells occur in the human embryo on the axis of stem cell migration and precede lymphocyte migration.

Strophantin-induced Enhancement ol ItLA.DR Expression

Tm~mcttAvsr~" I., govA~O H., ZAmU~e"KOVA M. Department of Gnotobiology, MB(.] t~SA V, Novy Hr~dek

Strophantin (ouabain) increases intracellular Na § concentration similarly to lipopolysaccharide (LPS). We found that both substances enhanced the amount of HLA-DR in the cell membrane. As regards human lymphocytes, the optimum effect was achieved with ouabain concentration of 0. I ~tmol/L given together with LPS (10 rag/L). Enhanced expression is manifested also by increased frequency of HLA-DR § cells after 12 h cultivation with a peak after 24 h. Ouabain increases simultaneously the number of cells with membrane IgM. Similar, but less expressed effects are indilced by quercetin (it increases [Ca2+]i) and valinomycin (it decreases [K+]i).

18 5th IMMUNOLOGICAL CONGRE~ Vol. 35

c ' ~ ~ n o . . ,.. mt.A4>/1~ kkn, nty

Pommsi<A S., Bt~ M., Bm~covA M., Nvtn.Assy ~., ~T~XNOW~: J. LF UK, ZL/P VOLB, Bratislava

Induction of cytotoxie lymphocytes (CTL) is controlled by genes of the I-ILA complex. These cells are generated provided the responding and stimulating individuals differ in gene products of the HLA-D locus. There exist several exceptions from this rule. When examining a husband and a wife before kidney transplantation, we found that this couple differed in class I HLA antigens, hut was identical in HLA-DR antigens. Negative MLC test confirmed the serological results. However, surprising was the positivity of the CIVIL test. Subsequently, the PLT test confirmed that the proliferation stimulus was provided in this case not by HLA-DR but by DP antigens.

ot ~bnodon~ A n ~ k 8 ~ / ~ m n ~ o ~ e ~ k e u s

Ho~.~l V., Ka~roFovA H., B~hL V., ~T~XNOVA I., ~ r t ~ O V A P., I-Imo~aT I. OMG CSAV, Prague

In the past 4 years, we prepared and characterized monocionai antibodies (MAb) against a number of already recognized surface antigens of human ieukocytes: B2M-01 and -02 (l$-2-microgiobulin), MEM-09 and -40 (Ig-x), MEM-50 (IgM), MEM-60 (Ig-k), MEM-12, -24G, -32B, -36 (HLA-DR), MEM-15 and -18 (CD14), MEM-28 (CD45), MEM-56 and -93 (CD45R), MEM-31 (CD8), MEM-32 (CD5), MEM-57 (CD3), MEM-59 (CD43), MEM-75 (receptor for transferrin), MEM-78 (CD10), MEM-25, -30, -83 (probably CDlla). Most of these MAb can be used for practical purposes instead of the corresponding commercial MAb. The MEM-57 MAb might be probably used even therapeutically as an immunosuppressive agent similarly to OKT3 MAb. In addition, furhter MAb recognizing obviously new, not yet described antigens, were prepared: MEM-43 (glycoprotein 18-25 kg/mol), MEM-48 (glycoprotein 110 kg/mol), MEM-53 (glycoprotein 40 kg/mol). These antigens were isolated and characterized biochemicaUy. MAb MEM-55 and MEM-58 recognize saccharide epitopes of antigen CIM5.

Detenn/maon o! LCA (CD451 bo/otms and Tbek DMerenl~b/Expres~n am Human Neopim~ l temtol~eae Cell Lines

Sm>t.Ax J., D t ~ J., Pt.~KovA I., CMOBVATH B. OEO SA V, Bratislava

Expression of isoforms of leukocyte common antigen (LCA, CD45), i.e. 2 - 4 polypeptide chains in the region of 170-220 kg/mol, on various human neoplastic hematopoietic cell fines was studied using Western blotting with anti-LCA monoclonal antibodies Bra55 (IgGt) and Brali (IgGt). Visualization of the bond was done by immunoperoxidase staining of ~I-radiolabelled protein A. Non-T, non-B ALL cell lines (NALL 1, REH 6, KM 3) and B-cell lines (JOK 1, U 698, DG 75, BALL 1) expressed LCA isoforms with higher molar mass compared with T-leukemic (MOLT 4, SKW 3, Jurkat) and myeloid leukemic cell lines (HL 60, K 562).

Le~oofle C D ~ Anagen V ~ Ret-ol~l~l by Monoaou/Amgbod~

B,~n. V., I'/n.G~T I., K~roFovA H., Ho~,m~l V. 0MG L'~SAV, Prague

Monoeional antibodies (MAb) reacting with various forms of giycoprotein CY)45 were prepared and characterized, g A b MEM-28 recognizes an epitope that is common to all forms (M -- 220, 200,190 and 180 kg/mol), MAb MEM-55 and MEM-93 react specifically only with forms with M--220 and 200 kg/mol, expressed on B lymphocytes and a subpopulation of T lymphocytes (CIM5R). MAb

1990 ABSTRACTS 19

MEM-55 and MEM-58 recognize saccharide epitopes occurring on different leukocyte subpopulations and are present in various forms of glycoprotein CD45. A similar saccharide epitope is recognized also by antibody UCGL-1, reacting, however, with the 180 kg/mol form of C'D45 only. Significant/n v/tro functional effects of MAb-55 and btEM-58 (Kraft eta/., Leukocyte Typing Ill, 1987, 864) suggest that corresponding saeeharide epitopes might be of functional importance and that these MAb can define new subpopulatious of leukocytes, differing from those characterized by expression of forms CD45R and UCHL-1.

r ot Lu tecy te Amalee/ t lK~.4J

~ T ~ V A I., ILm~OFOVA H., HIt~mT I., Hot~.~I V. OMG CSA V, Prague

Murine monodonal antibody (MAb) MEM-43 (IgG2a) recognizing an antigen that is present (expressed) on all peripheral leukocytes (most strongly on granulocytes), erythrocytes and several cell lines (e.g. HPB-AI~, MOLT-4, HL-60), but is absent in several other lines (e.g. U937, Naim-6, Daldo, Rail), was prepared. MAb MEM-43 precipitated an antigen (molar mass 18-25 kg/mol) from the lyzate of superfidally labelled HPB-ALL cells that was noncovalently associated with a further subunit (80 kg/mol). Using repeated immuno-affinity chromatography on immobilized MAb MEM-43, a glycoprotein (M -- 18-25 kg/mol), composed of a 14 kg/mol polypepfide chain modified by N-glycosidically bound saecharide chains of the complex type, was isolated from various sources. The sequence of 17 N-terminal amino acids had no similarity with any protein sequenced so far. The function of the new leukocyte antigen as well as the nature of the 80 kg/moi associated subunit are under study; at the same time, it is attempted to clone the corresponding eDNA.

De t e t~n of Bevarne/~erenelaaen ~ by /14emdem/Anabed~

~ r ~ , R., SnvJON M. OFHZ SA V, Ko~ice, Laboratory lvanka pri Dunaji

Monodonal antibodies (MAb) of the IgG isotype were obtained after fusion of murine SP2/0 cells with spleen cells of BALB/c mice immunized with bovine lymphocytes. Immunofluorescence and FACS analysis revealed that 9 MAb recognized monomorphous antigenic determinants in a certain part (20-50 %) of the lymphocytes only. Two MAb detected 90 % of adherent (B) and 5 % of nonadherent (T) population. Further MAb reacted with 40 % of B and 8 % of T cells. In remaining 6 MAb no significant difference in their reactivity with T or B cells could be demonstrated. These MAb bound preferentially to B cells but they reacted also with 30-60 % of the T-cell population.

E t l e ~ ol E a m ~ on ~ ! ~ a t . l ~ t Uter~ ~ i V q i m

BUKOVSK't A., ~ J. ~ , Prague

MHC I molecules were detected in the rat uterus mucous membrane during the whole ovarian cycle; in the vagina, however, they disappeared in the preovulation period that is characterized by high circulating estrogen levels. Ovarectomy resulted in disappearance of MHC I molecules from the uterus mucous membrane and in their significant expression in the vagina. Estradiol administration to ovarectomized female rats led to MHC I expression in the uterus and to MHC I disappearance from the vaginal mucous membrane. These results suggest that MHC I expression in the tissues tested was modulated by the effect of estrogens that stimulated the presence of MHC I in the utex-~Js and st~ppress~d its presence in the vagina. It was also found that estradiol administration in the postnatal period alte, r-~,~ thz ability of adult female rats to respond rapidly by chac.ges in the expression of l'~-IC i i~ *he ':t~ :~: ,'.,6 ",~T':~': ~Stel" ovarec~c:~y, which favours the epigenetic establishment of the mechanism ~.,::~; :~ir ~ ~.~LC ~ ,~.::~-~~ss~,~a i:a th~*~ tissues.

2 0 5th IMMUNOLOGICAL CONGRESS Vol. 35

The F, ffea of lmmusomod~ators #,, HLA-H AnSge, Ex~,t, saon

Mu~ozovA H., Russ G., Buc M. LF UK, Institute of Virology SA V, Bratislava

Changes in the HLA- I antigen expressivity were stt~died by the RIA method. Interleukin-2 enhanced the expression of all HLA-II antigen classes in both the stimulated and nonstimulated cells. Vermiculin enhanced the expressivity of this antigen at low concentrations, high concentrations were immunosuppressive. Novozir and tetracycline decreased the density of Ia-antigens on cell surfaces. The immunosuppressive effect of Labetalole was observed in HLA-DO antigens only; density of HLA-DR and HLA-DP antigens remained unchanged. Determination of changes in Ia-antigen expressivity can be employed in studies of the role of the HLA complex in the origin of autoimmune diseases.

Biochemica .ha Im~unolo#caf ~radles of the Expression of Cer t~ DMerent~tion A.tigem ot ltmmm Leukem/c and Lymphonm Ce//s ~ in Vitro/ndm-ed D~erenl~don

DURAJ J., SEr)LAK J., POLAKOVA K., PLr~KovA I., KARPATOVA M., C-MORVATB B. UEO SA V Bratislava

Expression of cell surface differentiation antigens (LCA, CALLA, HLA-DR, transferrin receptor) and changes in their levels in an in vitro differentiation induced by 12-Q-tetradecanoylphorbol-13- acetate (TPA) in non-T, non-B REH6 (ALL), HL60 (APL) and U937 (histiocytic iymphoma), was studied by indirect immunofluorescence, single- and double-spaced SDS-PAGE of glycoprotein im- munoprecipitates, visualization of proteins after transfer to NC membrane, pulse-chase assays - using the corresponding monoclonal antibodies. The analysis confirmed a profound decrease of CALLA (CD10) and increase of HLA-DR antigens, as well as a slight increase of certain myelomonocytic antigens in REH6-TPA cells. Immunoblotting and pulse-chase immunoprecipitin studies of the common leukocytic antigen (LCA, CD45) showed the presence of various LCA isoforms and their different expression and maturation in cells studied following induction with TPA.

lmmunogeneffe StarVes m Hypertro~ ~ y o l m t b y

SArOLOVA H., GREOOR P., IVAgKOVA E., C"ERVENKA V., KtWKOVA L., W~D~SK~r P., VtgEK V., KAgLIK J. IKEM, FH UK, Prague

A total of 12 families with 48 patients suffering from hypertrophic cardiomyopathy were examined. The occurrence of class I and II HLA antigens was followed with respect to clinical and morphological criteria according to the NYI-IA classification, myocard thickness and the presence or absence of obstruction. The following antigens were found frequently: B21 (p < 0.001, RR 7.1), A23 (p < 0.05, RR 5.8) and B17 (p < 0.05, RR 2.9); in unrelated patients the following antigens occurred: B21 (p < 0.001, RR 4.9), DR2 (p < 0.05, RR 2.6)and B17 (p < 0.05, RR 1.8). The nonobstructive form of cardiomyopathy occurred mostly in carriers of antigen B17 (p < 0.0001, RR 5.9). The group with stage NYHA I + II included primarily patients with antigens B21 (p < 0.001, RR 6.4), B17 (p < 0.01, RR 3.6), AI (p < 0.05, RR 2.1) and DRw6 (p < 0.05, RR 2.4), while patients with NYHA stage I I I + IV were Awl9-positive (p < 0.05, RR 3.2). The results document the relationship of certain HLA antigens to hypertrophic cardiomyopathy which can be exploited in clinical practice.

H L A - C W 4 - a R/sk Factor in Venom Thrombosis

KUPKOVA L., PREROVSK'<r I., IVAgKOVA E., SAJDLOV./~ H., FArrOvA A., ~ R ~ V., SLAV~K M. Orthopedic Clinic, IKEM, Prague

We followed 150 patients after a total hip replacement where venous thrombosis is a common post-operative complication. Venous thrombosis was diagnosed by a test employing labelled fibrinogen

1990 A BSTRACTS 21

125 (FUT). The patients were examined for 41 class I HLA antigens. In the group studied, 44.6 % of patients had a positive FUT test. HLA-Cw4 occurred in 33.3 % of patients with positive FUT as compared with 18.3 % in FUT-negative patients (p < 0.001, RR 2.8; the total Cw4 occurrence in the group of patients was 15.38 %). HLA-B35 was present in 27.5 % patients with thrombosis and in 16.4 % patients without thrombosis. The results of the present prospective study are identical with those obtained in three independent retrospective studies that proved association between HLA-Cw4 and the throm- boembolic disease.

Coe~kia - a I00 % HLA-Assoaated Di~eme

IVA~;KOVA E . TLASKALOVA H., FRIC P., SAJDLOVA H., DVO~,AK M., KUPKOVA L., SLABY J. IKEM ; MBU C~SA V; Department of Gastroenterology, FVL UK, Prague

Coeliakia is a disease with a significant hereditary predisposition and with a known association with the major histocompatibility system. We examined class I and II HLA system antigens in 28 patients. HLA DQ2 was found in all patients (p < 0.001); A1, A23, B8, DR3 and DR7 occurred frequently, B7, DQ1, DR2 rarely. The table shows the occurrence of these antigens in patients (P), controls (C), the statistical significance and the relative risk (RR) :

A1 A23 B8 DR3 DR7 B7 DQ1 DR2

P, % 50.0 14.3 67.8 89.3 60.7 7.1 17.8 0 C, % 28.1 2.0 16.7 16.1 30.7 23.3 54.2 27.7 p < 0.05 0.01 0.001 0.001 0.01 0.05 0.001 0.001 RR 2.5 8.0 10.0 43.2 3.4 0.2 0.2 -

All patients were carriers of DR3 or DR7, sometimesof DR3 + DR7. A frequent occurrence of 138, DR3 and decreased occurrence of B7 and absence of DR2 corresponds to the findings in insulin-dependent diabetes. A 100 % occurrence of DO2 points to the etiological dependence of this disease on HLA.

Novotee CP 1000 Computer.Ass~ed Ertdenee ~ d F~..~-~ ~lion of Sara

KupKovA L., TSCrm~OSTEa E., IVAgKOVA E., SAJDLOV~ I-I. IKEM, Prague

Sufficient supply of sera with various specificities is a prerequisite for functioning of a laboratory for immanogenetics. Sera containing antibodies used for HLA antigen determination are mostly obtained from women after delivery. Selection and production of ABC and DR sera is in principle similar. However, obtaining sera with DR specificity is much more difficult and time-consuming. All sera obtained are analyzed and registered and selected characteristics of these sera are recorded. Finally, suitable sera are selected into the typing panel. A program for evidence and selection of sera was elaborated, employing the data base system dBASE HI Plus (Aston-Tate Company). The program media make it possible to store information about individual sera, perform their actualization and print the stored data. Thus the program permits selection of sera according to previously selected criteria and properties.

B - ~ ~ i l t e d RL~l~l~On O! Jmmgno/'~lG~l'~/y tO ~ m ~ k ~

C ~ A M . , l ~ , ~ ~ P., P ~ M., 7 _ ~ o I., G A S ~ M., S ~ I S., ~ v ~ D I G. Institute of Microbiology and General Pathology, University of P/sa (Italy); Department of/mmunolo- gy, MBU CSA V, Prague

C57BL/6 mice infected with 2 x 107 CFU of Mycobacter/um bov/s strain BCG Pasteur (BCG) either i.v. or s.c. were challenged with PPD in the hind footpad 14 d later. Mice infected s.c. were able to mount

5th IMMUNOLOGICAL CONGRESS Vol. 35

a specific delayed-type hypersensitivity response (DTH), whereas those infected i.v. were not. Multiple- step spleen cell transfer studies were done to establish the cellular basis underlying the suppression, which turned out to be mediated by at least three cell types: idiotype-positive (id § anti-PPD B lymphocytes ( 1st day after infection), anti-idiotype (anti-id) B lymphocytes (4th day after infection) and T lympbocytes (Ts id +, antigen-specific). These cells arising at different times during the infectious process, appear to belong to the same immunosuppressive circuit. In cell transfer studies, splenic B and T lymphocytes involved in suppression of DTH to BCG were also found to be capable of inhibiting the granulomatons response to this microorganism.

Vlsm,/~lton ot N e m - M m e s ~ / ~ m f of Ac t~ /a C e ~ ~ t ~ / s , mm,e SyRem

Zs R., ROMANOVSK~" A., MAC~ J., HABROVA V., PALECEK J., NEDVIDEK J. Department of Animal Physiology and Developmental Biology, I~F UK, Prague

Lymphocytes contain mainly two nonmnscle forms of actin, NMI5 and NMy. Antisera obtained by immunization of rabbits with synthetic peptides, coding the N-terminal parts of both isoforms, where the maximum of sequence differences is located, were used for their Io~liT~tion by immunofluorescence. Paraformaldehyde (3 %) and Triton X-100 (1%) were used for optimum fixation of the blood cell smears. With cells of the lymphocyte line, both forms were homogeneously distributed in the cytoplasm; as to macrophages, sfress filaments stretching in the direction of cell pseudopodium formation were observed.

New ~ for Dymum~ Stmks of ~ mwI Ly~pImtle Tlmw ta Vkm

~TFJ~L I., ~ ~ O V A I., F ~ o v A A., PosrL~ M. VUE, Prague; Department of Immunology, MBU CSA V, Prague

The ACUSYST system (USA) permits studies of cell cultures, tissues and small organs under continuous culture conditions. The dynamics of TGL (thyreoglobulin), T3 (L-3',5'-triiodothyronine) and T4 (L-tyrosine) formation was studied. In addition, fiberation of cAMP from cells after a pulse stimulation of hypofunctional adenoma of the thyroid after TSH in a 2- and 6-day culture was examined. Under the given conditions, not only the stimnlatory, but also the inhibitory phase of the substance tested can be followed. A similar system was employed for the production of LAK cells by using recombinant lymphokines (rlL-2, interferons). Comparison of activities of cell populations obtained under stationary and perfusion conditions favored the perfnsion system because of the use of lower stimulatory doses given as pulses to the cells. Activated lymphocytes can be obtained also on "hollow fiber" carriers.

RossP~,l,~ P., Bt.qcovsxr A., MA'rob'~owc K., HOLUe M., IOOa. J. MBU r V ; UPMD ; IKEM, Prague

Proteinuria and oedema were induced in rats (strain AVN) by 10 s.c. injections of the aminonucleoside puromycin in I-day intervals. Both the light and electron microscopy revealed a well-known picture of podocyte pedicel fusion with partial polyanion depletion, detectable by colloid iron. A panel of 7 monoclonal antibodies of the MRC OX type were used in a two-step immunoenzyme histochemistry in order to prove class I and II antigens, differentiation antigens (Thy 1, thymocvte glycoprotein) and lymphocyte epitopes ((pan T, CD8). In nephrotic rats, depletion of MHC + in intrnglomerniar cells was found while the number of Ia + cortical elements was incrensed. A significant increase of pan T and CD8 lymphocytes in the interstitium was observed. Expression of MHC I and differentiation antigens was unchanged. The aminonucleoside nephropathy, which represents a classical model of a non-immune damage of the kidney, is characterized by significant anomalies in the local expression of surface antigens.

1990 ABSTRAC'I~ 23

T Lymplmcyta ia Pstimts ~ Cstaseom LeiWmmsta~

JAao~ovA L., VLAg~ Z., S'raXNsr'l, J., S ~ M.M. Department o[ Immunology, MBU ~ A V, Prague; ,41 ~ ! ~ Hospital, Kuwait

The parasites Le/shmarda major or Le/s/unun/a trap/ca are etiological agents of various forms of cutaneous leishmaniasis (CL) occurring in Kuwait, characterized primarily by the type of skin lesions and course of the disease. T lymphocytes were determined in the peripheral blood of patients using monoclonal antibodies (OKT3, OKT4, OKTS) and by formation of active and total rosettes. The T-lymphocyte profile was found to be normal in the so-called simple CL characterized by early uicerating skin lesions of the "wet" type. On the other hand, a significant decrease of active E-rosettes and T4 § helper cells and increased "1"8 § suppressor cells was found in the peripheral blood of patients with poorly healing skin lesions (late-ulcerating of the "dry" type), and in recurrent leishmaniasis. The lowest 1"4 § § ratio was found in chronic recurrent leishmaniasis of the "iupoid" type (duration 1-10 years). With patients with vast skin lesions, persisting over 9 months, a decreased number of active T lymphocytes was found also in cell extracts from the skin lesions.

Sereee/q H d ~ e t t ~ ~ a ~ A,-~b~ ~ , ~ Symtm

NOUZA K., ~;0x.A K., SvoaooovA M., DvolO~ R., MOT w ~ J., Soao'rKovA E. (JEM t~SA V, Prague

A multitest system (MTS), making it possible to determine in SRBC-immunized animals the weight and cellularity of the thymus, spleen and lymph-node cluster, delayed hypersensitivity, antibody formation, phagocytotic activity of peritoneal macrophages and blood leukocytes, responsiveness of splenic lymphocytes to "T" (PhA, CanA)and "B" (LPS) mitogens, was elaborated for inbred mice. The changes of immunity parameters are determined at two time intervals after immunization in normal mice and in mice with selectively suppressed T system (anti-Thy- 1.2 MAb), B system (cyclophosphamide) and macrophage system (silica) and finally in mice treated with dexamethazone or by whole-body irradiation. The MTS detects selectively and with high sensitivity the effects of immunosuppressive and immunostimulatory substances and allows a partial analysis of the immunomodulatory (immunotoxic) effects.

Compsdson of Vs/ses ot Cd/-Me~sted/mmm/ty Tests s t t~ t ~ / m m u o u o d ~ t a t T m s t s ~ t

~OLA K., NOUZA K. (/FAW ~ A V, Prague

The monoclonal anti-Thy-1.2 antibody suppressed significantly the GVI-IR in mice after treatment of both the donors and/or the recipients. On the other hand, DTH was suppressed only when antibody was given immediately before the sensitization period; otherwise DTH was stimulated. The positive immunomodulator (dsRNA)-always suppressed the DTH, stimulated G VHR in irradiated mice, but was without effect in nonirradiated animals. Immunomodulators of bacterial origin have no unequivocal effects. Some of them (e.g. Coryaebactetium parvmn, LPS, preparations from various Klebsiella pneumoniae strains) significantly suppress DTH and rather stimulate GVI-IR, while others (ADDP) influence neither GVHR nor DTH. The response to immunomodulators is always dependent on the dose, time schedule and route of administration used. The two tests of cellular immunity (DTH, GVHR) illustrate different features of the complex immunomodulator effect and cannot be therefore mutually substituted. These tests detect more reliably the effects of negative rather than positive immunomodulators.


The Effects ot lmm~aomN)o'n/ators on a IM~4e| E g p e r / s l l e l l b l / ~ Jf~ecl~oa

MALINA J., HOFMAN~ J., FRAN~ J. UEM (3SA V, Prague

The application of a complex experimental biological model for preclinical evaluation of IM substances (host-resistance assay) serves two purposes: 1. To establish the resulting changes of the immune system as a whole, 2. to evaluate alterations of individual factors of immunity in the context of the current immune response. In order to meet these requirements we elaborated a model of inhalation infection elicited by Klebsiella pneumoniae strains of medium and very high virulence. Such an arrangement and comparison of stimulatory effects of IM preparations with the intensity of the specific immune response induced by a homologous K. pneumoniae vaccine, made it possible to objectively compare the IM potency of the listeria extract Ei, LPS and synthetic ADDP.

/ntmunopharmaco/ogka/Properffes of Bacteria//mmunomodu/stors in an A n / n ~ Model and in Vitro Experimuts

PR0CHOVA J., VIaXovA E., MARA M., O~E~A~KOVA J. SUKL, Prague; FVL ilK, Prague

The effect of the Propionibacterium ache vaccine and of its lipid-free phenol component on the tbc infection was studied in guinea pigs without or with BCG vaccine. The weight of the spleen and Feldman's index (FI) were determined. Both substances a) enhanced the nonspecifie immune response when given in one dose immediately prior to tbc infection; repeated doses did not further increase the response, b) enhanced weakly, but statistically significantly (5 %) the activity of the Soviet BCG vaccine, c) the lipid-frece phenol residue was more effective than P. ache in increasing both the spleen weight and the FI.

Comparison of the Effects of &)me lmmunomodu/ators

VE.mORA 0., POSTUPA J., Hmscn M., PEKA~K J., JAR? J., KAL~A E. LF UK, Hradec Kr~lovd; USOL, Prague; VSCHT, Prague; OUNZ K/adno

Using in vitro and in vivo systems, we tested the Transferfactor (Sevac), Soicoseryl and substance IMUNO 101. According to 02 consumption by a liver homogenate, the highest stimulatory effect was found with substance IMUNO 101. Using a model experimental Cand/da afbicaas infection, prolonged survival of infected mice was observed after treatment with Transferfactor that was prepared as a dialyzate of the homogenate of leukocytes obtained from the peripheral blood of donors with a positive skin test to candidin.

The Effect ol Hsnmmm Thruster Factor on the Tick-Borne Eacep/m/~ Experiments//m/ecdou

KOPECK~ J., TOMKOVA E., ~ J. Institute of Parasitology, C~SA V, ~r Bud~jovice ; (]SOL, Prague

Administration of the specific human transfer factor (TIC), obtained from reconvalesceuts after tick-borne encephalitis (TE) protected C3H mice from lethal infection with the TE virus. The effect of TI c on individual mechanisms of antivirus immunity (interferon, NK-cells, neutralizing antibodies, cytotoxic T-lymphocytes) was evaluated.

1990 ABSTRACTS 25

PossiMUties at Immunomodu/stion in Long-Term Hospitat/zed Patients

V~tOLA F., VRaOVA E. HIE, Prague

The wide use of antibiotics in human and veterinary medicine as well as in agriculture has some disadvantage.s. Besides the spread of bacterial resistance, some antibacterial drugs possess an immunosuppressive effect, particularly after a long-term treatment. Finally, antibiotics may affect adversely the microbial ecological balance. Our abundant experience with the treatment of staphylococcal and pseudomonad infections has shown that a combined chemo- and immunotherapy is more rational than chemotherapy alone. Such a therapeutical regime shortens the duration of hospitalization, limits the occurrence of bacterial resistance and maintains the host's immune system. Vaccines STAVA and PSAEVA, used in our therapeutical regime, influence positively the specific immunity and, in addition, possess an immunomodulatory activity. The vaccines are mostly given in chronic staphylococcal and pseudomonad infections without antibiotics; moreover, they can be successfully used in the prevention of some nosocomial infections.

hnmunomodu/atoty EHect of Bscter/a/L/p/ds

MARA M., MENCIKOVA E., JtnAz J., OCENA~,KovA J., gR Z. Department of Microbiology and Epidemiology, FVL UK, Prague; S(JKL, Prague

Lipids of L/steria monocytogenes obtained by a chloroform-methanol (2:1) extraction in the cold, protect mice both from listeria infection and infection with G - and G+ bacteria. Lipids were prepared from 40 bacterial species and injected to mice i.p. 2 days prior to infection. Lipids obtained from Bacillus f/rmus, KlebsieUa pneumoniae, Proteus vulgaris, Proteus mirabilis, some listeria species and from some other strains possessed a significant protective antilethal effect. Other strains and species were ineffective. The spectrum of fatty acids of individual lipids, determined by chromatography, confirmed the known differences among individual species, but failed to explain differences in the immunostimulatory activity.

Effects o ! / m m u o m o d ~ t o r s of B a a e t ~ Odg~ on Cytoei~me P-450/,ere/

M., O~NA~KOVA J., B ~ N ~ M., I.~DV~A J. Department of l~crobiology and Epidemiology, FVL UK, Prague; UOCHB ~SA V, Prague

Bacterial immunomodulators (endotoxins of various origin, peptidoglycans, glucan and listeria factor Ei, Propionibacterium ache vaccine and its components) decreased after an i.p. administration the content of liver cytocbrome P-450 in mice. MDP and its synthetic analogue ML-276 did not induce inhibition. In addition, activation of the RES, determined by hepato- and splenomegaly, occurred later after administration of immunomodulators.

Bio~emical ~ of Reg~tory Molec~es bvolred in A~alection lmmani~

KovA~ovA H., S~I~LIK J., MAC~A A. Institute o~ Biochemistry, LF UK, I-lrad~ Kr~lov6 ; VL VDU JEP, Hradec Kr~ilov~

The aim of the present study was to define REGULON as a space-limited idiotypic and antiidiotypic regulatory unit of the immune response. REGULON includes interaction of recognition molecules with the antigen leading to the production of antigen-specific and antigen-unspecific molecules. An analysis of newly synthesized proteins during a Francisella mlarensis infection was performed using the SDS-PAGE gradient method and autoradiography in the supernatants of mouse spleen cells or cell cultures of purified lymphocytes. The de novo synthesized proteins were detected from the 5th up to the 10th day after infection. After eluting these proteins from the gel, a macrophage-activating factor was detected by


a functional test in the regions of M = 14-16, 26-30 and 47-55 kg/mol. Using the Western blot method, immunogiobulin chains could be detected from the 5th day after infection. We are going to identify further proteins to obtain a characteristic map of induced protein production after antigen stimulation.

Ti~ Effea o/S. tmem ~ t K Prom~m~ oa in ViUo ,~admaoa M Hmmm L ~ W m ~ e s

PROKEgOVA L., Poawrr-BOaR Z., BARAU K., Jom~ C. Institute of Medical Microbiology and Immunology, FVL UK, Prague; Institute of Molecular Biology, Jagiellon University, Krakow

The $. aureus serine proteinase (SP), used in a wide concentration range (0.1-100 mg]L), does not possess a cytotoxic effect on human lymphocytes, does not stimulate lymphocytes in an in vitro culture but it influences the effect of a number of polyclonai activators. SP (100 mg]L of the culture) completely inhibits the blastic transformation induced by B-cell mitogeus (NDCM, S. aureus, strain Cowan and Wood 46, E. co/i) and decreases the Mastic transformation after lymphocyte stimulation with PWM, protein A and ConA; however, it does not influence the mitogenic effect of PHA. In addition, SP (100 mg]L) completely suppresses Ig formation in cultures stimulated with PWM, NDCM, strain Wodd 46 and E. co//. SP influences lymphocyte stimulation when added to cultures together with the mitogen, or 18 h after mitogen addition. It thus follows that SP does not influence the initial stage of stimulation (when interacton of the stimulator with the cell surface takes place), but influences later events occurring in the cell cytoplasm.

/mmmm~,g~tory Effesf of V ~ / m Mode/Amtm~mm~ Dkeme

ROVEUSK'? J., GmmovA J., FOSKA J., PoKom~A D., LA~OWC"KA J., PEKARF_.K J. ~OI(L, Bratislava ; VOFB, Prague; CHTF SV~T, Bratislava ; USOL, Prague

Recently, the effects of the antibiotic Vermiculin (Vrm) have been successfully tested in an autoimmune disease model. Vrm is a macrolidic agiycosidic antibiotic isolated from Pem'c////um vermiculaaun DAuo. It was shown that Vrm prevents the development of the spontaneous autoimmune disease in NZB/W F1 mice. In addition, Vrm inhibits the development of adjuvant arthritis in rats (25 mg/kg, 3x a week, 21 days per os). Improvement of both clinical and immunological parameters wa~ observed. Vrm represents a new generation of immunoregulatory substances which might be used in the therapy of inflammatory rheumatoid diseases.

St~m~doa of JL.1 Prodgetioa w;~ Solmble ~ d lmoluble 1,3o[@.D-Glmmm

~tROVA M., ~ I., H o ~ Z. MBU (~SA V, Prague

An in vivo injection of glucan results in an accumulation of macrophages at the site of injection and the overall symptoms correspond to the effects of IL- 1. We tested the effect of 1,3-iS-D-giucan isolated from the walls of S. cerevisiae in the soluble and insoluble form on in v/tro IL- 1 production in routine peritoneal macrophages. A 24-h incubation of noninduced macrophages with soluble or insoluble giucan results in a slight increase of IL-1 expression on the cell membrane but does not stimulate its secretion. Mercaptoacetate-induced macrophages increased significantly IL-1 production but most of the activity remained cell-bound. Both giucan forms exhibited a certain inhibitory effect on the cell activity when given in doses exceeding 10 mg/L

1990 ABSTRACI~ 27

~ t o r y F, f i n of O m m

(~av'~AgovX L., W ^ c ~ o v A J., D~vAT L., TmmovBc T. V(JL, Nlodta ; (.JEF, CFV SA V, Bratislava

The effect of a single i.e. and s.c. administration of carboxymethyl-l,3qS-D-glucan (obtained from cell wall glucan of S. cetev/s/ae) on in vivo humoral response and hematopoiesis was studied. Glucan was given in doses of 5, 10, 50 and 100 mg/kg. The following changes were observed: stimulation of total (IgG + IgM) serum hemagglutinafing antibodies against sheep red blood cells and an enhanced ability of the hematopoietic stem cells to form endogenous CFU in the spleens of sublethally whole-body-irradiated mice. The immunomodulatory effect depends on the mouse strain used, on the dose, route and time of glucan admires" tration.

~ R a g t ~ ot C d - M a t ~ e d lmm~i ty a~i tl~ I m ~ a e b ~ W ~ ot tl~ Glmc~ ldmmte

Sm)LhC~g D., P~ax~ V., Kmo^a V., S T ~ - a A. C~ic of Infectious Diseases, KUNZ-FN, Plzeit

The authors compared the results obtained with the Immunmkintest (USOL, Prague) with reactions to corpuscular and soluble ghicans in human volunteers. As compared with the Immunmkintest, the glucam yielded positivity more frequently in both healthy volunteers and in patients with presumed deficiency of cell-mediated immun3ty. Histological and immunohistochemical analysis of the excised tissue from the site of i.d. glucan injection showed a round-cell infiltrate corresponding to a delayed-type hypersemitivi- ty. In cooperation with the Institute of Physiology and Genetics of Farm An/ma/s CSAV (Prague), we studied the influence of corpuscular glucan on antibody formation to T-dependent antigens in goats and chickens. A significantly higher antibody level was recorded after glucan treatment in both species.

~ e Stt~atatory Effect e l Glacm oa ,.~k~ted ~ Parametm is Goats

HO~PF.S J., BENDA V., PALISA V. CFGHZ CSAV, Prague; LFUK, P/zed

The effect of corpuscular glucan on the formation of antibodies to HSA, phagocytotic activity and leukocyte dynamics in goats was studied. A significantly higher level of specific antibodies was found in animals treated simultaneously with the antigen and with glucan. Changes of phagocytotic activity were not significant; on the other hand, neutrophilia and lymphopaenia occurred already 24 h after glucan injection. The clinical condition was not affected by glucan.

;m,~woa~mmb~ d C~hr~ ~

SANGRLrr M., BAJOVA V., Hn'IKovA V., MoJ2I,f, ovA J., VIL(3~.K ~., VRTIAK O.J., Kt~-A V., BReZ~AN T., ~ o v g M. VSV, Ko~ice ; UEVM, Ko~ce

The immunostimulatory effect of glucan was studied in 3- to 4-months-old calves (the Slovak spotted cow breed). Experimental animals received a single dose (5 mg/kg) of glucan s.c. and the immunological profile was examined at various time intervals during a 3-week period, including the properdin level, bactericidal activity, phagocytosis, changes in the blood cells, MIF, ADCC cytotoxicity, interferon formation by ieukocytes and IgG levels. Statistical analysis of the results proved the importance of combining several immunological parameters.


Brief Stress and NK and IFN System Activities

LACKOVlt V., BORECKY L., VIOAg M., KOCIgKOVA D., MIS'IR~KOVA J., TATAR P. Institute of Virology SA V, (fEE SA V, Bratislava

We followed the effect of hyper- and hypothermia and of submaximum physical stress (induced on a bicycle ergometer) on the cytolytic activity of NK cells and of the IFN system. Both the somatotropin level and NK activity increased significantly in 8 out of 18 volunteers exposed to a hyperthermic bath (39 ~ 30 rain). Even hypothermia (4 ~ 30 min) induced an increase of these parameters togehter with noradrenaline elevation. The physical stress induced a 40 % increase of NK activity which could be further potentiated by means of IFN (500 U/mL). No IFN could be detected in the blood of stressed volunteers but their leukocytes produced higher amounts of IFN after a virus stimulus (VDN).

Porcine Alpha-Fetoprotein: a Time. and Dose.Dependent im,nunomods/sffon

glMAN P., KovAl~O H., KovAk0 F. MBU C~SA V, Nov~ Hr~dek

The time- and dose-dependent dependence of the immunomodulatory effect of pig alpha-fetoprotein (AFP) on concanavalin A (ConA) activated porcine thymocytes was studied. Determination of 3H-thymidine incorporation by a culture of tested cells showed that the highest inhibition (up to 90 %) of milogen-stimulated proliferation was obtained with relatively low AFP concentrations. However, this was the case, only when lower ConA concentrations were used than are optimal for the induction of lymphocyte proliferation. The results do not indicate the involvement of the ConA-ConA binding substance in the events observed.

The lmmunoregulatory Activity of Alpha.Fetoprotein ( AFP )

KovAI~O F., KovAkO H., TLASKALOVA H., ~;TEaZL J., V~'WC~A V., ~;IMA P. MBU CSA V, Nov# Hr~dek and Prague

Following the finding that immunosuppressive activity is associated with the ConA-binding isoform of porcine AFP, glycosylated (gly) derivatives of pig albumin were prepared using glucose or mannose. The gly-albumin inhibited the polycional activation of both T and B pig lymphocytes. The intensity of inhibition induced by gly-albumin was comparable with the AFP-produced suppression. Similarly to AFP inhibition, the gly-albumin-induced suppression was associated with the early interaction with the surface of activated lymphocytes and with a significant involvement of surface enzymes. In addition, regulation of antibody formation by murine iymphocytes in v/tro was tested in the presence of porcine or rat gly-albumins and AFP. The gly-albumins inhibited also phagocytosis of monocytes and granulocytes.

Isolation of Biologically Aclive Peptides from the Thymosin Fraction 5

RAHM J., PROCHAZKA V. Department of Immunology, KUNZ Ustl n.L. ; VOFB, Prague

The thymosin fraction 5 contains besides T-lymphocyte-stimulating substances (thymosins ct and 13) also inactive substances (nucleotides, lipopelatides, polypeptide [il) and free and bound amines (derivatives of spermine and spermidine) that inhibit nonspecifically the process of differentiation. The following possibilities of separation are discussed. 1. Removal of ineffective components and inhibitors 2. Separation of immunosuppressive thymosin ct7 from other thymosias possessing a stimulatory activity 3. Isolation of individual thymosias ctt and [~ in the aim to prepare a preparation with increased specific efficacy for clinical purposes.

1990 ABSTRACTS 29

]~e Tmm/er Factor in MKek's l)iwase

LEgNiK F., VAJDOVA E., KOPKA M., Jtm~NA A., LEVKUT M. VSV, Ko~ice

Association of immune complexes with glomerulonephritis of the membrane proliferative type was described in Marek's disease (MD) of fowl, caused by a herpesvirus of the genus Thetalymphocryptovirus. Dialyzable extract from peripheral blood leukocytes of noninfected roosters decreased significantly (p < 0.01) after an i.v. injection the occurrence of MD virus antigens in epithelial cells of feather follicles between the 56th and 77th day after infection; in addition, a decreased titre of antibodies against the A antigen of the MD virus was also observed (~ 1:4.6 vs. 1:13.1). As regards the level of circulating complexes, no difference was found in comparison with the control group.

Use of the Specific Tramfer Factor in Ant/rob/us Vscd~ffon

O~D~KA R., MLYNAR~KOVA K., SVR~K S., SAN~RJ~T M., VRT~K OJ., ZAV~OVA J., BE~EK Z., S0~IOVA J. V~V, Ko~ice

A technological procedure for the production of the specific antirabies specific transfer factor (SATF) was proposed and possibilities of its use in antirabies vaccination were determined. The SATF was prepared by a modified method of Lawrence, the preparation was lyophilized. Its efficacy was tested in laboratory animals and in cattle using the skin test and the secondary immune response. It is recommended to use SATF particularly in emergency vaccination in the focus of infection for acceleration of immunity induction.

lmmunomod~aoa in Exper/meata Ascada~

BORO~KOVA Z., BENKOVA M., DUBAJ J., SZ~CHENYI ~. Institute of Helminthology, SA V Koff/ce; Bioveta Nitra

Using the rosette methods (E, EAC) the authors studied the effect of preparations Giucan and Mialor G (Bioveta, Nitra) on T and B lymphocytes in experimental ascariasis in guinea pigs. Mialor G significantly increased the number of both B and T lymphocytes (p < 0.01) already during the early phase of the invasive process and maintained relatively high values of both cell types up to the 22nd day after invasion. The two preparations differed in their protective effect,, evaluated by the reduction of the numbers of migrating ascaria larvae in guinea-pig lungs (Giucan - 18 %, Mialor G - 50 %).

Therapy of Cellul~ immunity INsorders ~4th tile Transfer Fsdor

Gt~AAN M., Hm~HOROVSK~? M. Laboratory of Immunology and Allergology, 4th Clinic of Internal Medicine, FNsP Ko~ice

The following criteria were chosen for administration of TF to patients. A. Clinical picture. Recurrent bacterial infection, other diseases with known or suspected involvement of immune mechanisms. B. Laboratory findings. Absolute or relative deficit of T lymphocytes, A-ERFC or other deficit of cell-mediated immunity. Fifty-two patients, followed for 3 - 30 months, received 328 doses of "IF (Sevac, Prague) and the short- and long-term effects on the clinical condition of patients and on laboratory values of natural, humoral and cell-mediated immunity were determined. The effectiveness of the preparation was found to vary between 0 and 100 %, depending on diagnosis. The schedule of administration of the first 4 ampoules was highly individual (1 -6 months); quite often, clinical improvement preceded normalization of laboratory values. Certain differences were found in patients with a positive or negative


response to the "IF therapy. After cessation of TF therapy, a decrease of laboratory values occurred in the first place, followed by progression of the disease.

Tte laheace ol Petoral Admlai~ttsa~m ot Xeml lemm T n m ~ fsctor oa Pet~plm~ Biood L y m p ~ r

KRAL V., Rto-rra~ J., RAHM J., Jttax D., ZEmct~ovA M. Department of Immunology, KI-IS (Js6 n.L.

The transfer factor was prepared by a modified classical procedure from sterile porcine blood and was administered according to a common scheme (basic therapeutic procedure: 6 p.o. doses in one-week intervals; 1 dose of TF corresponds to 5 x 108 ieukocytes). The following criteria were used for initiation of the therapy: decreased responsiveness of patients in skin tests and decreased values of cell-mediated immunity parameters. Control examination performed during and at the end of therapy showed a significant increase of E-rosettes, active rosettes (E.) and T3 (CD3) positive lymphocytes. The relationship between the positive clinical effect and diagnosis is discussed. The results confirmed the scarce literary data concerning the use of xenogenous TF as an immunomodulatory agent.

In Vitro and ia Vivo EHects of Thymmla

KRAt. V., Rton'mz J., ~ Ate~ J. Department of Immunology, KI-IS (]sti n.L.

When developing the separation procedure for thymosin, the in v/tro efficacy and therapeutical use of the preparation was tested. The products of separation (fractions 4 and 5) were tested using a model of induction of receptor expression for sheep erythrocytes on spleen cells of infant mice. Both fractions possessed the inductor activity. In humans, fraction 5 increased significantly the expression of receptors for sheep erythrocytes (Tl l , CD2) on human peripheral lymphocytes (determined in a test according to Wara and Amman). Thymosin was used for treatment of patients with secondary immunodeficiency (tumours, chronic infections). Following administration of 20-30 mg of thymosin (fraction 5) during 4 - 6 weeks, a significant increase of T3 + (CD3), T11 § (CD2) and T4 + (CD4) lymphocytes was found. The authors presume that commercial production of this preparation should improve considerably the immunomodulatory treatment.

~ ~ ~ Evoha~

glMA P., SLIrKA J., BIL~ M. Department of Immunology, MBU t~SA V, Prague; LF UK, Plze~

The basis of incompatibility reaction in the simplest metazoa (Porifeta) is the affinity of cell surface glycoproteins. The preliminary step of adaptive immunity in endothermic vertebrates ate quasi-immune reactions of lower invertebrates (Coelenterata). Qualitatively higher is the primordial cellular immunity of invertebrates possessing a secondary body cavity (coeiom) and highly differentiated mesenchymal tissue (Anne//da, Echinodermata). It persists in vertebrates in the form of the T component. Im- munoevolntion reached its peak in the class of vertebrates. In addition to the T component they possess circulating antibodies of immunoglobulin nature (the B component). The complexity of the cell architecture of the lymphoid system that is responsible for the homeostatic immune regulation including the T and B immunity cooperation, increases from fishes to mammals.

1990 ABSTRACTS 31

Tu~ovA L., ReJNe~ J. Department of Immunology, MBU CSA V, Prague

The ability to recognize "self" and "non-self" (foreign) is present in all animal species and it has been perfected during evolution, as is evident from the divergence of IgG classes and subclasses with specialized effector functions. Presumably, during the early stage of phylogeny, a structural unit, corresponding to one Ig domain was formed, from which a family of Ig-like molecules had developed as a basis of self and non-self recognition. As one of the common properties of Ig-like molecules is the ability of mutual interactions, one can imagine that the primitive Ig domain secured originally interactions with identical molecules on the surface of other cells and later on, under the influence of gene duplication and divergence, interaction with similar but nonidentical structures occurred. This led to the origin of a primitive variable domain with a primitive binding site and later, under the selection pressure of microbial and viral antigens, to the origin of the necessary repertoire of antibody and receptor molecular specificities as occur in the vertebrates.

1~e A&ptb, e R e ~ n of A m e / ~ dter A d m / a J s h ~ ot ~ and M e t = Awffffem

Tuc'xovA L., ~ J. Department of Immunology, MBU (~SA V, Prague

Neither lymphocytes nor antibody-like molecules can be found in invertebrates. However, most of these animals can reject specifically an allogeneic graft in a reaction with brief immunological memory. Parenteral stimulation the annelids Lumbricns terrestris and E/sen/a foetida with proteins (ARS-BGG, ARS-HSA, HSA) and St BC led to a significant increase of protein concentration in the coelomic fluid. A slight increase could be also seen after administration of phosphate saline. However, also a protein, binding nsI- or peroxidase-labelled antigen, could be detected in the ceelomic fluid of stimulated animals. The maximum binding activity was found on the 8th day after the primary, and on the 6th- 10th day a~ter the secondary dose. The highest binding activity was obtained when the same antigen was used for both stimulation and detection. However, using RIA or ELISA tests, binding of other proteins was also found. Thus, it seems that specific binding is lower than in antibodies or in specific receptors for lymphocyte antigen.

Outraetedstt~ el J~limg M o k c , ~ Fermed tuner , q ~ m ~ w/~ Preteb Amigem t-

RmNEK J., TUC"KOVA L., Kos'rKA J. Department of Immunology, MBU (~SA V, Prague

Administration of proteins (ARS-HSA or ARS-BGG) to Lumbricns terrestris results in the formation of binding molecules that react with the antigen used for stimulation. SDS-PAGE analysis of coelomic fluid obtained from stimulated animals combined with enzyme- or radio-immunoassay, as well as affinity chromatography of isolated molecules showed that the binding protein has a molar mass of 50 kg/mol similarly to the previously described protein present in the coelomic fluid of unstimulated annelids that reacts with the fast migrating sheep and goat IgG. The concentration of molecules binding antigen or IgG increased after antigenic stimulation. Both these activities are probably carried by different molecules, because the IgG-binding molecules are present also in nonstimulated individuals. The fact that the antigen-binding protein reacted with antiidiotypic antibodies directed against the idiotype of porcine anti-ARS-BGG antibodies suggests the possible relationship between the binding molecule of annelids and the V-region of antibodies.


l~agocytotic Activity o! Am~//d Coe/omocytes after Sem/tfzat/ou wft/t Antigen

BXLEJ M., ~IMA P., VffrvlC'XA V. Department of Immunology, MBU C'SA V, Prague

Free cells of the coelomic cavity, coelomocytes, play a key role in the defense reactions of annelids. Contrary to mammalian immunocytes, all types of coelomocytes (acidophils, basophils, neutrophils and granulocytes) can ingest HEMA particles. The highest phagocytotic activity was observed in granulocytes. The phagocytotic activity changes after presensitization with the antigen (ARS-HSA and ARS-BGG). These changes can be induced by destabilizing ions in the coelomic fluid after "immunization" and thus by disturbing the balance of the internal environment.

U / ~ s s t m ~ ot P ~ e s o! the Coe/om and mood o! A~e//ds

TREBICHAVSK"I' I., ~IMA P., Bn.~ M., TRUXOVA E., VRTVIC'KA V. MBU CSA V, Nov# Hr~dek and Prague

Phagocytosis of cadmium and HEMA particles, SRBC and E. coil, injected into the coeiomic cavity of Lumbricus terrestris was determined in the coelome and blood cells 3-60 h after injection. In addition, phagocytosis was studied after peroral administration and after an in v/fro cultivation of coelomocytes in L. terrestris and Eisenis foetida. It was found that granulocytes and basophils were the main type of phagocytosing cells while acidophils were opportunistic phagocytes and neutrophils did not possess phagocytotic activity. The ultrastructure and characteristics of these cell types were described; their nomenclature, not corresponding to mammalian types, was taken from Cooper's classification.

New T'rssue Lectim from Caterpillsrs of Galleria melonells sad Their Role in Recognition

MA'['HA V., GRUBHOFFER L. Institute of Entomology, CSA V ; Institute of Parasitology, CSA V, Cesk6 Bud~jovice

The immunity factors of caterpillars of Galleria melonella were studied. Proteins with lectin activity were isolated and characterized. A lectin specific for the sugars L-Fuc and o-GaiNAc (molar mass 70 kg/mol) was isolated from the cuticle of the caterpillars by affinity chromatography. Another lectin, found in caterpillar hematocytes had a specificity for D-Clc, D-Man and zymosan; SDS-PAGE under non-reduction conditions yielded a high-molar-mass fraction (80 kg/mol), a major fraction (32 kg/mol) and a minor fraction (25 kg/mol).

The Development of/mmnm),:ompetence in Pig Fetuses

KovAl~O F., Kov~O H., TLASr,~a.OVA H., TaEmCHAVSgV L, KOT_~KOvA H., Z~mAD~C'KOVA M., ~PLI~a_ I. Department of Gnotobiology, MBU C'SA V, Nov~ Hr~dek and Prague

The pig fetus can form antibodies after intrauterine immunization after 55 days of gestation. In addition, an increased number of lymphoblasts and medium lymphocytes including activated cells of the erythroid and myeloid line were found in young fetuses (55-70 days after intrauterine immunization). Plasma cells occurred in older immunized fetuses (75th day of gestation). The intrauterine immunization induced expression of IgG receptors and class II MHC antigens. Lymphocytes of immunized fetuses possessed an increased activity of y-glutamyl-transpeptidase, alkaline phosphatase and Mg2+-ATPase and, on the other hand, decreased activity of 5'-nucleotidase which are the markers of cell maturation. Lower levels of AFP were found in the blood serum of immunized fetuses.

1990 ABSTRACrS 33

immuologiesl Profile ot Adult Germ.Free Pi~

T~VNiC'~K J., ] ~ E L L., T~aic-'UAVSrV I., TLASKALOVA H., Dvol~z~ P. Department of Gnotobioiogy, MBU CSA V, Nov~ Hr~dek ; UFGHZ, Lib6chov

Comparison of germ-free and conventional pigs revealed the following differences: lower numbers of leukocytes and decreased % of neutrophil granulocytes, no immature cell forms, lower total content of serum proteins, low levels of y- and m-globulins, increased albumin and [3-globulin, significantly decreased individual IgG class levels determined by radial immunodiffusion or by sensitive enzyme immunoassay. Cells with surface IgA predominate over IgG cells.in mesenteric nodes and in spleen. Lamina propria contains a high number of IgA-containing cells, IgG-containing cells being rare. In the peripheral blood, cells possessing the IgM surface isotype occur most frequently. The number of blood T lymphocytes is doubled.

ImmunocompeteKe ot Newborn Calves

T o ~ M., K ~ J ~ J., MF~IK P., ~T~PA~x J. VUVL, Brno

The activity of cells of the immune systems of calves was followed during the first month of life. Immediately after birth the activity of peripheral leukocytes was low; however, it increased already 24 h after delivery. The phagocytotic activity increased sharply up to the 3rd-5th day, then kept gradually decreasing and on the 14th-28th was low. The proliferative activity of lymphocytes increased up to the 7th day, reached the level of fully immunocompetent animals and persisted at this level with minor changes. As regards colostrum-deprived calves, the onset of proliferative activity was slower and did not reach the level of colostrum-fed animals. These results indicate the advantages of immunostimulation 1 day after birth or on the 14th-21st day of life when calves are placed in a stable.

Loq.Term lm~ence on the Derelopment o! Lymphatic Tissaes in lists ~ Ussstmsted Fstfy Acid DeBcie~zy during Esdy Postmtal Ontog~y

~TI~PANKOVA R., Dvo~to: B., TREBICHAVSK'~ I. Department of Gnotobiology, MBU C~SA V, Nov~ Hr~dek

We designated two types of milk diet for artificial feeding of young rats from delivery up to weaning: diet I(50A (42.6 % of essential fatty acids - EFA) and LNaH (0.7 % EFA). One-month-old animals fed the LNaH diet had significantly lower numbers of small lymphocytes in spleen and in thymus. The LNaH-fed rats had significantly higher numbers of lymphoblasts in spleen and in thymus as compared with breast-fed and K50A-diet-fed animals. Polychromatic and basophil erythroblasts were also significantly higher in LNaH-fed rats.

The EHect ot Unsstmsted Fatty Adds on the Rat Tissue SOsctsre dsr /~ Bre~-Feemm~

DvorAK B., gT~ANKOVA R., TREBICHAVSKY I. Department of Gnotobiology, MBU t~A V, Nov~ Flr~dek

Higher unsaturated fatty adds are an essential part of the phospholipid component of cell membranes. To the group of these substances belong also the so-called essential fatty acids (EFA) that are precursors of prostaglandin formation. We prepared artificial milk diets differing in the content of EFA for studies of the influence of EFA on the physiological development of rats during the first month of life: diet K50 with a high EFA content and diet LNaH deficient in EFA. The diets were delivered by hand feeding. In LNaH-fed rats a decreased mass and changes in ultrastructure of the thymus were found. The epithelial stroma of the thymus was vacuolized and mitochondria were altered. The phospholipid fraction of the


thymus has a decreased content of arachidonic acid and an increased content of oleic acid as compared with breast-fed and KS0-diet-fed rats. Similar changes in the phospholipid composition were found in liver tissue but the size of the liver remained unchanged. The spleen of LNaH-fed rats was enlarged.

The ln~ence of Ontogenetic Development on the Antibody Response in Wen and Poorly Respom~g Morse Strsim

V~x' ]~A V., i~dHOVA B., VomcovA J. Department of Immunology, MBU C~SA V; P~F UK, Prague

We compared the primary and secondary response to SRBC in well (Aft) and poorly (B10) responding mouse strains during ontogenetic development. We found that the number of IgM PFC after primary and secondary immunization was equal in both strains in all age groups (5, 10, 15 days, 3, 15, 19 and 23 months). Different results were obtained in IgG formation: in very young mice (5- and 10-day-old), more IgG PFC was found in strain B 10 while in adult B 10 mice 10 times less IgG was found. This amount did not further decrease with progressing age, while in A/J mice there was a sharp decrease during ageing, up to the age, of 15 months. Thus, the poor responsiveness of strain B10 occurs on the 15th day of life and persists up to the 15th month when the IgG formation becomes stabilized.

The Etlect of ~ z and Genetic hctors on Post-$~b~blfoo C b ~ e s of Hematologtcai Pmmnetem in Fowi and The/t Rda~omh/p to A~Obody Formation

HAMPL A., TRTKOVA K., PETROVSKY E., BENDA V. UFGHZ C~SA V, Prague

The aim of our experiments was to prove whether the hematocrit and the leukogram may serve as an easily available marker of the activity of the immune system that might be used for breeding. The values of both parameters were determined in four groups of hens and roosters differing genetically. The first determination was performed before, and further four determinations in several-day intervals after an i.m. immunization with SRBC and Brucella abortus. At the same time, antibody levels against the antigens used were determined. The following changes were recorded in the post-stimulation period: changes in antibody titres, significant shift in the relative proportion of individual types of peripheral blood leukocytes and changes in the ratio of blood plasma to erythrocytes. The sex dimorphism was not manifested in any of the parameters studied. The effect of genetic factors varied in individual parameters.

lmmHotoxlc Effect of Xenob/odcs din/rig the Per/~ts/Per/od

DOSTAL M., SOUmWOVA D., PRAUS R., I-~CMANOVA B. UEM CSA V, Prague

The embryotoxic effects of cadmium, vitamin A and E and cyclophosphamide on the postnatal development of the immune system (IS) was studied in randomly bred mice ICR. As regards substances lacking a specific 6ffect on the IS, the sensitivity of IS does not differ substantially from the susceptibility of other morphogenetic systems. Obviously, most substances possessing a teratogenic effect would influence the development of the IS during fetal and perinatal development.

~ t ~ E~eL'tS Of CIdminm on (be ~ e | m m ~ System

SOUKUPOVA D., DOS'rAL M., HEJCMANOVA B. UEM CSA V, Prague

Offspring of randomly bred ICR mice that received an s.c. injection of C_.d 2§ (2.5 mg/kg) on the 16th day of pregnancy, had an identical birth weight, an increased body mass at the time of weaning, a higher

1990 ABSTRACI~ 35

relative spleen mass at the age of 4 weeks and an enhanced humoral response to SRBC and an increased activity of peritoneal macrophages. The delayed-type hypersensitivity reaction to SRBC was decreased after sensitization on the 4th week and after repeated stimulation during the two following months of life.

Clmages la ~ene tT te Po/ydoaa/At'h~lloa ~ by Xeaoblel~ dining ~ Prelim/Peidod

PRAUS R., SOUrU~VA D., DOSTAL M., H o ~ q o v A V. UEM CSA V, Prague

Offspring of randomly inbred ICR mice that received 5 000 ILl of vitamin A on the 12th day of gestation and 0.5 nag of cyelophosphamide (CPM) on the 13th day or 1.2 rag CPM on the 16th day, developed enhanced basal proliferation of splenoeytes during the 3rd-12th week and increased activation induced with concanavalin A, phytohemagglutinin and lipepelysaeeharide on the 3rd-8th week. All parameters normalized till the 16th week with the exception of decreased activation with lipopolysaeeharide under the influence of CPM in the 3rd and 5th week.

of In/eeliaa ~ A//re

Lo~AJ J., Jor~ C. Department of Clinical Immunology, FNsP Brno ; Institute ot ll/Scrobiology and Immunology, FVL UK, Prague

Immunology was born in association with problems of infection and has not abandoned it so far. Immunological methods help to uncover new infections agents, to characterize more precisely the already known agents, and permit analysis of their interactions with the host. The infections agent turns the immune system against itself: it induces immunodeficiency states, allergic reactivity and damaging autoimmunity. The, antibiotics influence not only the microbes but also the host. The antimicrobial therapy is now combined with immunomodulation. New types of vaccines are being developed - prepared by recombinant technology, encoded in the vaccinia virus, synthetic peptides, idiotypic vaccines. Despite this progress there remains a considerable debt of immunology to infections.

Retai~fy of ~e /4mma Orga/sm to t le LDH Vlms

VEJBORA O., JANDOVA A., DVOI~KOVA M., POSTUPA J., HIRSCH M., ~MORANC P., RY~KOVA O., D ~ : K. LF UK, Hradec Kr~lov~ ; 2nd Clinic of Obstetrics and Gynaecoiogy, FVL UK, Prague; Cl/nic of Psychiatry, FVL UK, Prague

We compared the results of the ELISA test in patients suffering from malignancy or schizophrenia with positive LAI test to specific LDH virus antigen. In addition, we examined the occurrence of circulating immune complexes. The results have shown that the positive proof of specific antibodies (determined by the ELISA assay) did not always correlate with the results of the LAI test. The latter test, however, can detect changes in the reactivity of the organism to the antigens tested in the course of the disease.

Reat~rlty C'~aqes of C ~ [ - / ~ a/ter Interae~n ~ the LDH Vtms

VEmORA O., POSTUPA J., JANDOvA A., HIRscI4 M., MOTYCKA K. LFUK, Hraded Krdlov~ ; 2nd Clinic of Obstetrics and Gynecology, FVL UK, Prague ; UHKT, Prague

Purified virus antigen was prepared for specific determination of the LDH virus infection and for examination of the dynamics of changes in infected mice: body mass, spleen index, activity of spleen and peritoneal macrophages, 02 consumption in the tissue homogenate of the liver and spleen, activity of lactate dehydrogenase (EC 1.1.1.27) in the serum. Changes could be detected already on the 2nd day


after infection, the highest serum LDH activity was found on the 7th day; at that time, material was obtained for preparation of the antigen.

I n t ~ ol T ~ - B o m e ~ ~ with ~PS: the Effec~ of ~ ,4~tibod~ lad L e a ~

GmmHO~Ea L., KOVEC~ J., TOMKOVA E., MAIP, A V., VEg~ J. Institute of Parasitology, C~A I/; Institute of Entomology, r V, C2sk6 Bud~joviee

The effect of subneutralizing dose of specific antiserum and a selected group of lectins (ConA, LcL, PsL, SBA, HPA, PHA) on the interaction of the tick-borne encephalitis virus (Flaviviridae) with murine peritoneal mact~phages was studied. A subneutralizing dose of specific antiserum enhanced the percentage of infected MPS cells. Using the above mentioned lectins, characterization of membrane receptor structures of macrophages was performed by direct fluorescence. On the basis of these data some ieetins were selected for comparison of their enhancing effect with the effect of antibodies.

PrepJ~on u d ~ of Monoebm/Andlxxi/es ~ the Bo~Be l~e~om (Bin)

RemoLcovA O., ZA~^C J., Zu~-eA T. Bioveta Nitra

Forty-two hybridomas, producing antibodies against the BIR virus, were prepared by fusing the myeloma cell line Sp 2/0-A6 with cells isolated from the spleens of inbred BALB/c mice. The specificity of antibodies was determined by direct ELISA test and by indirect fluorescence with 6 strains of the BR virus (BHV 1, BHV 2, BHV 3, BHV 4) and the Aujeszki disease virus (AV). Four antibodies reacted with the AV, the remaining antibodies were strictly specific for the BIR virus. Neutralizing activity was found in two monoclonal antibodies in a plaque-reduction test.

(Jtmmabn of Monodoml Antibod~ in the ~ of Bo~e infe~ms ~ (BIR)

Zm^c J., REJnOLCOVA O., ZU~A A. Bioveta Nitra

When comparing the classical ELISA test with the immunoenzyme ELISA test with monoclonal antibodies and with neutralization and immunodiffusin tests for the detection of antibodies against the BR virus, the ELISA with monoclonal antibodies turned out to be the most sensitive and specific serological diagnostic method for BIR detection. The use of monoclonal antibodies permits a fast and specific detection of the virus but the sensitivity of the method is limited by the virus content in the matedai examined.

Determiulfen o / ~ / g M Aaltbedles aga/ast ~ b / a ~ Warn

~'rRPANOVA V., HORAL'~EK J., CHC/LKOVA V. Department of Virology, KIdS Hradee Kr~lov~

The solid-phase immunosorbent technique (SPIT) for detection of IgM antibodies to rubella virus was modified using domestic immunosorbents. Specific IgM antibodies could be detected in sera already 1 day after the occurrence of exanthema, the highest titres were reached from the 9th day after exanthema appearance; antibodies persisted for 3 -4 weeks. The presence of IgM antibodies correlated with positive conversion of hemaggiutination-inhibiting antibodies in sera of children and pregnant women. The method is of diagnostic value particularly if the first serum sample was obtained from pregnant women later after exanthema occurrence or after contact with rubella-infected individual, i.e. when seroconver- sion of HI antibodies yields negative results.

1990 ABSTRACTS 37

prepm~tims of Cow's ~ with s ~ Contemt ot Anffbodles sqp~st Rotav~s u d Its ~ in t ~ lYeveBtinn ot Roinv~s lntectlom o! C ~ e s sled P i g . s

I O ~ l J., ~TIWANF~K J., BARTL M., FRANC J. VOVL, Brno

Repeated systemic and local intramammal immunization of pregnant cows before and at regular intervals after delivery induced a long-lasting secretion of high specific antibody levels into the milk. Preventive administration of such a milk in a dried or lyophilized form prevented the development of infection in experimentally infected calves and SPF piglets. In addition, the milk possessed a positive therapeutic effect.

AaSerence AcUity o! E. coil SWabs Isolated f~om the Stoob ol Infants and Theik Motlms

SLAVIKOVA M., ~rlKOvA-LOD~OVA R., ~ N L.A., ~ o v A M. OPMD, Prague; Department of Clinical Immunology, C.-6tebor$

The presence of adhesins and the ability of dominant E. coil strains to adhere to cell clones HT-29 was studied using the hemagglutination technique. The E. coil strains were obtained from breast-fed and bottle-fed infants, artificially colonized with E. coil strain 083 and from control infants and their mothers. In the colonized infants, E. co//strains possessing fimbriae of the I type and the serotype 083, 04, 08 and 09 predominanted; strains with P fimbriae, serotypes O1, 03, 04, 06, 08 and O18 and strains lacking adhesins, serotypes 07, 077 and O13 ocurred less frequently. E. co//strains possessing P fimbriae had a more expressed adherence activity detectable in cell line HT-29 than E. co//strains with fimbriae of type I. Minimal adherence activity was found in strains lacking type I and P fimbriae. The E. co//strain 083 possesses fimbriae of type I.

l ~ m m o ~ in Gm~tob/offr ~ ~ by I B t ~ t m / l ~ a e a s

TALAFANTOVA M., MANDFL L., T~nCrtAVSK~, ' I., ~;'r~vAl~,~ovA R. Department of Gnotobioiogy, MBU CSA V, Nov~ Hr~dek

Gnotobiotic colostrum-free piglets can be compared in some parameters with patients suffering from immunodeficiency. After the peroral or rectal monoassociation of piglets with one of 10 species of intestinal bacteria, translocation of these bacteria into the lungs occurred in most cases. Depending on the bacterial species and their properties (e.g. toxins), damage of the lung tissue or even death of animals occurred. Intratracheal administration of E. coil results in multiplication of bacteria in the lungs (depending on the dose used). However, when similar experiments were performed in conventional piglets, the lungs were found to be sterile 24 h after bacterial administration. Similar findings were obtained after an i.v. injection of E. coll. The same finding applies to gnotobiotic rats. Increased numbers of alveolar macrophages expressing class II MHC antigens were found in all monoassociation experiments. Massive hemorrhage, necrosis and locally adherent bacteria were found in massive infections with fatal outcome.

Pseudomonns aeruglnosa Andgens and Immulty

Ktystc"zovA D., TESAI~ A., ~OLTISOVA D., JANDOVA D., PILLICH J., VRm3vA E., V~OLA F., TOUFAROVA D. Institute of Biophysics, C~3A V, Bmo ; IHE, Prague

Selection of suitable bacterial and phage strains of P. aemg/nosa allowed the preparation of phage lyzates (PL) with immunomodulatory properties. The basis of these preparations are antigenic components released by lyzing the selected strain with a virulent polyvaient phage. PL and autolyzates (AL),


prepared from strains with proteolytic and elastolytic activities and producing exotoxins, were compared with iyzates from strains deficient in the production of certain extracellular enzymes. PL prepared from mucous P. aerug/nosa strains was compared with PL from KlebsieUa pneumoniae. Antigenic profiles of PL and AL were evaluated electrophoretically and chromatographically (HPLC). Some PL exhibited stimulatory activity in the blast transformation tests. The highest stimulation indexes of pseudomonas and klebsiella lyzates were found after a 2-day incubation with mouse spleen cells. The results of experiments with the spleens of athymic mice indicate that PL stimulates primarily proliferation of T cells. In addition, PL potentiated to various degree the phagocytotic activity of human phagocytes determined by the luminol-dependent chemiluminiscence.

Cro~.Relct/ag A~tigeas of Echinococcus gnmalosus

K u ~ < o V.V., Ltn~g ~;. Skryabin All-Union Research Institute of Helminthology, Moscow; Institute of Parasitology, (TSA V, Ceskd Bud~jovice

Infection with Echinocoocus granulosus (hydatidiasis) represents a diagnostic problem in the tropics as well as in the mild weather zone. The required serological proof is often complicated by cross-reactions. We performed an analysis of the echinococcus fluid from the sheep cyst before and after fractionatibn on DEAE-Sephadex using SDS-PAGE and transfer to nitrocellulose. Purification of the antigen with ion-exchanger removes the cross-reactivity with $. mansoni (two antigens in the region of 43-50 kg/ tool). Cross reactions with Alveococcus multiocullaris and Opistorchis felineus remained (studied with human monoinvasive sera).

Common Antigens of Tspeworms Genw TaeniA

Lux~_~ ~., ~ J., G ~ I.E. Institute of Parasitology, CSA V, Cesk~ Bud~jovice ; Skryabin All-Union Research Institute of Helmin- thology, Moscow

Studies of common antigens in cestodes is of importance (a) for the removal of nonspecific and cross reactions in serodiagnostics, (b) for obtaining sources of alternative antigens for certain medically important species. Using SDS-PAGE and immunoblotting on nitrocellulose, antigens of Taenia saginata, Ofsticercus bovis, C. crassiceps, Taenia hydatigena, T. taeniaeforrnis and T. pisiformis were studied. Replicas on nitrocellulose were analyzed using antisera of cattle with cysticercosis and sera of mice invaded with T. crassiceps.

Compllrisou of the Immune Respo~e in V~c~ l t ed ~ d Nontsccimted Mice sfter/~ect/on w;;~ Tsenis cr~iceps

V~ovA L., K ~ A K., PRo~o~ J. Institute of Parasitology, C~A V, ~skd Bud~jovice

The antibody and cytotoxic responses of mice (strain |CR), vaccinated with a cell suspension prepared from the larvae of Taenia crassiceps (5 • 104 cells per mouse) and of infected mice (10 larvae of T. crassiceps) were compared. On days 33, 43, 55 and 65 after immunization and/or infection, one mouse of each group was stimulated with 3 larvae of T. crassiceps. The blood and spleens were obtained 12, 34 days after vaccination/infection and always 24 and 48 h after stimulation. The cytotoxic response of immunized mice was always higher than that of infected mice during any period tested. The antibody response, measured by the ELISA assay, was higher in infected mice particularly after stimulation with larvae of T. crassiceps. The high cytotoxic response in immunized mice (contrary to infected mice) and the fact that despite high antibody levels in infected mice the development of the parasite was normal, indicates that cellular immunity plays a key role in the host defense against parasitic worms.

1990 ABSTRACI'S 39

The Lyme Disease

MLADA L. Department of Immunology, KHS PIze~

We examined 442 patients with suspected Lyme borreliasis using indirect immunofluorescence test with Borrelia recurrentis antigen. Positivity of IgG antibodies was found in 5.7 % cases (titres 256 and more), a suspect finding in 6.3 % (titres 64 to 128), a negative finding in 88 % (titre up to 32). Seventy-seven patients (17.4 %) had a typical clinical picture of erythema chronicum migrans. In this particular group, positive titres were found in 5.2 %, suspect findings in 9.1% and 85.7 % were negative. The control group included 50 healthy blood donors, 22 % of them had titres up to 32. Serological diagnostics of the Lyme disease is in its initial stage and it is therefore necessary to concentrate on the dynamics of antibody titres and the serological findings must be interpreted in correlation with the clinical picture.

Detem~i~tion ot An t ibod~ against Bovine El~ootie Lem4~om in the ~ u d its UtiBzstion in the

HOFiREK B., JAGOg P., Pm, rr0c"EK B., S~r.JSr, AL J. V$V, Brno ; District Veterinary Centre Brno-country

Enzootic bovine leukosis (EBL) was diagnosed by determination of anti-EBL antibodies in the milk using the ELISA assay. The aim of this experiment was to prove the suitability of this diagnostic approach for limitation of this disease in cattle breeds. Determination of anti-BVL antibodies was performed in milking cows and nonlactating cows and young cattle in the serum three times a year. After a 2.5-year duration of the experiment, when all positive animals were discarded from the breed, the occurrence of ieukosis decreased in the whole district from 0.41% in 1985 to 0.068 % towards the end of 1987. In addition, the number of loci of infection decreased from 4 to 1 and, finally, the number of farms with sporadic occurrence of positive animals decreased from 11 to 1. The desribed diagnostic method and restriction of EBL is economically advantageous and useful in practice.

lmmHoffmphylnis ot Salmonellosis in Calres

MIKULA I., ~ C E., BULiK J., PISTL J. Depanroent of Microbiology, Immunology and Zoohygiene, V~V, Ko~ice ; Imuna Y~ari~sk~ Micharany

On the basis of results of immunoprophylaxis of salmonellosis of calves we recommend the use of the following preparations: (1) vaccines (attenuated, inactivated, subceUniar), (2) specific leukocyte dialyzate and (3) specific intravenous Ig. The advantages and risks of the afore-mentioned preparations are discussed. In addition, results of testing the living salmonella vaccines in association with postvaccination complications and contamination of the environment are presented.

Treatment of ~ o n e l l o ~ ot CJlves with the Trawler FKtor

PXSTL J., MIKULA I., ~ C E., DOBRnCOVA E., BAtCSVOVA B. Department of Microbiology, Immunology and Zoohygiene, V•V, Ko~ice

A group of 12 calves suffering from salmonella infection was treated with the specific leukocyte dialyzate (LED); the efficacy was compared with a group of control calves. Calves aged 21 days received a triple i.v. dose of LeD and were then infected p.o. with a virulent strain of Salmonella typhimurium. Comparison of treated animals and controls revealed significant differences in the clinical condition, numbers of salmonellas in the stools and parenchymatous organs, in the phagocytotic activity of leukocytes, number of rosettes and MIF production.


Prevention o! ~ m o n e k ~ s ~ th ~ Serum and Tnmtet Fsaor

~;i~.4x F., GRANATOVA M., KRF_JCI J., Jingo J. VUVL, Brno ; Bioveta lvanovice na Han~

A biopreparation for passive immunization of calves against salmonellosis was prepared from the blood and bovine lymph nodes of animals immunized with selected salmonella strains in complete Freund's adjuvant. The lyophilized preparation was irradiated with ~~ (25 kGy). The efficacy and harmiesness was tested in white mice and calves, After a triple i.p. injection to mice at 1-2 day intervals, enhanced values of phagocytosis (measured by the INT test) were observed after 48 to 72 h (index MA > 1.5). In addition, foot pad induration in the delayed-type hypersensitivity test and a 80-100 % protection after an i.p. challenge with LD,0o of S. typhimurium strain 10 312 was observed up to the 7th day. The LMI values were found to be 22-45 % after both subcutaneous and peroral administration. A 100 % protective effect was observed after p.o. challenge with 10~-108 S. typh/mutium 10 312, after the challenge dose of 109, 60-80 % calves survived for 4 weeks after the infection.

lrbe/nnnent~ o! 1~ymk ~ d o a s on ~be Course o! E z l s e r l m ~ a ~ / n ~ ~ Taem~ (Zeder 1800) Am ICR Mke

~ K J., PROKOPIC J. Institute of Parasitology, (~A V, Cesk6 Bud~jovice

The immunological status of the host plays an important role in the pathogenesis of parasitic infections. Using the model system of mouse - Taenia crassiceps, we attempted to test the effect of immunomodulatory substances of the thymic origin (T-activin and Thymulin) on the course of experimental invasion. A single s.c. administration of 100 [tg of T-activin, given at various intervals before and after invasion, led to a significant decrease in the numbers of Taenia crassiceps in the abdominal cavity of experimental mice (by 47.1 to 93.6 %) as compared with control animals. After the s.c. injection of T-aedvin and i.p, injection of antigen (a full homogenate of Taenia crassiccps cysticerci), the decrease of cysticerci was more pronounced than in the group of mice that received T-activin only or the homogenate (by 76.8 % against 65.5 % and 17.0 %, respectively). The protective effect of individual combinations of T-activin with the homogenate did not correlate with the level of specific antibodies in sera of immunized mice (detected by indirect ELISA method). It thus f611ows that the thymic preparation can be used for enhancement of anthelminthic vaccine activity.

Cro~-Re~/i~ Ant/bo~es ~ VArm~ of Genus Paramyxovirns

FRA~r~OVA V., HOLUBOVA J., GRUBHOFFER L., KAgOVA V. Department of Microbiology and Epidemiology , FVL UK, Prague; HIE, Prague ; Institute of Parasitol- ogy, (~SA V, Cosk~ Bud~jovice ; Department of Virology, KH$ Usti n.L.

Using the ELISA method with various antigen types of the parotitis (Vpar) and parainfluenza virus 3"3 (Vpi3), sera of 34 individuals with parotitis and 74 sera of patients with parainfluenza were examined for the presence of IgM and IgG. 37.5 % patients with parotitis were IgM-positive with surface antigen Vpi3 and 30.6 % individuals with parainfluenza were IgM-positive with the whole virion antigen Vpar. Cross-reactions, observed primarily in elderly individuals, were eliminated by using the nucleocapsid antigen Vpar or labelled Vpar in the ELISA test. Infections with parainflueaza viruses TI or T2 were regularly accompanied by positive IgM against the most frequently occurring T3 and often even by high antibody titres against T3 in HIT. The results illustrate the dependence of the immune response on previous experience with microorganisms bearing similar surface epitopes.

1990 ABSTRACTS 41

llbe Rote of IfM l~ term~t iou in the ~ of Rmbella

P^v~K E., FRAg*KOVA V., H~KOV~ V., KU~OVA H. Department of Microbiology and Epidemiology, FVL UK, Prague; Department of Virology, Hygienic Station Prague

Rapid diagnosis of rubella is of high importance in pregnant women with suspected rubella infection. We developed a kit for an indirect ELISA test employing a purified whole-virion antigen for the determination of IgM in a single serum sample; the test proved to be reliable and sensitive. The accuracy of results was confirmed by an increase of specific IgG in the ELISA test and positive antibody titres in HIT and CFR in 50 paired patient sera, and by mostly negative results in sera of a large group of control individuals. Using indirect IF, paired sera of 42 patients and 32 healthy individuals were examined. The proof of IgM in the patient's sera was less sensitive than the results obtained by the ELISA assay. Specific IgM could be detected by indirect IF in about 40 % of sera of patients witn an acute infection. However, the indirect IF appeared to be suitable as a practical method for confirming contradictory results of HIR and CFR by determining the IgM and IgG antibody response in paired sera.

z . . ~ ~ of Eq.ine l . t ~ Anemb

C ~ ~R V. VSV, Brno

Equine infectious anemia (EIA) represents a classical example of a persisting infection accompanied by depression of hematopoiesis, hypergammaglobulinemia, formation of immune complexes and their deposition in glomeruli and by decreased C3 complement component. We examined precipitating antibodies against EIA in the blood serum. The spleen pulp of experimentally infected horses served as material for antigen preparation. Precipitating antibodies were detected already 20 days after experimental infection with subsequent permanent persistence. The immunosuppressive effect of dexamethasone on antibody formation and on activation of the chronic form of EIA is discussed. Determination of precipitating antibodies is the basis for EIA surveillance in Czechoslovakia.

lmpommce of New Deve/opments/n Tm:omr lmmm~bgy

EovxAtx J., LAc~r~ovA L., Mc~z~ovA M., B ~ x J. Department of Immunology, IMV VUI~O, Brno

The proof of existence of turnout-specific or tumour-associated antigens, studies on specific and nonspecific antitumour defense in human neoplasia, gradual understanding of regulatory immune mechanisms including their modulation and the hybridoma technology opened new possibilities of tumour diagnostics, monitoring of the disease and optimization of therapy. The contradictory results of ceil-mediated immunity tests and of TAA turnout markers in the diagnostics of the primary disease are still being discussed. Quantification of TAA in the body fluids is utilized in the diagnostics of metastases and recurrence. Of unequivocal value is the typing of differentiation antigens in the diagnostics of lymphoproliferative malignancies and the TAA phenotyping in the histopathoiogical diagnostics of solid turnouts. Immunoscintigraphic and immunocytochemical localization of tumour cells is promising for the diagnosis of early tumour stages. As regards modern oncoiogical therapy, immunomodulators of the lympho- and cytokine type, conjugates of monoclonal antibodies with toxins, cytostatics o[ radionuclides and antiidiotypic antibodies are used.


/ m m ~ o d i a g n o ~ of Ovsr/nn and Uterus Turnouts

To~r~_~ J., LAONOVA V., LOUDA B., KRF_.JSEK J., TURKOVA M. Clinic of Obstetrics and Gynecology, 2nd Clinic of Internal Medicine, LF UK, l-Iradec Kr~lov~

The tumour antigen CA 125 is significantly enhanced at the time of primary diagnosis only in women with ovarian epithelial carcinoma (x = 326.5 U/mL). As regards women with carcinoma of cervix uteri (x = 93.5 U/mL) and carcinoma of endometrium (x = 56.2 U]mL), the CA 125 values did not differ significantly from those of controls (x = 35.6 U/mL). In addition, nonspecific immune complexes, C3, prealbumin, transferrin and ct-2-macroglobulin are similar in the controls and in women with tumours before initiation of therapy. On the other hand, orosomucoid, ceruloplasmin and CRP are significantly higher in women with ovarian carcinoma. With the exception of CA 125 and orosomucoid in women with ovarian carcinoma, the diagnostic value of these parameters is low. Their changes are more important for determination of tumour recurrency.

Determin~on of Surface Cell Antigens in H u n Uveal Melanoma by Monoclonsl Antibocks

F., KI.~E~)A J., Novo~A E., ~ N A K A. (_lEO SA V, Bratislava ; Clinic of Ophthalmology, LF UK, Bratislava

Murine mydoma cells Sp 2/10 were fused with the spleen cells of mice immunized with freshly obtained cells of human uveal melanoma. The hybridomas obtained were subsequently examined (using the RIA test and indirect immunofluorescence) for their ability to bind to cell membranes of uveal melanoma and negative binding to cell membranes of carcinoma ceUs, fibroblasts, cells of the retina and skin of healthy donors. The results showed that antibodies produced by ten clones bound strongly to

�9 antigens of cells of human uveal melanoma (UMEL-H, UMEL-K). Weaker binding was observed with the uveal melanoma cell line VUP-1 and with cell lines of human skin melanoma (HMB-2, BHM-8, BOWES). Binding to control cells was negative. At present, studies of these monoclonal antibodies are in progress with the aim to provide more accurate analysis of antigenic profiles of uveal melanoma cells in comparison with profiles of other normal and malignant tissues in order to further improve the diagnostics of this disease.

|mml/DoreIelfJ~ It4onJtor~ in Padents w/th Ms~,mmcy

PEKAREK J., PROCHAZKovA J., RYgKovA O., Turn, ovA M. FN and KHS-KUNZ and LF UK, Hradec Kr~/ov6; (./SOL, Prague

The aim of the present study was an early diagnosis of recurrence of the disease and the use of immunotherapy in a complex therapeutic regime. A list of immunological tests is presented; TF Sevac and TF'FS were used for immunotherapy. Since 1982 we have obtained 492 blood samples from 145 patients for immunological examination. A total of 412 ampoules of TF Sevac and 74 TF'FS were given to 38 patients. Recurrence or progression of the disease was detected by laboratory tests already 6 - 1 2 months prior to clinical symptoms almost in 100 % cases. Laboratory and clinical improvement was observed in 80 % patients after "IF therapy.

Interactlon of Interferon with Primary Tumour Cells and with Some Types of lmmunocompetent Cells in Patients

KUBEg M., go~'i~KovA D., FUOtSBEROER N., BOREC~ L., HOR~AK M., GOD.M.A., ZVARA V. Institute of Virology, SA V, Clinic of Urology, NsP LD, UKO, Bratislava

Using DNA synthesis determination by 3H-dThd incorporation, we examined the susceptibility of tumour cells obtained from the carcinoma of individual patients, on the antiproliferative activity of human

1990 ABSTRACTS 40

interferon. In addition, the effect of interferon on peripheral blood NK-cells was studied in the same patients using the cyt0toxicity test with radioactive chromium. The NK activity against autoiogous tumour cells was also measured in patients by a modified cytotoxicity test based on competition of unlabelled tumour cells with K562-1abelled cells. The results of antiproliferative tests revealed different individual susceptibility to the effects of interferon which might contribute to better selection of patients and more exact indication of interferon therapy. Similar individual differences in NK activity were observed in patients with tumours.

Stimalation ot Haman Lymphocyte L'~totoxicity ia a Two.Day Culture

CHUDOMEL V., SOUC-'EK J., HRUBA A., SCHW~Q,Z J., JEIL/~BEK J., SMETANA K. UHKT, Prague; IHE, Prague

Peripheral lymphocytes were cultivated in the presence of IL-2 or extracts of human myeloblasts (F-3) prior or after 10-s treatment with direct electric current (intensity 5 mA/cm 2 of the electrode area). The cytotoxic activity of K 562 cells or myeloblasts was determined by trypan blue, 59Cr release and formation of conjugates. Enhanced cytotoxicity was found in both the IL-2- and F-3-treated cells; electric current was more effective and the combination of the current with IL-2 or F-3 was the most effective. Stimulation of lymphocytes was confirmed by nucleolus activation and by changes in soluble protein spectrum.

Rad/ores/sta~e of LAK Cell-Mediated L'ytotoxir Knnaf

J|RA M., Hogk~ J., STREJ~'ES: J. 1st Clinic of Internal Medicine, LFH UK, Prague ; II-IE, Prague

Lymphocytes isolated from venous peripheral blood were irradiated from a cobalt source prior to stimulation with recombinant interleukin 2 (IL-2). DNA synthesis (measured by 3H-thymidine incorporation) decreased significantly together with increasing radiation dose. However, decreased cytotoxicity to NK-resistant cell lines was apparent only when doses higher than 20 Gy were used. These results do not support the original observation of Rosenberg and Grimm who consider radiosensitiv/ty to be one of the typical characteristics of LAK cells. On the other hand, the results of our study favour the hypothesis of close developmental relationship to NK cells which are also radioresistant. Provided the in vivo situation is analogous to that in vitro, immunotherapy of malignant tumours with IL-2 and LAK cells could be combined with radiotherapy without decreasing the effectiveness of immunotherapy.

Association of IILA Antigens with Sm~ivai in Amte Leukemia

PRA~g J. Department of Transplantation Immunology, 0I-IIr Prague

We followed the association of HLA antigens with sex, duration of survival and diagnosis in patients suffering from acute leukaemia (AL). The frequency of HLA antigens was examined and statistically evaluated in 80 patients with AL (56 myeloid, 24 AML and 24 lymphocytic ALL leukemias); 300 blood donors served as a control group. The following methods were used: biphasic microlymphocytotoxicity test, statistical evaluation - X 2 test with Yates' correction, Fischer's exact test, relative risk (RR). No significant association with HLA was found in AL and AML, a significant increase of HLA-B5 was found in ALL (X 2 - 11.58, Fischer's test Pc - 0.046, RR - 4.64). In females, compared to males, an insignificant elevation of A28, B17 and decrease of B35 was found. Patients possessing antigens A3, A10, B12 had poor prognosis. Prolonged survival was observed in patients with antigen A2, however, the results were not statistically significant. One may conclude that the presence or absence of HLA is not an indicator of survival in AL patients.


Eszymk Character/s/ks of Lymp/m- and Mye/opto~erafive Diseases

KLOBU~XCK~ M., Hm~AKovA A., BmaUgtKOVA O. (.lEO SA V, Bratislava

Cytochemical, biochemical and immunological analysis of malignant lymphoid and myeloid cells were the components of a multicenter phenotypic study of human ieukemias. We studied the relative importance and specificity of certaln~er/zymic markers of leukemic cells in the diagnosis of leukemia. The immunophenotype of leukemic cells was determined by a panel of monocional antibodies. For cytochemical marker analysis, a broad spectrum of enzymic reactions (KF, DPP IV, ANAE, 5'-NT, MPO, SBB, ANBE, CHAE) was used. The presence of purine metabolism enzymes (ADA, PNP) was determined by the radiochromatographic method. The results of cytochemical tests correlated closely with the results of immunochemical studies. Analysis of enzymes of purine metabolism was significant in acute leukemia of the T phenotype for the distinction of acute myeloid leukemia from leukemia with morphologically uncertain cell types. Comparative cytochemical, biochemical and immunological studies stress the importance of a complex approach to characterization, classification, diagnostics and therapy of hematological malignancies.

Ssppres~n ot Gnmst~r te u d Mscrepimge ( O ~ - G M ) Proge~tor Ce/b by the H s m u Myele/d Leskemk Line ML-2-Dedved/nk/b/tory Aearity

C~K~OVA V., NEuwnt'r J. U/-/KT, Prague

Cells of the ML-2 line release a high-molar-mass substance into the medium that inhibits the entry of CFU-GM into the S-phase of the cell cycle. The inhibitory effect of this factor was measured using the 3H-thymidine suicide method. The inhibitor was partially purified by gel filtration chromatography on the Sepharose 6B column. Molar mass of the active protein was estimated to be 310-440 kg/mol. The inhibitory effect of the factor can be neutralized with antiserum against human placental ferritin. A functionally and immunologically similar inhibitor was found in media, conditioned by cells of patients with the myelodysplastic syndrome having enhanced numbers of blasts who are prone to transmission of the disease to acute leukemia.

Conunon L e u k o ~ Antigen in Acute L e e k ~ : Deletion by Monock)m/And/~ly ~ 28

KozA V., CE~OVSKY P. 1st Clinic of Internal Medicine, LF UK, Plze~

The common leukocytic antigen was determined by indirect immunofluorescence using monoclonal antibody MEM 28 in 9 cases of acute myeloid leukemia and in 6 cases of acute iymphoblastic leukemia (ALL). According to the FAB classification of acute leukemias, false negativity was found in 2/2 M 1, 0/3 M 2, 1/2 M 4 and 0/2 M 5. In ALL with common antigen positivity, negative results were obtained in 3/4 cases, in T-ALL in 1/2. The antibody can detect the antigen in the myeloid, monocytic and lymphoid lines. However, the negative finding does not exclude the possibility of hematological malignancy, particularly in dedifferentiated cells.

Stimulatory F ~ e ~ of Vurtons hnmMomod~tors on N K - C e / / A ~ y

S o u ~ J., C ~ V. UHKT, Prague

The effects of interleukin 2 (IL-2), anti-T-monocional antibody (~SAV (MEM-57) and an alloantigen prepared from leukemic cells (F3) on the killer activity of peripheral lymphocytes of normal donors or of

1990 ABSTRACTS 45

leukemic patients, was tested in a short-term culture. After a 3-7-day cultivation of normal lymphocytes with IL-2 or MEM-57, stimulation of cell proliferation occurred accompanied, particularly with IL-2, by enhanced cytotoxicity against the target cells K-562. The F~ preparation enhanced the stimulatory effect. The onset of the stimulatory effect of IL-2 or MEM-57 was delayed in cells of leukemic patients, Le. after a 7-14-day cultivation. While IL-2 induced significantly the NK activity, MEM-57 influenced it only slightly. Even in this case, F3 had a stimulatory effect.

Pand/el Use of Moaockma/A~d/wd~ mid Pimlroe~olk ;,, die W.~m,dm,doa d ~ F_~m~#es

ZEMANOVA D., MtmZAROVA M. IMV, VOKEO, Brno

Moneclonal antibodies (MAb) permit to identify various cell types; phagocytosls is one of the parameters for evaluation of macrophage functional activity. We combined determination of the cell phenotype with phagocytotic activity tests in an attempt to obtain more accurate analysis of exudates in breast and ovary carcinomes. Phagocytosls was examined using Cd microcrystnls. Cell samples prepared on the cytospin and phenotypic characteristics of cell populations were tested by immunochemicai indirect methods with peroxidase- or alkaline phosphatase-labelled conjugates. Selection of suitable MAb and combination with other methods makes it possible to determine simultaneously the phenotypic markers of various cells (on the basis of different staining) and the phagocytotic activity in a single preparation. We employed MAb specific for epithelium differentiation (BA16, DA7, DC10, HMFG2) and for cells of the macrophage type (3,9', BRA55 and MEM18). Transparent Cd particles are clearly visible in diffusely stained cytoplasm of phagocytes. The above procedure facilitates accurate differentiation of exudate cells.

Re/a/iota/alp o/Pkumm Prote/m to CA 125 A~//g~m tn Women w/~ Tamom~

T o ~ a J., LACrNovA V., IO~sex J. Clinic of Obstetrics and Gynecology, 2nd Clinic of Internal Medicine, LF UK, Hradcr Krdlovd

Using the monoclonai antibody, we found increased levels of CA 125 antigen in 98.4 % women with active ovarian epithelia carcinoma, but also in 33 % women without malignancy. Simultaneous examination of orosomucoid (a-1-acid giycoprotein) enhanced the sensitivity of CA 125 determination from 65 % to 82.1%. No correlation between CA 125 and orosomucoid, preaibumin, transferrin and nonspecific immune complexes could be found in women with ovarian carcinoma. "Falsely positive" CA 125 finding occurs quite frequently in women with endometdosis and adenomyosis. In these patients, a significant correlation between CA 125 and transferdn (r = 0.67) and circulating immune complexes (r = 0.80) was found.

Cell Stance M~rters in HenmtologJml

BABU$|KOVX O., KONIKovA E., Har~X~ovX A. OEO SA V, Bratislava

During studies of the cell surface markers in hemoblastosis, we studied the association of the immunological phenotype with enzymes of purine metabolism in T-ALL The T-cell phenotype ALL is associated with poor prognosis. Its determination and selection of patients with T-ALL is of prognostic and therapeutic importance. We determined the cell type in 30 patients using a panel of 11 monoclonal antibodies against various differentiation antigens of the T-type by means of the E-rosette test. The enzymes of pmine metabolism (ADA, PNP) were determined by radiochromatography. A typical finding for the T-ALL group was characterized by a significant increase of ADA activity accompanied by decreased PNP activity as compared with the controls. When determining the relationship between the immunological phenotype and typical enzyme activity in T-ALL, a correlation was found in 27/30 of cases


(the T-ALL cells expressed in 6 cases also cALLA, rarely even the Ia antigen). In 2/30 cases the typical T phenotype was associated with atypical enzyme activities. In 1/30 patients with non-T phenotype a typical pattern of enzyme activities was obtained similarly to T-ALL Studies of purine metabolites stressed their diagnostic value in the detection of patients with unfavourable T-ALL phenotype.

Innnunorestomtire Effect ol Levu~sol in l~ts with Tnmsplsnted Syngeneic Turnouts

KUSENDA J., KALAFLrr F., NOVOTNA E., KLOBU~ICKA M. UEO SA V, Bratislava

The immunorestorative effect of Levamisol was studied in rats with syngeneic B-77 tumours that were simultaneously immunized with sheep red blood cells. Tumour-bearing rats had depressed cell-mediated cytotoxicity, delayed-type hypersensitivity and antibody production against sheep erythrocytes. While the suppressed cytotoxicity could not be restored with Levamisol, the delayed-type hypersensitivity and antibody formation returned to (or above) normal values. Turnout growth was significantly inhibited after Levamisoi administration. The presence of the tumour induced changes in the uptake of 3H-uridine-labelled lymphocytes in the spleen and peripheral blood. The restorative effect of Levamisol on T lymphocytes differed in different organs. The differences in the effects of Levamisol on aH-uridine-labelled spleen and peripheral blood lymphocytes may be due to functional heterogeneity of immunocompetent cells. The results help to further clarify the mechanism of the immunostimulatory effect of Levamisol.

Occurrence of HLA.DR Antigens on the Epithelium of the Nl~umary Gland: Clbuges during D/fferent/at/on and Turnout Tnmstormstion

PE'r~K M., BARTEK J., VOYI'~EK B. Department of Microbiology and Immunology, LF UP, Olomouc ; VUKEO, Brno

Using immunohistochemical methods, the occurrence of HLA-DR (MHC II) antigens in the epithelium of the mammary gland (MG) was studied at various stages of its differentiation in 50 benign and 72 malignant MG tumours. HI.A-DR antigens occurred neither in resting MG nor in the proliferating epithelium (cultivated organodis). During pregnancy and lactation, part of the epithelium becomes HLA-DR-positive with subsequent decrease of expression after cessation of lactation. Induction of I-ILA-DR antigen expression occurs quite frequently in tumours; the percentage of positive tumour cells is variable (0-20 % benign, 0 - 9 0 % in malignant lesions). The observed dynamics of HLA-DR antigen expression during differentiation and tumour transformation of the MG, as well as their presence on the epithelium, i.e. cells outside the immune system, is in correlation with the concept considering the class II MHC molecules to be-differentiation antigens participating in a number of immunological events, e.g. antigen presentation during the immune response against the tumour.

A n ~ sis of Human Leukemic Cell Markers Using Monoclonsl Antibodies

KOUBEK K., STARY J., I-L'xuZNER P. UHKT, Prague ; 2nd Clinic of Pediatrics, FN Prague; Department of Immunology, Prague

We determined the membrane markers on various morphological types of leukemic cells isolated from peripheral blood and bone marrow of patients with various blood diseases (CLL, ALL, AML, AM/VIoL), using immunofluorescence methods. Individual phenotypes of leukemic cells were determined by monoclonal antibodies according to the international CD classification. Individual types of leukemic cells express on their surface antigenic structures to various degree which can be employed for more accurate classification of individual differentiation stages of hematopoiesis.

1990 ABSTRACTS 47

Immnnoem~/mic Cell I ~ e n o ~ in Pedpheml Blood n d Bone-MmTow Sinews by Mesm of Mom~doml A n a ~ e ,

KOZA V., C-'errrovsrV P. 1st Clinic of Internal Medicine, LF UK, Plzefi

The authors described an indirect immunoperoxidase method permitting immunophenotyping of cells on dry smears of peripheral blood or bone marrow. Domestic monoclonal antibodies, diagnostically important in lymphoproliferative diseases, were employed: HL40, TEC01, MEM 78, MEM57, MEM 32, MEM 31, MEM 28, MEM 18. The described procedure allows examination of stored unfixed preparations, determination of morphology of positive and negative cells and also retrogade evaluation of the findings.

Time-Dependence of Put/he MetaboBtes Effects on Antibody FormslAon to Sheep Erythroc~es

PA~AJOTOVOVA V., NOUZA K. VOFB, Prague; OEM C~SA V, Prague

We tested the effect of the original domestic antipurine preparation Cloturin with antitumour activity in comparison with standard antipurines 6-MP and azathioprin. All the substances tested depressed significantly the formation of antibodies against sheep red blood cells 2 days after antigen; however, 6-MP when administered 2 days before antigen enhanced the antibody formation. Cloturin depressed the antibody response, azathioprin had no significant effect. Thus, 6-MP and azathioprin manifested primarill an antimetabolic activity while Cloturin behaved like a cytostatic drug of the 3rd class. The monofunctional alkylating component of the molecule obviously participates in its immunomodniatory activity.

lmmm~mod~tion of Turnout AIIogr~ Growth

Soao'rxovA E., ~tX.A K., NouzA K. ~EM C'~A V, Prague

Humoral, cellular and phagocytotic responses were suppressed in both syngeneie and allogeneie recipients during the development of a tumour graft Sarcoma I. One of the mechanisms responsible for the suppression is the presence of suppressor macrophages and T lymphocytes that can be eliminated by repeated administration of silica or anti-Thy 1.2 monoclonal antibody. Using the method of adaptive transfer to syngeneie recipients a gradual decrease of the tumorigenic activity was demonstrated while in allogeneic ~eeipients, the tumorigenic activity was found both in the early and late periods. This model can be used for analysis of the effects of positive and negative immanomodulators. Determination of the nature of immunomodulatory activity requires detailed studies of the effect of syngeneic and aliogeneic tumours. Both immunomodulator types can induce, depending on the time and mode of administration, antitumonr and protumour effects.

~]be Stmte ot lmm~m/ty in Psmproteinemiss

ZAVI~_.AL V., SEBEROV~ E. OK/A, l~r P/zefl

Positive proof of myeloma proteins in sera of immunologically examined subjects has increased at least five-fold over the past 20 years. The finding of paraprotein is unexpected in 71% of cases; in only 29 % the diagnosis was known or suspected. The age distribution keeps gradually shifting towards young individuals so that today 50 % of patients are younger than 50 years. We attempted to solve the problem of prognosis of benignity by testing the immunological status of patients. It was shown in a group of 130


patients that not only the malignant but also the benign forms are accompanied by immunodeficiency and possess characteristics that are present in other malignancies. Nevertheless, the total number of immunological aberrations is significantly higher in malignant forms. Although the immunological examination is of limited value for distinguishing between the two forms, it is of importance for the follow-up of individual prognosis during dynamic examination.

o! LAK C d . M e ~ t e d Cytotoxic

J~,A M., Hoar~, J., STREJC'F.K J. Ist Clinic of Internal Medicine, LFH UK, Prague;/HE, Prague

The mechanism of lymphokine-activated killer (LAK) cytotoxicity is not sufficiently known. In our study, lymphocytes were separated from the peripheral blood of healthy donors and the cells were irradiated with a cobalt source prior to activation with recombinant intedeukin-2 (IL-2). DNA synthesis decreased together with increasing radiation dose (measured by the incorporation rate of tritiated thymidine) ; however, the decrease of cytotoxicity against NK-resistant lines became significant only when doses higher than 20 G-y were used. The results point out the fact that similarly to the NK activity, even the IL-2-induced cytotoxicity of peripheral lymphocytes does not require DNA synthesis. Provided the in rive situation is analogous to that in vitro, immunotherapy of malignant tumoun with IL-2 and LAK cells could be combined with radiotherapy without decreasing the efficacy of such a combination.

Amoe~tloa o! ~e/mmmmolol4es/P/,eaotype with Psrise Metsbollsn F2,zys~ b Acute L y m p & ~ i s ~ Le~emlss

BAaugI~ovA O., Halv~ArovA A., UmAzv P. (.IEO SA V, Bratislava

The T cell phenotype ALL is associated with poor prognosis. Its recognition and selection of patients with T-ALL is of prognostic and therapeutic importance. We determined the cell type in 30 patients with T-ALL using a panel of 11 monoclonal antibodies against various differentiation antigens of the T type in an E-rosette test. The pudne metabolism enzymes (ADA and PNP) were determined by radiochromatography. A significant increase of ADA accompanied by decreased PNP activity was a typical finding in patients with T-ALL. When comparing the relationship between the immunological phenotype and typical enzyme activity in T-ALL patients, correlation was found in 27/30 cases (the T-ALL cells expressed even cALL in 6 cases, rarely also the Ia antigens). In 2/30 cases the typical T phenotype was associated with atypical enzyme activity. In 1/30 patients with non-T phenotype, a typical enzyme activity, similar to that in T-ALL was found. The research of purine metabolism enzymes stressed the diagnostic importance of detection of patients with unfavourable T-ALL phenotype.

/mm~oge~d0, o1 the .4dt~gr~ Tri~er/~ oi the Trs,~hmtsaon Rese6o=

HA~,KOVA V. Department of Immunology, IKEM, Prague

When the donor and the recipient differ in the major histocompatibility complex (MHC) the allotransplantation reaction is rapidly and effectively triggered by direct Tn lymphocyte activation, brought about by MHC molecules of the donor that are present on dendritic cells or corresponding cell elements. These cells express MHC I molecules and particularly MHC II molecules depending on the conditions, e.g. the presence of interferon. The so-called "passenger leukocytes" of the donor tissue play also an important role. When these cells are removed from the graft or inactivated, e.g. in the tissue culture, the immunogenicity of the graft may decrease. Recognition of foreign molecules of the allograft "on the background" of self MHC molecules of the recipient, similarly to the response to conventional

1990 ABSTRACTS 411

thymus-dependent antigen, occurs only on the condition that MHC molecules are released from the cell or when non-MHC molecules are involved. This is a less effective pathway. IL-1 and IL-2 are involved in all these processes.

Immunomodu/s/ion in Sterile Women with Hydrocorttsone

ULC"OvA-GALLOVA Z., MRAz L., PLAm~'XOVA E., MAC'K0 F. Clinic of Gynecology, UK, Plzed ; Pharmaceutical Department, KONZ PIzeit

Local sperm-agglutinating antibodies (LSAA) present in the ovulatory cervical mucus may be the cause of primary sterility in 2 - 3 % of infertile women. A group of women, suffering from long-lasting infertility with LSAA in the cervix uteri, in whom previous occlusive condom therapy and repeated intrauterine insemination was without effect, were treated with hydrocortisone that was applied on the ectocervix for at most the period of four cycles. LSAA were determined by the sperm-mucus capillary penetration text, microagglutination and mixed antiglobulin reaction (MAR) in 4 patients after termination of the treatment. LSAA disappeared completely in 16 infertile patients, nine of them gave birth to healthy children, the 10th woman is at present pregnant. No undesirable side-effects were observed during local administration of hydrocortisone to the ectocervix.

The Effect ot Sperm Anffgenir Fmcffom on T Lymphoeytes

UL~OvA-GALLOVA Z., PANZN~R P., ZAVAZAL V., KnAUZ V. Clinic of Gynecology and OKIA, UK, P/zed

We examined the ability of erythrocyte binding to the CD2 receptor of lymphocytes preincubated with various fractions of spermatozoal antigens. In further experiments, lymphocytes were preincubated in the presence of a high-molar-mass spermatozoal fraction. The results were compared with the E-rosette test without preincubation in primary infertility, allopecia, immunodeficiency, chronic infections, turnouts and some other diseases. Inhibition of E-rosette formation by the high-molar-mass spermatozoal fraction was regularly observed in all lymphocytes tested. Statistical analysis (by means of a paired t-test) revealed significant E-rosette inhibition in sterility (p < 0.05), chronic infections and other diseases (p < 0.01).

Imm//no/oSk~l/R~pO/Me/~ore ~ ~ter ~ ]r'r~lml~tl/Jon

KAgldK J., KO~ANDRLE V., FAmAS J., MACUROVA H., KOR~AKOVA L. IKEM, Prague

The immunological response was examined in 74 candidates for heart transplantation. The following results were obtained :

~, Lymphocytes/L 26/28 $ CD4+/CD8 + index 25/63 ~, CD3 + lympho/L 28/44 ~, skin test index 60/65

CD4 + lympho/L 46/63 1' cytotox, antibody > 20 % 4/72 CD8 + iympho/L 32/63 DR 6 antigen 5/66

No correlation between the decreased immune response and the cause of cardiac failure could be found. Thirteen patients were immunologically monitored for at least 6 months after transplantation. The patients received CyA and corticosteroid immunosuppressive therapy. Only three incipient rejection reactions were diagnosed by endomyocardiac biopsy. Thus, the whole group may be classified as low responders with effective immunosuppressive therapy. However, the absolute lymphocyte number and the CD4+/CD8 + lymphocyte index were found to be within the range of normal values. CD3 + lymphocytes decreased after transplantation and tended to normalize towards the 6th month. A constant


profound decrease of peripheral blood leukocytes without serious rejection episodes was a regular finding in patients after transplantation. This criterion is used for monitoring patients treated with CyA.

Immuogenedr Problems o! Boae.MmTow Tmmpl~mlJon

Buc M., Nw,~ M., BLAHA M., VA~ASEK J. LF UK, Bratislava ; VL VDU JEP, l-Iradec Kngov6

We examined 39 families (altogether 102 siblings) in order to select suitable couples for bone marrow transplantation (BMT). We found 20 HI_A-identical couples, in 10 of them a potential donor for BMR could be selected. Identity between the parents and the child (patient) was observed in two cases. 20 HI.A-identical couples represent 19.6 % of HLA identity at the average of 2.6 children per family. The following hapiotypes were the most frequent in the families examined: HLA-A1, B8- 0.096 I, HLA-A3, ]37 - 0.0513, HLA-A2, B12 - 0.0449 and I-ILA-A2, B15 - 0.0385. Recombination was not observed.

Assoekl/Jon between//LA Typ/q u d MLC Rese/~n/n Bone Marrow Donms ~ d Rec/p/ents

S o u ~ J., M ~ s ~ A., P ] ~ K J., NovJac LT. OHKT, Prague

Within the frame of the transplantation program of f3HKT, suitable donors of bone marrow were selected among the parents and siblings of 50 hematological patients. The results of the MLC reaction and HLA typing were compared. Among 56 siblings, 38 (67 %) completely matching donors were found. Among 11 tested fathers two suitable donors (18 %) were found while no mother was suitable out of the 11 tested even though there was a match in class I HLA antigens with the child. Positive MLC was observed in five I-ILA-matching donors while in I-ILA non-matched donors the MLC reaction was negative in five cases. The association between HI..A, DR examinations and MLC is discussed.

T.Lympl~Tte Sabpol~stiom in Pstlents sfter Kidney Tnm~phmtstlon

KOPECT~ O., PROCHAZKOVA J., KREJSEK J., FIXA B., ERBEN J. Department of Medical Immunology, KI-IS I-lradee Krdlov6; 1st Clinic of Internal Medicine, FN l-Iradec Krdlov~

We followed the dynamics of changes of CD3 +, CD4 + and CD8 + T-lymphocyte subpopulations in a group of patients (12 males and 6 females) who received a cadaverous kidney graft, at regular 3-4-d intervals. The dynamics of changes of T-lymphocyte subpopulations in three patients with four rejection episodes that followed after a period of adequate function was highly similar to that found during the period of rejection (AR). An increase of CD3 § and CD4 + lymphocytes was observed 3 - 9 days prior to clinical symptoms of AR. CD8 + lymphocytes were predominant at the time of AR. In 6 patients with a delayed onset of the graft function (14-21 days after kidney transplantation), the immunological findings were diverse.

Regm4mtlon of Tmnspl~tstlon Immunlty by Mes~ of MOnOClOBI Andbod;es ~ T.Lympbocyte ~bpopslstlom

HoL~ V. 0MG I~SAV, Prague

Recipients of skin allografts, incompatible in antigens encoded by genes of individual regions or by a part of the major histocompatibility complex (MHC), were treated with monoclonal antibody against T-lymphocyte subpopulations. According to the graft survival it was found that the grafts incompatible in class I antigens, were rejected by Lyt-2 + (CD8 +) cells while rejection of allotransplants differing in class II

1990 ABSTRACTS 51

antigens only (i.e. Ia antigens) was mediated by L3T4 + (CD4 § cells. The suppressor effect of monoclonai antibodies against T-lymphocyte subpopulations was comparable with the effect of antibodies directed against the whole T-lymphocyte population. The results show that monoclonal antibodies against T-lymphocyte subpopulations may replace antibodies against the whole T-lymphocyte population, their effect, however, being highly dependent on the genetic barrier between the donors and recipients of the allograft.

C]u,mcteAstks o! Pept/des Liberated/n A//oguek CeR Reac~om/n Mk~

KorAn0 H., KozAKovA H., KovAR~ F., RYBA M., ~IMAN P., ~ V., BA~YgOVA M. MBU, UOCHB, FgU CSA V, Nov,f Hr~dek and Prague

We examined the effects of peptides (molar mass 1-2.5 kg/mol) released during the allogeneic reaction of brain cells (90 rain) or allogeneic spleen cells (4 h). An immunosuppressive effect of peptides on mitogen-stimulated lymphocytes was recorded according to enzyme changes and 3H-TdR incorporation. The peptides inhibited the activity of 5'-uncleotidase of brain cells and slightly accelerated the cell cycle of neuroblastoma cells. BPCL analysis of spleen peptides indicated a lower degree of heterogeneity in fraction 1, probably with two dominant components. The activity of isolated peptides was compared with the activity of peptides with defined structure (~ 1 kg/moi).

The Elfecf ol L~e/oq~r/n A on ~e Fur and ~ymm o! nu/ns Mke

SLAV~V A., HOLUla M. Department of Immunology, IKEM, Prague

In addition to the immunosuppressive effect of cyclosporin A which is probably caused by a selective blockade of helper and effector T lymphocytes or by inhibition of IL-2 activity, the influence on fur growth was also observed. We found that administration of CyA (10 mg/kg p.o.) induced in athymic mice, having abnormal keratinization of hair follicles, hair growth up to the almost regular thin fur coat all over the body with the exception of the abdominal region. Simultaneously, changes in the dysgenetic thymus occurred, suggesting the effect of CyA on certain thymus epithelial cells.

Follow.up Study of MonoclonsI.Antibody.Treated Patients

MOKRA R., HA~KOVA V., KAgLIgOVA J., KAgLiK J., NEME.qgALOVA V. Department of Immunology, IKEM, Prague

Xenogeneic polyclonal and monoclonal antibodies against human T lymphocytes have been used as immunosuppressive substances in patients with transplanted organs. However, foreign antibodies may induce sensitization. Therefore, we monitored patients treated with murine monoclonal antibody OKT3 (Ortho, USA) against the recognition structure of T lymphocytes. So far, we followed (by means of the ELISA test) the occurrence of antibodies against the murine monoclonal antibody used and against the nonspecific murine IgG in 8 patients who had received 5 mg OKT3 perdiem for 10 days. Antibodies did not occur in any of the treated patients during the course of therapy. Antibodies were detectable in 75 % of patients up to one month after termination of the therapy. In most of them, the titre against OKT3.was higher than that against nonspecific murine IgG. By immunizing rabbits we prepared our own polyclonal antibody for determination of serum OKT3 level which can detect concentrations of the order of ng/mL. Retrospective determination of OKT3 levels was performed in the patients' sera. No significant decrease was recorded in any patient during the therapy.


Creebe~ntk Aat/tbymoc~e

Jmk~'KOWt L., K.~LlXOV~ J. /KEM, Prague

Antithymocyte globulin (ATG), a polyclonal antibody against human T lymphocyte.s, has been used in the IKEM since 1980. It is obtained by isolation from sera of rabbits immunized with human thymocytes obtained at necropsy or after operation. It is employed for the therapy of rejection that is resistant to therapy with steroids or classical immunosuppressives, and for prophylaxis in heart transplantations. Experience was obtained with two immunization schemes yielding qualitatively and quantitatively different ATG.

Moe~tion of AIIommsj~taebn Tolermme ~ by Means el lteeoamlg~ut bterle~dm-I u d laterle~tcm.2

HOLA~ V. UMG (~SAV, Prague

Transplantation tolerance was induced in newborn mice by injecting hematopoietic cells from semiallogeneic donors. Naonatally treated animals received allogeneic skin graft at the age of 7 - 8 weeks. Part of animals with neonatally induced tolerance was treated on days 0, 3 and 5 after birth or after transplantation of the skin graft with 1 000 units of recombinant interleukin-1 (rIL-1) or interleukin-2 (rlL-2). The results showed that the treatment of newborn animals with rIL- 1 completely abolished their susceptibility to tolerance induction, and that treatment of tolerant animals with rIL-2 and rIL-1 at the time of transplantation decreased the proportion of animals tolerating skin allografis. However, such modulatory effects of interleukins were detectable only in strain combinations with a high degree of genetic difference represented by incompatibility in the whole major histocompatibility complex or in the K-end of the H-2 complex, where a relatively high degree of resistance to neonatal transplantation tolerance induction was present.

/Odney Tramphnta~on-/~or

MACU~OVA H., JmxA J., IVA~KOVA E. Department of Immunogenetics, IKEM, Prague

Using a cumulative method, we followed the survival of the kidney graft (transplantations performed in the CVO IKEM from 1984 up to the end of 1987) in patients in association with HLA compatibility, presence of cytotoxic antibodies before transplantation and the method of immunosuppression. The results confirmed data obtained in other transplantation centers, that even when cyclosporin was used, HLA compatibility had to be respected, particularly in ~ensitized patients.

New ~ h, rise ~ ot l e k e m ~ ~ , m d ~

2_.rr~,~ D., Dos'rAL C. V(]RCH, P/e~fany; VUCHR, Prague

The etiopathogenesis of certain rheumatic diseases and diffuse diseases of the connective tissue is closely related to mechanisms of autoimmunity. The causative relationship between the origin of individual types of autoantibodLes and nosological units or syndromes has been intensively studied throughout the world. The number of autoantigens, both intraceUular and others (phospbolipids, fihronectin), keeps permanently increasing which allows to define certain disease variants (e.g. CREST, antiphospholipid syndrome etc.). In addition, the knowledge concerning the mechanisms of cell- -mediated immunity and the complement system (inborn defects, membrane receptors) has also increased

1990 ABSTRACTS 5,1

considerably. The role of virus infection as a significant stimulus for the development of newimmunogens involved in autoimmunity is now again appreciated. Finally, new approach, given by molecular biology, genetics and immunogenetics helped to clarify the genetic basis of the above mechanisms by means of recombinant DNA techniques and other methods.

Antibodies Comb/a/rig w/tb the Natfimr Mater/a/o! ~ t / a l g C e b

~'r~AN D., LUKAC J., CF~ECAUF.R L., Lur~CowCovA E. VURCH, Pie~fany

Using indirect immunofluorescence, the reactivity of HEp-2 and He-La cells with sera of 84 patients with diffuse sclerodermia (SD), 100 patients with systemic lupus erythematosus (SLE), 24 with dermato- and polymyositis (DM-PM) and 15 patients with a mixed connective tissue syndrome (MCTS) was determined. In SD patients, positivity to HEp-2 and HeLa was found in 99 % (compared to 67 % found in liver sections): in more than 2/3 of cases, granular reactions occurred. Nucleolar reactions (combined or isolated) as well as centromere reactions dominating the complete or partial CREST syndrome were observed in approximately one-fifth of the cases. In SLE with antinuclear activity, over 90 % of homogeneous or fine granular reactions were present. Granular and nucieolar reactions were more frequent in HeLa cells, in 2 cases (SLE+SD) centromere reactions were recorded. As regards DM-PM, the positivity of proliferating cells exceeded 40 % (only 17 % in the liver sections) and homogeneous and granular reactions predominated. MCTS sera were positive in all substrates and yielded the granular type of nuclear reaction. The advantage of proliferating cells resides in the possibility of detection of antibody against centromeres.

AnUboe'y tmm~Tty t~ Prtmsy Goee

HRNO_R Z., KRUPAR V., BRADNA P., TICHY M., ZAYDLAR K. 2nd Clinic of Internal Medicine, LF UK, Hradec Krdlov~

Serum levels of IgA, IgG, IgM, IgD, IgE, 13-2-MG, IC, IgM RF/LFF, IgM RF/HAT and ANA/IF were determined in 77 patients with primary gout (16 in acute gout attack, 61 off attack), 30 healthy individuals, 30 patients with IgM RF+ rheumatoid arthritis and 20 with IgM R F - RA. Using the pace discrimination analysis, markers allowing distinction of the groups examined were selected and their classification validity, permitting classification of randomly chosen individuals, was determined. The following symptoms are of clinical importance: acute gout attack- IgM RF+ RA (IC and [~-2 MG), gout off attack - healthy subjects (IgA, IgG, IgM, IgD, IC, IgE). IgM RF was found in 5/77 patients with gout, ANA/IF was not detected.

/m~ao~me~k A s s o ~ t i o ~ a d P u t ~ e ~ ot ~ Antibody L e v ~ s p ~ t t/~ E p ~ - ~ V/ms is F ~ c4 Psdents ~ kom R~.smstold A r t b ~ (RA~ ssd S ~ m d c t~pm Ery~emstosw (SLY)

DotAL C., Von-~ovsz~ K., IVASKOVA E., MACUROVA H., lIANA I., ZvARovA J. VUCHR, Center of Epidemiology, HIE, HLA Laboratory, IKEM, Section of Clim'cal Immunology ILF, Department of Medical/nfonnat/cs, UK, Prague

According to our previous findings significantly increased antibody levels against EBV were present in patients with RA and SLE in the Czech Socialist Republic. The comparison of haplotypes in sibling pairs suggested the possibility of genetic association with antibody levels against VCA-EBV not holy in patients, but also in some healthy members of these families. A highly significant match in one haplotype was found in pairs from families with RA, in SLE patients match in both haplotypes was observed. Simultaneously, antibodies against the cytomegalovirus were determined. These observations further


extended the finding of genetic association with RA in sibling pairs from 83 families, published in 1986 and they point to the possible involvement of EBV in the pathogenesis of RA,

Relstinnsl6p betweeu Antibodies to Polyaucleotides u d Pbospbolipids

CEBECAU-E]Z L., P O V F _ ~ A.M. VURCH, Pie~fany ; Institute of Medical Radiobioiogy , Ohninsk, USSR

The earlier surprising find.ing was confirmed that monoclonal antibodies against DNA can cross-react with cardiolipin (Lafer et al. 1981). Normal lgG separated on ionex (QAE-Sephadex) and normal serum fractionated on Sephadex G200 at acidic pH (4) acquired the ability to react with DNA, deoxyribonucleoprotein and cardiolipin in an immunoenzyme assay. Thus, it follows that normal sera contain immnnoglobulins that are able to react with unusual antigens, such as polynucleotides and phospholipids. Under normal conditions their reactivity is eliminated by mutual interactions and, obviously, at least part of these immunoglobulins react with the above molecules on the basis of charge properties, i.e. nonspecifically. It seems that in systemic connective tissue diseases characterized by the presence of such antibodies (anti-DNA, anti-cardiolipin) a disorder- whose model are the separation procedures- may be present rather than antibody formation induced by an antigenic stimulus.

The Role of Anfiperinocl~r Antibodies in Some Rheumatic Diseases

GOETZOVA A., BARDI~ELD R., TRNAVSI~ g. VUCHR, Prague

Using the indirect immunofluorescence method (epithelial cells of human buccal mucous membrane served as the antigenic substrate) we examined antiperinuclear antibodies (APNAb) in sera of 369 patients: rheumatoid arthritis (RA) - 168, systemic lupus erythematosns (SLE)- 61 and juvenile chronic arthritis (JCA) - 140 ; 53 healthy individuals served as controls. High specificity of APNAb (87 %) was found in RA and, in contrast, low specificity was observed in SLE (19.6 %) and JCA (10.7 %). No APNAb were detected in healthy subjects. The main contribution of this study resides in the fact that APNAb were found to be more specific for RA and were frequently observed also in seronegafive patients.

immalaologk~ Profile of l~mozygous TwOs with Systenlk Lupo$ Erythenmtoses ($LE)

Lur, Ae J., CEBECAUER L., BogAK V., 2xn~AN D. VT_]RCH, Pie~fany

The authors report a case of SLE in female twins; their identity was determined from foetal membranes (monoamnial, monochorial) and on the basis of probability of zygosity calculation from genetically determined polymorphous markers (HLA, blood groups, haptoglobin). Initial clinical symptoms of SLE appeared in one patient at the age of 13 years: fever, episodic arthritis, iymphadenopathy, immunological positivity of LE cells, complementemia and tendence to recurrency. Clinical symptoms started to appear in the second twin 1.5 year later: photosensitivity, disturbances of acral circulation and discoid facial morphs. Laboratory immunological examination revealed leukopenia, anti-dsDNA and anti-ENA positivity, hypergammaglobulinemia and rheumatoid factor. Such cases allow studies of genetic and immunological aspects of SLE and their impact on the clinical pattern of the disease.

1990 ABSTRACTS SS

Pulse Therapy ot Systemic Lupus Erythemstosus with Methylpredn/so/one

I-h~c~ Z., BR~mA P., I ~ N V. 2nd Clinic of Internal Medicine, LF UI(, Hradec Kr~lov~

Pulse -therapy with methylprednisolone (1 g i.e., 18 pulses in 5 series, 3 - 5 pulses each) was performed in 5 cases of active systemic lupus erythematosus (SLE), classified according to ARA criteria 1982, in two patients with lupoid nephropathy and three cases of pan- or bicytopenia. General symptoms of SLE activity disappeared in all patients. Significant decrease of nephritic urinary syndrome occurred in SLE patients with lupoid nephropathy; in SLE and pan- or bicytopenia cases, restoration of anemia and leukopenia and improvement of thrombocytopenia were recorded. The most promising method appeared to be the determination of anti-dsDNA antibodies (Farr test) in 12 ANA specificities examined; a significant decrease of binding was observed in all cases.

Systemic Lupus Ery~emuttos~ ($LE) sad lnterleukim

MIs~KovA J., I ~ c ~ o ~ V., B o ~ c ' ~ L., ~ A ~ D., L ~ J. Institute of Virology, SA V, Bratislava ; V(]RCH, P/eg~any

We examined 67 patients with SLE at various stages of activity of the disease and 10 healthy individuals. The ability of 1L-2 production was determined in a non-adherent fraction of separated peripheral blood cells (T lymphocytes) after stimulation with ConA; IL-1 production was studied in the adherent cell fraction (macrophages) after LPS stimulation. IL-1 detection was performed according to Friemel et al. (1986), IL-2 according to De Vos and Libert (1984). In comparison with healthy individuals, IL-2 production was decreased in 26.6 % of SLE and IL-1 in 48 % patients. T lymphocytes obtained from patients having at the time of sampling interferon in their blood (14.7 %) did not produce IL-2 after ConA treatment. Production of IL-2 was inhibited also by autologous serum obtained from 15.7 % of SLE patients (in comparison with inactivated calf seruth - 10 %) regardless of the presence of interferon in the serum.

Unusmll Course o! Sepsis in L-bronic ht/ents during Long-Term/nummosuppresdon

BAKOSOVA M., DRAarovA J., DOS'rAL C., BARDFELD R., I'~NA I. ROZS Prague; V~CHR, Prague; Section of Clinical Immunology, ILl:, Prague

On the basis of a follow-up of six patients the authors demonstrate the origin of the septic state in patients with an autoimmune disease (MG, SLE) undergoing a long-term immunosuppressive therapy (corticoids, Imuran). The onset of sepsis was inconspicuous without a significant body temperature increase with progressive anemia, without leukocytosis and in the absence of an obvious focus. The clinical manifestation of sepsis occurred abruptly and was accompanied by a fast respiratory and kidney insufficiency ; at this stage the response to therapy was poor.

An~Jio l ip in Antlbodies in ~me Rheumatic Diseases

P~t~ ,ovA A., VE~covs~ J., BAZOFI~ R., TRNAVS~ K. VOCHR, Prague

Anticardiolipin (antiphospholipid) IgG and IgM antibodies were determined by the ELISA method (employing bovine cardiolipin as antigen) in sera of patients with some rheumatic diseases, diffuse connective tissue diseases and in healthy individuals. As a limit of positivity served the mean anticardiolipin antibody level of 39 healthy subjects, increased by four standard deviations. Positive anticardiolipin antibodies were found in 33.9 % of patients with systemic lupus erythematosus (19/56), in


22.6 % patients with rheumatoid arthritis (12/53), 47.8 % patients with reactive arthritis (11/23) and 13.8 % patients with juvenile chronic arthritis (4/29) as well as in 2.6 % healthy individuals (1/39).

lmmllDoc~e/nJc~ Pl'oot of ~ J[~'adlt~On Products ill the Syllov~d FklJd

KRsovA D., KRAJlC~OVA J., TRNAVSK~' K. VFJCHR, Prague

proteoglycans of the cartilage matrix are in some rheumatic joint diseases degraded and their fragments are released into the synovial fluid. These fraginents can be detected by the ELISA method. The results of our study revealed differences not only in proteoglycan concentration in the synovial fluid in various diseases, but differences were recorded also within the group of patients with rheumatoid arthritis. These results indicate the heterogeneity of the diseases, the degree of the disease progression and further destruction of the cartilage.

Effects of Serum Fmcton at Pmtteuts with Systemic Connective Tissue Diseases on Cbemilumiuescence

RAUOVA E., LUKAC J., GREOU~KA O. VFflRCH, Pietifany

Chemiluminescence is employed in assessing the metabolic activity of phagocytotic cells. Formation of antibodies and antigen-antibody complexes are involved in the pathogenesis of systemic lupus erythematosus (SLE). Following the contact with polymorphonuclear (PMN) leukocyte these substances induce their metabolic activation which results in the formation of highly reactive oxygen radicals that can be detected by chemiluminescence. We studied the degree of activation of leukocytes obtained from venous blood of healthy donors during incubation with sera of patients suffering from various connective tissue diseases (SLE, PSS, RA). The results showed that sera of SLE patients increased the chemiluminescence of normal leukocytes. Presumably, the main cause of activation are circulating immune complexes. Association of the results with clinical and immunological characteristics of the disease was analyzed.

(~uant~@ve ~ r o s r A n ~ v ~ of IZpeptidyl Peptidase ! ( DPP !) Activity in H m m Lympbocytes

JULIa; I., LCUDA Z., ALU~IK ~;., TRNAVSK~ K. Laboratory of Histochemistry, FVL UK, Prague ; V(.ICHR, Prague

DPP I is a cysteine proteinase present in the lysosomes of approximately 20 % of circulating human T and B lymphocytes. We determined the fluorescence emission intensity of the reactive product (Gly-Arg-MNA served as a substrate) using a computer-assisted microphotometer. We examined blood samples of healthy individuals and of patients treated with cytostatic drugs for rheumatoid arthritis or systemic diseases. The enzyme activity was low in healthy individuals; as regards steroid-treated patients, higher percentage of positive iymphocytes was found and, in addition, the DPP I activity had wider variability. The highest values were found in patients undergoing combined therapy. The differences among groups were highly significant (p < 0.001 ) and reflected the influence of therapy. Microfluorimet- ry of DPP I contributes considerably to the determination of functional activity of lymphocytes in various diseases.

Antibodies to Topobomen~e ! ( ANYI.SCL 70) in Profre~ve Systemic Sdems~

KozAKovA D., LUKAC J., CEBECAUER L., Tt,U~A J. V~RCH, Pier,any; Department of Biochemistry, PF UK, Bratislava

When analyzing the group of 156 patients with progressive systemic sclerosis we identified (in addition to other antibodies) a frequent occurrence of antibodies directed against the nuclear antigen -

1990 ABSTRACTS 57

topoisomerase I (SCL 70). The presence of these antibodies was confirmed by determining the inhibition of topoisomerase I activity by plasmid pBR327. Its detection by common immunological reactions is not reliable. Immunoprecipitation employs fresh tissue extracts purified by ultracentrifugation or by other methods. However, topoisomerase I is extremely unstable. Using chromatography on hydroxyapatite for its isolation, we obtained a functional, sufficiently pure and stable antigen that could be used for immunoenzyme determination (ELISA method) of these antibodies.

C-ReKflve Protein m $efa lind Synorial Fluid

MAH~ F., CEBECAUER L., LUgAC J., ONOaAgiK M., GREGU~gA O. VURCH, Pie~fany

The authors studied the relationship between the CRP concentrations in paired samples of synovial fluid (SF) and serum (S) in patients with rheumatoid arthritis (R A), psoriatic arthritis (PsA) and osteoarthrosis (OA). CRP levels were determined by radial immunodiffusion. Patients with RA had significantly lower CRP levels in SL than in the S, no significant differences between SL and S CRP levels were found in OA.In addition, the ST/S ratio of the following proteins was determined: orosomucoid, c(-1 antitrypsin, ceruloplasmin and c(-2 macrogiobulin. A regression equation was computed on the basis of their molar mass. It was found that the ST/S CRP values were significantly lower than the expected ones. Thus, selective binding of CRP in the synovial tissue or its specific consumption in the synovial fluid is presumed.

Mononucleat Cell Subpopu/st/ons in ~ e Syaovial E~ds te and Pedl~eml Venous Blood in Progressive

JIRA M., FIAusNEa P., STREJCmK J., ZAVADIL Z., REJHOLEC V. 1st Clinic of Internal Medicine, LFH UK, Clinic of Oncology, FVL UK, Department of Internal Medicine, FP KUNZ of Central Bohemian Region, Prague

Differentiation markers of mononuclear cells of synovial fluid (SF) and peripheral venous blood (PB) were determined in 18 patients with progressive polyarthritis. Using an immunofluorescence method the following cell markers were determined by microscopy and partially by flow cytometry: CD2, CD3, CD28, CD5, CD4, CD8, CDw29, CD45R, CD25, Ia +, CD9, CD19, IgM + and IgD +. The findings in the synovial fluid were highly variable without significant association with the activity or stage of the disease. As compared with PB, SF exhibited a higher proportion of CD25 + or Ia + cells (mostly together with CD4 + and CD8+). An interesting finding was a frequent decrease of CD28 + cells; a part of the CD28- cells remained CD4 + or CD8 +. Within the subpopulation of C'T)4 + cells, an increase of CD45R was recorded while CDw29 + were decreased, both in CIM + and CD4- subgroups.

CompaAmn of Vmfom l ~ t h o l lor Determ/mt/on ot Antlbo~es against Keratin

VORCH, Pie~tfany

Antikeratin antibodies (AKAb) appear to be a useful diagnostic marker of both the seroposifive and seronegative rheumatoid arthritis. A standardized procedure of AKAb determination is based on indirect immunofluorescence of the rat esophagus. The results of AKAb determination by immunoenzyme techniques are presented.


HLA System Antigens in Rheumatoid Art~t is

Bo~;AK V., MIC"EKOVA D., ONDRAgtK M., HREBiK I. VURCH, Pieg(any

We examined HLA antigens of loci A, B, C, DR and DQ in a group of 64 unrelated patients with rheumatoid arthritis (RA ; 13 males, 51 females aged 32 to 71 years) and in 29 family probands with RA (altogether 108 family members). An association between RA and HLA-DR4 antigen was found (see the table) which is in agreement with findings in other Caucasian populations. The results of detailed immunogenetic analysis in RA as well as the results of family studies - "affected sib pair analysis" - are presented.

RA DR4 frequency, % X 2 P~o,, Relative risk

RA Siovak population

Seropositive (42) 52.4 12.5 < 0.01 4.0 Seronegative (22) 36.4 21.4 1.5 - 2.1 Total (64) 46.9 11.2 < 0.01 3.2

Method for Modification of Hybrldoma Biological Properties

JANDEJSEK J., ~KVOR J., HEDVa~AKovA P., KOTRnOVA A. USOL, Prague

Hybridoma HP-03 cannot be employed for industrial production of monoclonal antibody against horse-radish peroxidase because it would not grow in the mouse strain BALB/c. The hybridoma was used as a model for elaboration of a modification of an unsuitable biological property method. By means of fusion of gently killed hybridoma myeloma line, selection in the HAT medium, repeated passage in BALB/c mice and cloning, a new hybridoma was constructed which produced monoclonal antibody of identical specificity and continually multiplied in BALB/c mice. The described method may serve as a general procedure for modification of certain biological properties of hybridomas.

Contemporary Possibilities of the Departments of Clinical Biochemistry in the Diagnosis o! lm- munolmtldes

O~DRU~ I., SARTOmS M. OKB OUNZ, NsP Trnava ; NsP Dunajsk~ Streda

The general concept of the Ministry of Health (MH) concerning medical immunology is at present realized mostly at the level of hospitals with policlinics of the IIIrd type (NsP); therefore, physicians-immunologists (clinical immunologists) or physicians who are responsible for patients with immunopathies, treated at the level of Of./NZ-NsP of the IInd type, suffer from lack of laboratory diagnostic background. The new MH concept of clinical biochemistry allows the establishment of departments of immunology as a part of Departments of Clinical Biochemistry (DCB) of NsP type II, which opens the possibility to determine humoral components of nonspecific and specific immunity by biochemical methods. The authors present an algorithm which can be employed for the determination of disturbances of humoral immunity and recommend a panel of tests that can be performed in all DCB NsP, type II.

1990 ABSTRACTS 59

Use of Chromogem in ELISA Systems Emplayiq Horse Radish-Labelled Amtibody

Vo~tmA T., PR0CHA M., RAKOSNm P. Department of Immunology; VTJHEM, Prague

Chromogen 1,2-phenylenediamine is photosensitive. The alternative ABTS (azinobisethyi- benzthiazolinesulfonic acid) is light-stable and the slowly proceeding reaction need not be terminated. ELISA ~-HCG was used as a control. The sensitivity of both chromogens is comparable. Determination of coiour intensity was performed with ABTS after 2 h at 414 nm, in some cases after 24 h and at 2-3-times lower ABTS concentrations. In parallel sample reading the variation ABTS coefficient was found to be 11.3 %, in OPD 15.4 % (n = 40). The advantage of ABTS is a low background (0.05). The concentrations of H202/lsed were comparable, the optimum being 0.01 to 0.001%. The concentration exceeding 0.05 % inhibited the substrate reaction.

Use o! Monocloul Antibodies against Human lmm~aglobalin* ~ Their ,~bcl2sses tar Ommtitstive Determlm,tian of Serum and Secretory lmmano~bnllnr ~ An~hol~$

TLASKALOVA H., BARTEK J., ~IMEC~OVA J., VANC~PKOVA, Z., DRAHORADOVA I., ROZPmMOVA L., RADL J., MANCAL P. MBU C'SA V, Prague; U / ~ O , Bruo; []SOL, Prague; OK/] Thomayer Hospital, Prague; Institute Organization for Health Research, TNO, Rijswijk

Monoclonal antibodies (MoAb) against the secretory component of human polymeric immunoglobulins (Ig) and against heavy (l~ and a) and light chains of human Ig were prepared. These MoAb were used for specific and sensitive detection of serum and secretory Ig in body fluids by the microELISA test. In addition, these MoAb and other antibodies directed against Ig subclasses were also used for rapid, semiquantitative determination of Ig levels by the "dot" technique: the samples were applied to the nitrocellulose membrane (Synpor) ; after drying and blocking, the nitrocellulose strips were incubated with MoAb and, after washing and adding a conjugate of antiserum against routine Ig-peroxidase, the samples were developed. Finally, classes and subclasses of antibodies against food antigens were determined in sera and saliva of patients by MoAb.

Utlli~tioa of the ELISA Method tar the Deterrent/on of Specific Antibedies against Trepoaema pallidum

J|LEK D., KRAL V., RANA M., NI~MEC T., KNAPPOVA M., RICHTER J. Department of Immunology, KHS Ust[ n.L. ; USOL, Prague; NRL for Lues, Prague

The authors attempted to determine antibodies against Treponema pallidum using a common ELISA method. Sonicated suspension of treponema, strain Nichols, served as the antigen, P-plates (KOH-I- NOOR, DaleS'in) served as the solid phase. The authors present the optimum conditions of the test; selection of a suitable treponema antigen proved to be the most critical factor for obtaining reproducible results. The results of a comparative study of 70 sera by means of ELISA and TPHA provide a guideline for syphilis diagnosis in serological field laboratories.

Utilization of the EUSA Teclmiqae for the Diagno~s of Bscterisl l~ectiom

~VARr L, S ~ 2 I. V~I'RN, Prague

More and more attention is being paid to indirect diagnostic methods for the detection of infectious agents that are mostly based on the determination of specific antibodies. We introduced an ELISA


method with automated reading for the determination of specific IgG antibodies against mycobacterial antigens (sonicated), strain H37Rv. Antibody titres were determined in 100 patients with proved tbc of the respiratory tract, 50 patients with lung cancer and 100 blood donors (control). The sensitivity of the method proved to be 86.5 %, specificity 92.5 %. In addition, it was shown that administration of corticoids, certain wide-spectrum antibiotics and cytostatic drugs, as well as professional pneumoconiosis and age over 85 years, were usually accompanied by decreased formation of specific antibodies.

Ot Helmin(ho~ by DOT-J~

L ~ S . Institute of Parasitology, CSA V, ~sk6 Bud~jovice

Simplification of diagnosis of helminthoses in human and veterinary medicine is a prerequisite for its wider use. The nitrocellulose membrane appears to be the best carrier of antigen for obtaining reproducible results. A 90-100 % sensitivity of the method was found in both experimental and spontaneous parasitic infections. We tested domestic Synpor filters for DOT-ELA and suitability of chromogens 4-chloronaphthol, 1-naphthol and 1,4-phenylenediamine with peroxidase-labelled conjugates. The method is useful for screening of bovine fascioliases, trichinellosis of pigs and for cysticerciasis of cattle (provided a specific antigen is available). Another advantage of the method lies in the minimum amount of antigen required, high effectiveness of laboratory work, possibility to prepare nitrocellulose with antigen in advance. Various methods of conservation of prepared membranes and their stability were tested.

Deign o! Itnto~dtmd/es ~ SMP, F/-AT/~ ComlNex and/~P ~ L/v~- Disess~ by F_~USA Method

I., KULIgKOVA L., ~ O V A A., IWUBVL B., DOSTALIK Z., gEFLovA J., MACHAta~ J. Department of Immunology and Allergology, K(JNZ-KHS Ostrava ; Department of Internal Medicine, KONZ and MUNZ Ostrava

Certain liver diseases are associated with the occurrence of autoantibodies. We prepared three antigenic complexes (AC) from rat liver that are frequently used in diagnosis and monitoring of the course of liver diseases with a suspected autoimmune underlying mechanism. (1) Mixture of inner and outer mitochondrial membranes - called submitochondrial particles (SMP), (2) F1-ATPase complex (ATPase) and (3) the liver-specific protein (LSP). The AC were standardized and their use in the diagnosis and monitoring of radons forms of hepatitis was tested by the 1~.! .ISA. Cross-reactivity with all the AC used was found in sera of most positive patients. This may be due to the fact that all AC contained probably some common structures as revealed by electrophoresis in polyacrylamide gel.

|mmEI lO~ ~ for ~be Determ/~lton of ~ ~ A ~ A~dboSel

VANCIKOVA Z., TLASKALOVA H., KOCNA P., TUCKOVA L., AND~tA L., FmC P., Dvo~J~z M. MBU (~SA V, Prague ; Laboratory for Gastroenterology, FVL UK, Prague

Using the immunoblotting method, we examined the occurrence of class IgA and IgG antibodies against gliadin and individual isolated gliadins in sera and saliva of patients with coeliacai sprue. Gliadin proteins were separated by SDS electrophoresis in polyacrylamide gel. Following electrophoretic transfer to the nitrocellulose membrane, we examined the reactivity with individual gliadin fractions using an enzymic assay. Similarly, we examined also rabbit hyperimmune sera and routine monoclonal antibodies prepared by immunization with gliadin. The strongest reactions were obtained with gliadin proteins of molar mass of about 30-40 kg/mol.

1990 ABSTRACTS 61

Detection of Monomedc lmmunoglobulin M in 5era by Rocket lmmunoselection with Phytohenmggluti. nin Technique

KRALOVA E., NOVOVNA H., LOr, AJ J. OKB and OKI, FNsP Brno

Under physiological conditions, IgM is present in blood as the pentameric macroglobulin. However, low-molar-mass subunits (monomers) occur in sera during certain pathological conditions. Their determination requires the use of sophisticated methods, e.g. ultracentrifugation, chromatography, isoelectric focusing. Our detection method is based on different reactivities of pentameric and monomeric IgM with phylohemagglutinin. During electrophoresis in a PHA-containing gel, complete IgM molecules are immobilized while monomeric molecules migrate and form rockets during the reaction with anti-IgM serum. Using this method we demonstrated low-molar-mass IgM in the serum of a child with hypergammaglobulinemia M and in 10 patients with liver diseases. Sera of 25 healthy controls yielded negative results.

Follow-up of the Immune Response stter Admlnisttstion of Antirsbies Vaccine by Two Methods

VRZAL V., CHUMELA J. Bioveta Ivanovice na Hang

Animals immunized against rabies can form virus-neutralizing, lyric, complement-fixing and other antibodies. We examined the sensitivity of the virus-neutralizing and complement-fixing tests for the evaluation of humoral immunity of vaccinated dogs in a long-term experiment. Comparison of complement-fixing and virus-neutralizing antibody titres showed a higher sensitivity of the latter test. The complement4ixation test can be employed for a quantitative evaluation of antirabies antibodies particularly during the early significant response to vaccination. However, it is not useful for detection of low antibody titres.

Ut///zat/on of LPS from E. coil O75 and Upid A from Adnetobacter calconceticus for Detection oi Chron/c lntecthms by Pamive Hemolysis

BOaKOVA M. Agrogcn, JZD AK Slu~ovice

Lipopolysaccharides of all G - bacteria possess one common component, heptose-2-keto-3-deoxy- -D-manno-octonic acid that forms the determinant group of the LPS antigen. Sera of patients who had overcome an infection caused by G - bacteria contained specific antibodies against the common component of LPS. The presence and titre of antibodies and titre of haemolysis can be determined on the basis of passive hemolysis. The titre of hemolysis is determined by the amount of antibodies still causing hemolysis of sensitized erythrocytes. Serum dilution, causing 50 % hemolysis, represents one hemolytic unit. The tests were performed in microtitre plates, complement was obtained from guinea-pig sera, the optimum dose of LPS and lipid A varied within the range of 20-80 ~g/0.2 mL of the original erythrocyte suspension. The method is suitable for detection of chronic infections caused by G - bacteria.

Qusntitative Determlnation ot Porc/ne Serum/mmunog/obu/ins by Mono~ions/Ant/bodies

D v o ~ K P., F~UBAN V., MADR P., T~S~LOVA H. UFGHZ CSA V, Lib~chov ; MBU C~SA V, Prague

Murine monoclonal anti-lgM antibody (LIG 4) was used in a sensitive ELISA test for accurate determination of IgM levels in sera of pigs from birth (2 ~,g/mL) up to the age of 65 days (1.9 mg/mL) and in adult pigs of various breeds (3 .6-4 mg/mL). The shape of IgM increase curve was similar to that


described by other authors but IgM values found in our experiments in young and adult pigs were substantially different from the published data. A simple binding test on nitrocellulose was used for rapid semiquantitative determination of serum IgM. In addition, IgM was determined by radial immunodiffusion using precipitating antibody LIF 4. The minimum amount of IgM detected by the ELISA test was 1 ng/100 I~L, by the binding test on nitrocellulose 2 ng/2 ~tL and by radial immunodiffusion 300 ng/10 ~L. The ELISA and the binding test on nitrocellulose were also modified for accurate semiquantitative determination of porcine IgA using anti-IgA monocional antibody LIG 33.

Economic Eleetrophoresis Method

SAR'roms M., ONDRUg I. OKB, OUNZ NsP Dunajsk~ Streda ; OKB, OONZ NsP Trnava

The most common method of immunoelectrophoresis used in Czechoslovakia is the micromodification according to ~;kvafil; the kit IEP-2 SEVAC is manufactured by OSOL, Prague. Using this kit the cost of one determination is reported by various laboratories to be 160 to 260 CSK. The authors developed a simple and enconomical method with comparable reproducibility and sensitivity which required 3 days compared with 5 days fo the original method ; in addition, the cost is reduced by 75 %. The basis of our method is a newly designed stencil with two long and two short throughs for application of sera, and 10 starting wells for application of samples and reference material. The examination is performed on a single transparency slide. The cost of material for one examination amounts to 38.20 CSK. This method reduces consumption of antisera by 76 % and allows to perform 4 determinations instead of one with the same amount of serum.

Utilization of the lmmunoskintest SEVA C in Pneumology

KOLEK V., KOHOUTEK M. Department of Radiotherapy, Clinic of TRN, FNsP Olomouc

Skin tests have been traditionally used in pneumology in the diagnosis of tbc, mycobacteriases and mycoses. Simultaneous administration of several antigens that induce the immune response of the cellular type can be used as an in vivo model for determination of ceU-mediated immunity. Our experience obtained with the kit of 5 recall antigens used in approximately 500 patients is reported here. After reading the induration after 48 h, the skin test was compared with other immunological tests. The following cases were included : (1) respiratory tract diseases, (2) lung symptoms in systemic diseases, (3) malignancy. Decreased skin reactivity was found in patients with recurrent infections, chronic bronchitis, sarcoidosis, collagenoses and chronic renal insufficiency. The prognostic value of the test was verified in lung carcinoma. It is recommended to use the Immunoskintest in all pneumoiogical departments where tuberculin is routinely used, because the skin test is simple, available and easy to perform.

Uti~stion of the PHA $~n Test in Calves

SAr~ORE'r M., BREZ~AN T., PEYRR.LOVA D., VRT1AK O.J. V$V and f.IEVM, Kogice

We studied the possibility of cell-mediated immunity determination in calves using phytohemagglutinin (PHA) ; 3- to 4-months-old calves (Slovak spotted or black spotted breeds) were used throughout the experiments. PHA was injected intradermally at various concentrations in amounts of 0.1 mL. Skin induration was measured at the site of PHA injection with a micrometer. The results showed that the optimum PHA dose was 100 ~tg/mL, the reading should be done 20 h after PHA administration.

1990 ABSTRACTS 63

in Vivo Testing of hnmunolog/ca/Reactivity

POSTUPA J., VF_JBORA O., VINOGRADOV V.V. LF UK, Hradec Kr~lov6, Institute of Physiology, Siberian Branch AMN USSR, Novosibirsk

Determination of in vivo immunological reactivity was performed by following the dynamics of granulomatous tissues formation using an original experimental model. A plastic sponge was implanted subcutaneously to rats and various morphological changes were examined 3 to 28 days after implanta- tion : capsule formation, penetration of blood capillaries, occurrence of fibroblasts and macrophages, their mutual ratios and differentiation. These criteria make it possible to distinguish between the states of activated and suppressed immunological reactivity.

Comitogenic Test As a Proof of Adjuvant Activity of lmmunomodulators

HI~dBALOVA V,, BARNET K., PEKAREK J., JE:g~EK J., STRAKA R., LEDVINA M., MARA M., HOFMAN J., ROTrA J., ZAORAL M. IHE, USOL, (_IOCHB CSA V, FVL UK, MBO OSA V, Prague

Using a set of muramyl-dipeptide-derived substances we showed that their ability to enhance the proliferative response of thymocytes stimulated with T mitogens in vitro, was related to their in vivo adjuvant activity tested in a model of experimental allergic encephalomyelitis. Contrary to the assumption (Iribe, Koge 1984) that the comitogenic effect is present in muramyl peptides only, we were able to demonstrate it even in other immunomodulators, e.g. listeria E~ factor, lipopeptidopolysaccharide isolated from Listeria monocytogenes and in the dial.v.zed leukocyte extract (Transfer Factor SEVAC).

Determination of EBL by Colloid Gold in P ~ n $ectio•

LEVKI_rr M., KOSTROUCH Z., LF2,NIK F., ~KARDA R. V~V, Kogice ; UK, Prague

We studied the possibility of histological EBL determination with colloid gold. Samples were fixed in paraformaldehyde and embedded in paraffin. The direct immunocytochemical method with homologous antibody was used. For antigen detection, the gold complexes were furhter treated with silver.

Preparation of Monoclonl Antibodies aga/mt the Stmctm~ Peptide of the H m a n lmmunodeficiency Virus HIV- I

STOCKBAUER P., NOVAK J.T., C.MTHAMLOVA H., PALUSKA E., KRCHIqAK V., HRKAL Z., MACHO. UHKT, Prague ; L6~iva Prague ; UMG CSA V, Prague

The diagnosis of HIV- 1 infection is at present based on the determination of antibody against the virus in the serum. However, in some cases, determination of the presence of viral proteins in the material examined is required. With viral proteins an alternative method was chosen for the preparation of monoclonal antibodies. It employs synthetic peptides representing protein antigenic determinant predicted from nucleotide sequence of individual HIV-1 isolates. Mice (strain BALB/c) were immunized with synthetic peptides derived from immunogenic regions encoded by gag, pol and env HIV-1 genes without binding to the high-molar-mass carrier. Spleen cells of immunized mice were fused with the murine myeloma lines Sp 2/0, P3OI or FO and the resulting lymphocyte hybridomas were selected for production of antipeptide antibodies by the ELISA method, and cloned by the limit dilution technique. Several lymphocyte hybridomas producing antibodies reacting with HIV-1 in the ELISA assay were obtained.


Experience with Serological Diagnostics of Ounpylobacteriasis

JtLEK D., PULCHARTOVA E., KRAL V., CECH M., CHRAMOSTOVA K., LAZAROVA D. Department of Immunology, KHS Osti n.L. ; Department of Immunology, OIlS Liberec ; Department of Infectious D/seases, OONZ Liberec ; Department of Infectious D/seases, KUNZ Osti n.L.

The antibody response against Campylobacter jejuni was determined by the ELISA method employing the outer membrane protein of C. jejuni. We examined 233 paired sera of patients hospitalized for acute diarrhea. A four-fold increase of antibodies was found in 12.4 % cases, the maximum occurrence being in the age group 0 - 3 years (17.6 %). This finding is in agreement with already published data concerning C. jejuni as an etiological agent of diarrheal diseases. The question remains as to the 15 % cases where increased antibody titres (more than ~ + 2 SD of the control group) in both samples were present.

MonoeJoH/Antibodies agl/nst LegionoUm

~KVOR J., STRNAD V., JANDEJSEK J. (]SOL, Prague ; FDL UK, Prague

The occurrence of legionellas in water samples in Czechoslovakia in association with some clinical cases stressed the need to improve the diagnosis. Because of Difficulties with their bacteriological determination and identification, monoclonal antibodies (MoAb) against their surface antigens were constructed. By fusing spleen cells of BALB/c mice immunized with killed legionella with the Sp 2/0 cells, four hybridomas were obtained that produced MoAb reacting with certain serovars of L. pneumoniae belonging to the most frequent etiological agents of the disease. The reactivity of MoAb with type strains and domestic isolates was determined in the CELISA and IFA tests.

Experience frith the l~reIamration of ~ d e r i ~ ~bosomaas

ZAHRAO~K F., ZEMANOVA Y. Institute of Microbiology, LF UK, Plze~

Ribosomes were obtained by fraction centrifugation. The efficacy of various disintegration methods was tested in klebsiella. Osmotic shock and repeated freezing and thawing methods were selected. Osmotic shock makes it possible to treat large amounts of biomass, but a drawback resides in the large suspension volumes required. Repeated freezing and thawing requires lower volumes but aggregation of particles occurred during this procedure and this could not be relieved by addition of detergents. When combined with mechanical disintegration of aggregates, the method is suitable for preparation of the basis of ribosomal vaccines.

The Effea of Monodonal Antibodies j g a m t Catala~ on Chem/lmninesceKe

MACHA J., DvotZAK V., ROMANOVSK'i' A., HABROVA V., NEDVIDEK J. Department of Animal Physiology and Developmental Biology, P~F UK, Institute for Toxicology and Forensic Chemistry, FVL UK, Prague

We prepared monoclonai antibodies against bovine liver catalase by using myeloma cells FO and spleen cells of BALB/c mice immunized with bovine liver cataiase. Two antibodies were selected for further testing: UK-CAT which reacts with native nad dodecylsulfate-denatured catalase, and UK-CAT-2, reacting with native catalase only. Specificity was determined by blotting and fluorescence of the liver tissue where fluorescence was observed in peroxisomes. Both antibodies enhanced chemiluminescence but only when purified antibodies were used. Admixtures present in the ascitic fluid or serum may enhance or suppress the reaction by several orders of magnitude.

1990 ABSTRACTS 65

Determhtatton of Gentamidn by Competft/re ELi$A Method

BUKOVSKY M. Department of Biochemistry and Microbiology, FaF UK, Bratislava

Therapeutic use of gentamicin requires the control of optimum blood level (2-10 rag/L) in order to minimize the risk of ototoxic and nephrotoxic effects. We elaborated an immunoenzymic method allowing a rapid and reliable determination of gentamicin in blood serum.

Program ot Immunological ~ c Prqmmtions Prodwctton in Biogem~ Cejknv

TALAN P., MUCHA J. Biogema, W Z BTA Cejkov

Selected immunological diagnostic preparations (ELISA-DNP, colloid gold) have been produced in Biogema since the year 1985. We elaborated a program of development of further production for the next two years to cover the needs of developing clinical immunology. In 1988 production of the EID-IgE kit for enzyme-immunodiffusion determination was started. In addition, production of gold-labelled protein A and selected gold-labelled monoclonal antibodies (MEM 31, MEM 57) was started recently. The future program includes kits for the determination of antibodies to n-DNA by the ELISA method, a kit for determination of gliadin (immunodiffusion - ELISA). Production of kits for ELISA thyreogiobulin determination, ELISA rheumatoid factor and secretory IgA determinations will begin in 1989.

Experience with the EID-lgE Kit

STROJ~? L., MUCHA J., TAt.AN P. Biogema, W Z BTA Cejkov

In 1987, testing of the kit for determination if IgE by enzyme-immunodiffusion (EID) was begun. The kit was developed in Biogema in cooperation with the Department of Clinical Immunology FN in Plze6. Altogether 9 lots were tested in several clinical laboratories and the clinical interpretation of the results and technical requirements were evaluated. The final form of the kit is based on the suggestions of users. Optimization of concentrations of basic components and calculation of the optimum size of wells made it possible to decrease the lower detection limit to 65 U/mE The reproducibility of the kit expressed as VK is below 5 %, the dependence on dilution is linear, recovery varies from 90 to 110 %. In addition, a panel of sera was produced and their long-term reproducibility was determined.

lnterprerstion of Remits Obtained by Meus of ELISA. VET-n6-DNP Kit

MUCHA J., TALAN P., C-~BECAUER L. Biogema, W Z BTA Cejkov ; V(JRCH, P/egfany

The ELISA-VET-anti-DNP kit for determination of antibodies to deoxyribonucleoprotein has been manufactured in Biogema in cooperation with VORCH since 1986. We describe the comparison of the results obtained with the kit using a calibration curve obtained on the basis of an internal standard VORCH KM 56. A total of 14 lots of kits were tested. In addition to limiting values of negativity and positivity, other parameters important for the long-term reproducibility of the kit (panel of sera, dependence on dilution, nonspecific binding) were also tested. The results confirmed the long-term reproducibility of the kits and provided hints for further innovations.


Application ol the laboratory Infocamtion System In Immunology

HoNzovA S., "]~r..ASAL P., MATRJO M. Ist Pediatric Clinic, FN Prague 5-Motol

A version of the laboratory information system for the immunological laboratory was elaborated. The program facilitates administrative routine work of the laboratory, such as printing laboratory protocols, results and cumulative findings as well as allows data storage and data recruitment. In addition, it allows statistical treatment of results, laboratory calculations of reference values for individual examinations. When printing the results, the value found is compared with the reference value, it can be expressed in standard deviations and the pathological value is noted. This system permits automatic comments on the examinations performed. Connection of a microcomputer with an automatic photometer MR 66 allows automation of certain laboratory methods. The program was designed for the Apple lie computer in the Dos 3.3 system using Basic.

Experience with the Serae lmmunoskintest

SUCHAr,~K I., W~.DERg_Art~ D., WIEDEaMANNOVA D., KADL~AKovA E. Clinic of Pediatric Infectious D/senses, FDNsP Brno ; Department of Pathological Physiology, UJEP, Brno

The authors performed skin tests for the determination of in vivo cell-mediated immunity. After obtaining the blood sample for immunological examinations, five antigens (tuberculin, tetanus antigen, toxoplasmin, diphtherin, candidin) were injected intradermally to 150 randomly selected children. The skin test index was correlated with various parameters of cellular immunity, obtained by other tests. Absolute and relative numbers of T lymphocytes did not correlate with the results of the skin tests.

Urli~tion of a New Cell Culture for /mmunotgqochem/ca/ Determ/unaon of the Molluscum contagiosum Watts 0Poxviridae)

KOUKAL M., TREBICHAVSKY L, SMOLOVA M. MBU L~SA V, Novy 14r~dek

Molluscum contagiosum (MC) is difficult to cultivate both in vivo (only in eye lids of primates) and in vitro in cell cultures (CC) where proof of serial multiplication cannot be obtained. In contrast to the vaccinia virus, the MC virus cannot propagate in eggs which prevents the possibility of titration. Using a fragment method, we derived a new CC from condyloma accuminatum cervicis which, besides other changes, exhibited a cytopathogenic effect (CPE) in the presence of the MC virus up to the dilution of 1 : 1000. The CPE can be used for determination of anti-MC antibodies in children who had not been vaccinated against orthopoxviruses (variola).

Sem/quant/tst/ve lgM and lgA Determi~tion in Pig Seca by the lmmunoenzymic Test o n Nitrocellnlow (Doting)

I~DR P., TLASr,.ALOVA H., Dvo~tgg P., HRUBAN V. UFGHZ L"SA V, Libdchov ; MBU L~SA V, Prague

We modified an immunoenzymic test on nitrocellulose (Synpor 6) using monoclonai antibodies LIG 4 (anti-IgM) and LIG 33 (anti-IgA) for the rapid semiquantitative IgM and IgA determination in pig sera. Two modifications of the method were employed: in the first method the serum examined was first applied to nitrocellulose, while in the second method the monoclonal antibody bound first. We calculated the correlation coefficient of the two methods (r = 0.87 and r = 0.91, respectively). The absolute sensitivity was found to be 10-20 ng of antigen in 2 ttL.

1990 ABSTRACI'S 67

An~/sis of Antibody to Tetanus Yozoid by Affinity lmmunoblotting

~IMEC'KOVA J., KNISLEY K.A., RODKEY L.S. Department of Immunology, MBU CSAV, Prague; Department of Pathology and Laboratory of Medicine, LIT Medical School, Houston

The kinetics of antibody (Ab) isotope expression in sera was studied by isoelectric focussing (IEF) followed by affinity immunoblotting (AIB). Outbred rabbits were immunized with tetanus toxoid (TT) or TY/anti-TT complexes. Sera were obtained from individual animals at regular time intervals during the immune response, separated by IEF and the distribution of clonotypes producing anti-TT Ab was analyzed by means of AIB on nitrocellulose membranes with bound TT or its C fragment. Clonal heterogeneity of anti-T[ Ab was demonstrated on AIB replicas ; changes in the expression of individual clonotypes producing anti-Tl" were observed during immunization. Certain Ab clonotypes dominating at the beginning of the immune response, disappeared later and were replaced with others. IEF in combination with AIB appears to be a suitable technique for examination of clonal heterogeneity of the immune response and for analysis of fine specificity of individual Ab-forming clones and for comparison of purified Ab preparations.

Monoelomwl Antibo~es against Cmrcinoembryonic Antigen

~KVOR J., JANDEJSEK J., HEDVICAKOVA P., PEKAREK J., CAPEK P., KOTRBOVA A. USOL, Prague; FVL UK, Prague

A suitable reagent for quantitative CEA determination in the human serum is difficult to obtain due to the antigen complexity and to the large number of determinants carried by macromolecules in human tissues. Over 80 clones producing MoAb against CEA were obtained from 15 fusions. Specifically reacting antibodies were further characterized by means of ELISA with non-CEA antigens and immunohistologically. Some MoAb reacted with CEA in ELISA only, the majority of them were directed against epitopes present simultaneously in some non-CEA. The most promising appeared to be MoAb 5/A3 that reacts with CEA only and is suitable for both the ELISA and immunohistological determinations.

~ t s Of lmm~li~ in C~]ndhood ~]d Adulthood

FO~KOVA T., KASAL P. OKI, FN II, Ist Pediatric Clinic, FDL UK, Prague

Since 1952, when the first immunity defect (Bruton's agammaglobulinemia) was described, other aspects of the problem have gradually emerged and, in addition, even the methodological approach has changed considerably. The following stages of immunity defect evaluation can be distinguished. 1. Clinical description of observed syndromes. 2. Recognition of pathogenetic mechanisms, development of immunodiagnostics, increasingspectrumof

laboratory methods. 3. Attention to secondary and associated defects. 4. Development of immunotherapeutic and immunomodulatory methods. All these aspects are being continually developed. In order to further increase the quality of the work of immunologists the following conceptual requirements should be met. 1. Standardization of description of clinicai units. The present incompleteness leads to many errors and

confusion. 2. Elaboration of interpretation of mutual relationships between the results of laboratory tests for

increasing the effectiveness of immunodiagnostic~. 3. Obtaining further information concerning the genetically determined reactivity and dynamics of

immunological changes, the effect of therapy, etc. 4. Search for criteria for individualization of therapy.


$~emized Noraendature In Immunology

KASAL P., HoNzovA S., PErR0 V., (?ol,ovA M. 1st Pediatric Clinic, 2nd Pediatric Clinic, FDL UK, Prague

Systemized nomenclature of medicine - SNOMED is a system of hierarchic codes for medical terms at 6 levels: topography, morphology, etiology, function, syndrome, procedure. Their combination leads to an economical and highly effective description of every situation in medicine. For clinical immunology, a complex of terms which can be used not only for treating groups of data, but also as an effective tool for classification, unification and determination of the term apparatus, was elaborated. The specialized Czech version originated on the basis of selection of terms related to clinical immunology from the original general vocabulary SNOMED (English and German versions) containing 80 000 indexes for the entire medicine. Using a computer, the system allows natural and simple formulation of knowledge rules within the frame of the expert system, and, in addition, with respect'to the existence of synonyms and word roots, it permits also communication with the computer in the natural language without limiting "menu". SNOMED contains indexes for symptoms, drugs, examination and therapeutic procedures and can be used also for transformation of data for common medical documentation.

Immunodenc/eney w/th Hyperimmunoglolmlinemia M

LOKAS J., BAsra M., KRALOVA E., BURSA J., LrrZMAN J. OKI and 1st Pediatric Clinic, FNsP Brno ; OKB, FNsP Brno-Bohunice

Hypedmmunoglobulinemia M is a primary immunodeficiency caused by a disturbance in the "switch" during the formation of individual immunoglobulin isotypes. Our patient (female) born in 1983, suffered since the 2nd year of life from recurrent lymphadenitis imitating a malignant process, otitis media, purulent rhinitis and bronchitis. Immunological examination revealed increased polydonal IgM (3.48-6.68 g/L), IgD (163 IU/mL), IgE (87.9 IU/mL), decreased IgG (0.35 g/L) and IgA (0.13 g/L). Using immunoselection with the PHA technique, monomeric IgM was detected in the serum. Lympho- cyte subpopulations (E-rosettes, M-rosettes) were within reference values, the immunoregulatory index was significantly decreased (CD4 :CD8 = 0.4). The metabolic activity of granulocytes was undisturbed, complement components lay within normal values. The presence of an inflammatory process was reflected in acute phase proteins. Intravenous administration of immunoglobulius mitigated the infectious processes but it did not influence the concentration of IgM and other Ig isotypes. Ig administration had to be terminated after a year of therapy because of undesirable side effects caused by anti-Ig antibodies. No immunological abnormalities were found in the parents and the brother of the patient.

lmmunologicsl Diagnosis ot lgA Deficiency

PRu~r~c P., BARrOOOVA E., VALEmOVA A. OKI, FN Bratislava

Selective IgA deficiency is the most common type of immunodeficiency with a prevalence from 1:500 to 1:700. The occurrence of IgA deficiency in patients treated at the Department of Clinical Immunology is naturally higher. In our department, we found in 1987 10 patients with IgA deficiency out of 1563 patients with a suspected immune defect ; five deficiencies occurred in adults (out of 1346 - 1:269) five in children (out of 217 - 1:43). The deficiency was defined as a state when repeated examination by radial immunodiffusion showed zero IgA levels (i.e. below 0.1 g/L, which is the lowest limit of sensitivity of this method). All 10 patients suffered from recurrent respiratory infections. Substitution therapy (gammag- Iobulin) or blood transfusion represents the risk of an anaphylactic reaction in these patients. Thus, it is necessary to establish whether the IgA level is really nil or whether IgA is present in at least minimum amounts. We therefore elaborated a highly sensitive method (ELISA) permitting to detect the presence of IgA in serum below 0.1 g/L.

1990 ABSTRACTS 69

/mmunolo#t'a/Parameters in Children with IgA Deficiency

KOSSAROVA K., ~;USTROVA M. Allergological Ambulance Unit, UNZ Bratislava ; OKI, DFN Bratistava

In our ailergological ambulance unit (Petr~alka District of Bratislava) we examined in 1985-1988 the incidence of IgA deficiency in children with various diagnoses. We followed 32 children aged 3-10 years with IgA levels decreased below 0.2 g/L and examined the association of IgA deficiency with changes of other immunological parameters. We determined IgG, lgA, IgM, IgE, T,, To, B, LTI', FA, bactericidy, CIC, CRP, orosomucoid and in v/vo skin tests using common inhalation allergens. We obtained the following results: decreased IgG levels in 7 children (22.4 %), increased IgM in 2 patient~ (~.4 %), increased IgE in 15 patients (48 %), dedreascd To in 8 children (25.6 %). No significant changes of phagocytosis and acute phase proteins were recorded. According to the severity of the disease, children were treated with immunopreventive and immunomodulatory drugs. Clinical improvement was recorded in most patients but no significant changes of IgA levels were obtained. With respect to increasing numbers of IDS in the population, 1.7 % of patients with IgA deficiency (out of 1870 patients) require a complex approach to diagnosis and therapy.

$eleai,e IgA Defdency in Chl/&~en with Various D(seases

~;USTROVA M., KOSSAROVA K. OKI, DFN Bratislava ; Allergological Ambulance Unit, UNZ Bratislava

In 1985- t987 we examined 7 300 children. Hypogammaglobulinemia A (below 0.05 g/L) was found in 125 children, aged 3 years (1.7 %). Selective IgA deficiency (IgAundetectable) was found in 39 children (0.53 %), 16 children (41%) suffered from autoimmune diseases (DM I., JCA, sclerodermia, ITP), 7 children had allergic diseases (17 %) and 9 children (23 %) had recurrent respiratory infections. In the latter group further clinical and immunological examination revealed the allergic basis of the disease, in one patient a combined immunodeficiency. Furthermore, complete IgA deficiency was found in 2 children with ALL, 2 with myocarditis, 1 with aplastic anemia and in 2 clinically healthy children. Two children (1 with ITP and 1 with RRi) were treated outside our department with i.v. immunoglobulins and developed subsequently an anphylactic shock. The whole group of pediatric patients, particularly those with complete IgA deficiency, are being currently followed and immunologicaily monitored (T and B lymphocytes, Fa, bactericidy, Ig, IgE, complement, BAF). Obviously, the increasing occurrence of IDS m the population requires a complex approach.

OinRsl and lmmHologksl Aspects of Immune D e f c i e ~

ZAVAZAL V., SEBEROVA E., MRAeKOVA E. OKIA, FN Plzefi

Recently, a significant increase of individuals, particularly children, suffering from immune defects can be observed. In addition, such an increase can be recorded even in elderly subjects because of their increasing proportion in the population. Part of the populations has latent disturbances, the lower but steadily increasing proportion suffers from manifest inflammatory diseases of the respiratory tract, skin, fever or some kind of immunopathology. The immunological pattern is diverse, individual deviations are of different intensity. The clinical picture itself usually does not make it possible to characterize the changes and further analysis employing approximately 50 immunological and biological tests is required. Although in the patients registered so far the involvement of the HIV virus cannot be excluded, nevertheless the clinical symptoms of immune deficiency are in many parameters identical with those of AIDS and therefore a specific examination of HIV antibodies is required. Only lymphadenopathy is rare in this group of patients. The therapy is successful in the case of cellular immunity disturbances. Minor


defects are observed in the deficiency of humoral immunity. Thus, the differential diagnostics of AIDS is inevitable in the near future which requires an adequate equipment of laboratories and appropriate techniques.

Immunological Findings in Children and Adolescents with Recurrent lnteeh~ns

gEDZVA A., VEar, mROVA E., JANOUgKOVA A., RF~NSTEIUOVA H. 4th Pediatric Clinic, FVL UK, Prague

We present an evaluation of immunological examinations of children and adolescents with recurrent infections which have been performed since 1986. We performed a complete examination of humoral and cell-mediated immunity in 230 children. Since 1987 we have also examined cellular immunity by determining lymphocyte subpopulations by means of monoclonal-antibody-labelled cells using flow cytometry. Humoral immunity disturbances (most frequently decreased IgA - 40 %) were found in most children. The defects of cellular immunity were mild and were discovered when a detailed aqd accurate analysis was performed. Surprisingly, a significant negative association between the number of B lymphocytes and the age of children was found. Besides decreased IgA, no typical abnormalities of laboratory findings, that could account for recurrent infections, could be found.

Laboratory and Clinical Response of Pat/ents Treated with NORGA or PJbcebo

KRYfflOFKOVA O., HAUSNER P., POCH T., Fuc"movA T., VosEc~ M., BARTf.n~OVA J., JANATKOVA I., KRUPZCKA J., SKOPOVY P. OK/, FVL UK, Prague ; Laboratory of Oncology, FVL UK, Prague

Recently, a prospective double-blind study of alternate administration of low doses of NORGA or placebo was performed in 29 patients with clinical symptoms of immunodeficiency with hypogammaglobulinemia or dysgammaglobulinemia. During the follow-up study, clinical symptoms were evaluated on a five-grade scale together with the examination of certain immune mechanisms. Statistical evaluation of these parameters confirmed the placebo effect of the first NORGA administration on subjective clinical symptoms and on phagocytotic activity enhancement. Other parameters were influenced with a latency exceeding 2 months. Therefore, we followed the further fate of patients who had received gammaglobulin preparations repeatedly. We took into account the frequency of infections as a manifestation of immune deficiency, symptoms of tolerance of the preparations and changes in parameters of immunological examinations. The results were compared with previously obtained data in the same group of patients.

ImmuaologieM Profile in Patients wfth CEroaie Ur/muy T m a l~eeffon

KAMiNKOVA J., HORACKOvA M., MOgNOVA H., POLACKovA M. Laboratory for Research of Infectious Diseases, FDL UK, Prague ; 2nd Clinic of Internal D/seases, FN Prague-Bulovka ; Institute of Medical Microbiology, FDL UK, Prague; Department of Immunology, FN Prague-Bulovka

The aim of the study was to determine the immunological profile in 18 patients with chronic pyeionephritis and 12 patients with recurrent urethrocystitis. During the remission of the disease we examined repeatedly the white blood cell count, number of active and total T iymphocytes, phagocytotic activity of peripheral neutrophils, monocytes and eosinophils, serum IgG, IgA, IgM and acute phase proteins. The E-rosette test, phagocytotic assay with HEMA particles and radial immunodiffusion were employed. Significant changes were found in both groups, particularly in patients with pyelonephritis: decreased active and total T lymphocytes, increased IgG and IgM levels, increased neutrophil and eosinophil number per ttL. No changes in leukocyte phagocytotic activity could be demonstrated in the two groups.

1990 ABSTRACTS 71

Detects of Cell.Mediated Immunity in Patients with Chronic Lymphedenm of Lower L/robs

VRUBEL J. IKEM, Prague

We examined 30 patients with chronic secondary lymphedema of lower limbs. Of 30 patients, 26 suffered from repeated attacks of erysipela. Examination of cellular immunity revealed decreased CD3, CD4 and CD8 T lymphocytes in 18 patients and decreased phagocytotic activity of leukocytes in 17 patients. Onl)' in 2 patients were the findings within normal values. Of 4 patients without erysipela, decreased T lymphocytes were found in one, while decreased leukocyte phagocytotic activity occurred in the second. All 30 patients had a decreased immunoskin index below 1.0, in 12 total anergy was observed.

Ciinicmi Findings in Dispensadzed Patieuts with i ~ V Seropositivily

STANKOVA M. 3rd Clinic of Infectious Diseases, FVL UK. Prague

Towards the end of 1985, a center for therapeutic and preventive care of individuals with HIV seropositivity was established at the 3rd Clinic of Infectious Diseases, FVL UK (Bulovka Hospital, Prague). So far, 51 HIV seropositive patients (37 Czechoslovak citizens, 14 foreigners) were treated in our ambulance unit. The asymptomatic form of the carrier state was diagnosed in 42 patients, ARC in 3, and full-blown AIDS in 5 patients (1 died). The most frequent risk factor appears to be homosexuality (32). As regards clinical symptoms, the most frequently occurring were lymphadenopathy, diarrhea, loss of weight, herpetic diseases, pneumonia, recurrent catarrhs of the upper respiratory airways and liver disturbances.

Immunological Findi~s in BgV-Positive hlients

KAMINKOV./~ J., VACEK Z., HAVLIK J., STAIqKOVA M. Laboratory for the Research of Infectious Diseases, FDL UK, Prague; Ist Clinic of Infectious Diseases, FDL UK, Prague; 3rd Clinic of Infectious D/seases, FVL UK, Prague

The group included 12 male homosexuals and 1 female. The mean age was 34 years (23-56). 7 patients were HIV-positive carriers, 2 with ARC and 4 with full-blown AIDS. The white blood count, active and total T lymphocytes, TH (CD4 § "Is (CD8 § lymphocytes and the T , :Ts index were examined in all patients using E-rosette tests and monoclonal antibodies (Beckton-Dickinson). The results showed decreased percentage of total lymphocytes and low Tn in all patients. The percentage of "Is was increased and the Tn :Ts index was decreased in all patients, the most profound changes being found in patients with full-blown AIDS. Interestingly, the blood of most patients contained up to 40 % of atypical lymphocytes. A shift to the left was present in all patients. The results represent the first experience with HIV patients in our country.

Immunity Parameters in HIVoNegative Homose~sb

FmRLOVA E., ZAV.~ZAL V., BR0C"KOVA M. Clinic of Dermatology and OKI, LF UK, PIzefi ; IHE, Prague

Cell-mediated, humoral and nonspecific immunity was examined in 46 homosexuals. Despite the fact that all homosexuals were HIV-negative, immunological disturbances similar to those found in AIDS patients were recorded: decreased TH/Ts lymphocyte ratio, increased Ig, presence of acute phase reactants, [3-2-microglobulin in particular. On the basis of these results one can assume that homosexuality together with repeated virus, bacterial and parasitic infections predispose patients for immunodeficiency and probably even for HIV infection.


Mechanisms of A#ople Reaction

ZAVAZAL V. OKI, LF UK, Plze~

Following the discovery of IgE, a large number of studies provided further explanation of the atopic response; however, the problem appears to be more complicated. The process of mediator release itself is not induced by binding the antigen to IgE only but involves a number of chemical bonds in other parts of the molecule. In addition, the outcome depends also on the ability of cells to release mediators, Thus, a complex.of immunological reactions is decisive for the initiation of the response. Gradually, the sites on the e-chain have been discovered that are responsible for binding to various receptors present on cells other than mastocytes and basophils. The delay of mediator functions explaining the wide variety of biological functions is being recognized. Regulation of IgE formation can be explained at the cellular and organismic levels and we are approaching the turning point when a control of IgE formation will be possible. However, despite all new discoveries, the basis of atopic response and particularly the diagnostic and therapeutic procedures remain in the area of experience and practice which had been known long before IgE discovery as an atopic antibody.

Sadimun and Histamine Release from Mast Cells

DRAnEROVA L., STERZL J. Department of Immunology, MBU C~SA V, Prague

Sandimun (Cyclosporin A, CS) is an immunosuppressive substance employed in clinical practice. We studied the influence of CS on histamine release from rat mast cells. We found that CS itself (1) did not induce histamine release from mast cells up to the concentration of 100 ~tg/mL, (2) CSis a strong inhibitor of histamine release induced by the substance 48/80, the calcium ionophore A 23187 and concanavalin A + phosphatidylserine (ConA + PS), (3) did not influence ConA binding to the surface of mast cells~ (4) suppressed the release of intracellular Ca 2* induced by A 23187, (5) increased the entry of 45Ca into mast cells treated with A 23187. Our results indicate that CS inhibits histamine release from mast cells at the Ca 2+ level.

M ~ e u d ~ / g E Ab/llty ot L e c ~ BhzNng

g ~ u z V., Z A v ~ V., T ~ s ~ _ o v A H. OKI, LF UK, Plzeit ; Department of Immunology, MBU C~A V, Prague

Properties of murine and human IgE were compared using the reaction with plant lectins. Heating to 56 ~ for 4 h did not influence the precipitating activity of the two IgE preparations. Heating to higher temperatures led to denaturation of the protein component of the IgE molecule, resulting in loss of precipitating activity with the antibody but precipitation with lectins remained intact. No changes were observed after IgE digestion with papain, bromelain, ficin, a slight decrease of precipitating activity was observed after oxidizing the sugar moiety with sodium periodate. Digestion with trypsin removed the precipitation with lectins, the reaction with antibodies remained intact. Similarly, formaldehyde treatment reduced significantly the precipitation with antibody while precipitation with lectins decreased only slightly. Thus it follows that the structures of the two molecules seem to be similar including the sugar moiety.

1990 ABSTRACTS 73

Compm/son of the Effects of Var/ous Methods of Po//en Affergy Hyposens/t/zsaou

ZAVAZAL V., KRAUZ V., SEBEROVA E., Mm~t'XOVA E. OKIA, FN Plzeh

Evaluation of pollen allergy hyposensitization requires permanent and wide-spectrum examinations of clinical and laboratory parameters. Previous experiences were re-evaluated after a 6-year therapy with native allergen including combined immunomodulation with the transfer factor or zinc, depot allergens Sevac and Pollinex and peroral allergen therapy alone or together with the transfer factor. All treatments used resulted in total IgE decrease but only native allergens and Pollinex stimulated specific IgG4. Both depot treatments led to a significant increase of immune complexes. Native allergens and Pollinex yielded a better clinical response than the perorai treatment. No significant changes in T cell and'Is numbers were obtained after a single simultaneous immunomodulatory treatment. A significant decrease of subjective symptoms, skin test positivity and drug consumption were observed after treatment with Pollinex ; on the other hand, no beneficial effect of a long-term therapy with domestic depot allergens could be demonstrated. One can conclude that the most effective method remains the administration of native allergens or depot treatment with Pollinex. Other types of treatment are useful at the beginning of the disease only and in highly sensitive patients.

The llypedmmunoglob,dinemls E Syndrome

LOKAJ J., MATU~KA J., LITZMAN J., BURSA J. OK/and the 1st Pediatric Clinic, FNsP Brno

The hyperimmunoglobulinemia E syndrome (Job's syndrome) is a rare immunodeficiency which is manifested already in early infancy by purulent processes of the skin and lungs. We present an immunological characteristic and therapeutic experience obtained in a 5-year-old boy. We found permanent increase of IgE concentration (5-10 KU/mL), a mild decrease of IgA, unchanged IgG, IgM and IgD and increased circulating immune complexes. No changes of the complement system were found. The number of B lymphocytes (M-rosettes) was at the same level as in healthy individuals, the number of T lymphocytes (E-rosettes) was near the level of reference values. The phagocytotic function, measured by chemiluminescence, INT-test and ingestion of methacrylate particles was normal but a defect of spontaneous and chemotactic leukocyte migration was present. The patients required permanent antibiotic treatment. Ketotifen, inosiplex, dialyzate of leukocyte extract, high doses of intravenous normal gammaglobulin were without clinical effect. A transient beneficial effect was obtained by plasmapheresis with substitution of antistaphylococcus plasma. The lung lesions had to be treated surgically.

Cbmnges of IgE Levels Following Transfer Factor Administration

CEmtOVA M., P E r ~ K J., ~CH K., ZAVAZAL V., Htnvn_ M., govA~KovA P. OONZ Sokolov ; ~JSOL, Prague ; OK~A, FN Plzeh

We examined a group of patients with cell-mediated immunity deficiency associated with the dermorespiratory syndrome. According to laboratory results, the patients can be divided into two groups with different IgE levels. The first group was characterized by low IgE levels (below 50 U/mL), the second group had high levels (above 1 000 U/mL). Long-term administration of transfer factor had an immunomodulatory effect, i.e. low IgE levels increased and, on the other hand, high IgE levels decreased. This decrease was dependent on the frequency of transfer factor administration.


Detenninmtion of IgG, lgM, lgA, lgE n d lgG4 Specific Antibody against Pollen Allersem in the Sets of Ped/a~r/c Pst/ents dur/ng Immunotherspy

POLACKOVA A., TLASKALOVA H., ~PI(?AK V., Pot.AO~ovA M., MAN'('AL P., RADL J. MBU C'SA V, FYF UK, Department of Pediatrics, FN Prague-Bulovka, OKI, FN Prague-Bulovka, USOL, Prague; Institute Organization [or Health Research TNO, Rijswijk

The effect of hyposensitization therapy with Pollinex and dynamics of all specific antibody classes against pollen allergens (including IgE and IgG4) had been followed in pediatric patients with pollinosis during a one-year period. While IgE-specific antibodies reached maximum levels in sera of patients towards the end of the pollen season, subclass IgG4 antibodies reached their peak immediately after termination of the hyposensitization therapy, i.e. in May. Afterwards, their concentration decreased slightly during the season. The results were obtained by using a modified immuno-enzyme test elaborated for the sensitive detection of specific antibodies against pollen allergens.

The Effect of Pollinex Administration of T Lymphocytes rand Plmk, ocytotlc Aclivity of Periphetsl Lenkocytes

KAMINKOVA J., ~PICAK V., POLACKovA M. Laboratory for Research of Infectious D/seases, FDL UK, Prague; Department of Pediatrics, FN Prague-Bulovka ; Department of Immunology, FN Prague-Bulovka

The aim of the study was to evaluate the effect of Pollinex administration on the white blood cell count, number of active and total T lymphocytes and phagocytotic activity of peripheral blood neutrophils, monocytes and eosinophils. In the course of a year 6 examinations were performed in 44 children suffering from pollinosis in the following periods: (1) March - before therapy, (2) May, (3) September, (4) November, (5) January, (6) March of the following year. E-rosette test and phagocytotic assay with HEMA particles were employed. During the year of observation, eosinophils increased from 34 % to 53 %. A significant decrease of active and total T lymphocytes was recorded in all children. No improvement was observed after a year of therapy. Phagocytotic indexes of leukocytes were normal. In addition, dynamic changes in the numbers of phagocytotic eosinophils and particularly monocytes were observed. Before therapy, an increased number of phagocytosing monocytes were present in 36 % of children, one year after theral3y in 51%. In most cases changes of cellular immunity were not manifested by increased morbidity during the winter period or by deterioration of subjective seasonal symptoms.

Morbidity of Bremt-Fed Infants with High lgE Level in Cord Blood

TOBOLKOVA M., POUSTKA S., ZAV~aU. V. Department of Pediatrics, Hospital Karlovy Vary ; OKI, FN PIze~

Increased cord blood IgE levels were recorded in 13 infants out of 1 570 newborns examined in the Karlovy Vary district in 1986_ In addition, high IgE levels were found in mothers of these infants. At the age of 12 months, enhanced IgE levels were found in 2 infants only, together with symptoms of atopy. Polyvalency of skin tests without clinical manifestation was found in other 2 infants with normalized IgE levels after 12 months. Thus, it follows that a growing percentage of child atopics with age is probably caused by high antigenicity of the environment in association with family genetic predisposition.

1990 ABSTRACTS 75

l)etermins~n of l~sma Hlslzmlae by ~ C'nrommtolrraphy

PAUL~K M., NovAKovA J., EHL J. Department of Allergology, FN 2 with FP, ONZ of Prague

Plasma histamine was determined by reverse isocratic high-efficiency liquid chromatography by the pre-column derivation with o-phthalaldehyde (OPA). Fluorescence was monitored at excitation wavelength 350 nm and emission at 450 nm after elution with methanol-acetonitrile mixture containing phosphate (0.1 mol/L) with triethyleneamine at laboratory temperature. The retention time of OPA- derivatized histamine was 6.5 rain, the duration of chromatographic analysis was 30 rain, the detection limit 0.1 ng/mL. The advantage of this method appears to be its high specificity, a drawback is the need of sophisticated apparatus.

Re/e~e Test/n A J I ~ Pract/ce

YmAuz V., Vot.~ M. OKI, LF UK, P/zeft ; Department of Physiology, LF UK, Plzed

We examined histamine release from leukocytes in subjects reporting various degree of hypersensitivity to drugs. Half of the patients were treated also for allergy of other types. The most frequently used drugs included Acylpyfin, Alnagon and penicillin. Acyipyrin induced histamine release in 46 % of subjects, penicillin in 33 % and Alnagon in 30 %. When evaluating a panel of drugs, allergin reactivity was found in 42 % of individuals with clinical symptoms. Therefore, determination of histamine can be recommended as an auxiliary test for the determiiaation of drug and food allergy. Wider use of histamine liberation determination is hampered by high requirements for purity of laboratory glass and by complicated method.

T.Lymphocyte &~blmp~tlons in O~roair At'~v,e/-lelml~Is

FUC'iKOVA T., BRODANOVA M., SKOPOV'? P., BARTO~OVA J., HAUSNER P., KRUPIC'KA J. OKI, FVL UK, Prague ; 1st Clinic of Internal D/seases, FVL UK, Prague ; Research Laboratory for Oncology, FVL UK, Prague

T lymphocytes represent a heterogeneous population of immunocompetent cells and their quantitative and functional disturbances may be involved in the pathogenesis of many diseases. Chronic active hepatitis belongs to diseases where T lymphocytes play an important role in the initiation and progression of the disease. We examined T-lymphocyte subpopulations and their activation markers in patients with chronic active hepatitis with respect to the activity of the disease, therapy and the presence of HBsAg in the serum. We found quantitative changes in the proportion of individual subpopulations in 33 patients; decreased C-'D3 + lymphocytes were found in patients not undergoing immunosuppressive therapy. As regards HBsAg § patients, increased CD4 + lymphocytes were found compared with HBsAg- patients. The symptoms of activity of the liver disease correlated with the presence of the CD8 marker.

Cbsnges of the Lymphocyte Funct/ona/Ac//v/ly/n Padents w/th Psrodont/ds

B~TovA J., T[~sv,~ovA H., F~_JSA O., ~ L , ~ s Lo, B ] z ( ~ J. V(.IS, Prague; MB(J C"SA V, Prague; INSERM, Montpellier

The in vitro ability of suppressor cells to inhibit polyclonal activation of PWM-induced immunoglobulin formation was studied in patients with the mild and terminal stage of parodontitis and in control subjects. Autologous suppressor cells were stimulated with ConA. A decreased ability to inhibit polycionai activation of IgM, IgG and IgA classes was found in patients with terminal parodontitis while in patients with the mild form of the diseases and in controls the immunoglobulin formation was significantly inhibited. Using monoclonai antibodies, we detected in both groups a decreased number of CD8 + lymphocytes by means of immunofluorescence.


T.Lymphocyte ~bPolml~tions in Insulin.Dependent lYmbetes

NIKg M., M~CHALKOVA D., ~'I'EFANOVIC J., BIRCAK J. Department of Microbiology and Immunology, LF UK, Bratislava ; 1st Pediatric Clinic, LF UK, Bratislava

Relative proportion of CD3, CD4 and CD8 T lymphocyte subpopulations was examined in the peripheral blood of 27 children with clinically fully developed insulin-dependent diabetes (type 1). The values of T-lymphocyte subpopulations were found to be within normal values. No correlation between individual subpopulations and the duration of the disease or actual compensation of glykemia could be found. The long-lasting disturbance of compensation, accompanied with increased glycosylated hemoglo- bin was associated with decreased relative numbers of C'D8 T lymphocytes and with changes in the CI~/CD8 ratio, CD4 cell being prevalent (r = 0.48, p < 0.05). "]'he decrease of relative number of suppressor/cytotoxic CD8 T lymphocytes in association with long-lasting disturbances of glykemia might explain the role of glucose metabolism disturbance in the development of the disease and points to the significance of early and effective compensation of glykemia as a factor of natural immunomodulation in patients in the initial stage of insulin-dependent diabetes.

Immuaologlo~ Chaages in Demyeliai~tion Diseases of the CNS

RvgtovA O., NVPLOvA H., CHROMOVA L., VEmORA O. Institute of Microbiology and Immunology, I_J: UK, Hradec Kr~lov6 ; Clinic of Neurology, FN Hradec Kr~lovd

Cell-mediated immunity was examined in patients with various degrees of multiple sclerosis (MS) using Immunoskintest (containing tuberculin, toxoplasmin, candidin, tetanus and diphtheria antigens) and leukocyte migration inhibition test. The decreased skin reactivity was observed primarily in patients with chronic and progressive MS; on the other hand, leukocyte migration inhibition (with basic en- cephalitogenic protein) was not observed in most cases. Patients examined at the beginning of the disease (suspected MS) had normal skin reactivity and positive leukocyte migration inhibition. The preliminary results show that patients with various stages of MS had different immune response to intradermal antigens and in vitro response to stimulation with the basic protein,

Lr~mng~ in ~ll/alr/mmujndty/n Splenoc'to/nJzed Pal~nts

SEOL~X D., ~;TUKSOVA T., NOVAK g . PA[JSA V., K ~ A R V., STAJ~ A. Clinic of Infectious Diseases, OKtt, Surgical Clinic, KONZ-FN Plze~

The authors studied changes in immunological parameters in two groups (20 subjects each) of splenectomized patients. An increased absolute number of leukocytes, lyrnphocytes, % of active lymphocyte nucleoli, enhanced PMN phagocytotic activity and FW were found in both groups. In addition, a decreased number of T lymphocytes (p < 0.05), NK cells and skin test reactivity (Immuno- skintest OSOL, Prague) was found. An increased percentage of B lymphocytes was found in 50 % of posttraumatic splenectomies. The CRP level was higher in patients with hematological diseases in comparison with posttraumatic splenectomized patients (p < 0.05). A high incidence of infections of upper respiratory airways was observed in both groups.

1990 ABSTRACTS 77

lmmuolo#od Exmntntion o! Hemophllises

LrrZMAN J., BURSA J., KUKLINEK P., PENKA M., BLA~OVA E., BALCARKOVA H., LOKAJ J. OKI, OKH, 1st Pediatric Clinic, FNsP Brno

Within the framework of a complex dispensary care of hemophiliacs we performed laboratory examination in 18 adults and 8 children (HIV-negative) with hemophilia. As compared with healthy control individuals we found a decreased relative (not absolute) number of T lymphoeytes(determined by E-rosette test), increased relative proportion of CD8 + lymphocytes and decreased immunoregulatory index CD4+/CD8 +. The phagocytotic activity of isolated granulocytes, measured by the INT test, was normal. Concentrations of IgG, IgM and circulating immune complexes (CIC PEGIKEM) were significantly higher in adult patients than in control subjects. The difference between circulating immune complex concentrations in adults and children were statistically significant. In addition, we found a positive correlation between the coagulation activity of factor VIII or XI and the immunoregulatory index. These results stressed the need of immunological monitoring of patients with hemophilia.

Involvement of Bacteria of Dental Plaque in Immm~logiod Meehmnisms of Psrodontsl Diseases

BARTOVA J., TLASKALOVA H., KREJSA O., POSVi~IL M., Mma.AS L., SKAm.ANIrr P., KOMPERT O., ~iaovA M. VUS, Prague; MBO CSA V, Prague ; Ist Clinic of Stomatology, Prague

Bacteria of the dental plaque play an important role in the etiology of parodontitis and they behave as important polyclonal activators. Using in v/tro tests, we studied immunoglobulin formation after stimulation of peripheral blood lymphocytes with Actinomyces v/scosus and anaerobic bacteria isolated from the dental plaque of patients with the mild and terminal stage of parodontitis and from healthy individuals. No significant differences were recorded among individual groups after stimulation. However, contrary to healthy individuals, increased IgA and IgM formation was found in patients with the terminal stage of parodontitis after stimulation with E. coll. Following stimulation with PWM, a significantly higher formation of antibodies against A. viscosus was found in tissue cultures. Finally, enhanced formation of autoantibodies against collagen HI in tissue culture supernatants in patients with the terminal stage of parodontitis after stimulation with A. v/scosus was found.

Autouti/M~v sgsimt the Bass/Ce//s of the Parot/d Dud

NO0~XA Z. Institute of Pathological Anatomy, LF UK, Hraclec Kr~lov~

IgG class antibody that can bind to basal cells of the parotid duct can be found in about 0.5 % of sera, examined for the presence of autoantibodies by the two-step immunofiuorescence method. We presume that these antibodies are directed against a certain cytokeratin fraction. The possible association of these antibodies with some systemic diseases is being studied.

T Ce//s m~ The/r ~btypes ~ Cm/m's D~sse

KOMAaXOVA O., FLXA B., KOPECK~ O., I~,mSEK J. 2nd Clinic of Internal D/seases, LF UK,/-/radec Kr~lov~

We determined the absolute lymphocyte count and relative and absolute number of mature lymphocytes, helper and suppressor cells in the peripheral blood of 21 patients with Crohn's disease. The determination was done by indirect immunofluorescence using anti-CD3, anti-CD4 and anti-CD8 monoelonal antibodies. The relative numbers of CD3 lymphocytes were decreased in 16 patients, CIM lymphocytes in 10 patients. CD8 lymphocytes were decreased in 8 and increased in 6 patients. Changes in


absolute numbers of T lymphocyte subpopulations were less expressed in individual subgroups. Values of the immunoregulatory index (CD4+/CD8 § were highly variable with two extremely high and two low values. These findings were compared with clinical parameters and with the results of other immunological tests. Although abnormalities of circulating T cells and their subpopulations were found in many patients their association with the actual process in the intestine could not be demonstrated.

Diapmsis of Enteroinvasive E. coil in Paltents with Ulcerative Co~ts Using Collcln Is

BURF~ J., HORAK V., FIXA B., KOMARgOVA O. 2rid Clinic of Internal D/seases, Department of Microbiology, FN and LF UK, Hradec Kr~lovd

Enteroinvasive E. coil strains (EIEC) are able to penetrate into the large intestine mucous membrane and induce an inflammatory reaction in the lamina propria. The disease is characterized by the colitis syndrome, admixture of blood and mucus in stool. It is difficult to determine diagnostically ulcerative colitis (UC), particularly in its initial phase. EIEC determination (Sdreny's test) is not routinely available. Hor:ik et al. designed a simple screening test for EIEC determination by means of colicin Js. The test is based on the susceptibility of EIEC to colicin Js. Using this test, the authors examined patients with UC. The test was in all cases negative and the diagnosis of UC was confirmed. It is recommended to use the simple screening test in all patients with UC, particularly at the beginning of the disease.

Involvement of Immunological Mechanisms in the Psthogenesis of lchbpsthic Nep&rotic Sym~me and the PossibiBties of Therapy

LISgA J., VARVAROVSr,.A J., SLOVACEK R., ZAVAZAL V., MRAC"XOVA E. Pediatr/c C/in/c, FN P/zet] ; OK/A, FN P/zer]

The authors summarized the results of immunological examinations performed by means of 30 tests in 42 children with idiopathic nephrotic syndrome (INS), in the acute phase and during remission. Disturbances of cell-mediated immunity, particularly of lymphocyte transformation, were found in primary attacks and relapse of INS. In addition, a significant decrease of IgG was observed during the acute phase of the disease. In 60 % of patients, a long-lasting increase of IgG was present, and this finding correlated with increased frequency of positive allergological skin tests to basic and food allergens (73 % vs. 26 % in normal population).

The Course of Chronic Graaalomatom Dtscase is a 5- YeK-Old Boy

WIEDERMANNOV./~ D., LOKAJ J., WIEDERMANN D., HOLCIKOVA A., ZARO~SKA E., BUCEK J. Clinic of Pediatric Infectious D/senses, Brno ; OKI KUNZ ; Department of Pathological Physiology, UJEP ; 2nd Institute of Pathological Anatomy, UJEP, Brno

The disease was manifest at the age of 3 months as a septic form of salmonellosis accompanied with osteomyelitis. At the age of 5 years, after the second attack of bronchopneumonia, liver abscesses and later granulamatous lesions occurred in the lungs, kidney, lymph nodes and meningae. Staphylococcus aureus and Candida species were found in the lesions. The diagnosis was based on examination of granulocyte metabolic activity by the INT test that showed the inability of phagocytes to kill engulfed catalase-positive microorganisms. A progressive decrease of some serum proteins was recorded.

1990 ABSTRACTS 79

Cell-Mediated Immunity Parameters in lmmunolo#cal Practice

PAnZNER P., SEBEROVA E. OKIA, FN PIzed

The aim of the study was to re-evaluate the diagnostic value of determination of T lymphocyte subpopulations in the peripheral blood of patients who are routinely examined in the immunological and allergological ambulance unit. 104 patients were divided into 4 groups according to the diagnosis. These groups were compared with one another and also with a control group of 32 healthy individuals, using the following parameters : T8 § and T4 § cells, T4/T8 ratio and theophylline-sensitive cells. No statistically significant differences in theophyiline-sensitive cells and T8 § lymphocytes were found. As regards T4 § cells, a significant difference between the control group (41%) and the group with immunodeficiency (36 %), tumours (32 %) and treated immunodeficiency (30 %) (;t = 2.04) was significantly different from that found in atopic diseases (~ = 2.03), tumours (;~ = 1.42) and autoimmunity (~ = 1.20). In addition, differences in the immunoregulatory index between the immunodeficiency group (1.73) and autoimmunity (1.2) were present. No significant abnormalities in atopic patient were observed.

I lLA Phenotypes in Recently Di~guosed Type-1 l~be tes and Assodntion with Metabolic Pmm~ters

HUL~SKY I., IVAgKOVA E., KUPKOVA L., KMLtK J. IKEM, Prague

We examined the functional secretory reserve of Langerhans islet cells by measuring the C peptide in 17 patients (7 females, 10 males) aged 17 to 30 years with the clinical diagnosis of insulin-dependent (type-1) diabetes during a one-year period. We attempted to establish the correlation between the HLA phenotype and disappearance of the secretory reserve. Twelve patients possessed phenotype DR 4 (the frequency of this phenotype in a healthy population in the Czech Socialist Republic is 18.4 %), two patients had the DR 2 marker (controls 14.9 %). All patients possessed the DQ 3 marker, 15 patients had BW 6 marker. These markers correlated positively (BW 6, DR 4, DQ 3) or negatively (DR 2) with the occurrence of type 1 diabetes; correlation with metabolic parameters was not found.

M o a o d o . a A.tfbo~es atndmt Hmmm Heart Myada

MACHA J., ROMANOVSKY A., LUKACOVA D., ANGYAL R., HAJKOVA H., HORAKOVA K. Department of Animal Physiology and Developmental Biology, P~F UK, Prague ; CFV SA V, Bratislava

The content of light subunits of heart myosin (LC) is directly proportional to the size of lesion in myocardial infarction. Hybridomas producing antibodies against LC of human heart myosin have been constructed with the aim of their use in clinical tests. The antigen was prepared by denaturation of heart myosin with guanidinium chloride. Hybridomas were prepared from the myeloma line SP2 by a standard technique in normal BALBc mice and mice rendered tolerant to subcomponents of skeletal myosin. So far, 16 hybridomas producing monoclonal antibodies against LC have been isolated. Most of them react with both the LC1 and LC2, one with LC1 only.

The Gsrdner-Dismond Syndrome - D ~ r b s n c e of Erythro~e Membnme PbospboJ~ds A s y m ~ y by A~d~ry

MACHA J., STRUNECKA A., KRPEJgOVA L., MATUROVA M. Department of Animal Physiology and Developmental Biology, I~F UK, Prague; FN Prague 10

The rare Gardner-Diamond syndrome is manifested by purpura and cutaneous hypersensitivity to phosphatidylserine (PS). PS was demonstrated on the erythrocyte outer membrane by fluorescence and trinitrophenylation in one female patient. Asymmetry disturbance can be induced by the patient's serum


in normal erythrocytes ; induction can be abolished by heating at 56 ~ or by filtration through a sorbeut with anti-IgE. Similarly, hypersensitivity transfer to guinea pigs can be inhibited. Two minority proteins are missing in erythrocyte membranes of patients and thus the erythrocytes have an enhanced deformability.

lmmunoloffieM Parameters for Monitodnff of the Psrodontitis

POLEUIK P., ZAVAZAL V. Department of Stomatoiogy, LF UK, Plzed ; OKIA, FN Plzed

Parameters of cellular and humoral immunity were compared with the stage of the disease in patients with parodontitis. Correlation analysis revealed direct statistical correlation between the depth of parodontal pockets and concentrations of C3, ceruloplasmin, IgE, immunocomplexes (after PEG precipitation) and relative proportion of theophylline-sensitive lymphocytes. In addition, a direct correlation between values of the gingival index and concentrations of ceruloplasmin and immune complexes was recorded. Finally, immunocomplexes levels correlated with parodontai index values.

lsmms~logical Monitoring u d Therapy Attempts/n Recurrent Uveitis

I-~NA I., BOGUSZAKOVA J., STARA J., DOS'rAL C., TESAREK B., BARDFELD R., PEKAREK J. Section of Medical Immunology, ILF ~ Department of Opthalmology, 1LF ; VUCHR ; USOL, Prague

The authors followed 15 patients with recurrent anterior uveitis for the period of 9 months. Selection of patients was performed according to the frequency of the disease attacks; the group included only patients who had suffered at least three attacks of iridocyclitis within one year, one patient suffered 26 attacks during 5 years. The patients were submitted to repeated ophthalmological, rheumatological and immunological examinations. The immunological examination included HLA typing, quantitative determination of lymphocytes and T lymphocytes, IgG, IgA and IgM, circulating immune complexes, C3 and C4 complement components, antinuclear antibodies, rheumatoid factor, ASLO, CRP, anti-dsDNA and skin tests. Seven patients with rheumatoid symptoms possessed HLA-B27 marker. All 15 patients suffered from anterior uveitis, 2 patients had also posterior uveal disorder and one had episcleritis. It seems that absolute T lymphocyte number yields the most valuable immunological information. The patients were submitted to a long-term therapy with transfer factor according to previously elaborated schedule. Only 3 recurrencies occurred during the 9-month treatment - two cases of anterior uveitis and one episcleritis, all three in a mild form.

]~le El~ect ot lmm~jt~ DL~order on PsrodontM ~ / n lmmunodeflcient Psdents

C a ~ O ~ E C ~ D., I ~ l ~ J. 2rid Clinic of Stomatology, FVL UK, Prague; OKI, FP 1, Prague

The condition of the parodont is influenced by the immunological background of the patient. We attempted to establish what immunological parameter influences significantly the pathological changes of parodontal ligaments. We examined serum levels of IgG, IgA, IgM, C3, CIC PEG and E-rosettes. Gingival bleeding was evaluated by the gingivai test, the degree of parodont destruction by the parodoutal index. Using a gradual regression analysis, we found a highly positive correlation between the IgA level and the gingival index. Patients with low IgA level had also lower gingival bleeding.

1990 ABSTRACTS 81

Results of lmmanohemtological Examation in O~l~en with Type 1 Diabetes, Their Parents and Sibling8

VALNIL"KOVA Z., KtmiKovA A., GAILLYOVA R., KALINA 7_. Department of lmmunogenetics, IMV, VOP, Bmo

Preliminary results of immunohematoiogical examination of children with type 1 diabetes and of their family members are presented. Association of the HLA system and cell-mediated immunity with the clinical course of the disease was examined. The frequency of HLA-A9, B8, B15 and B18 antigens was increased, reactivity to nonspecific mitogens PHA and ConA was decreased.

Histological and Antibody Responses in Experimental Controlled (Auto) Immune ThyrokHtis in Guinea

VELICK~ J., TnIBACH M., Lus'nG R., LHOTOVA H., JARNIK M., RAgKA I. OEM CSA V, Prague

Changes after immunization with thyreoglobulin (Tg) with CFA or with a suspension of thyroidal cells plus CFA were examined in guinea-pig serum and thyroid gland. Using dot immunodetection and fluorescence, antibodies against Tg were found in the sera of all animals. In one case, even antibodies against calcitonin were found after immunization with cell suspension. Other morphological changes were demonstrated.

Chunges Occurring during Immunization with Insulin in Guinea Pigs

TITLBACH M., MOLLEROVA M. OEM CSA V, Prague

Guinea pigs immunized with insulin in complete Freund's adjuvant or with an extract from iisteria (Ei) exhibited a significant increase of giykemia during immunization. Animals given physiological saline with CFA or Ei remained normoglykemic. Morphology of Langerhans islets revealed polynesia, B-cell degranulation and increased number of A-cells. These changes were more significant after immunization with insulin plus CFA than after insulin with Ei.

Immunological Study in 35 PedSatrie Hemophiliacs

NOVAKOVA J., MISAt~OVA Z., KOMRSKA V. Department of Pediatrics, ILF, Prague ; 2nd Pediatric Clinic, FN Prague

Selected determinations of cell-mediated and humoral immunity were performed in 35 children with hemophilia. Statistically treated results with respect to HIV-positivity are presented. In addition, a computer-assisted evaluation, by a cluster analysis program was performed. The patients were divided into groups according to 9 selected criteria, HIV seropositive patients formed a single homogeneous group.

Epideminlogy and Development of Allevgoses in lndustritl and Urban Areas

MRAt2"XOVA E., ZAVAZAL V., KRAUZ V., PANZNER P., SEBEROVA E. OK/A, FN P/zer]

Epidemiological examination of allergy occurrence was performed in 1 000 school children (aged 4-16) in various areas of Plzefi and Prague. We evaluated the allergological questionaire, results of skin tests with assorted pollen and house dust and selected immunological parameters including IgE. The results have shown an at least two-fold increase of allergy occurrence compared with originally reported

$2 5th IMMUNOLOGICAL CONGRESS Vol. 35

12-15 %, significantly increased pollen allergy (mean 14 %), pathological high levels of IgE in adults, and IgA deficiency in 1/3 of children. The findings obtained in individual localities confirmed the validity of the working hypothesis concerning the influence of air pollution on the occurrence of allergic respiratory diseases.

State of lmmmRy in l O-Yemr.Oid CkUdren IJiCmg Permm~..tly in Poihted Arem

l~tALIg J., KANIA A., ~ O V A I., BAS'rL R., PRvszczovA M. OHS Karvin~

The Karvimt district belongs to the Ostrava-Karvina industrial agglomeration with heavily polluted air; the pollution index ranges from 3 000 to 10 000. In 1987, we repeated the immunological examination of a selected group of 296 10-year-old children in spring and in autumn. We examined IgA, IgM, IgG, acute phase proteins and other 13 serum proteins. In addition, we examined the salivary immunity, evaluated the results of calmetization, and the skin test was performed with the Skintest in selected tuberculin-negative individuals. We found changes of IgA, IgM, IgG, C3 in autumn ; changes of orosomucoid, ceruloplasmin and ct-2-macroglobulin were found primarily in boys.

t]mnges ia ~ ~ Psttem hi ~ e D i s a ~ ol Ltberee ~ o v A J. Allergoiogical Ambulance Unit, OUNZ Liberec

Pollen allergy has been followed since 1959. About 1% of the whole population of the district has allergy but we presume that the number is substantially higher because not all patients require medical care. The incidence is higher in young individuals; recently, about 100 new patients per year have been reported; on the other hand, the decline of allergy cases is negligible. In the years 1960 - 1980 the peak of the season was in June-July but since 1980 the main occurrence of allergy has shifted to March (sometimes even February) and April. According to our results hypersensitivity to the pollen of birch, alder, aspen and sallow has increased considerably. In approximately 10 % this allergy is isolated, in 30 % of cases it appeared after treatment of summer rhinitis. We consider this development to be the consequence of changes in ecology, particularly the decay of cultivated conifers and excessive growth of weed woods whose pollen is extremely aggressive. This situation must be coped with by the production of new allergens for hyposensitization in the Institute of Sera and Vaccines.

Assec~a~en el Peach Hypersem~vtty w/~ the Overg~w~ o! Cera~ P lu t s

POCrA L., RmOFJ~ M., B~qotn~ovA L., ~'r~pA~'rovA E., B~,w_~ J. Center for Allergens, USOL, Prague; Department of Allergology , FN Prague I 0

Using a computer-assisfed analysis, the results of intradermal skin tests with 37 pollen allergens were evaluated in I 142 patients at the Department of Allergology (FN Prague 10) treated during the period of 1982-1986. The skin reactions were expressed by the urticarial iesion-to-erythema ratio. The concentrations used were 10 PNU and 100 PNU. The field trial was performed during the vegetation season of 1987 on the area of approximately 100 km 2, mostly in the urban district of Prague 10. The results mostly supported the suitability of the contemporary composition of the test panel of pollen allergens used in this department. Monitoring of hypersensitivity of the population together with changes in vegetation induced by ecological factors enables the physicians and producers of allergens to provide a high-quality diagnosis and suitable hyposensitization therapy without exposing the patients to undesirable allergens. The computer program used in tl~e present study is available at the USOL.

1990 ABSTRACTS 83

Fonow-up of $aeaed ~ PL-t~#en dn~tg ~ l ~ . r~y /u ttte l t ~ Tatm M o ~ t a m

SocmxwovX M., Kmg.~KOVX J., OLEKBAKOVA M. VOI'IB, Bratislava ; Czechoslovak State Spa, ~trbskd Pleso

The aim of the study was to establish the effect of a complex climatic therapy at a high altitude (1 355 m above sea level) on some immunological parameters. The following examinations were performed in the venous blood of 49 patients: IgG, IgM, C3, cernloplasmin, orosomucoid (determined by single radial immunodiffusion). CIC were determined by the PEG method, total and active T lymphocytes by the E-rosette test. The examination was performed immediately after initiation of climatic therapy and after 5 weeks. After a 5-week therapy, a significant increase of T iymphocytes and decrease of IgG, IgM, IgA and orosomucoid was recorded. Changes in CIC, ceruloplasmin and active T lymphocytes were insignificant.

Food A//ergy hi Patients w/th P ~

ZMRBALOVA J. VRANovA A., Ku~RA P., ~EnE.~;OVA M., BENEg J. Department of Allergology, FN Prague 10

We examined the occurrence of food allergy in patients with pollinosis. Ananmestic data were obtained by means of a questionnaire. Pollen allergy testing including pollen of birch, hazel and hornbeam was performed in 366 patients. In patients with symptoms of hypersensitivity to certain food components related with pollen allergens (mainly birch and hazel), tests with food allergens (e.g. apples, potatoes) were also performed. Hypersensitivity to birch, hazel and hornbeam pollen was found in 80 % of patients. Symptoms of pollinosis in the early pollen season were reported by 145 patients (39.6 %), symptoms of food allergy were present in 131 patients (35.8 %). A significant association between food allergy and symptoms of pollinosis in the early season was found not only on the basis of skin tests with the above allergens. On the other hand, the skin tests with food allergens were not correlated with anamnestic data and thus they cannot be considered as reliable. We would like to point out the high occurrence of food allergy in patients with pollinosis in the early season and stress the high incidence of sensitization with pollen allergens of birch, hazel or hornbeam.

An Allergologfst's Approach to Professioml Resphstory Allergosls o! Fsrmers

SEnEaOVA E., MAtNEROVA J., ZAvAzm. V. Department of Immunology, KHS Plzed ; OKIA, FN PIze~

Farmers belong to a high-risk group with respect to professional allergosis. This is caused by massive exposure to aggressive biological dust, bacteria and fungi in animal production, the main source being fodder. Allergic diseases affect most frequently the respiratory system (bronchial asthma and its equivalents, exogenous allergic alveolitis, chronic rhinitis) and the skin (atopic eczema). The joint approach of allergologists, immunologists and occupational medicine is required for an initial diagnosis determination, examination of immunity and allergy and determination of the pathogenetic role of the above factors.

Lymphocyte ~bpopu/adom in Recurrent Infections of Children

SCHNEIDEROV./~ H., WmOERMA~N D. Clinic of Pediatric Infectious D/seases, FDNsP Brno ; Department of PathologicaI Physiology, LF UJEP, Brno

The aim of the study was to determine the mean values of B cells in children of various age and to establish their association with immunoglobulin levels. B cell detection was performed by direct


immunofluorescence. The mean value of B cells in the whole group was found to be 7.91 +_ 0.20 %. The relative number of B cells decreased with age while the total serum immunogiobulin level gradually increased. No correlation between the levels of individual immunoglobulins and number of B cells could be demonstrated.

The Effect of Eovirortmeaal/Factors on the Immune Profile of C-bildreu with Atopic Demmtitis

VELtCKA Z., ~;USTROVA M. Pediatric Dermatological Clinic, Bratislava ; OK, I, DFN Bratislava

Atopic dermatitis occurs in 3 - 1 0 % of children and recently its frequency has obviously increased. The clinical manifestation in genetically predisposed individuals depends on environmental factors. Our group of patients included 85 % children from urban areas, 15 % from the country. Disorders of cell-mediated immunity were more frequent in city children ; no significant differences in humoral immunity (IgG, IgA, IgM) were recorded.

The Role of Klebsiells Infection in Infant Morbidity

VARVAI~OVSKA J., JAN1CK)V. ~, V., TtmKOVA S., PUZOVA H. Pediatric Clinic, FIe, PIze~ ; Department of Hygiene, Epidemiology and Microbiology, LF UPJ~, Koffice ; Department of Microbiology, KHS PIzed

We summarized the results of the follow-up study of 52 children (aged 1 month to 14.4 years) with positive bacteriological Klebsiella pneumoniae finding in the respiratory airways. The microbe was found in 100 % of laryngeal smears while pharyngeal smears were positive in 15 % only. The predominant clinical manifestation was bronchitis responding poorly to ATB therapy. 80 % of children with altered immunity and longlasting disease were affected by klebsiella, particularly children receiving corticoid therapy; in such cases klebsiella produced severe complications of the primary disease Type 2 Klebsiella pneumoniae and other strains that could not be classified according to the scheme used, were predominant. 23 % of children suffered from nosocomial klebsiella infection.

Non-Specific Hypo~mit~tion vd~ H~t~gloM,

PAZDIORA A. Regional Division of Hygiene and Epidemiology, Plzefi

With respect to good previous experience with Histaglobin (histamine + gammaglobulin) in various allergic conditions, we used this therapy in chronic urticaria and transient edema. Complete disappearance of symptoms was observed in 8 of 22 patients, considerable improvement was observed in 10 individuals. Histaglobin was administered in increasing doses followed by maintenance therapy (1 mL each dose).

ltyposemttiutlou with ltCD - the Product ot O$OL, Prague

BORYSOVA L. Allergological Ambulance Unit, Ostrava-Poruba

Peroral hyposensitization was performed in 60 patients (49 females, mean age 44 years ; 11 males, mean age 39 years) using the stock vaccine HDC manufactured by 13SOL (Prague). This preparation contains the following bacterial strains: Staphylococcus aureus, S. albus, Streptococcus alpha- haemolyticus, Neisseria pharyngis, Diplococcus pneumoniae; concentration 0.1-1000PNU. The therapy was applied to 19 patients with bronchial asthma accompanied with chronic bronchitis, 4 patients with bronchial asthma and generalized eczema, 3 patients with chronic tracheitis, 1 patient with

1990 ABSTRACTS 85

bronchiectasia, 7 with chronic bronchitis, 18 with chronic rhinitis, 2 with chronic urticaria, 1 with chronic eczema and 5 with decreased resistance of the organism to infection. Bacterial hypersensitivity was found in all patients by means of a skin test. The patients received the stock vaccine in concentrations of 0.1 PNU to 10 PNU in increasing doses from 1 drop to 20 drops three times a week, 100 and 1000 PNU twice a week. The mean duration of the therapy was 4 months. No side effects were recorded during the therapy. Considerable improvement was found in 15 patients (25 %), moderate improvement in 19 (31.7 %), no improvement in 26 patients (43.3 %). Evaluation of the effect of therapy included data reported by the patients (number and degree of asthma attacks, cough, expectoration, drug consumption, resistance to infection), laboratory, physical and lung examinations. Examination of cell-mediated and humoral immunity was performed in patients with decreased anti-lnfections resistance only. No significant changes before and after therapy were recorded in these patients. Beneficial therapeutic results with HCD were obtained in 56.7 % patients with allergic diseases based on bacterial allergy. The preparation exhibits good tolerance and minimum time requirements in the therapy.

Treatment of CTJil~en's Astlmm Broncbisle with Levs,nkole

MArkKA J., I_~KAJ J., K ~ K P. 1st Ped/atr/c Clinic and OKI, FNsP Brno

Levamisole belongs to immunomodul~itory preparations stimulating T-lymphocyte precursors and regenerating the effector function of peripheral T lymphocytes and phagocytes. It has been successfuly used in the therapy of children suffering from recurrent infections of the respiratory tract and in immunodeficiencies. We administered levamisole (preparation Decaris) to children with asthma bron- chiale (AB) having a decreased relative number of peripheral blood T iymphocytes (determined by the E-rosette test). The group of 69 children included 28 children with the infectious type of AB, 26 with the mixed type AB and 15 with the dermorespiratory syndrome. Clinical improvement was recorded after a one-year observation in 38 (55 %) children, i.e. in 19 (68 %) with the infectious type of AB, in 15 (54 %) with mixed type AB, but only in 2 (13 %) with the dermorespiratory syndrome. According to our experience, levamisole had a beneficial clinical effect in children in whom the infectious component and disorder of cell-mediated immunity were involved in the pathogenesis of the disease while in children with atopic predisposition the therapy was without effect.

CllluIges of lmmuaJ/y in Vaflous J~gh-RJsk l)tolessJons

S~aEROVA E., ZAVAZAL V., KRAtrz V., ToM~movA A., PANZNER P. Department of Immunology, KHS Plze~ ; OKIA, b-N Plze~

In cooperation with other laboratories we followed clinical and immunok~gical manifestations of immunopathology in various groups of individuals working in a high-risk environment. We examined e.g. workers of the chemical plant in Sokolov where acrylic substances are manufactured, workers of the power plant ~;koda where low-pressure gasification of brown coal is carded out, farmers exposed to the effects of insecticides and fodder and workers of the research institute involved in the production of cytostatic drugs. Healthy blood donors served as controls. The results document enhanced perturbation of the immune system by professional noxious substances which was manifested by changes of inflammatory proteins and by initial activation and later suppression or even total breakdown of specific immunity. Increased occurrence of allergoses was found in some groups. The increased turnout risk could not he proved by immunological examination. Determination of the immunological profile allows one to exclude workers with unacceptable risk factors and to take preventive measures.


/mmamm~bistory EHec~ ot 31crowsve Rsdbffom

I_~.K A., ~ESNF.X J., BULAWOVA H. Institute of Biophysics, OSA V, Brno

Recently, the effect of microwaves (MW) on the immune system has been demonstrated although the results are still incomplete. The aim of this study was to extend our previous findings concerning the effect of MW on the blast transformation and plaque-forming activity of murine lymphocytes. In the present study, we examined changes in spontaneous E-rosette formation of human iymphocytes exposed in vitro to MW radiation. Mice and cell suspensions were irradiated with MW at the frequency of 2.45 GHz (absorbed dose rate 200-300 W/k8), i.e. much higher doses than those used in previous experiments. The high MW doses having a pronounced thermal effect inhibited the above parameters. These results, compared with the data of other authors, confirmed the assumption that high absorbed doses with a thermal effect had rather an inhibitory effect. On the other hand, low doses depressed cellular immunity while humorai immunity was stimulated.

T ~ Effect of Ionized Air on ~ Immm~4olicsl Psrmueters

F ~ o v ~ J., ZvoN~ J., P~p I. VOHB, Bratislava

The quality of air influences considerably many physiological functions of the organism. To the most important criteria of air quality belongs the presence of ions, mainly their number and ratio of positively to negatively charged ions. Ionized air containing 70 000 small negatively charged ions in one mL was applied to patients with bronchial asthma in a 2-bout session during 4 consecutive days. As control sewed patients who had received placebo therapy with normal air (not ionized) 3 weeks before the ion therapy. As compared with placebo inhalation, ionized air induced a significant increase of CHs0, C3 and (:4 in the blood serum. In contrast, the ability of INT reduction by phagocytotic peripheral granulocytes and the amount of circulating immune complexes decreased. These results suggest that ionotherapy obviously influences the activity of certain components of the immune system.

Effect ol Some Mkrodlmste Fsaon on the D e v ~ t ol ~ im

SPLgAKOVA E., S m ~ B., PAVKOVCEKovA O., KUCHTA M., TISCHLER V., KLUKA V. Pe.~atric CtiMc, FNsP Ko.~ce

We performed an anamnestic analysis of risk factors in 244 children with allergy, divided into three groups. (1) 105 children with pollinosis; flowers, animals and birds were present in the household in 71.4 %, smoking in 58 families (52 %). Positive tests to pollen were found in all 105 children, positivity to hair in 15 (20.5 %), feathers in 50 (68 %) and dust in 63 (86.3 %). (2) Children with asthma bronchiaie: presence of flowers, animals and birds in 45.2 %, smoking in the family in 61.6 %, positivity to pollen in 44 children (60.3 %), hair in 15 (20.5 %), feathers in 50 (68.5 %), dust in 63 (86.3 %) (3) 56 children with the dermorespiratory syndrome: presence of flowers, animals and birds in 83.9 %, smoking in the family in 76.8 %, positivity to pollen in 39.3 %, hair in 14.3 %, feathers 75 % and dust 85.7 %. These risk factors play an important role in the pathogenesis of allergic diseases of children.

Effect o / S o t ~ Stress om Aummdy FormslJom in Fow/

T~axovA K., HAtm'L A., PE'mOVSKY E., BENDA V. OFG/-/Z ~ A V, Prague

The effect of social stress on antibody formation was examined in white leghorn (WL) and Rhode Island (RI) chickens. The chickens were divided into three groups. (1) WL and RI chickens were housed

1990 ABSTRACTS 87

in pairs per one cage; (2) WL and (3) RI housed individually. The chickens were immunized with two antigens, sheep erythrocytes and Brucella abortus, intramuscularly. Antibodies were determined before immunization and 5 and 7 days after antigen injection. It was found that WL chickens housed individually produced higher antibody titres; on the other hand, higher antibody titres were obtained in RI chickens housed in pairs. A significant difference in the'intensity of antibody response between both breeds was found which was highly significant in individuals housed in pairs.

Effects ot Deemotrepk ~lmmmes

JlaovA D., NlXOLOV ̂2 , Fns~J~ S., TEISn~OER J. /HE, Prague

When evaluating the irritating effect of substances contacting the human skin, it is recommended to use the screening determination of the in vitro cytotoxic effect in the tissue culture of human peripheral iymphocytes and rabbit splenocytes. Changes of cell proliferation, functional activity and enzymic changes are recorded.

Loaf.Term Follow-up ot Workers F_~msed to the Risk of Asbestods. !. Clumps ot ~ lmmmtity

PROCHAZKOVA J., HANOVCOVA I., KOeECI~ O., MARE~ D., KR~SF~ J., OPRAVn.OV,~ H., S ~ V., KuBtKovA K., Mush. M. Department of Medical Immunology and RL for Immunotoxicology, KttS Hradec K~lov~

Exposure to asbestosis belongs to important carcinogenic risks. We examined workers with direct exposure and retired workers without symptoms of the disease. The results were compared with the laboratory control and with results obtained in workers without asbestosis risk. Statistical analysis revealed significant differences in the risk group: decrease absolute and relative number of E-rosettes, spontaneous and stimulated BIT values, PMNL chemotaxis, bactericidal activity of the blood. On the other hand, increased stimulated INT reduction and phagocytosis of yeasts were found. T-Lymphocyte subpopulations were determined in 28 subjects with a long-lasting exposure: the CD4/CD8 index was decreased in 9 patients. The results indicate repeated stimulation of the immune system which may result in disorders o~f the immunity balance.

Long.Term FoUow-~p of Workers Erpesed to the iibl~ ot Asbestos& IL C h t ~ ot the ltbmmrsl Response

~ o v A V., TURKOVA M., PROCHAZKOVA J., KuaL~ovA K., SPa V., MUSh. M. Department of Medical Immunology and RL for Immunoto3a'cology, KHS Hradec Kr~/ov~

We examined a group of workers professionally exposed to asbetos. We determined Ig serum concentrations, antibodies to DNP, selected acute-phase proteins and CIC. A comparison with the workers of other factories and blood donors was performed. We also evaluated the results obtained from retired workers suffering from asbestosis, retired workers without symptoms of asbestosis and compared with a corresponding control group. The asbestos-exposed workers had a significant increase of IgG values, increased orosomucoid, C3 and ceruloplasmin. A characteristic finding in retired workers with asbestosis was significantly enhanced C3, ceruloplasmin, CIC, lysozyme and ct-1-antitrypsin, while retired workers without symptoms had higher celuplasmin and CIC. In addition, higher % of individuals with anti-DNP antibodies was found in the group of retired workers with asbestosis risk.


The EHect of Profess/oJm/Env/ronment on ~ O r / g ~ of P t o l e s s l o ~ Broncb~ Ast/uns

VrlovA V., ~;[MCIKovA B., OHNUTKovA E., LOKAJ J. Clinic of OccupationaI D/seases, Department of Allergology, Department of Immunology, KONZ Brno

The aim of the present study was to establish the effect of the presence of noxious factors in the professional environment on health of workers, the effect of therapy and the state of health after leaving the workplace. During 1975-1980 54 cases of professional asthma bronchialc were diagnosed. Most of the patients were employed in food industry (hypersensitivity to flour) and chemical industry (production of foam urethane) and suffered from an irritating form of asthma. In 1987, control examination of the whole group was performed: internal and allergological examinations, antibody immunity and lung function determination. Our results showed that exclusion of the effect of professional noxious factors did not prevent progression of hypersensitivity in patients being in remission. It is necessary to exclude the patients as soon as possible after occurrence of initial bronchospasms. As regards preventive measures, it is important to employ individuals without anamnestic allergosis, inflammatory diseases of the upper respiratory airways in the above-mentioned risk occupations and to treat individuals with positive family anamnesis, and to perform appropriate adaptations in production technology.

Exsmintion of lmmmdaty und Aller~ in Workers n d Apprentices involved in Cotton Treatment

JARO• F., ZAVAZAL V., TOMgiKOVA A., J~ENSr, A Z. Department of Occupational Medicine, OUNZ Trendin ; Department of Immunology, FIN P/zed, V(_]PL, Bratislava

We examined the influence of the professional environment in a cotton factory on the occurrence of nonspecific respiratory tract diseases and parameters of immunity. Bacteriological and mycological examination of air, cotton and sputum of workers was performed. Antibody against micromycetae was determined in sera. Chronic bronchitis was found in 78 (27.2 %) and bronchial asthma in 3 (1.1%) workers. A significant increase (p< 0.05) of IgE, AIAT, IgM and orosomucoid and decrease of C3 (p < 0.05) was found. In addition, significantly increased antibody titres against A. fumigatus and versicolor were recorded (p < 0.01).

Cbsnges of Some Immunity Pmm~ters in Workers F_~l~Oyed in Granulated Fodder Produc@on

CAP J., VRLiK M., SOMORA D., ZAHRADN~' V., M~zo D. Department of Medical Immunology and Clinic of Occupational Medicine, FNsP Martin

In 1987 and 1988 we examined repeatedly 30 workers employed in granulated fodder production, mean age 47.5 years, duration of exposure 5.7-3.6 years. One-third of the workers suffered from recurrent respiratory tract diseases. Positive skin tests to the following antigens were found : fowl mixture - 34.8 %, sheep - 39.1%, cattle - 13.0 % and professional dust - 60 %. Increased IgG, C3 and IgE (in 23.3 %) were found. Our observations support the view that work in the granulated fodder production affects the immune system of employees.

Working Conditiom sad l ~ r i ~ of Workers in Fodder ~ Production in tl~ Cenlrsl Bohe~u Re,ton

ZNOJEMSKA S., JANECKOVA V., WAGNEROVA M., DOUSOVA L., MAgovA L., ~;VAbZ)OVA E. KHS SKNV, II~, Prague

We performed a complex examination of the professional environment and the state of health of 278 workers in 16 fodder mixtures production plants. The dust concentration exceeded 5 - 7 x NPK, the microclimate did not differ from the outer one. Concentration of pathogenic fungi was 500-5 000

1990 A BSTRACTS 89

colonies per m a. The ventilation capacity index was found to be 16.2, i.e. by 60 % lower than in the same workers 10 years ago. As regards 12 parameters of humoral and cellular immunity, significant differences in ct-2-macroglobulin, ceruloplasmin and IgE concentrations were found in comparison with the controls. Smokers had lower IgG nad IgA. 6.5 % of subjects had lgE levels over 580 IU/mL. lmmunoskintest positivity was increasing together with the duration of exposure. Our results may serve as a basis for improvement of the professional environment.

Loug-Term FoBow-up of immunologic~ Panuneters ot Workers lm, oh, ed in the Produc~n of.~vomh"

JAHNOVA E., JANOTA S., VARGOVA M., KELEOVA A., KOHANOVA M., FLAgKAROVA E., NYULASSY ~. V~JLB, UNZ Bratislava ; VOPL, Bratislava

We examined cellular and humorai immunity in 45 workers exposed to mankozeb during the production of Novozir. We found a significant increase of IgG, IgE and o-2-macroglobulin levels. On the basis of our previous examination of the workplace, preventive measures were taken in order to decrease exposure to dust. The examination was repeated after 2 years ; in addition, the above parameters we also examined phagocytosis, lymphocyte subpopulations, detailed studies of LIT and the effects of Novozir on the immune system were performed using various in vitro systems. The IgG and IgE levels decreased considerably but IgE levels were still higher than in the controls. Significantly higher immunoglobulin production in vitro was found in exposed individuals. Immunological examination and monitoring appears to be a suitable method for determination of potential toxic effects of industrial noxious substances.

Dy, mmics ot C~m~es of the lmmm~ Response in Workers Exposed to Styrene

HALVOVA Z., ZNOJEMSKA S., STL~ Z., PALEK V. OKI, ZUNZ of Uranium Industry Ph'bram ; KI-IS SKNV, Prague

A group of workers exposed to styrene during fiberglass production has been followed for 3 years. Styrene is a potential carcinogen ; a retrospective study has shown a three-fold increase of incidence of tumours in exposed individuals. During the 3-year period we recorded decreased skin test responsiveness, E-rosettes, phagocytosis and secretory IgA and increased serum IgA levels. The lowest values of skin tests, E-rosettes, serum and secretory lgA were found in workers who had been exposed to styrene for up to one year. The association between the immune suppression at the beginning of exposure with subsequent adaptation or occurrence of delayed effect is discucced.

Immune Response in Medical Workers Arbnlnktering Cytostat~

~KOVA V., HANOVCOVA I., TURKOVA M., KOPECKY O., MAR~C"EK D., PROCHAZKOVA J., CERMA~ovA A., SRB V., KUB|KOV.~ K. Department of Medical Immunology, RL for Immunotoxicology, KHS Hradec KrMov~

Cellular and humoral immunity was examined in 70 medical workers administering cytostatics. In addition to hematological examination we determined the number and functional activity of T lymphocytes (E-rosettes, blastic transformation, lymphocyte subpopulations CD4 and CD8). Phagocytosis of leukocytes was determined by INT reduction, uptake of S. cerevisiae, bactericidal activity against E. coli strain $346/40, chemotaxis and chemiluminescence. Basic Ig classes, selected acute phase proteins, lysozyme, ~2-microglobulin (132M) and CIC were determined in serum. In addition, 132M and lysozyme were determined in urine. In the departments of radiology, tuberculosis and respiratory diseases and gynecology (with higher exposure) significantly higher numbers of lymphocytes were found. Enhanced phagocytosis of S. cerevisiae and bactericidal activity of blood was found in employees of the radiological clinic: 27 % of the examined individuals had a decreased CD4 :CD8 ratio. Humoral tests showed an increase of ct-2-macroglobulin and ceruloplasmin in the whole risk group.


Outages o//-/aaaa~ l ~ a / ~ , and O~ogeaelk ~ / a Workm ot G~m/od/sdumy

PETRAgOVA D., K~'nv^ M., KALrSA I., KotL~ J., K ~ A H., OmmUgEKOVA A., TOrnovA E. Institute of Experimental Meal/c/he, LF UPJ;~, KoffJce; Department of Medical Biology, 2nd Clinic of Internal D/senses, FNsP Kog/ce

The authors examined 30 workers of the chemical factory CHEMKO Stni~sk6 exposed professionally to various chemical substances (aldehydes, triarylphosphates, benzene etc.). We performed dynamic examinations of immunological (IgG, IgM, IgD, Prea, Trf, ORM, cttAt, o.2M, C3, (?4 complement components, CPL, CIC, CSI) and cytogenetic (frequency of chromosome aberrations in peripheral lymphocytes) parameters. We found changes of serum IgA and ORM and high variability of CIC and increased values of the carcinogenic index. These results correlated with the results of cytogenetic analysis of peripheral lympbocytes. The decrease of ascorbic acid level was significant.

The Effect ol Metal Waste on the Imm, me Respome

REICHRTOVA E., TAgAC V., HoRvA~JovA J., PA~.~tKovA 0., ORAVEC C., VESELA A. OF, BE SA V, Bratislava ; ILF, Bratislava ; OEO SA V, Bratisiava ; PF IrK, Bratisiava

We examined the response of the organism to inhaling metal waste in a nickel forge in the P and F1 generation of rabbits. We found increased accumulation of Ni and Cr in the lungs, liver, myocard, muscles and kidney. Using fluorescence we detected the presence of tissue immunocomplexes in the myocard and lungs in the P generation and increased level of circulating immunocomplexes in the serum of the F1 generation. The tumoricidal activity of the blood serum increased in the P generation by 8.2 % and in the F1 generation by 14 %. Following inhalation exposure to metal aerosol in the chamber (50 mg/m 3) the presence of particles in the myocard together with ultrastructural changes were found in Wistar rats. Rejection of the aliograft was delayed after an i.v. injection of particles to rats.

Liver Dm~ge and Regenemdon alter Repeated Tetmchlotometlume Administmlion in l~ts

KANTA J., CHLUMSKA A., KVAS~KOVA E., BARTOg F. Department of Physiology, LF UK, Hradec Kr~lov~

Female Wistar rats received a low dose of CCI, (125 ~L/kg, i.g.) in olive oil, the controls received the oil only. After 20 days, all animals were given CCL, (125 ttL/kg). Liver damage was examined after the second CC'h dose on the basis of serum levels of transaminases ALT and AST, regeneration was evaluated according to 3H-thymidine incorporation into the liver DNA and according to the mitotic index of hepatocytes. It was established that the liver damage was the same in control and experimental animals but regeneration was by 40 % lower in CCh-pretreated animals. We assume that this phenomenon facilitates proliferation of connective tissue and contributes to the origin of liver cirrhosis.

C~umg~ ot ! ~ Re~'tit#y in ~ 360 l~ys alter Whole-Body lnsdbtion

VA~ovA J., ] ~ - ~ r P. VL VD(] JEP, Hradec Kr~iov~

We examined the long-term effect of ionizing radiation on hematopoiesis and immunological reactivity in mice. It was shown that although after the whole-body irradiation with a sublethal dose of 7 Gy a complete restoration of hematopoietic cells of the bone marrow (CFU-S) occurred within 30 days, residual disturbances and a profound decrease of C'FU-S occurred after 360 d. Similarly, the immunological reactivity of 7 Gy-irradiate mice was decreased 360 d after irradiation. The titre of antibodies against sheep erythrocytes decreased by 50 % despite the fact that the number of nucleated cells in the spleen of irradiated animals was the same as in control animals. No differences in numbers of PFC in the spleen and

1990 ABSTRACTS 91

in antibody titres between young (60 days) and old (420 days) non-irradiated mice could be detected. However, the CFU-S number in the bone marrow was significantly lower in old than in young mice.

/mmmm/ogkd Parameters of Wor~ers/n Ant/o~4ut Prodsct/on

BARLOC~VA S., HuRBANKOVA M., VESELA O., BAUMANOVA E., KUaOVA J. V(.IPL, Bratislava

A two-year immunological study of workers in the production of antioxidant CD is presented. We examined 12 parameters of nonspecific humorai and cellular immunity in 89 workers exposed during a one-year period. It was found that chemical noxious agents modulated the immune response. The changes of immunological reactivity were mostly of immunostimulatory character (increased % of active T lymphocytes, nucleolar activity of lymphocyte.s, lysozyme level in saliva, etc.). However, decreased percentage of total lymphocytes and suppressed PMN phagocytotic activity was observed at the same time. The increased proportion of theophylline-sensitive T lymphocytes (< 10 %) and increased IgE levels correlated in most cases with the occurrence of contact hypersensitivity in the anamnesis. A complex assessment of the influence of professional factors in antioxidant CD production on the immune system requires a long-term follow-up of the immunological reactivity and confrontation of the results with morbidity.

O m ~ s o! lmmuologtc~ Pmmeters im WoOers Pmlessiomny ~posed to Styrene u d Metl~y;. ~ t lm~y~ te

HANZL J., KAmCXOVA M., KLEMES'rovA H. KHS Brno ; OHS Jihlava

We followed 28 workers exposed to styrene-methylmethacrylate, age 37.6 + 9.44 years, exposure 5.5 + 3.9 years. The concentration varied around the NPR-P values, only exceptionally were higher. We found significantly decreased lysoz3,rne levels, low a- 1-antitrypsin, (24 complement component and other multiple aberrations of the immune profile. In the group of exposed individuals, 46.42 % of workers had three and more abnormalities (the control 11%).~A high incidence of abnormal Ig levels (IgG - 46.42 %, IgM - 64.26 %), CPL (71.4 %) and AIAT (24.99 %) was also found. The results of these examinations correlated with cytogenetic analysis of peripheral lymphocytes (5 % ABb).

Effects of the Mode/sml Nstms/Stress on Sdeaed/mmmm/og/cs/Parsmeters in Stress.Ssseeptib/e snd S t r e s s - R ~ t Pigs

ROZKOgNY V., ROZACO~NA A., GRANATOVA M. VEWL, Brno

A model stress of genetically defined, stress-susceptible pigs (breed Belgian Landrace - BL) induced a decrease ed peripheral blood leukocyte phagocytotic activity by approximately 45.94 % while in the stress-resistant pigs (breed Durok) by 19.7 % only. Using the mitogen-induced blast transformation of peripheral lymphocytes test, activity in BL decreased by 27.65 %, in Durok pigs by 12.15 % only. Similar results were obtained when natural stress was used.

C l ~ q ~ el l ~ r in B~ery Workers

G u ~ M., BEI~ovA K., ~OROVSK~' M. Laboratory of Immunology and Allergoiogy, 4th Clinic of Internal Diseases and Clinic of Occupational Diseases, FNsP Ko~ice

The authors examined 109 probands working in a bakery and a sweetshop; 62 workers were exposed to dust, 47 were not exposed. In the group of exposed probands, 30 % reported subjective symptoms. Using


a battery of skin tests (inhalatory, bacterial, professional and fungal allergens) a 36 % positivity was found in unexposed, 83 % in exposed workers without symptoms (n = 42) and 95 % in 20 exposed workers with symptoms. No differences in IgG levels were found between unexposed and exposed without symptoms (12.31 and 12.09 g/L respectively). In exposed individuals with symptoms the values were higher (14.35 g/L). IgE levels were comparable in all groups. Mean values of 93 KU/L were found in unexposed, 127 KU/L in exposed without symptoms and 347 KU/L in exposed with symptoms. Differences in the distribution of C3, cry-AT and IgM were also recorded. The percentage of clinically ill individuals increased together with the duration and degree of exposure to dust.

Importance ol Lysozymurla in Clinical and Preventive Medi~ne

RICHTER J., KRAL V., JiLEK D., Tio-r~ P. Department of Immunology, KHS Osti n.L.

On the basis of our previous research of lysozyme, we have concentrated on the occurrence of lysozymuria for many reasons. We present examples of lysozyme excretion following exposure to heavy metals, ionizing radiation, organic compounds etc. We described the presence of lysozymuria in top-performing sportsmen, depending on the degree of stress. In addition, a number of examples of lysozymuria in various diseases (uremia, leukemia, tumours) were observed.

Protessional Risk ol Carcinogens

KUKLOVA D., BALCAROVA O., DANDOVA S., ZAVAZAL V. Department of Occupational Hygiene and Occupational Diseases, LFH UK, Prague; Group of Occupational Hygiene and Occupational Diseases, ILF, Prague; Department of Hematology, FN Prague 10; OKI, FN Plze~

We summarized the results of a study concerning the risk of professional exposure to substances with cancerogenic and immunoregulatory effect in research workers working with cytostatics (A) and in the production of Ni-Cd accumulators (B). We examined the working conditions and performed a repeated laboratory examination including selected immunological parameters. Group A included 53 exposed workers with high occurrence of allergic diseases (49 %) and leukopenia (24.5 %). Group B included 18 females with inflammatory elements in urine (30 %) and [~-2-microglobulin in urine (15 %). Examina- tion of selected immunological parameters revealed a number of abnormalities in both groups, some of them being statistically significant.

]be Effect of Some Hygienically Topical Elements (Cr, Ni, Se ) on Immunity

P ~ R J., P ~ R I. Department of Immunology, KHS (]sti n.L. ; Presidium of C~SA V, Prague

We summarized immunological findings (decrease of phagocytosis, antibody response, increased sensitization, etc.) obtained in patients with professional exposure to Ni and Cr. Increased emission of these metals requires to extend the studies of their noxious effect even in professionally unexposed population. Adverse effects of Se deprivation are also discussed. Deprivation of Se, possessing antioxidant and detoxicating activities, induces a number of disorders including defects of immunity. Increased occurrence of tumours was described in individuals with Se concentrations less than 45 Ixg/L. The concentration 85 I~g/L is considered to be a satisfactory protective serum level. Vitamin E can functionally and immunologically compensate for Se deprivation. Se deficiency results primarily in decreased cell-mediated immunity. In some countries, supplementation of fodder and fortification of food componetns is done in regions with Se deficiency. According to trials performed in this laboratory, Czechoslovakia belongs to regions poor in Se.

1990 ABSTRACTS 93

Pass/re Smoking anti Immunity

RIo-rrF_a J., PFEU~R I. Department of Immunology, KHS Usti n.L. ; Presidium of C~SA V, Prague

While there is no doubt about the adverse effects of active smoking, passive smoking has long been considered to be only unpleasant. However, recent epidemiological studies and chemical analysis proved a health risk. A number of noxious substances (CO, polyeyclic aromatic hydrocarbons, nitrosoamines etc.) occur in the side smoke stream at much higher concentrations than in the main stream. Their effect is particularly significant in closed spaces (workplace, means of transportation, mainly passenger cars). Increased infant morbidity (mainly up to the first year of life) was described in families of smokers. Asthma-type conditions and increased risk of malignancy (tumours of lungs, ovaries, brain and esophagus) were described in passive smokers. The risk of professional sensitization occurs in the following order: smoker-passive smoker-nonsmoker. As regards immunological parameters, reduction of immunoglobulin titres, complement, T cell function, induced by both direct and indirect (decreased ascorbic acid level) mechanisms were described.

Contribution ot Immunology to Monitoring the Effect of Extenml Environment on Population Health

RICHTER J., KRAL V., KASAL J., PFEIFER I., J|LEK D., PAROHA L. Department of Immunology, KHS Usti n.L. ; Presidium of C~A V, Prague ; Institute for Environment, Usti n.L.

Monitoring of adverse effects of complex environmental factors on the health of the population by immunological methods has begun in the seventies. Various methods for the detection of changes of the immune response have been gradually completed with respect to the possibility of massive use and diagnostic validity. In the first place, analysis of proteins was employed as a parameter of changes of the systemic and local immune response. In the present study we concentrated on the use of wide-scale tuberculin testing in order to obtain data about the cellular responsiveness in the population of North Bohemian Region.

Therapeutic Apheresis As ~ Component of lmmnnotherapy

FIXA B., KOMARKOVA O. 2nd Clinic of Internal D/senses, FN KUNZ Hradec Kr~lov~

Therapeutic apheresis serves the removal of harmful substances from blood. The most frequently used methods include plasmapberesis (plasma exchange), sorption techniques and cascade filtration. From the immunological point of view, a number of substances (e.g. immunoglobulins including autoantibodies, complement components and circulating immune complexes) are removed during apheresis. This determines the therapeutical indication of apheresis. Beneficial effects were described mainly in Waldenstr6m's macroglobulinemia, Goodpasture syndrome, systemic lupus erytbematosus, myastbenia gravis, polymyositis, dermatomyositis, acute polyradiculoneufitis Guillain-Barr6, amyotrophic lateral sclerosis and a number of other diseases. Apheretic methods are mostly combined with immunosuppres- sire therapy. The mechanisms of immunotberapeutic procedures are complex and unclear so far. Besides removal of potentially harmful substances, such a therapy represents a serious impact on cellular immunity, phagocytosis etc. We present changes of basic immunological parameters in sera under the influence of plasma exchange and bemoperfusion, based on our preliminary experience.


The Reslmme of Lymidmtic ~ to FrKtiomte ln~thtioa and Relocated Bone Mm'tow Tnmspi~ts. tioa

Ho~r_a M., TKADLF.~X L., VIKUCKA ~;., ~ Z. Institute of Biophysics, CSA V, Brno

Mice were irradiated with fractionated doses of y-rays (3 Gy) in 4-day intervals up to the total dose of 24 Gy. Haft of the mice were injected with 106 of bone marrow cells after each fraction dose. The number of lymphocytes in peripheral blood, thymus weight, density of lymphocytes in sections of the thymus cortex, mass of the spleen white pulp, number of lymphocytes in the spleen and bone marrow of the femur were examined. In addition, the morphology of lymphatic tissues including their ultrastructure was studied. Samples were obtained 4 days after the last irradiation; after receiving total doses of 18, 21 and 24 Gy, new experimental groups of animals were formed that were allowed a longer (9 days) interval for regeneration after the last irradiation. Repeated transplantation had a significant positive effect on lymphatic tissues up to the 24th day of experiment. Particularly in mice with the 9-d regeneration interval after last irradiation, a significant difference in individual parameters of lymphatic tissue was observed in recipients as compared with repeatedly irradiated animals.

Pa~mt loa and Cbnl~teri~tioa oi Moneelonel A a ~ for ~ t m t i o m Tmatmemt ot the Bone l~4~rrow

HRUaA A., KOUUEK K., SOU(~.K J. UHKT, Prague

Cell populations involved in the graft-versus-host reaction can be inactivated by means of monoclonai antibodies. Using indirect immunofluorescence, cytotoxiclty test, MLC reaction and formation of hematopoietic CFU-GM colonies we examined monoclonal antibodies against membrane antigens of human lymphocytes that were constructed in this institute together with some other antibodies. Antibody MEM 57 CSAV iulfibited the MLC reaction after a short incubation in the presence of complement. Our antibody MG-7, directed against the C3d receptor, inhibited MLC in the absence of complement. Formation of hematopoietic colonies was not inhibited under similar conditions.

Ptq~mtion B d ~ ot LAK OIIs

I ) ~ , ~ v A V., Poatmsr~ S., Buc M., ST~ANOVIr J. Department of Microbiology and Immunology, LF UK, Bratislava

Human peripheral blood lymphocytes (PBL) were incubated in vitro in the presence of IL-2 without an exogenous antigenic or mitogenic stimulus and the formation of LAK cells was examined. IL-2-containing supernatants were obtained by stimulating human PBL, lines JURKAT and MLA 144. The specificity of lysis was tested in a nonseparated population of cultivated LAK cells and in isolated clones. The LAK activity was tested in autologous and allogenei~: PBL, allogeneic and xenogeneic turnout cells, including the NK-sensitive line K 562 and NK-resistant line 1"-24. LAK lyzed neither autologous nor allogeneic PBL, various tumour cell lines were lyzed rapidly while xanogeneic cells did so weakly or not at all. The activity of cloned LAK cells was lower or similar.

Effects o1 Recomb1~mt Lymp~Idnes u d Vmccine ~ on NK.Cdl Act1~y

Fi~EaovA A., Pc~pl~n. M., HUAN N., ~ I., KuaaYCm J. Department of Immunology, MB(_I t~SA V, Prague ; VUE, Prague

The aim of our study was the preparation of LAK cells and examination of the combined effect of the vaccine Coparvax (Cotynebacterium parvum with rIL-2 activity) on the in vitro activity of NK-cells. In

1990 ABSTRACTS 9w

patients with malignant pleural erddate, the NK activity became normal 21 days after the vaccine administration, and after an in v/tro cultivation of lymphocytes in the presence of rIL-2 it increased more than twice. Our model of obtaining activated cytotoxic cells (LAK) is based on the addition of molecules released by EDTA-supplemented medium from the surface of lymphocytes stimulated with rlL-2 and rIFN-o.2. These molecules not only enhanced the NK-cell activity, but also the number of E-rosetting lympbocytes, particularly when added in the autologous system. After separating the EDTA-released molecules by HPLA, no correlation between the cytotoxic activity and the number of E-rosettes could be demonstrated in the active fraction. The nature of molecules inducing enhanced E-rosette formation and involvement of the E-receptor in the recognition by NK-cells are under study.

Possible Therapeutic Use of Monoeloml Antibody MEM-57 ( C'D3)

HILGERT I., FRAN~ F., KAgLIK J., HOLUB M., KORCAKOVA L., IOJ~OFOVA H., Ho~.Jgt V. UMG CSA V, Prague;/KEM, Prague

We compared the properties of monoclonai antibodies (MoAb) MEM-57 and MoAb OKT3 which are commercionally produced for therapeutic p ~ s . Both MoAb are of the IgG2a isotype, bind near to the epitopes of the T-lymphocyte antigen CD3, the pl value of MoAb MEM-57 is 7.6-6.8, that of OKT3 is 6.6-6.8. Both MoAb possess a comparable mitogenic effect on human lymphocytes (the stimulatory index at 1 ng/mL is 19 for MEM-57 and 20 for OKT3). Neither MoAb reacts with lymphocytes of the baboon and the rhesus monkey but both bind to gorilla lymphocytes. The MoAb MEM-57 preparation, purified by Rivanol, diluted in phosphate buffer to an Ig concentration of I mg/mL and stabilized with Tween 80, was called A1-CD3. When injected i.v. to a gorilla, significant lymphocyte depletion occurred in the blood: 1 h after injection their nflmber decreased from I 312/ttL to 338/p.L. As both MoAb possess similar biological properties both in vivo and in vitro, one may assume that the A1-CS3 preparation might replace commercial MoAb OKT3 for therapeutic purposes in organ transplantation.

P/asma Prote/n ~anges during Therapeu6e P/asmaphere~ (TP)

OPAa-Rbri' K., JR., OPATRNY K., ~ V., KOVARmOVA P., CERHovA M., RUS~AZOVA H. Ist Clinic of Internal Diseases, LF UK, P/zer~ ; OKB Sokolov ; OKB Plzed

The authors performed 23 TP in 13 patients using the filtration method in the A 2008 PP (Fresenius) and filters Plasmaflux P 2 (the same company). The following plasma proteins were examined: IgG, IgA, IgM, IgD, transferrin, prealbumin, C3, C4, ct-1-antitrypsin, orosomucoid, C-reactive protein, ct-2-macroglobulin, haptoglobin, ceruloplasmin and 13-2-microglobulin. The blood samples were obtained before TP, immediately after TP, after 24 and in some patients after 48 and 72 h. The blood samples were withdrawn from the inlet and outlet tubing of the filter, the filtrate sample was obtained 15 and 90 rain after initiation of TP. Our measurements made it possible to evaluate not only changes of plasma protein concentrations during TP but also according to "sieving" coefficients and clearance at various stages of TP.

Treatment of Pure Erythro~iesis A p l a ~ with Antithymocyte Globulin

gVOJGROVA M., g t ~ r r o v A J., P A N 2 ~ P., C ~ A K V. Ist Clinic of Internal Diseases and OKIA, KUNz, FN Pized

The case of a 59-year-old female patient with lymphocytic lymphoma of the B type and pure aplasia of erythropoiesis of autoimmune etiology is discussed. Combined r therapy, immunosuppression with cyclophosphamide, Prednison or Azathioprin did not lead to clinical improvement. Treatment with antithymoofle globulin (Atgam, Upjohn) induced a complete restitution of erythropoiesis persisting for over 1 year. A temporary decrease of the relative number of T lympbocytes (OKT-4 and OKT-8) occurred during ATG therapy. ATG therapy had noeffect on humoral immunity parametes.


lmmunopharmacological and Physical Methods for/nBuenchtg/mmun/ty/n Asthma

7~V~AL V. OKIA, KONZ, FN Plze~

The complexity of mechanisms and causes involved in the origin of asthma results in diagnostic and therapeutic confusion. Therefore, classical hyposensitization together with symptomatic therapy usually cannot influence all processes involved in this disease. Thus, other means, e.g. physical, immunological, prophylactic and pharmacological, must be used to cope with the disease. Allergen elimination can be achieved by climatic and spa therapy, by changing the environment or placing the patient in the allergen-free units in hospitals. The frequently occurring immunity disorders as well as the associated inflammatory processes should be normalized at an early stage of therapy. Beneficial effects were obtained with Zaditen or other immunomodulators. Administration of intravenous immunogiobulin G positively influences IgE regulation and provides the missing IgG subclasses. Immunosorption and new preparations depressing bronchial reactivity are also promising.

lmmunomodulatory Therapy of Children. L The Effect of Transfer Factor and Levamisole on the Clinical Condition

~IMONEK I., KM~.K A., PENNIGEROVA S., FUOKOVA T. 3rd Pediatric Clinic, OKI, FV-L IlK, Prague

We treated two groups of children with clinical symptoms of immune deficiency (recurrent respiratory infections, mainly recurrent bronchitis). One group was treated with the transfer factor (I~ISOL) according to the recommended schedule, the second group with levamisole (Decaris) in commonly used doses. The effect of therapy was evaluated by comparing the frequency of symptoms before and after therapy. We found that both preparations improved the clinical condition of patients and decreased the frequency and severity of recurrent infections.

Immunomodulatory Therapy M Ch//dren. IL Effects of Transfer Factor and Levamisole on Cell. Med/ated and HumorM lrmmlulJty

~ : A., g~n~'EK I , FuL"IKovA T., HAUS~R P., BARTOI~KOVA J., KRYb~OFKOVA O., JANATKOvA 1. 3rd Pediatric Clinic, OKI, FVL UK, Prague

We treated two groups of children with clinical symptoms of immunodeficiency with a confirmed defect of immunity by laboratory examination. One group was treated with the transfer factor (I~SOL), the second group with levamisole (Decaris). We examined T. and Ts lymphocytes, B lymphocytes, IgG, IgA, IgM, C3 complement component and CIC before and after therapy. The immunomodulatory therapy with transfer factor and levamisole led to a slight improvement of cellular immunity parameters, mainly to normalization of T lymphocytes. No changes of humoral immunity were observed.

/mmunophamacogenet/r Study of TheophylUne

~Au~ovA E., NYULASSY $., GAZDiK F., BENCOVA M, JANOTA S.) CHREI~IOVA S., KELEOVA A., JAHNOVA E. ZLIP VULB ; UNZ Bratislava

Selected immunological parameters were examined in 24 theophyiline-treated patients before and after a 2-month therapy, and association with MHC was compared. We found the following significant associations: decrease of total T-y with B18 (p < 0.05) and with B12 (p < 0.05), increase of total T-7 with B40 (p < 0.05), increase of C~ complement component with A19 (p < 0.05). Our results confirmed the assumption that MHC genes may influence the immune response of theophylline-treated patients.

1990 ABSTRACTS 97

/ ~ ~ e l Aeydevir

VA~"UnOVA M., Jlt.mr P., Fm's'reat~ V., PnozttAzKovA J., St.A~FJc T. FaF UK, RL for Immanotoxicology, Hradec Kr~iov~ ; Lachema, Bmo

We compared the effect of acyclovir Lachema with acyclovir Welicome on the immune system. Acyclovir was administered to CBA/0 la mice (50 mg/kg s.c. 2 x a day for 5 days). We examined the body weight, weight of the spleen and other organs, number of peripheral blood leukocytes, differential leukocyte count, blast transformation of spleen cells, number of plaque-forming cells in the spleen and delayed-type hypersensitivity. Using an in vitro system the effect of acyclovir (0.01-10 ttmol/L) on the viability, number and antibody response of routine spleen cells was studied in tissue culture. Finally, the influence of acyclovir on an in vitro blastic transformation of human peripheral mononuclear cells was established. The results have shown that both substances were comparable and at concentrations used did not influence significantly the above parameters.

Co/ns~t/on at t he /n t e s t~ /Trac t w/t~ s N o ~ l m ~ c E. co/i S t n ~ ss s P m v ~ ot Gmtm- intestiml ~ ot Pro-Term sad Fd.Term N e . ~

~ADNIKOvA-LODINOVA R., KORYCH B., BARTAKOVA Z. UPMD , Prague; Institute of IV~crobioiogy , LF UK, Prague; Nursery Plzed

The E. coil strain 083 is nonpathogenic and possesses suitable antigenic and genetic properties. Its extraordinary ability to persist for a long period of time in the intestional tract was utilized in clinical practice for preventing alimentary infection of patients at risk. We colonized 1 112 full-term and 129 pre-term infants perorally with a suspension of living E. coli 083 (3 x a week, 1 mL, l0 s of bacteria). The presence of E. co//083 in the intestine prevented the invasion of pathogens and significantly reduced the number of already present pathogenic bacteria. Colonization also induced a complex local and systemic immune response.

B~'qcovA M., NYULASSY ~., ~^u~avA E., JANOTA S., JAHt~VA E., I(m ~ovA A. 711P VOLB ; UNZ Bratislava

We examined a group of 27 patients with chronic recurrent pyelonephritls before and after a 6-month antimicrobial treatment with M-H. We examined HLA-A,B,C, changes in 33 immunological parameters and immunogenetic associations. The following significant associations were found: decreased C4 complement component in AI-negative patients (p < 0.05), increased CA in Al-positive individuals (p < 0.05), increased absolute T lymphocyte number associated with B14 (p < 0.005), increased T-~/ lymphocytes with B13 (p < 0.005), increased LGL number with B17 (p < 0.005), decreased lgA level with 138 (p < 0.05). The results support the hypothesis that genetic factors of the major histocompatibility system (HLA) might influence some parameters of the immune response in M-H-treated patients. This idea is based on the fact that genes regulating CA and IgA levels as well as the IS (immune suppression) genes are localized in the HLA supergene region on the 6th chromosome of the human genome.

s at Aseerbk A d d Pml~ylKffe Adm/a/stmffem om S d e e t d / a m w m / e l t ~ Parameters sml on tlm F ~ oi Oumm3somsl A ~ m tn ~ Ezpowd to Bl~k Cosl Tst

MACHALEK J., DOBIA~ L., ~ N I. Department of Immunology and Allergology, KUNZ-KI-IS Ostrava ; Department of Genetic Toxicolo- gy, KHS Ostrava

We examined the effect of prophylactic ascorbic acid (AA) administration on selected parameters of immunity: serum IgG, IgA, IgM, ct-l-antitrypsin, prealbumin, orosomucoid, transferrin, ct-2-macro-


globulin, C-reactive protein, rheumatoid factor and cancer serum index (CSI). Simultaneously, the antimutagenic effect of AA was examined by evaluating the chromosomal abberation frequency (AB.B) by cytogenetic analysis of peripheral lymphocytes and by a short-term mutagenicity test according to Ames. After a 3-month administration of 1 g of AA we found increased preaibumin concentration from 0.37 :t: 0.08 g/L to 0.,t8 + 0.08 g/L (P < 0.01), decrease of CSI from 2.28 + 0.88 to 1.76 + 0.50 (P < 0.01) and decrease of ct-2-macroglobulin from 3.40 + 0.95 g/L to 2.06 + 0.39 g/L. The frequency of AB.B decreased significantly from 5.07 % (+ 0.52) to 1.77 % (+ 0.21). These results confirmed the beneficial effect of AA on the above parameters in exposed individuals.

-Experience w/th A/ded~ ht OWMreu amf A d d s

~;Pxd:AK V., ZAVAZAL V. Department of Pedistrics, FN Prague-Bulovka ; Department of Clinical Immunology, FN Plzed

We describe our experience with inhalatory Aldecin in pediatric and adult allergic patients suffering from poilinosis, permanent rhinitis and asthma. After a 2 - 3-months treatment (dose 50 mg 4 • a day) the course of disease was evaluated by means of nasal symptoms score (secretion, abstruction, burning, sneezing) and by asthma symptoms (number and intensity of attacks, FEVt and drug consumption). The score improved during therapy in all groups by about 50 to 66 %, the starting values (with maximum symptoms) being 87 %. The undesirable side effects (irritation and burning of mucous membranes) were rare.

In Vil~'o Effect o; Lsbets/ol on the Immune System

ZVERKOVA D., NYULASSY ~. VULB, UNZ Bratislava ; Clinical Department gPOFA, Prague

Coreton (VULM) Labetaloi (LB), a j3 + ct blockator is an antihypertensive drug. In a clinical study, we found decreased lymphocyte proliferation in the blast transformation test (LBT) after a 6- and 12-month therapy; increased classical pathway of complement activation (hemolytic activity - CHs0) was found after a 12-month therapy. The aim of the present study was to elucidate these changes in an in v/fro model. We used (1) LBT micromethod with 3H-thymidine incorporation, (2) determination of PMN leukocytes activity after stimulation with Z y ~ n using a Berthold luminometer, (3) CH 50 and AK 50 titration in a microsystem. Labetaioi was toxic in LBT at 100 mg/L ( i = 6 000 vs. 140 000 cpm), 10 mg/L significantly decreased the LBT response (106 000 vs. 129 000); the same concentration inhibited metabolic activation of PMN. LB at wide doses spectrum did not interfere with CH 50 and AK 50. We conclude that LB causes a significant decrease of LBTin hypertonic patients and we assume a stimulatory effect on 'Is lymphocytes. It also affects the metabolism of neutrophils which can partially explain the changes found in treated patients. We do not consider the degree of changes to be clinically immunotoxic.

The F~ect of Ergot ~ on l ~ m e F ~ - t i o ~

JILEK P., VANC~'ROVA M., N'i'VLTOVA M., P~mYLOVA L., PROC~AZKOVA J. FaF UK, RL for Immunotoxicology, Hradec Kr,~lovd

We examined the immunomodulatory activity of certain ergot alkaloids administered for 14 days to mice. Dihydro-a-ergokryptin (aEKP) and dihydro-j3-ergokryptin (bEKP) induced primarily a decrease of spleen cellularity, of antibody response (PFC number in the spleen, serum anti-crythrocyte antibodies) was not influenced. Cell depletion in the spleen was not proportional as suggested by increased PFC numbers and significantly higher mitotic activity (PWM) in tissue cultures of aEKP-treated animals; administration of bEKP induced a significantly increased response to PHA in vitro. Dihydroergocornin

1990 ABSTRACTS

(ECO), dihydroergocristin and dihydroergotoxin were without effect. An interesting finding was a decreased activity of serum lysozyme after bEKP and ECO administration. We conclude that the immunomodulatory potential of ergot alkaloids is relatively low at therapeutic doses; nevertheless, it should be taken into consideration, particularly in geriatrics.

The lnfleeuce of ~xo r~ on the Immune System 0! Patients wli~ Benign l~perbi~sblnen~

KELEOVA A., JANOTA S., HOLOMAI~ J., JAHNOVA E., KOHAUOVA M., FLAgKAmOVA E., NYULASSu ~. V~LB, ONZ Bratislava ; ILF, Bratislava

Zixoryn (m-tdfluoromethyl-a-ethylbenzhydrol) is an inducer of the monooxygenase system in liver cells. We studied its effect on the immune system in patients with Gilbert's disease (11 males) during a 2-month therapy. We recorded a number of changes of immunological parameters such as increase of the relative proportion of active lymphocytes and T? iymphocytes after a 2-month therapy, and decrease of OKT4-positive lymphocytes after a 1-month therapy. Increased phagocytotic activity was found after termination of the therapy. An increased hemolytic activity of complement (activated by the classical pathway) was recorded after a 2-month administration. The results have shown that Zixoryn influenced the distribution of lymphocyte subpopulations, decreased probably the proportion of iymphocytes inducing immunosuppression and increased the number of cytotoxic T lymphocytes. An interesting finding is the increased phagocytosis (ingestion) or its normalization with respect to an assumed mild phagocytosis defect in patients with benign hyperbilirubinemia. The mode of action of Zixoryn on the immune system remains obscure.

lramHolo#e~d Fiud~gs in Dislyzed Pstients Treated w;t~ Rocsltrol

BARTONKOVA J., SUI_KOVA S., HAUSNER P., Pi~, P., FU~KOVA T., PosovA H. OK/, FVL UK, Prague ; Artificial Kidney Unit, Prague-Strahov ; Laboratory of Oncology, FVL UK, Prague

We followed the effect of therapy with Rocaltrol (l,25-dihydroxycholecalciferol, 0.25 ~tg per day) in patients with kidney insufficiency on Ca -P metabolism and immunological parameters of humoral, nonspecifie and specific cellular immunity. Blood sampling was performed before and 2 and 6 weeks after therapy. We found significant changes in all compartments of immunity. The trend of changes was different after a 2-week administration (stimulating effects) from those after 6 weeks (suppressive changes). As regards C a - P metabolism, only a decreased ALP activity was found. A role of vitamin D3 in the regulation of immunity is suggested.

The EHect ot a Single.Dose Adminislmtiou of Tnmslet Fsctor on LympkxTte $ubpopmbtions

Fuc'IKOVA T., PosovA H., HAUSNER P., VLAa-nmSK~" M. OK/, FVL UK, Prague

We administered a single dose of transfer factor (TF) to patients with protracted recurrent infections of upper respiratory airways in an attempt to establish its possible effect on lymphocyte subpopulations in peripheral blood. The patients were examined immediately before 'IF administration and 1 and 3 weeks after "IF. Examination of lymphocyte subpopulatibns was performed by flow cytometry. The foildwing monoclonai antibodies (MoAb) were employed for identification of lymphocyte subpopulations: MEM 57 - total lymphocytes, OKT4 (CD4) - T helper lymphocytes, CD8 - T suppressor iymphocytes (MEM 31), 2H4 (CD4 in CD45R) - suppressor inducers, IgD and IgM (13SOL) - B lymphocytes. We found gradual decrease of B lymphocytes in most patients. Further discrete changes were found in CD4 and CD8 lymphocytes which led to normalization of the originally decreased immunoregulatory index.


Peflen as .Allerl~z~u~ Agent

1 ~ M., KoP.~OVA M., ~'r~.~'KOVA E., B~.HO~OVA L., KOFRO~VA O. Center for AJlergens, ~]SOL, Prague; Laboratory of Ultrastmctural Morphology, MB~ CSA V, Prague

A large proportion of our population suffers from pollen allergy and the number of patients is steadily increasing. Therefore, the requirements for production of poolen allergens in the ~JSOL are also increasing. As compared to 1980, twice as many allergens (Pollinare mixture) were manufactured in 1987 (for 56 515 patients): 11 x more Betula pondula allergen, 3.6x more Artemisia vulgaris etc. Moreover, the spectrum of required allergens has changed considerably during the past years (1980-1987). At present, the demand for pollen allergens of spring woods (mainly birch) and autumn plants (mainly Canada goldenrod) has increased. This trend is probably associated with changes in the reactivity of our population and with the wide distribution of these species in the nature. A new panel of pollen allergens is being prepared in the USOL which will be extended by some new types of pollen allergens, and the exact species specification of further allergens will be determined.

Determ~tion oi Aliefgu Extract Onllty by the ELI.fA Me ted and lmmfmoblottiq- ,.~udmcl~t~m ot A ~ e ~

Dsm~ORADOVA I., TLASKALOVA H., POLACKOVA A., ~PICAK V., VAh~k(OVA Z., POLACKOVA M., PoCr^ L., RINGER M., ~M/DOVA V. ~]SOL, MB~] CSA V, I~F UK, FN Prague-Bulovka

Determination of allergen quality is insufficient; therefore, we attempted to employ some im- munochendcal methods for that purpose. We used the inhibition method in the ELISA test with hyperimmune rabbit sera and IgE-containing sera of patients suffering from pollen allergy. The method is based on inhibition of previously determined serum concentration with various concentrations of the allergen examined. The inhibition is expressed as a percent decrease of activity of corresponding serum. Another method which is suitable for characterization of allergens is the SDS electrophoresis and immunoblotting. The allergen extract, separated in the electric field, was transferred to nitrocellulose by electrophoresis. After incubation with corresponding rabbit hyperimmune serum or serum of the patient with allergy and application of the peroxidase conjugate, fractions of extracts reacting with corresponding antibodies can be detected after developing. The ~I.ISA method is suitable for the coarse detection of allergen activity while immunoblotting allows to determine even the specificity and purity of extracts.

immwoptophy/a~ a Sheep Plem~paeamoa/s Cused by Adlaobsdllm plemopaeamoalae

~NmC J., GoI~ M., PODMANICI~ D., GUBRAN D. Bioveta Nitra ; V~/L , Bmo ; District Veterinary Center Kom~no

We examined the immunity against pleuropneumonia in sheep using three chemically inactivated immunogens. Individual immunogens contained bacterial cultures of Actinobacillus pleuropneumoniae, serotypes 1, 2 and 1+2. Groups of sheep (15-20 kg) free of specific pathogens were immunized i.m. twice in a 14-day interval with 3 mL of immunogen. 28 days after the first vaccination all immunized sheep together with control animals were infected with 2 - 3 x 101~ KTJ. All control sheep died with symptoms of peracute or acute form of pleuropneumonia. Immunized sheep developed transient fever and mild hyperventilation which returned to normal after 20 h.

1990 ABSTRACTS |01

~vi~ ~ Vmcc~ mpimst ~ Jml~m ot ~ mmd ~mr Canmlv~Nm Aahm~

ZU~A T., Knosor P., BOueKovA V. Bioveta Nitra ; svU, Liberec ; VELAZ Prague

We developed monovalent and combined living vaccines against rabies, parvovirosis (CPV), infections laryngotracbeitis (CADV 2) and parainfluenza (Claw 2). Vaccination of dogs and foxes of various age categories induced general seropositivity of medium to high titres of HI-antibodies against CPV, CADV 2 and CPIV 2 viruses and neutralizing antibodies against the rabies virus. Higher titres were obtained in young pups than in adult dogs. Passively administered and actively produced antibodies interfered with the active antibody response. No differences in the antibody response against individual viruses were recorded in animals vaccinated either with the monovalent or combined vaccine.

hu~vated V m ~ e A ~ u k ND+GO+IB q ~ s t ~ (NDV), t . t e ~ o ~ k n t # s (IBm) ~ d latectiow Bronchitis ( IBr ) in Fowl

LOPA~OVSK~ E., ZAJAC J. Bioveta Nitra

We have developed and tested a tervaient inactivated vaccine against NDV, IBu and IBr for immunization of chickens before initiation of egg laying. The intensity of the immune response induced by vaccination was measured according to HI antibody content against NDV, and ELISA antibodies against IBu and IBr. A good immunogenicity of the vaccine was confirmed both in laboratory and in breeding conditions.

/nact/vated Vmcdne agm/mt Salmone/k~ ot Cm/ves

]ht.n~_.c E., M_no_a.A I., ROSOCnA J., ~Nmr J., I~_~vA E. Department of Microbiology, Immunology and Zoohygiene, V~V, Kogice; Bioveta Nitra

Calves, aged 21- days, after a triple i.m. administration of the vaccine, were injected p.o. with a virulent strain of Salmonella typhimurium (106 and 109 CFU). The vaccine which consisted of strains differing in various factors (adhesion, toxin production, seroresistance, invasivity, plasmid profile) yielded protection of immunized calves without postvaccination reactions.

/mzt/~ted, Concentrated, Parab~ Pm/s Ant/rab/es Vact/ae

FEn~AR V., BF.NI~EX Z., g v R ~ g., VRTtAK O.J., S0UOVA J., ZAVADOVA J., (3~'DmmCA R., M_L,tq, t m ~ o v A K. UEVM, Ko~ce

We proposed a technological procedure for the production of a cellular inactivated, concentrated, partially purified adjuvant antirabies vaccine for preventive immunization of domestic animals. The strain Vnukovo-32-107 was grown in a cell-line BHK-21, clone 135. The infections cell medium was concentrated by ultrafiltration, inactivated by [3-propiolactone and lyophilized. The vaccine is prepared in a water-oil (3:1) system. The composition of the oil adjuvant secures the efficacy of the vaccine - it is metabolized and does not possess ailergogenic activity.


/mnmnopro~yk~ of C - ~ s ~ r d ~ s m~ D k r o ~

Ktn~ZNA K., VA~ovA L., PROKOPIC.L lns~mte of Parasitology, ~ A V, Cesk6 Bud~jo~ce

Mice (strains ICR and NIH) were vaccinated with suspensions prepared from larvae of Taenia cras.~ceps. The mode of vaccine administration, the intervals and various doses were tested in three consecutive expedmer ~s. Doses of 5 x 10 s and i x 10 ~ of cells per mouse, administered in a 4-day span, yielded 100 % protection regardless of the mode of administration. Based on the same principle, we prepared a vaccine that was tested on a large scale in the breeding farm Spiksk~ Nov:i Ves. We vaccinated 3-month-old lambs against dicroceliosis with suspensions of cells prepared from adult liver-fluke Fasciola hepatica. The efficacy of the vaccine was 98 %. The results of vaccination indicate that the heterologons vaccine possesses sufficient efficacy.

lqbe Psidar ~, ed - a / N e w A//ergen ?

Bu.u~ovA A., VAaOA L. WOLURCH, Tatranskd Polianka ; Vf.R__J4, Zrolen

The aim of our study was to establish the reactivity of patients with pollinosis who reported worsening of subjective symptoms during the season of poplar seed maturation. In cooperadion with VOLH Zvolen (Research Station Gab~ikovo) we collected pollen and seeds from selected cultivars of poplar. Allergens for testing were prepared from the pollen and seeds in the USOL. So far, a control group of volunteers has been tested.

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