Chemically Modified Tetracycline 3 Prevents Acute Respiratory Distress Syndrome in a Porcine Model of Sepsis + Ischemia/Reperfusion–Induced Lung Injury

CHEMICALLY MODIFIED TETRACYCLINE 3 PREVENTS ACUTERESPIRATORY DISTRESS SYNDROME IN A PORCINE MODEL OF

SEPSIS + ISCHEMIA/REPERFUSIONYINDUCED LUNG INJURY

Shreyas K. Roy,* Brian D. Kubiak,* Scott P. Albert,* Christopher J. Vieau,*Louis Gatto,† Lorne Golub,‡ Hsi-Ming Lee,‡ Suraj Sookhu,*

Yoram Vodovotz,§ and Gary F. Nieman**Department of Surgery, Upstate Medical University, Syracuse; †Department of Biological Sciences, SUNYCortland, Cortland; ‡Department of Oral Biology and Pathology, School of Dental Medicine, State University

of New York, Stony Brook, New York; and §Department of Surgery, University ofPittsburgh School of Medicine, Pittsburgh, Pennsylvania

Received 20 Nov 2011; first review completed 9 Dec 2011; accepted in final form 9 Dec 2011

ABSTRACT—Experimental pharmacotherapies for the acute respiratory distress syndrome (ARDS) have not met withsuccess in the clinical realm. We hypothesized that chemically modified tetracycline 3 (CMT-3), an anti-inflammatory agentthat blocks multiple proteases and cytokines, would prevent ARDS and injury in other organs in a clinically applicable,porcine model of inflammation-induced lung injury. Pigs (n = 15) were anesthetized and instrumented for monitoring. A‘‘2-hit’’ injury was induced: (a) peritoneal sepsisVby placement of a fecal clot in the peritoneum, and (b) ischemia/reperfusionVby clamping the superior mesenteric artery for 30 min. Animals were randomized into two groups: CMT-3group (n = 7) received CMT-3 (200 mg/kg); placebo group (n = 9) received the same dose of a CMT-3 vehicle(carboxymethylcellulose). Experiment duration was 48 h or until early mortality. Animals in both groups developedpolymicrobial bacteremia. Chemically modified tetracycline 3 treatment prevented ARDS as indicated by PaO2/FIO2 ratio,static compliance, and plateau airway pressure (P G 0.05 vs. placebo). It improved all histological lesions of ARDS (P G 0.05vs. placebo). The placebo group developed severe ARDS, coagulopathy, and histological injury to the bowel. Chemicallymodified tetracycline 3 treatment prevented coagulopathy and protected against bowel injury. It significantly loweredplasma concentrations of interleukin 1" (IL-1"), tumor necrosis factor !, IL-6, IL-8, and IL-10. This study presents a clinicallyrelevant model of lung injury in which CMT-3 treatment prevented the development of ARDS due in part to reduction ofmultiple plasma cytokines. Treatment of sepsis patients with CMT-3 could significantly reduce progression from sepsis intoARDS.

KEYWORDS—Sepsis, acute respiratory distress syndrome, ARDS, lung injury

ABBREVIATIONS—ARDSVacute respiratory distress syndrome; BALFVbronchoalveolar lavage fluid; MMPVmatrixmetalloproteinase; CMT-3Vchemically modified tetracycline 3; TNF-!Vtumor necrosis factor !; ILVinterleukin; RMANOVAVrepeated-measures analysis of variance

INTRODUCTION

The acute respiratory distress syndrome (ARDS) affects

nearly 200,000 patients per year. Despite intensive research and

improved mechanical ventilation strategies, ARDS claims the

lives of nearly 30% of the afflicted individuals (1). Acute res-

piratory distress syndrome can be caused by either primary or

secondary lung injury. Primary ARDS is caused by pulmonary

processes such as aspiration or pneumonia, whereas secondary

ARDS is caused by systemic inflammation following severe

injury such as trauma, hemorrhage, or sepsis (1, 2).

The pathophysiology of secondary ARDS can be concep-

tually divided into an initiating inflammatory phase followed by

secondary tissue damage phase (2). In the inflammatory phase,

trauma, hemorrhage, and severe sepsis cause a massive release

of multiple inflammatory mediators that cause the clinical

manifestations of acute shock (3). In the tissue damage phase,

these inflammatory mediators trigger organ damage by pro-

moting a variety of proteolytic and oxidative effectors including

matrix metalloproteinases (MMPs), reactive oxygen species (4),

and damage-associated molecular patterns (5). Experimental

pharmacotherapies that target single mediators in the inflam-

matory phase of ARDS pathophysiology have not met with

success in the clinical realm. This failure of single-mediator

pharmacotherapy in ARDS is attributed primarily to the com-

plexity and redundancy of the inflammatory phase of shock and

also to limitations of the small-animal and cell culture models in

which these therapies were initially tested (6).

Pleiotropy is a pharmacologic term given to medications

that work on multiple targets rather than a specific one. We

hypothesized that a pleiotropic drug, acting on multiple targets

in both the inflammatory and tissue damage phases of secon-

dary ARDS pathophysiology, could succeed where prior drugs

targeted at individual mediators have failed. In theory, such a

drug should not only attenuate the release of inflammatory

mediators, but also inhibit the proteolytic and oxidative effec-

tors that cause end-organ damage as well. Chemically modified

tetracycline (6-demythyl-6-deoxy-4dedimentylamino-tetracycline;

424

SHOCK, Vol. 37, No. 4, pp. 424Y432, 2012

Address reprint requests to Shreyas K. Roy, MD, CM, Department of Surgery,

750 E Adams St, Syracuse, NY 13210. E-mail: [email protected].

All animal experimentation for this study was performed at SUNY Upstate

Medical University in Syracuse, New York.

This work was partially funded by the National Institutes of Health

(R33HL089076 and R42Hl065030) and by CollaGenex Pharmaceutical Inc.

G.F.N. is an inventor on the following patent: BMethod of Treating Sepsis-

Induced ARDS[ (serial no. 03768759.7-2123-US0335531).

DOI: 10.1097/SHK.0b013e318245f2f9

Copyright � 2012 by the Shock Society

CMT-3) is a nonantimicrobial, pleiotropic, anti-inflammatory

agent that inhibits multiple inflammatory and proteolytic

mediators including MMPs, tumor necrosis factor ! (TNF-!),

and interleukin 6 (IL-6) (7, 8). In a previous study using the

same B2-hit[ porcine peritoneal sepsis + ischemia/reperfusion

(PS + IR) model described herein, we demonstrated that CMT-3

administered prophylactically before PS + IR injury did pre-

vent ARDS (9). In the present study, we hypothesized that

CMT-3 would attenuate the lung injury associated with our

porcine PS + IR model and prevent the development of ARDS

when administered 1 h after the injury. In terms of clinical

time frame, CMT-3 can be delivered to trauma patients at

high risk for developing ARDS during the initial resuscitation

in the emergency department. Thus, the current study demon-

strates the proof of concept that CMT-3 can be used in a

clinically relevant time frame in patients at risk of developing

ARDS secondary to trauma or sepsis (9, 10).

The differences between the CMT-3Ytreated and placebo-

treated animals were dramatic. Chemically modified tetra-

cycline 3 treatment completely prevented ARDS as measured

by PaO2/FIO2 ratio criteria as well as by quantitative histol-

ogy. Chemically modified tetracycline 3 significantly lowered

plasma concentrations of IL-1", TNF-!, IL-6, IL-8, and IL-10,

suggesting that the mechanism of lung protection may have

been the drug’s pleiotropic reduction of systemic inflamma-

tion. The placebo group developed severe ARDS, coagulop-

athy, and histological injury to the bowel. These data clearly

support our hypothesis that CMT-3 prevents the development

of ARDS in this clinically relevant model of sepsis. Interest-

ingly, in developing this porcine model of sepsis and IR, we

found that the injury not only caused ARDS but also caused

injury to other organ systems (11), specifically, coagulopathy

and histopathologic injury in the intestine. These injuries were

prevented by CMT-3 in the present study as well.

METHODSAll techniques and procedures described have been fully approved by the

Committee for the Human Use of Animals at Upstate Medical University.

AnimalsDetailed methods describing this model have been published elsewhere

(12, 13). Briefly, healthy female Yorkshire pigs (21Y38 kg) were pretreated

with glycopyrrolate, tiletamine hydrochloride and zolazepam hydrochloride,and xylazine. After intubation, anesthesia was maintained using a continuousinfusion of ketamine/xylazine to maintain a comfortable plane of anesthesia.All animals were continuously monitored and cared for by the investigators forthe full 48-h duration of the experiment.

Tracheostomy and mechanical ventilationAn open tracheostomy was performed, and the animal was connected to

a Galileo ventilator (Hamilton Medical, Reno, Nev) with initial settings dur-ing the surgical preparation as follows: tidal volume of 12 mL/kg, respiratoryrate of 15 breaths/min, FIO2 of 21%, and positive end-expiratory pressure of3 cmH2O. Respiratory rate was titrated to maintain PaCO2 within the referencerange (35Y45 cmH2O); FIO2 was titrated to maintain O2 saturation of greaterthan 88%. Low-tidal-volume protective mechanical ventilation was not usedbecause we did not want to Bprotect[ the lung with the ventilator but rathermeasure the development of lung injury if it occurred.

Surgical preparationUnder sterile conditions, a carotid arterial catheter and two external jugular

central venous catheters were placed. A Foley catheter was inserted directlyinto the bladder for measurement of urine output and collection of urinesamples. All animals received a regimen of intravenous fluids and antibioticsat a dose and quantity established in our initial experiments (see Fluids andantibiotic management).

InjuryAfter placement of arterial and venous catheters, a midline laparotomy was

performed for placement of a gastric tube (Bard, Covington, GA) and induc-tion of a 2-hit injury, PS + IR, described in detail in prior publications from thismodel (12, 13). The superior mesenteric artery (SMA) was clamped for 30 minto induce intestinal ischemia. During the 30-min ischemic time, 0.5 mL/kg offeces was harvested from a cecotomy and mixed with 2 mL/kg of blood tocreate a fecal clot. After releasing the clamp on the SMA, the clot wasimplanted into the lower portion of the abdominal cavity. The abdomen wasthen closed with a running monofilament fascial suture and skin staples.

MeasurementsBaseline (BL) measurements were taken following vascular access before

injury. Time 0 (T0) measurements were taken immediately after the inductionof injury (i.e., removal of SMA clamp and placement of fecal clot) upon clo-sure of the abdomen. (For a full timeline, see Fig. 1.)

Treatment groupsOne hour after injury, the animals were randomized into two groups: group

1, CMT-3 treated (n = 7), the animals received an orally active dose (200 mg/kg)of the modified tetracycline CMT-3 (6-demythyl-6-deoxy-4dedimentylamino-tetracycline; CollaGenex Pharmaceuticals Inc, Newton, Pa) delivered per gas-trostomy; group 2, placebo (n = 9), the animals received the same dose of avehicle (carboxymethylcellulose) for CMT-3 delivered per gastrostomy.

Fluids and antibiotic managementRinger’s lactate was used to for fluid resuscitation and maintenance.

Maintenance fluid requirements were calculated on a per-kilo basis according

FIG. 1. Timeline of CMT-3 or placebo treatment. After the animal is under anesthesia, ‘‘surgical preparation’’ with tracheostomy, vascular access, and Foleycatheterization is performed; this takes 30 min. Baseline measurements are then performed. Then 2-hit injury is performed through a midline laparotomy viaclamping of the SMA and placement of fecal clot in the peritoneum; this takes 30 min. Upon release of the SMA clamp and closure of the abdomen, T0measurements are taken. One hour after T0, treatment with either CMT-3 or placebo is given via gastrostomy tube. For the remainder of the experiment, theanimal is treated according to intensive care unit standards of care with antibiotics and fluid management.

SHOCK APRIL 2012 CMT-3 AND ARDS IN PORCINE LUNG INJURY 425

to clinical guidelines. Fluid boluses were given as indicated by deteriorationsin hemodynamic parameters or decreases in urine output less than 0.5 mL/kgper hour. Broad-spectrum antibiotics were delivered intravenously followingclosure of the abdomen (ampicillin 2 g i.v. [Bristol Myers Squibb, Princeton,NJ] and metronidazole 500 mg i.v. [Baxter, Deerfield, Ill]). This antibioticregimen was repeated at 12, 24, and 36 h after injury.

Physiologic measurements and pulmonary mechanicsHemodynamic parameters were measured (CMS-2001 System M1176A,

with Monitor M1094B; Agilent, Bobingen, Germany) using Edwards trans-ducers (Pressure Monitoring Kit [PXMK1183]; Edwards Lifesciences, Irvine,Calif). Pulmonary parameters were measured or calculated by the Galileoventilator (Hamilton Medical).

Plasma and bronchoalveolar lavage fluid cytokinemeasurements

Blood was drawn every 6 h and spun at 3,500 revolutions/min at 15-C for10 min. The concentrations of TNF-!, IL-1", IL-6, IL-8, and IL-10 were deter-mined in the plasma and bronchoalveolar lavage fluid (BALF) using enzyme-linked immunosorbent assay according to the manufacturer’s recommendations.

Blood samplingMeasurement of blood gases and chemistries was made with a Roche blood

gas analyzer (Cobas b221; Basel, Switzerland). Clinical pathology and bloodcultures were performed on arterial blood samples by the Upstate MedicalUniversity pathology laboratory facility.

Bronchoalveolar lavage procedureAt necropsy, the right middle lobe was lavaged with 60 mL of normal saline

(three injections of 20 mL flushed into the right middle lobe bronchus and

aspirated out), and the volume collected was recorded. The BALF was spun for10 min at 3,500 revolutions/min at 15-C for 10 min.

NecropsyImmediately following early mortality or killing at 48 h, necropsy was

performed to recover tissue for analysis. Lungs were inflated to 25 cmH2Ousing stepwise increases of continuous positive airway pressure on the ven-tilator and clamped to maintain a consistent lung volume history in all animals.The lung was then unclamped, and the airway filled with formalin to a con-sistent filling pressure of 25 cmH2O reclamped and submerged in forma-lin. Following 24 h in the fixative, the lungs were removed, cut, and sent tohistology for processing. The following procedure was performed on the lastsix animals (three from each treatment group): the laparotomy was sub-sequently reopened, and 20-cm specimens of small bowel were removed fromthe proximal jejunum, distal jejunum, and distal ileum. These specimenswere preserved in 10% neutral buffered formalin for 48 h before histologicalsectioning.

HistologyHistological analyses (hematoxylin-eosin) were performed on all tissue

from necropsy. The histologist was blinded to animal group during morpho-metric measurements. A detailed description of our histological methods hasbeen previously described (12, 13).

Quantitative histologyThe quantitative histological technique described in Methods allows an

unbiased comparison of the degree of tissue injury to the lungs between thetwo treatment groups. As previously described (12, 13), the quantitative his-tological assessment of the lung was based on image analysis of 120 photo-micrographs (10 per pig), made at high-dry magnification following an

TABLE 2. Plasma cytokines

Cytokines Group BL 12 h 24 h 36 h 48 h P G 0.05

IL-6, pg/mL Placebo 316.4 T 28.5 1,430.0 T 186.5 1,294.4 T 315.8 2,662.0 T 548.0 2,773.3 T 861.2

CMT-3 277.3 T 49.7 775.0 T 104.3 638.6 T 175.6 868.4 T 216.3 1,851.5 T 655.2 *

IL-1", pg/mL Placebo 150.2 T 10.4 237.5 T 13.9 241.1 T 20.1 550.8 T 225.8 548.7 T 126.8

CMT-3 133.6 T 12.3 211.0 T 19.5 283.0 T 95.8 207.7 T 20.7 277.1 T 57.8 *

TNF-!, pg/mL Placebo 198.1 T 18.9 310.7 T 17.4 378.93 T 43.0 730.5 T 182.0 955.8 T 323.8

CMT-3 192.7 T 14.3 228.6 T 18.7 236.9 T 26.7 293.2 T 42.5 516.8 T 123.3 *

IL-10, pg/mL Placebo 124.5 T 13.2 147.5 T 11.1 137.9 T 11.5 161.7 T 17.6 175.5 T 16.0

CMT-3 109.4 T 8.3 127.7 T 11.2 117.4 T 11.2 121.7 T 12.5 139.4 T 15.5

*P 9 0.05 following RM ANOVA.

TABLE 1. Hemodynamic parameters

Parameters Group BL 6 h 12 h 18 h 24 h 30 h 36 h 42 h 48 h P G 0.05

HR, beats/min Placebo 138.7 T 10.4 103.2 T 9.5 97.9 T 15.3 75.8 T 7.1 74.5 T 6.7 94.3 T 12.4 84.7 T 6.6 94.0 T 10.6 123.3 T 28.8

CMT-3 126.4 T 10.1 134.9 T 10.0 82.6 T 4.6 78.9 T 7.0 72.7 T 4.2 70.9 T 4.3 76.9 T 7.4 80.3 T 9.1 74.6 T 7.3

MAP, mmHg Placebo 117.0 T 10.1 71.0 T 5.0 71.1 T 2.6 65.5 T 2.8 66.0 T 1.8 64.4 T 3.6 58.3 T 3.6 57.2 T 4.4 53.2 T 5.4

CMT-3 135.0 T 5.2 71.7 T 2.7 75.1 T 1.0 72.9 T 2.5 69.6 T 2.1 63.9 T 3.2 64.9 T 2.4 67.8 T 2.1 69.3 T 7.6

CVP, cmH2O Placebo 7.4 T 1.0 7.7 T 1.2 7.2 T 2.1 10.4 T 1.7 9.5 T 2.0 11.0 T 1.7 13.8 T 2.5 11.3 T 2.8 10.8 T 4.0

CMT-3 8.4 T 0.9 8.0 T 0.7 8.7 T 0.6 9.3 T 0.5 9.7 T 0.6 9.7 T 0.6 11.2 T 0.7 11.2 T 1.1 10.6 T 0.8

PAP, mmHg Placebo 27.1 T 2.6 25.8 T 2.1 28.8 T 1.2 26.4 T 1.8 24.9 T 2.5 28.7 T 2.0 29.5 T 2.3 27.2 T 4.6 26.0 T 10.1

CMT-3 29.3 T 4.2 21.1 T 2.2 25.2 T 2.5 24.3 T 1.8 27.0 T 1.8 26.1 T 1.6 26.3 T 1.4 29.5 T 1.7 30.3 T 2.5

PAW, mmHg Placebo 11.3 T 1.3 9.4 T 1.1 12.2 T 1.3 11.3 T 0.8 12.4 T 1.6 11.9 T 1.6 12.8 T 1.8 13.3 T 2.7 14.0 T 4.5

CMT-3 11.8 T 1.1 10.4 T 1.2 11.5 T 1.2 11.3 T 0.6 12.4 T 0.8 12.0 T 0.8 12.8 T 0.7 12.8 T 0.7 13.3 T 1.4

CO, L/min Placebo 4.53 T 0.71 2.51 T 0.43 2.18 T 0.32 2.31 T 0.46 2.67 T 0.38 3.51 T 0.58 3.44 T 0.70 3.12 T 1.30 3.29 T 1.93

CMT-3 6.61 T 1.03 4.37 T 0.53 4.17 T 0.62 4.45 T 0.60 4.98 T 0.80 4.74 T 0.62 5.22 T 0.83 6.00 T 1.9 5.96 T 1.29 *

Temperature, -C Placebo 36.1 T 0.3 35.8 T 0.6 36.2 T 0.6 36.6 T 0.6 36.9 T 0.6 36.6 T 0.5 36.5 T 0.7 37.4 T 1.3 37.9 T 0.7

CMT-3 36.6 T 0.4 36.6 T 0.6 36.7 T 0.5 37.2 T 0.6 37.4 T 0.4 37.5 T 0.7 38.2 T 0.6 38.2 T 0.5 37.3 T 0.5

*P 9 0.05 following RM ANOVA.HR indicates heart rate; MAP, mean arterial pressure; CVP, central venous pressure; PAP, pulmonary artery pressure; PAW, pulmonary artery wedgepressure; CO, cardiac output.

426 SHOCK VOL. 37, NO. 4 ROY ET AL.

unbiased, systematic sampling protocol. Each photomicrograph was scoredusing a four-point scale for each of five parameters: atelectasis, fibrinousdeposits, and blood in airspace; vessel congestion; alveolar wall thickness; andleukocyte infiltration.

Statistical analysisSurvival rates between the two treatment groups were compared using the

event time distribution functions and a log-rank test. A repeated-measuresanalysis of variance (RM ANOVA) with pig number and treatment as randomeffects was performed to compare differences within and between treatmentgroups for continuous parameters over time. Significance in the RM ANOVAis expressed as P for group � time meaning that there was a difference be-tween groups over time. A criterion of the repeated-measures analysis is thatthere can be no missing data points. This study produced severe critical illness,and certain animals suffered early mortality. To account for animals that failedto survive the full course of the experiment and therefore incorporate all ani-mals randomized in the study, a least squares regression model was used tocalculate the intra-animal predicted value for the repeated-measures analyses.Significant results from the random-effects RM model were further examinedat each post-BL time point following a Bonferroni correction to adjust formultiplicity. Quantitative histology data were analyzed using a Mann-WhitneyU test after testing for normality. P G 0.05 was considered significant. Allanalyses were performed using JMP version 5.1.1 (Cary, NC).

RESULTS

Sepsis/shockBlood cultures—Animals in both groups developed poly-

microbial bacteremia as assessed by qualitative blood cul-

tures. The bacteria found in these cultures were Escherichiacoli, streptococcal species, staphylococcal species, Serratiamarcescens, Pseudomonas aeruginosa, Klebsiella pneumoniae,

and Aeromonas hydrophila.

Hemodynamics—The mean arterial pressure (MAP) for the

placebo group at BL was 117 T 1 mmHg, and by T48, it had

dropped to 53.2 T 5.4 mmHg. The CMT-3 group’s BL MAP

dropped from 135 T 5.2 mmHg to 69 T 7.6 mmHg at T48.

These differences were not statistically significant. Central

venous pressure and cardiac output did not differ significantly

between the two treatment groups (Table 1).

Fluid balance—The CMT-3 group had significantly higher

urine output, lower resuscitative fluid requirements, and

correspondingly lower positive fluid balance over the 48-h

study (P G 0.05).

Plasma cytokines—Circulating levels of TNF-!, IL-1", and

IL-6 all increased significantly and progressively over the 48-h

course of the experiment (Table 2) in the placebo group. Chem-

ically modified tetracycline 3 significantly reduced circulating

cytokine levels versus placebo.

Bronchoalveolar lavage cytokines—Levels of IL-1", TNF-!,

IL-6, IL 10, and IL-8 were lower in the BALF of the CMT-3Ytreated animals than that of the placebo group; however, these

data did not show statistical significance (Table 3).

Plasma MMPs—Levels of MMP-2, MMP-9, and neutrophil

elastase were not different between CMT-3 and placebo group s

in this study (Table 4).

Survival—The placebo group experienced 40% survival at

48 h, whereas the CMT-3 group experienced 85.7% survival at

48 h. However, this difference was not statistically significant

(Fig. 2).

Wet-to-dry weights—There was no statistical difference be-

tween treatment groups in tissue edema of the lungs.

Lung injuryPulmonary mechanics—Peak airway pressures (Ppeak) were

initially similar in the two groups (CMT-3: 22.4 T 2.4 cmH2O

vs. placebo: 21.1 T 0.8 cmH2O). However, by 30 h into the

experiment, the placebo group had Ppeaks of 36.0 T 4.5 cmH2O,

whereas the CMT-3 group was nearly unchanged at 25.4 T1.6 cmH2O. At the end of the experiment, the protective effect

of CMT-3 on peak airway pressures was clearly demonstrated,

TABLE 4. MMP-2, MMP-9, and neutrophil elastase

Group T0 T24 T48 BALF

MMP-9, 2g/mL Placebo 89.3 T 4.9 85.0 T 6.5 71.5 T 7.7 59.6 T 12.6

CMT-3 103.3 T 7.1 76.7 T 6.0 68.1 T 5.7 147.5 T 29.9

MMP-2, 2g/mL Placebo 80.4 T 8.1 74.9 T 5.7 70.8 T 9.0 51.1 T 11.7

CMT-3 77.8 T 8.1 72.2 T 8.2 85.5 T 9.5 81.5 T 18.1

Elastase, 2mol/mg per 18 h Placebo 27.5 T 1.7 25.2 T 2.9 28.8 T 2.1 44.7 T 19.0

CMT-3 25.0 T 1.2 23.2 T 2.8 29.5 T 3.7 24.7 T 9.5

Columns reflect time in hours.

TABLE 3. BALF cytokine concentration

Group IL-6 IL-1 TNF-! IL-10

Placebo 1,564.0 T 470.5 365.8 T 67.9 213.7 T 53.1 112.4 T 6.5

CMT-3 785.1 T 414.4 564.1 T 135.0 407.8 T 113.5 101.5 T 9.3

FIG. 2. Effects of CMT-3 on survival. Kaplan-Meier survival curve ofCMT-3Ytreated (dashed line) and placebo-treated (solid line) animals; 60% ofthe placebo-treated animals died, whereas only 14% of the CMT-3 animalsdied before 48 h.


as the CMT-3 group had Ppeaks of 28.6 T 2.5 cmH2O, whereas

the placebo group was extremely difficult to ventilate at a

Ppeak of 34.5 T 5.6 cmH2O. Plateau and mean airway pres-

sures correlated to the trend seen in the peak airway pressures

(Table 5). In addition, the static compliance changes (Fig. 3)

corroborate the changes in lung pressures. The PaO2/FIO2 ratio

(Fig. 4) demonstrated that, by 30 h into the experiment, the

placebo group met the criteria for acute lung injury, and by

48 h, these animals were well within the range of ARDS. In

contrast, ARDS did not develop in the CMT-3 group.

Quantitative histology—The quantitative histological tech-

nique described in Methods allows a bias-free comparison of

the degree of tissue injury between the two treatment groups.

The specific histological lesions analyzed are considered patho-

gnomonic for ARDS when in the context of the appropriate

clinical course (14). Atelectasis, fibrin deposits, leukocyte in-

filtration, and alveolar wall thickness were all significantly

lower in the animals treated with CMT-3. Dramatic differences

were observed between groups in alveolar wall thickness,

which was nearly quadrupled in the placebo group versus the

CMT-3 group (placebo: 2.41 T 0.3, CMT: 3 0.49 T 0.10)

(Table 6). Dramatic differences were also observed in the

degree of fibrinous deposits and leukocyte infiltration, which

were both doubled in the placebo group as compared with the

CMT-3 group (Table 6). The histological lesions of ARDS

mentioned above are visually represented in Figure 5, sup-

porting the impression of protective effects of CMT-3 on the

lung. Figure 5A shows the lung of a placebo-treated animal

with classic stigmata of ARDS atelectasis, intra-alveolar

hemorrhage, fibrinous exudates, and leukocytic infiltrates.

Figure 5B shows the lung of a CMT-3Ytreated animal, with

preservation of nearly normal pulmonary architecture.

Hematologic derangements

Placebo-treated animals became progressively and pro-

foundly leukopenic, which is consistent with severe sepsis in

humans. Baseline white blood cell counts were significantly

higher in the CMT-3 group than in the placebo group (Table 7).

This difference between groups persisted throughout the course

of the experiment. There was a predominance of neutrophils in

TABLE 5. Pulmonary mechanics

Parameters Group BL 6 h 12 h 18 h 24 h 30 h 36 h 42 h 48 h P G 0.05

Ppeak, cmH2O Placebo 21.1 T 0.8 21.7 T 1.1 23.8 T 1.6 25.8 T 1.7 26.6 T 1.7 36.0 T 4.5 35.8 T 4.5 40.9 T 4.0 34.5 T 5.6

CMT-3 22.4 T 2.4 20.3 T 0.7 22.4 T 0.8 22.1 T 1.0 23.1 T 1.1 25.4 T 1.6 26.0 T 0.7 30.2 T 1.9 28.6 T 2.5 †

Pmean, cmH2O Placebo 6.8 T 0.5 7.9 T 0.3 8.4 T 0.7 8.8 T 0.5 9.2 T 0.6 12.1 T 1.9 14.6 T 2.3 15.5 T 2.9 15.2 T 3.4

CMT-3 7.1 T 0.4 6.9 T 0.3 7.3 T 0.5 7.1 T 0.3 7.3 T 0.3 7.7 T 0.5 8.5 T 0.6 9.2 T 0.8 9.1 T 0.8 *

Pplat, cmH2O Placebo 18.2 T 0.7 19.3 T 1.2 21.4 T 1.5 23.1 T 1.6 24.2 T 1.9 29.1 T 2.8 37.2 T 4.1 32.2 T 5.1 30.5 T 5.3

CMT-3 20.3 T 2.0 18.6 T 0.4 20.0 T 0.8 19.9 T 0.8 21.0 T 0.9 20.9 T 0.7 22.9 T 0.9 26.0 T 2.0 26.6 T 2.9 *

EMV, L/min Placebo 4.1 T 0.3 4.5 T 0.2 4.4 T 0.2 4.3 T 0.2 4.6 T 0.3 4.4 T 0.3 5.2 T 0.6 5.5 T 0.2 5.8 T 0.5

CMT-3 5.5 T 0.4 5.2 T 0.2 5.3 T 0.2 4.8 T 0.2 4.7 T 0.2 4.6 T 0.3 4.9 T 0.2 4.6 T 0.1 5.3 T 0.4

PCO2, mmHg Placebo 37.3 T 1.6 33.9 T 1.0 31.6 T 0.9 32.1 T 1.8 34.2 T 1.3 42.0 T 3.6 48.7 T 4.0 42.9 T 3.2 43.5 T 2.5

CMT-3 34.4 T 2.4 32.3 T 1.9 29.6 T 1.5 31.4 T 1.7 32.9 T 1.7 35.1 T 1.1 34.8 T 1.0 39.0 T 1.6 36.8 T 2.2

PO2, mmHg Placebo 103 T 5.2 101.2 T 6.6 167 T 33.5 139.1 T 20.3 166.5 T 43.8 103.9 T 14.6 136.8 T 28.2 147 T 42.5 93. 8 T 31.1

CMT-3 82.5 T 13.5 99.3 T 4.3 101.6 T 2.3 97.6 T 5.0 111.1 T 18.8 110.52 T 20.0 180.7 T 64.2 197.4 T 52.9 123.4 T 16.2

*P 9 0.05 following RM ANOVA.Ppeak indicates peak airway pressure; Pmean, mean airway pressure; Pplat, plateau airway pressure; EMV, expired minute volume; PCO2, partial pressureof carbon dioxide in arterial blood; PO2, partial pressure of oxygen in arterial blood.

FIG. 3. Effects of CMT-3 on lung compliance. Static compliance for theCMT-3Ytreated (dashed line) and placebo-treated (solid line) animals recor-ded hourly for 48 h. There was a significant difference (P G 0.01) betweengroups by group � time interaction.

FIG. 4. Effects of CMT-3 on oxygenation. PaO2/ FIO2 ratio for the CMT-3Ytreated (dashed line) and placebo-treated (solid line) animals recordedhourly for 48 h. There was a significant difference (P G 0.046) between groupsby group � time interaction.


both groups at T12. As placebo animals became leukopenic, all

subpopulations (neutrophils, eosinophils, basophils, lympho-

cytes) decreased equally. Chemically modified tetracycline 3

preserved all subpopulations of white blood cells equally.

Chemically modified tetracycline 3 exerted a significant,

protective effect on the coagulation system. Placebo-treated

animals became progressively coagulopathic, whereas CMT-

3Ytreated animals did not as evidenced by international nor-

malized ratio (INR) and partial thromboplastin time data

(Table 7) (P G 0.05). Chemically modified tetracycline 3 pre-

vented the profound thrombocytopenia observed in the pla-

cebo group (Table 7); these data were statistically significant.

Intestinal histology

The placebo group (n = 3; Fig. 6: center panel BFecal clot[)

exhibited grossly shortened and denuded villi with significant

sloughing of surface epithelium into the bowel lumen. Intes-

tinal glands showed areas devoid of cellular definition, sug-

gesting a loss of the epithelial barrier. In contrast, the CMT-3

group (n = 3; (Fig. 6: far right panel BFecal clot + CMT-3[)

exhibited villi of normal length with only focal denudation of

the surface epithelium; intestinal glands were normal in this

group. The far left panel of Figure 6 (BNaive pig[) is a non-

manipulated healthy pig used as a histological control for

comparison.

DISCUSSION

The central finding of the present study is that admin-

istration of CMT-3 immediately following injury in a clin-

ically relevant porcine model of lung injury prevents the

development of ARDS, supporting our hypothesis. Severe

sepsis and its complications have eluded attempts at single

mediator targeted therapies (15). Only activated protein C has

gained clinical relevance, but pharmacotherapy with activated

protein C is extremely limited in postsurgical patients because

of hemorrhagic complications (16). A major cause for the

failure of single-mediatorYtargeted medications is thought to

be the extreme redundancy of the inflammatory cascade (17).

Chemically modified tetracycline 3 is known to work through

a variety of mechanisms to reduce inflammation; it is a truly

pleiotropic anti-inflammatory agent (18). It is possible that

CMT-3 treatment produced clinically relevant improvements in

our large animal model because the pleiotropy of the drug’s

effects overcame the redundancy of the inflammatory response.

The present study is the culmination of a series of experi-

ments demonstrating the efficacy of CMT-3 in preventing

ARDS (9, 10). Early studies using small animal models (rats)

and acute pig studies demonstrated the potential for CMT-3

use in systemic inflammatory injury caused by cecal ligation

and puncture, cardiopulmonary bypass, and endotoxin (9, 10).

Steinberg et al. (9) established proof of concept in a similar

chronic PS + IR porcine model of ARDS, where CMT-3 was

given before the 2-hit injury was induced. The current study

advances the therapeutic potential of CMT-3 because we dem-

onstrated that CMT-3 is efficacious when delivered 1 h after

injury in a translational model. This suggests that patients at

risk of ARDS secondary to sepsis or other acute inflammatory

insults could be given CMT-3 in the emergency room or inten-

sive care unit to effectively prevent ARDS.

An intriguing corollary to the main finding of the present

study was that CMT-3 also reduced sepsis-associated coagul-

opathy, thrombocytopenia, and the histologically observed

bowel injury that were recently shown to be features of this

animal model (12, 13). In a subset of the animals in this study,

intestine was harvested for histological analysis. There was a

significant reduction in mucosal and submucosal inflammation

in the small bowel of the CMT-3 group. Our study suggests

that the pleiotropic anti-inflammatory properties of CMT-3

have a protective effect on multiple organ systems. We discuss

those effects below.

Effects of CMT-3 on inflammatory cytokines and proteases

Chemically modified tetracycline 3 caused marked reduc-

tions in TNF-! and IL-1" (Table 2), both of which are impli-

cated in the early sepsis-induced systemic inflammatory

response, and may be the mechanism of protection to lung and

bowel in this study. Treatment with soluble TNF receptorYbinding protein prevented lung injury and ARDS in a porcine

model of postpump syndrome (19). Treatment with IL-1"receptor antagonist prevented lung injury in a rat model (20).

In that study, the investigators also demonstrated that IL-1"

TABLE 6. Quantitative histology

Histological change Placebo CMT-3 P G 0.05

Atelectasis 0.40 T 0.13 0 *

Fibrinous deposits 1.60 T 0.15 0.59 T 0.18 *

Blood in airspace 1.34 T 0.15 1.01 T 0.16

Vessel congestion 1.77 T 0.14 1.00 T 0.13 *

Alveolar wall 2.41 T 0.13 0.49 T 0.10 *

Leukocyte infiltration 72.73 T 5.69 37.64 T 3.38 *

FIG. 5. Pulmonary histology. A, A representative section of lung in a placebo-treated animal shows significant ARDS with atelectasis, hemorrhagic andexudative airspace disease, and significant lymphocytic infiltrates. B, A representative section of lung in a CMT-3Ytreated animal with total preservation of normallung architecture, no atelectasis, minimal airway edema, and scant lymphocytic infiltration.


blockade reduced alveolar epithelial injury, pulmonary neu-

trophil sequestration, alveolar wall thickness, and inflamma-

tory infiltrate; these effects are similar to those shown here

with CMT-3 treatment. Because blockade of either TNF-! or

IL-1" can prevent ARDS, the fact that CMT-3 blocks the

production of both suggests that this may be a mechanism of

action offering lung and bowel protection in this study.

The majority of studies on the anti-inflammatory effects of

CMT-3 have shown that the drug’s efficacy is due in part to

inhibition of both MMP-2 and MMP-9 (7, 8). The current

study did not find any significant differences in plasma and

BALF concentrations of MMP-2, MMP-9, and elastase be-

tween groups (Table 4), although prior studies from our labo-

ratory demonstrated a sepsis-induced increase in MMP activity

that was reduced with CMT-3 (9, 21). In the current study,

MMP activity was not examined. Chemically modified tetra-

cycline 3Yinduced reduction of MMP activity may be respon-

sible for the positive outcomes observed and will require

further investigation.

Another explanation for the lack of change in MMP con-

centration between the CMT group and the placebo group

may be related to the use of ketamine in this study. Ketamine

has been shown to mitigate the effects of cecal ligation and

puncture in rats via inhibition of nuclear factor .B (22).

Nuclear factor .B signal blockade reduced the expression of

MMP-9 in a murine lung cancer cell line (23). Therefore,

TABLE 7. Coagulation, blood count, blood chemistry

Parameters Group BL 24 h 48 h P G 0.05

PTT, s Placebo 63.4 T 17.4 33.0 T 3.9 32.9 T 5.6

CMT-3 42.66 T 5.1 39.7 T 7.0 25.2 T 1.7

PT, s Placebo 13.2 T 0.2 17.6 T 0.5 25.6 T 2.1

CMT-3 13.4 T 0.2 16.4 T 0.3 18.4 T 1.3* †

INR Placebo 1.0 T 0.02 1.4 T 0.1 2.3 T 0.3

CMT-3 1.0 T 0.2 1.3 T 0.1 1.5 T 0.3* †

WBC, �103/L Placebo 11.6 T 0.9 10.7 T 1.4 5.1 T 3.3

CMT-3 15.8 T 1.3 * 17.9 T 1.2 * 12.8 T 3.3 †

Platelets, �103/L Placebo 303.9 T 43.7 128.0 T 17.4 73.8 T 16.2

CMT-3 365.3 T 19.8 226.1 T 32.8* 161.6 T 33.3* †

Total protein, g/dL Placebo 5.5 T 0.1 2.66 T 0.2 3.1 T 0.1

CMT-3 5.4 T 0.2 3.5 T 0.2 3.3 T 0.3

Albumin, g/dL Placebo 3.3 T 0.6 1.7 T 0.1 1.2 T 0.2

CMT-3 3.4 T 0.1 1.9 T 0.2 2.0 T 0.2

AST, U/L Placebo 23.0 T 2.5 115.4 T 41.2 124.5 T 46.5

CMT-3 29.5 T 7.2 81.4 T 17.1 187.8 T 93.2

Creatinine, g/dL Placebo 0.95 T 0.02 0.97 T 0.14 1.37 T 0.47

CMT-3 0.84 T 0.02 0.80 T 0.06 0.91 T 0.10

*P G 0.05 following post hoc analysis with Tukey test.†P G 0.05 following RM ANOVA.PTT indicates partial thromboplastin time; PT, prothrombin time; WBC, white blood cell count; AST, aspartate aminotransferase.

FIG. 6. Effects of CMT-3 on intestinal histopathology. Lu indicates intestinal lumen; Vi, villus; mm, muscularis mucosa; Sm, smooth muscle; Pp, Peyerpatch; me, muscularis externa. Left, ‘‘Naive pig’’ is a naive animal used as a histological control. Center, Representative section of midjejunum in a vehicle-treated animal demonstrating grossly shortened and denuded villi with significant sloughing of surface epithelium into the bowel lumen; intestinal glands showedareas devoid of cellular definition, suggesting loss of the epithelial barrier. Right, Representative section of midjejunum in a CMT-3Ytreated animal demonstratingnormal length villi with only focal denudation of the surface epithelium; intestinal glands were normal in this group.


ketamine may have acted via nuclear factor .B blockade to

reduce overall MMP levels. Lastly, it is possible that the

absence of MMP activity may not be related to ketamine but

rather to a CMT-3Yinduced reduction in the overall inflam-

matory status, such that MMPs were not upregulated and

released in the same concentrations as in previous studies (9).

Effects of CMT-3 on hematologic and coagulation function

The coaguloprotective effect of CMT-3 was unanticipated

(Table 7). Previous studies have noted a connection between

coagulopathy and ARDS in sepsis (24). A recent review

observed that disordered coagulation likely underlies the

alveolar fibrin deposition that is characteristic of ARDS (25).

In the present study, CMT-3 decreased alveolar fibrin deposi-

tion (Table 6), suggesting that modulating the coagulation

system may be yet another mechanism by which CMT-3 re-

duces lung injury.

The most important mechanism of sepsis-associated coa-

gulopathy is the direct alteration of the coagulation cascade

and attendant thrombocytopenia (26); CMT-3 prevented the

direct alteration in the coagulation cascade, evidenced by

normalization of prothrombin time and INR and the prevention

of thrombocytopenia (Table 7). The mechanisms underlying

these novel actions of CMT-3 are unknown, and further studies

are necessary.

Adequate platelet number and function are both necessary

for normal coagulation (27). Therefore, the ability of CMT-3

to prevent thrombocytopenia may be the central mechanism by

which this drug preserves normal coagulation. Additional

mechanisms may also underlie the coaguloprotective effects

of CMT-3. Three enzymes essential for platelet activation are

cyclooxygenase 2, thromboxane A2, and phospholipase A2

(27). Chemically modified tetracycline 3 is known to act on

cyclooxygenase 2 to inhibit both nitric oxide and prostaglan-

din E2 (28), which are essential to platelet activation and

degranulation.

Effects of CMT-3 on the gut

Kubiak et al. (11) recently showed that this animal model

causes injury not only to the lung but also to the kidney, liver,

and intestine. Chemically modified tetracycline 3 reduced

bowel histopathology as compared with placebo (Fig. 6). In

our animal model, the intestines are the organ system receiving

the most serious insult. The sepsis and IR injuries both target

the abdomen; the severe intestinal histopathology seen in the

placebo group reflects this. The prevention of detectable bowel

damage by CMT-3 provides compelling preliminary evidence

for this drug’s efficacy as an antiYsystemic inflammatory

response syndromeYinduced organ damage pharmacotherapy.

The mechanism by which CMT-3 protects the bowels is as

yet unknown, but might be related to the known effects of

CMT-3 on TNF-! (29). Monoclonal antiYTNF-! antibody

(Remicade) is the standard of care for reduction of intestinal

inflammation in the management of inflammatory bowel dis-

ease (30). The CMT-3Yinduced reduction in plasma TNF-!could be responsible for the protection of the bowel. Further

studies are needed to determine if the reduction of systemic or

local TNF-! by CMT-3 is the mechanism for bowel protection

by this drug.

SUMMARY

In summary, this study demonstrates that the modified tetra-

cycline CMT-3, given 1 h after injury, will prevent the devel-

opment of ARDS and intestinal histopathology in a clinically

relevant porcine model of established sepsis and gut IR. The

mechanism of this protection may involve a reduction in the

circulating levels of TNF-! and IL-1". However, CMT-3 also

prevented thrombocytopenia and reduced the coagulopathy

associated with this animal model, mechanisms that may also

play a role in the protective effect of CMT-3 to the lung and gut.

Thus, CMT-3 or related compounds may represent novel ther-

apies for prevention of ARDS.

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Chemically Modified Tetracycline 3 Prevents Acute Respiratory Distress Syndrome in a Porcine Model of Sepsis + Ischemia/Reperfusion–Induced Lung Injury

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