Lung Biomolecular Profile and Function of Grafts from Donors after Cardiocirculatory Death with Prolonged Donor Warm Ischemia Time.

The acceptable duration of donor warm ischemia time (DWIT) after cardiocirculatory death (DCD) is still debated. We analyzed the biomolecular profile and function during ex vivo lung perfusion (EVLP) of DCD lungs and their correlation with lung transplantation (LuTx) outcomes. Donor data, procurement times, recipient outcomes, and graft function up to 1 year after LuTx were collected. During EVLP, the parameters of graft function and metabolism, perfusate samples to quantify inflammation, glycocalyx breakdown products, coagulation, and endothelial activation markers were obtained. Data were compared to a cohort of extended-criteria donors after brain death (EC-DBD). Eight DBD and seven DCD grafts transplanted after EVLP were analyzed. DCD's DWIT was 201 [188;247] minutes. Donors differed only regarding the duration of mechanical ventilation that was longer in the EC-DBD group. No difference was observed in lung graft function during EVLP. At reperfusion, "wash-out" of inflammatory cells and microthrombi was predominant in DCD grafts. Perfusate biomolecular profile demonstrated marked endothelial activation, characterized by the presence of inflammatory mediators and glycocalyx breakdown products both in DCD and EC-DBD grafts. Early graft function after LuTx was similar between DCD and EC-DBD. DCD lungs exposed to prolonged DWIT represent a potential resource for donation if properly preserved and evaluated.

Effluent Molecular Analysis Guides Liver Graft Allocation to Clinical Hypothermic Oxygenated Machine Perfusion.

Hypothermic-oxygenated-machine-perfusion (HOPE) allows assessment/reconditioning of livers procured from high-risk donors before transplantation. Graft referral to HOPE mostly depends on surgeons' subjective judgment, as objective criteria are still insufficient. We investigated whether analysis of effluent fluids collected upon organ flush during static-cold-storage can improve selection criteria for HOPE utilization. Effluents were analyzed to determine cytolysis enzymes, metabolites, inflammation-related mediators, and damage-associated-molecular-patterns. Molecular profiles were assessed by unsupervised cluster analysis. Differences between "machine perfusion (MP)-yes" vs. "MP-no"; "brain-death (DBD) vs. donation-after-circulatory-death (DCD)"; "early-allograft-dysfunction (EAD)-yes" vs. "EAD-no" groups, as well as correlation between effluent variables and transplantation outcome, were investigated. Livers assigned to HOPE (n = 18) showed a different molecular profile relative to grafts transplanted without this procedure (n = 21, p = 0.021). Increases in the inflammatory mediators PTX3 (p = 0.048), CXCL8/IL-8 (p = 0.017), TNF-α (p = 0.038), and ANGPTL4 (p = 0.010) were observed, whereas the anti-inflammatory cytokine IL-10 was reduced (p = 0.007). Peculiar inflammation, cell death, and coagulation signatures were observed in fluids collected from DCD livers compared to those from DBD grafts. AST (p = 0.034), ALT (p = 0.047), and LDH (p = 0.047) were higher in the "EAD-yes" compared to the "EAD-no" group. Cytolysis markers and hyaluronan correlated with recipient creatinine, AST, and ICU stay. The study demonstrates that effluent molecular analysis can provide directions about the use of HOPE.

NDP-MSH treatment recovers marginal lungs during ex vivo lung perfusion (EVLP).

The increasing use of marginal lungs for transplantation encourages novel approaches to improve graft quality. Melanocortins and their receptors (MCRs) exert multiple beneficial effects in pulmonary inflammation. We tested the idea that treatment with the synthetic α-melanocyte-stimulating hormone analogue [Nle4,D-Phe7]-α-MSH (NDP-MSH) during ex vivo lung perfusion (EVLP) could exert positive influences in lungs exposed to different injuries. Rats were assigned to one of the following protocols (N = 10 each): 1) ischemia/reperfusion (IR) or 2) cardiac death (CD) followed by ex vivo perfusion. NDP-MSH treatment was performed in five rats of each protocol before lung procurement and during EVLP. Pulmonary function and perfusate concentration of gases, electrolytes, metabolites, nitric-oxide, mediators, and cells were assessed throughout EVLP. ATP content and specific MCR expression were investigated in perfused lungs and in biopsies collected from rats in resting conditions (Native, N = 5). NDP-MSH reduced the release of inflammatory mediators in perfusates of both the IR and the CD groups. Treatment was likewise associated with a lesser amount of leukocytes (IR: p = 0.034; CD: p = 0.002) and reduced lactate production (IR: p = 0.010; CD: p = 0.008). In lungs exposed to IR injury, the NDP-MSH group showed increased ATP content (p = 0.040) compared to controls. In CD lungs, a significant improvement of vascular (p = 0.002) and airway (Ppeak: p < 0.001, compliance: p < 0.050, pO2: p < 0.001) parameters was observed. Finally, the expression of MC1R and MC5R was detected in both native and ex vivo-perfused lungs. The results indicate that NDP-MSH administration preserves lung function through broad positive effects on multiple pathways and suggest that exploitation of the melanocortin system during EVLP could improve reconditioning of marginal lungs before transplantation.

Mesenchymal stem cell-derived extracellular vesicles improve the molecular phenotype of isolated rat lungs during ischemia/reperfusion injury.

BACKGROUND: Lung ischemia/reperfusion (IR) injury contributes to the development of severe complications in patients undergoing transplantation. Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) exert beneficial actions comparable to those of MSCs without the risks of the cell-based strategy. This research investigated EV effects during IR injury in isolated rat lungs. METHODS: An established model of 180-minutes ex vivo lung perfusion (EVLP) was used. At 60 minutes EVs (n = 5) or saline (n = 5) were administered. Parallel experiments used labeled EVs to determine EV biodistribution (n = 4). Perfusate samples were collected to perform gas analysis and to assess the concentration of nitric oxide (NO), hyaluronan (HA), inflammatory mediators, and leukocytes. Lung biopsies were taken at 180 minutes to evaluate HA, adenosine triphosphate (ATP), gene expression, and histology. RESULTS: Compared with untreated lungs, EV-treated organs showed decreased vascular resistance and a rise of perfusate NO metabolites. EVs prevented the reduction in pulmonary ATP caused by IR. Increased medium-high-molecular-weight HA was detected in the perfusate and in the lung tissue of the IR + EV group. Significant differences in cell count on perfusate and tissue samples, together with induction of transcription and synthesis of chemokines, suggested EV-dependent modulation of leukocyte recruitment. EVs upregulated genes involved in the resolution of inflammation and oxidative stress. Biodistribution analysis showed that EVs were retained in the lung tissue and internalized within pulmonary cells. CONCLUSIONS: This study shows multiple novel EV influences on pulmonary energetics, tissue integrity, and gene expression during IR. The use of cell-free therapies during EVLP could constitute a valuable strategy for reconditioning and repair of injured lungs before transplantation.

Effects of sodium citrate, citric acid and lactic acid on human blood coagulation.

INTRODUCTION: Citric acid infusion in extracorporeal blood may allow concurrent regional anticoagulation and enhancement of extracorporeal CO2 removal. Effects of citric acid on human blood thromboelastography and aggregometry have never been tested before. METHODS: In this in vitro study, citric acid, sodium citrate and lactic acid were added to venous blood from seven healthy donors, obtaining concentrations of 9 mEq/L, 12 mEq/L and 15 mEq/L. We measured gas analyses, ionized calcium (iCa++) concentration, activated clotting time (ACT), thromboelastography and multiplate aggregometry. Repeated measure analysis of variance was used to compare the acidifying and anticoagulant properties of the three compounds. RESULTS: Sodium citrate did not affect the blood gas analysis. Increasing doses of citric and lactic acid progressively reduced pH and HCO3- and increased pCO2 (p<0.001). Sodium citrate and citric acid similarly reduced iCa++, from 0.39 (0.36-0.39) and 0.35 (0.33-0.36) mmol/L, respectively, at 9 mEq/L to 0.20 (0.20-0.21) and 0.21 (0.20-0.23) mmol/L at 15 mEq/L (p<0.001). Lactic acid did not affect iCa++ (p=0.07). Sodium citrate and citric acid similarly incremented the ACT, from 234 (208-296) and 202 (178-238) sec, respectively, at 9 mEq/L, to >600 sec at 15 mEq/L (p<0.001). Lactic acid did not affect the ACT values (p=0.486). Sodium citrate and citric acid similarly incremented R-time and reduced α-angle and maximum amplitude (MA) (p<0.001), leading to flat-line thromboelastograms at 15 mEq/L. Platelet aggregometry was not altered by any of the three compounds. CONCLUSIONS: Citric acid infusions determine acidification and anticoagulation of blood similar to lactic acid and sodium citrate, respectively.

Influence of ex vivo perfusion on the biomolecular profile of rat lungs.

Despite increasing clinical adoption, biologic influences of ex vivo lung perfusion (EVLP) remain insufficiently elucidated. The aim of the current study was to investigate biomolecular changes induced by EVLP in rat lungs. EVLP was maintained for 180 min. Hyaluronan, mediators, and cells were assessed in the perfusate. Gene expression, signaling pathways, and ATP content were investigated in lung tissue. EVLP induced the release of medium-high molecular weight hyaluronan and transcription of hyaluronan synthases ( P < 0.001). Increasing concentrations of inflammatory mediators were detected in the perfusate ( P < 0.001). Perfused lungs exhibited a distinctive transcriptional signature compared with organs examined before or after surgery/procurement ( P = 0.003). Up-regulated genes were involved in inflammation and its regulation, apoptosis/survival, heat shock, and oxidative stress response ( q = 0). Down-regulated genes were related to lymphocyte function ( q = 0). The NF-κB, signal transducer and activator of transcription 3, ERK1/2, p38, Akt, and stress-activated protein kinase/JNK signaling pathways were modulated by EVLP ( P < 0.05). Most of these biomolecular changes were examined and confirmed in additional experiments that were performed in lungs procured from donation after cardiocirculatory death after 180 min of warm ischemia. The current study demonstrates that EVLP broadly affects the lung biomolecular phenotype. These findings improve our comprehension of the effects exerted by the procedure and encourage additional research in preclinical models to implement therapeutic interventions.-Lonati, C., Bassani, G. A., Brambilla, D., Leonardi, P., Carlin, A., Faversani, A., Gatti, S., Valenza, F. Influence of ex vivo perfusion on the biomolecular profile of rat lungs.

A standardized model of brain death, donor treatment, and lung transplantation for studies on organ preservation and reconditioning.

BACKGROUND: We set a model of brain death, donor management, and lung transplantation for studies on lung preservation and reconditioning before transplantation. METHODS: Ten pigs (39.7 ± 5.9 Kg) were investigated. Five animals underwent brain death and were treated as organ donors; the lungs were then procured and cold stored (Ischemia). Five recipients underwent left lung transplantation and post-reperfusion follow-up (Graft). Cardiorespiratory and metabolic parameters were collected. Lung gene expression of cytokines (tumor necrosis factor alpha (TNFα), interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), interferon gamma (IFNγ), high mobility group box-1 (HMGB-1)), chemokines (chemokine CC motif ligand-2 (CCL2-MCP-1), chemokine CXC motif ligand-10 (CXCL-10), interleukin-8 (IL-8)), and endothelial activation markers (endothelin-1 (EDN-1), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), selectin-E (SELE)) was assessed by real-time polymerase chain reaction (PCR). RESULTS: Tachycardia and hypertension occurred during brain death induction; cardiac output rose, systemic vascular resistance dropped (P < 0.05), and diabetes insipidus occurred. Lung-protective ventilation strategy was applied: 9 h after brain death induction, PaO2 was 192 ± 12 mmHg at positive end-expiratory pressure (PEEP) 8.0 ± 1.8 cmH2O and FiO2 of 40%; wet-to-dry ratio (W/D) was 5.8 ± 0.5, and extravascular lung water (EVLW) was 359 ± 80 mL. Procured lungs were cold-stored for 471 ± 24 min (Ischemia) at the end of which W/D was 6.1 ± 0.9. Left lungs were transplanted and reperfused (warm ischemia 98 ± 14 min). Six hours after controlled reperfusion, PaO2 was 192 ± 23 mmHg (PEEP 8.7 ± 1.5 cmH2O, FiO2 40%), W/D was 5.6 ± 0.4, and EVLW was 366 ± 117 mL. Levels of IL-8 rose at the end of donor management (BD, P < 0.05); CCL2-MCP-1, IL-8, HMGB-1, and SELE were significantly altered after reperfusion (Graft, P < 0.05). CONCLUSIONS: We have set a standardized, reproducible pig model resembling the entire process of organ donation that may be used as a platform to test in vivo and ex vivo strategies of donor lung optimization before transplantation.

Modulatory effects of NDP-MSH in the regenerating liver after partial hepatectomy in rats.

Melanocortins are endogenous peptides that exert protective actions on the host physiology. The broad modulatory effects of these molecules suggest that they might influence the mediator network induced during liver regeneration. The research aim was to determine if melanocortin treatment alters liver molecular changes induced by partial hepatectomy (PH). Rats under isoflurane anesthesia were subjected to standard 70% PH or sham surgery. Animals received a single i.v. injection of Nle4, DPhe7-α-melanocyte stimulating hormone (NDP-MSH) or saline 30 min before surgery. Sacrifice was performed at time intervals between 4 and 72 h. A preliminary screening based on TaqMan low-density array (TLDA) identified 71 transcripts altered by PH. Real-time PCR analysis revealed that NDP-MSH modulated the expression of a substantial proportion of these transcripts including several chemokines and their receptors. The critical signaling pathway interleukin-6 (IL-6)/signal transducer and activator of transcription (STAT)/suppressor of cytokine signaling (SOCS) was significantly enhanced by NDP-MSH. Further, peptide treatment considerably reduced the decline of IκBα protein caused by PH. Although the final organ regeneration was not substantially affected, NDP-MSH modulated expression of cell cycle mediators and exerted subtle influences on hepatocyte replication. Most of the changes brought about by NDP-MSH, a peptide approved for clinical use, should be salutary during liver regeneration.

Protective action of NDP-MSH in experimental subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is still a major cause of morbidity and mortality. α-Melanocyte stimulating hormone (α-MSH) and other melanocortin peptides exert potent neuroprotective action and they might modulate key molecules involved in SAH-induced vasospasm. The aim of this research was to determine whether treatment with the α-MSH analog Nle4,DPhe7-α-MSH (NDP-MSH) exerts protective effects in experimental SAH in the rat. Initial experiments examined effects of NDP-MSH on the basilar artery phenotype in the absence of injury. In these tests intrathecal injection of small concentrations (10ng) of the peptide induced a tolerant phenotype similar to that observed after ischemic preconditioning. Then the effect of systemic treatment with NDP-MSH (100µg i.v.) on experimental SAH was evaluated. SAH was induced by a single-blood injection into the cisterna magna. The basilar artery phenotype was examined at 4h and the artery caliber at 5days following SAH. Expression of 96 genes was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) using Custom Taqman Low-Density Arrays. Four hours after SAH, the transcriptional profile of the basilar artery was deeply disrupted. Transcript alteration included genes involved in inflammation, stress response, apoptosis, and vascular remodeling. Treatment with NDP-MSH prevented most of these transcription changes and decreased phosphorylation of extracellular-signal-regulated kinases (ERK1/2) and inhibitor protein IκBα. Vasospasm on day 5 was significantly reduced by NDP-MSH administration. These results combine with others on CNS inflammation to suggest that the melanocortins could be safe and effective therapeutic candidates to treat SAH-related complications.

Molecular changes induced in rat liver by hemorrhage and effects of melanocortin treatment.

BACKGROUND: Melanocortin peptides improve hemodynamic parameters and prevent death during severe hemorrhagic shock. In the present research we determined influences of a synthetic melanocortin 1/4 receptor agonist on the molecular changes that occur in rat liver during hemorrhage. METHODS: Controlled-volume hemorrhage was performed in adult rats under general anesthesia by a stepwise blood withdrawal until mean arterial pressure fell to 40 mmHg. Then rats received either saline or the synthetic melanocortin 1/4 receptor agonist Butir-His-D-Phe-Arg-Trp-Sar-NH2 (Ro27-3225; n = 6-8 per group). Hemogasanalysis was performed throughout a 60-min period. Gene expression in liver samples was determined at 1 or 3 h using quantitative real-time polymerase chain reaction. RESULTS: At 1 h, in saline-treated shocked rats, there were significant increases in activating transcription factor 3 (Atf3), early growth response 1 (Egr1), heme oxygenase (decycling) 1 (Hmox1), FBJ murine osteosarcoma viral oncogene homolog (Fos), and jun oncogene (Jun). These changes were prevented by Ro27-3225 (mean ± SEM: Atf3 152.83 ± 58.62 vs. 579.00 ± 124.13, P = 0.002; Egr1 13.21 ± 1.28 vs. 26.63 ± 1.02, P = 0.001; Hmox1 3.28 ± 0.31 vs. 166.54 ± 35.03, P = 0.002; Fos 4.36 ± 1.03 vs. 14.90 ± 3.44, P < 0.001; Jun 6.62 ± 1.93 vs. 15.07 ± 2.09, P = 0.005; respectively). Increases in alpha-2-macroglobulin (A2m), heat shock 70kD protein 1A (Hspa1a), erythropoietin (Epo), and interleukin-6 (Il6) occurred at 3 h in shocked rats and were prevented by Ro27-3225 treatment (A2m 6.90 ± 0.82 vs. 36.73 ± 4.00, P < 0.001; Hspa1a 10.34 ± 3.28 vs. 25.72 ± 3.64, P = 0.001; Epo 0.49 ± 0.13 vs. 2.37 ± 0.73, P = 0.002; Il6 1.05 ± 0.15 vs. 1.88 ± 0.23, P < 0.001; respectively). Further, at 3 h in shocked rats treated with Ro27-3225 there were significant increases in tight junction protein 1 (Tjp1; 27.30 ± 2.43 vs. 5.03 ± 1.68, P < 0.001) and nuclear receptor subfamily 4, group A, member 1 (Nr4a1; 91.03 ± 16.20 vs. 30.43 ± 11.0, P = 0.01) relative to sham animals. Treatment with Ro27-3225 rapidly restored blood pressure, hemogasanalysis parameters, and lactate blood levels. CONCLUSIONS: Melanocortin treatment significantly prevents most of the systemic and hepatic detrimental changes induced by hemorrhage.

The melanocortin system in control of inflammation.

Melanocortin peptides, the collective term for alpha-, beta-, and gamma-melanocyte-stimulating hormone (alpha-, beta-, gamma-MSH) and adrenocorticotropic hormone (ACTH), are elements of an ancient modulatory system. Natural melanocortins derive from the common precursor pro-opiomelanocortin (POMC). Five receptor subtypes for melanocortins (MC1-MC5) are widely distributed in brain regions and in peripheral cells. Melanocortin receptor activation by natural or synthetic ligands exerts marked anti-inflammatory and immunomodulatory effects. The anticytokine action and the inhibitory influences on inflammatory cell migration make melanocortins potential new drugs for treatment of inflammatory disorders. Effectiveness in treatment of acute, chronic, and systemic inflammatory disorders is well documented in preclinical studies. Further, melanocortins are promising compounds in neuroprotection. This review examines the main signaling circuits in anti-inflammatory and immunomodulatory actions of melanocortins, and the potential therapeutic use of these molecules.

The peptide NDP-MSH induces phenotype changes in the heart that resemble ischemic preconditioning.

alpha-Melanocyte-stimulating hormone (alpha-MSH) is a pro-opiomelanocortin (POMC)-derived peptide that exerts multiple protective effects on host cells. Previous investigations showed that treatment with alpha-MSH or synthetic melanocortin agonists reduces heart damage in reperfusion injury and transplantation. The aim of this preclinical research was to determine whether melanocortin treatment induces preconditioning-like cardioprotection. In particular, the plan was to assess whether melanocortin administration causes phenotype changes similar to those induced by repetitive ischemic events. The idea was conceived because both ischemic preconditioning and melanocortin signaling largely depend on cAMP response element binding protein (CREB) phosphorylation. Rats received single i.v. injections of 750microg/kg of the alpha-MSH analogue Nle(4),DPhe(7)-alpha-MSH (NDP-MSH) or saline and were sacrificed at 0.5, 1, 3, or 5h. Western blot analysis showed that rat hearts expressed melanocortin 1 receptor (MC1R) protein. Treatment with NDP-MSH was associated with early and marked increase in interleukin 6 (IL-6) mRNA. This was followed by signal transducer and activator of transcription 3 (STAT3) phosphorylation and induction of suppressor of cytokine signaling 3 (SOCS3). There were no changes in expression of other cytokines of the IL-6 family. Expression of IL-10, IL-1beta, and TNF-alpha was likewise unaltered. In hearts of rats treated with NDP-MSH there was increased expression of the orphan nuclear receptor Nur77. The data indicate that NDP-MSH induces phenotype changes that closely resemble ischemic preconditioning and likely contribute to its established protection against reperfusion injury. In addition, the increased expression of Nur77 and SOCS3 could be part of a broader anti-inflammatory effect.

Treatment with alpha-melanocyte stimulating hormone preserves calcium regulatory proteins in rat heart allografts.

Prevention of graft dysfunction is a major objective in transplantation medicine. Previous research on experimental heart transplantation indicated that treatment with the immunomodulatory peptide alpha-melanocyte stimulating hormone (alpha-MSH) improves histopathology, prolongs allograft survival, and reduces expression of the main tissue injury mediators. Because calcium-handling is critical in heart graft function, we determined the effects of transplantation injury and influences of alpha-MSH treatment on representative calcium regulatory proteins in rat heart allografts. Hearts from Brown Norway rats were transplanted heterotopically into MHC incompatible Lewis rats. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), protein kinase C epsilon (PKC epsilon), sarcoplasmic/endoplasmic reticulum calcium-ATPase 2 (SERCA2a), arrestin-beta1 (Arrb1), cholinergic receptor M2 (Chrm2), and inositol 1,4,5-triphosphate receptor 1 (InsP(3)R1) were examined in: (1) non-transplanted donor hearts; (2) allografts from saline-treated rats; and (3) allografts from rats treated with the synthetic alpha-MSH analog Nle4-DPhe7-alpha-MSH (NDP-alpha-MSH) (100 microg i.p. every 12h). Transplantation injury was associated with severe reduction in calcium regulatory protein transcription and expression level. NDP-alpha-MSH administration partly reversed inhibition of protein transcription and almost completely prevented protein loss. Finally, because certain effects of cyclic 3'-5'-adenosine monophosphate (cAMP) signaling on calcium handling in cardiac myocytes depend on activation of exchange protein directly activated by cAMP 1 (Epac1), we determined Epac1 mRNA and protein expression in heart allografts. Transplantation injury markedly reduced Epac1. NDP-alpha-MSH treatment significantly preserved both Epac1 protein and mRNA in the allografts. Administration of alpha-MSH or related melanocortins could reduce transplantation-induced dysfunction through protection of heart calcium regulatory proteins.

Inhibitory effects of the peptide (CKPV)2 on endotoxin-induced host reactions.

BACKGROUND: alpha-Melanocyte stimulating hormone (alpha-MSH) is an endogenous peptide that has remarkable anti-inflammatory and antimicrobial effects. These activities have been traced to the C-terminal tripeptide Lys-Pro-Val (KPV). A dimer composed of two KPV sequences connected with a Cys-Cys linker, (CKPV)2, is currently under clinical investigation for antimicrobial use. The present research was designed to evaluate effects of (CKPV)(2) on endotoxin-induced host reactions in vitro and in vivo. MATERIALS AND METHODS: Effects of (CKPV)2, KPV, and [Nle4-dPhe7]-alpha-MSH (NDP-alpha-MSH) on tumor necrosis factor alpha (TNF-alpha) production were determined: 1) in human peripheral blood mononuclear cells (PBMC) stimulated with lipopolysaccharide (LPS) in vitro, and 2) in rats injected with LPS i.v. and sacrificed at 1 h. In additional experiments, dialysis peritonitis was induced in rats by adding LPS to dialysis fluid. Net ultrafiltrate was calculated and concentrations of nitrite (NO2-) and TNF-alpha were measured in blood and peritoneal fluid at 7 h. RESULTS: (CKPV)2 inhibited TNF-alpha production by LPS-stimulated human PBMC. This small peptide was as effective as NDP-alpha-MSH and more potent than KPV. Similar effectiveness was observed in vivo: 1 h after LPS injection, the large increase in circulating TNF-alpha was markedly reduced by (CKPV)2 treatment. In LPS-induced peritonitis, (CKPV)2 restored net ultrafiltrate to control values and significantly inhibited concentrations of TNF-alpha and NO2- both in plasma and in dialysate. CONCLUSIONS: The remarkable capacity of (CKPV)2 to inhibit endotoxin-induced host reactions suggests that it may be useful in treatment of inflammatory disorders.