Organ blood flow after partial hepatectomy in rats: modification by endotoxin-neutralizing bactericidal/permeability-increasing protein (rBPI23)

BACKGROUND/AIM: Both maintenance of adequate perfusion and regeneration of the remnant liver are important in the recovery of liver function after partial hepatectomy. In previous experiments, we have shown that profound hypotension and liver injury can be attenuated by neutralizing endotoxins. The relative contribution of endotoxemia to changes in liver blood flow and blood flow to other major organs after partial hepatectomy is not known. The aim of this study was to examine the effect of endotoxin neutralization on individual organ blood flows including hepatic artery and splanchnic blood flow after experimental partial hepatectomy and its relation to liver cell proliferation. METHODS: Male Wistar rats underwent either two-thirds partial hepatectomy or sham operation. Treatment consisted of continuous infusion of recombinant N-terminal bactericidal/permeability-increasing protein (rBPI23) or control protein. At 4 h after surgery, organ blood flows were measured using the radiolabeled microsphere technique, and at 24 h, proliferation index in liver tissue was calculated. RESULTS: After partial hepatectomy, blood flows to virtually all organs were significantly lower as compared to values obtained in sham-operated rats. rBPI23 greatly improved hepatic artery flow (p<0.001) but not portal venous flow. These effects of rBPI23 on liver flow preceded an equally enhanced liver cell proliferation (p<0.01). Endotoxin neutralization led to significantly higher flows to some but not all splanchnic organs. Lung perfusion was significantly improved by rBPI23. CONCLUSIONS: Neutralization of endogenous endotoxins improves liver blood flow after partial hepatectomy and also periportal and pericentral liver cell proliferation. This proliferation effect may result from an increased hepatic artery flow. Lung, colon, spleen and pancreas flow but not kidney flow was greatly enhanced by rBPI23.     

Effect of the administration of recombinant hirudin and/or tissue-plasminogen activator (t-PA) on endotoxin-induced disseminated intravascular coagulation model in rabbits

We evaluated the effect of r-hirudin and/or tissue-plasminogen activator (t-PA) in a model of DIC in rabbits induced by i.v. infusion of 100 micrograms/kg/h/6 h endotoxin. Rabbits were treated with saline (endotoxin control group), r-hirudin at 0.3 mg/kg/h/6 h, t-PA at 0.3 mg/kg for 90 min and r-hirudin plus t-PA at the doses described above. The best results were achieved when r-hirudin and t-PA were infused together. This treatment reduced the consumption of platelets and protein C and attenuated the increase of PAI-1 more efficiently than r-hirudin or t-PA alone. r-Hirudin plus t-PA also resulted in the lowest formation of fibrin deposits in the kidneys. Finally, mortality at 24 h dropped from 70% in the endotoxin control group to 40%, 10% and 0% in the t-PA, r-hirudin and r-hirudin plus t-PA groups respectively. None of the t-PA-infused rabbits which had died by 24 h showed macroscopic signs of haemorrhage. r-Hirudin alone was better than t-PA alone, as was shown by fibrin deposits and mortality. We conclude that r-hirudin and t-PA given simultaneously were more efficient than either given alone in this model of DIC. Effective thrombin inhibition, which could influence other pathophysiological mechanisms apart from coagulation, together with the improvement in fibrinolysis, would explain these results.     

Aggravation of endotoxin-induced disseminated intravascular coagulation and cytokine activation in heterozygous protein-C-deficient mice

In the pathogenesis of sepsis and disseminated intravascular coagulation (DIC), dysfunctional anticoagulant pathways are important. The function of the protein C system in DIC is impaired because of low levels of protein C and down-regulation of thrombomodulin. The administration of (activated) protein C results in an improved outcome in experimental and clinical studies of DIC. It is unknown whether congenital deficiencies in the protein C system are associated with more severe DIC. The aim of the present study was to investigate the effect of a heterozygous deficiency of protein C on experimental DIC in mice. Mice with single-allele targeted disruption of the protein C gene (PC+/-) mice and wild-type littermates (PC+/+) were injected with Escherichia coli endotoxin (50 mg/kg) intraperitoneally. PC+/-mice had more severe DIC, as evidenced by a greater decrease in fibrinogen level and a larger drop in platelet count. Histologic examination showed more fibrin deposition in lungs, kidneys, and liver in mice with a heterozygous deficiency of protein C. Interestingly, PC+/- mice had significantly higher levels of proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and IL-1beta, indicating an interaction between the protein C system and the inflammatory response. Survival was lower at 12 and 24 hours after endotoxin in the PC+/- mice. These results confirm the important role of the protein C system in the coagulative-inflammatory response on endotoxemia and may suggest that congenital deficiencies in the protein C system are associated with more severe DIC and adverse outcome in sepsis.     

rBPI(10-193) is secreted by CHO cells and retains the activity of rBPI21

rBPI23, a recombinant N-terminal fragment of human bactericidal/permeability-increasing protein (BPI), kills Gram-negative bacteria and neutralizes endotoxin. rBPI21, a variant in which cysteine 132 is changed to alanine, retains the activities of rBPI23. Analysis of certain purified rBPI21 preparations revealed that some of the molecules had lost nine amino acids from the amino terminus. To explore the effect of this modification on structure and activity, we cloned and expressed a variant of rBPI21, designated rBPI(10-193), which lacks the first nine amino acids. A monoclonal antibody believed to recognize the amino terminus of rBPI21 cross-reacted with rBPI21, but not with rBPI(10-193) or full length recombinant BPI (rBPI). These results demonstrated that the antibody recognizes the first nine amino acids of rBPI21 and that this region of the holoprotein (rBPI) is inaccessible to the antibody (as suggested by the known 3-D structure). Purified rBPI(10-193) and rBPI21 were similarly potent in in vitro assays measuring bactericidal, endotoxin binding and neutralization activities. In a mouse model of lethal bacteremia, rBPI(10-193) and rBPI21 were similarly potent whereas in a mouse endotoxin challenge model, rBPI(10-193) appeared to be at least 2-fold more potent than rBPI21. In conscious rats, a rapid bolus dose of 40 mg/kg of rBPI21 caused a significant transient decrease in blood pressure while the same dose of rBPI(10-193) caused no blood pressure decrease. We conclude from these studies that the first nine amino acids of rBPI21 are not essential for the anti-infective and endotoxin-neutralizing activities of BPI.     

Neutrophil response to Neisseria meningitidis: inhibition of adhesion molecule expression and phagocytosis by recombinant bactericidal/permeability-increasing protein (rBPI21)

Polymorphonuclear neutrophil (PMNL) activation enhances microbial clearance but also contributes to the vascular damage and multiorgan failure associated with severe meningococcal sepsis. By use of a whole blood model of meningococcal bacteremia, loss of PMNL L-selectin and up-regulation of CD11b was observed in response to Neisseria meningitidis serogroups B and C, which is followed by opsonophagocytosis. PMNL priming with either Escherichia coli lipopolysaccharide (LPS) or FMLP prior to meningococcal challenge resulted in enhancement of both PMNL L-selectin shedding (1.5- to 4-fold) and phagocytosis (2- to 3-fold). Blockade of meningococcal LPS lipid A with recombinant bactericidal/permeability-increasing protein (rBPI21) resulted in partial inhibition of the PMNL activation and phagocytosis response to N. meningitidis. The effect of rBPI21 was reversed by excess E. coli LPS or FMLP. It is proposed that PMNL priming by N. meningitidis results in an exaggerated activation and phagocytosis response to the organism.     

Serum crosslinked fibrin (XDP) and fibrinogen/fibrin degradation products (FDP) in disorders associated with activation of the coagulation or fibrinolytic systems

Soluble crosslinked fibrin derivatives (XDP) in serum were determined by enzyme immunoassay utilizing monoclonal antibodies and compared with serum fibrinogen/fibrin degradation products (FDP) assayed by conventional techniques. In healthy subjects and patients with miscellaneous disorders not usually associated with activation of the haemostasis mechanism, mean XDP levels were 45 and 70 ng/ml respectively. However, elevated levels of XDP occurred in conditions commonly associated with intravascular and possibly extravascular activation of the coagulation system. Markedly raised mean XDP values (677-6900 ng/ml) occurred in treated pulmonary embolism, disseminated neoplasia, severe inflammatory disorders and complicated postoperative states, and lesser but significant elevation (mean 150-400 ng/ml) in treated venous thrombosis, uneventful postsurgical states, localized neoplasia, liver disease and symptomatic arterial disease. Levels during initial streptokinase therapy (mean 24 000 ng/ml) fell tenfold as treatment was continued. The degree of XDP elevation over normal values was significantly higher than that of FDP in conditions with a propensity for venous thrombosis (post-operative states, disseminated neoplasia and inflammatory diseases) than in liver disease, localized neoplasia or patients receiving heparin therapy for venous thromboembolism.     

Experimental endotoxemia in humans: analysis of cytokine release and coagulation, fibrinolytic, and complement pathways

Endotoxemia was evoked by bolus injection of Escherichia coli endotoxin (2 ng/kg body weight) in six healthy subjects to investigate the early kinetics of cytokine release in relation to the development of clinical and hematologic abnormalities frequently seen in gram-negative septicemia. The plasma concentration of tumor necrosis factor (TNF) increased markedly after 30 to 45 minutes, and reached a maximal level after 60 to 90 minutes. In each volunteer, the initial increase of plasma interleukin 6 (IL-6) concentrations occurred 15 minutes after the initial TNF increase, and maximal IL-6 concentrations were reached at 120 to 150 minutes. A transient increase in body temperature and pulse rate occurred simultaneously with the initial TNF and IL-6 increases, whereas a significant decrease in blood pressure occurred after 120 minutes. These changes were proportional to the changes in TNF and IL-6 concentrations. Coagulation activation, as assessed by a rise of prothrombin fragments and thrombin-antithrombin III complexes, was noted after 120 minutes, in the absence of activation of the contact system. A two- to sixfold increase in the concentrations of tissue plasminogen activator (t-PA) and von Willebrand factor antigen indicated endothelial cell activation. This increase started at 120 and 90 minutes, respectively. The release of t-PA coincided with activation of the fibrinolytic pathway, as measured by plasmin-alpha 2-antiplasmin complexes. The fibrinolytic activity of t-PA was subsequently offset by release of plasminogen activator inhibitor, observed 150 minutes after the endotoxin injection, and reaching a peak at 240 minutes. No complement activation was detected. These results show that in humans endotoxin induces an early, rapidly counteracted fibrinolytic response, and a more long-lasting activation of thrombin by a mechanism other than contact system activation. In addition, our data suggest that endotoxin-induced leukopenia and endothelial cell activation are mediated by TNF.     

Inhibition of coagulation,fibrinolysis, and endothelial cell activation by a p38 mitogen-activated protein kinase inhibitor during human endotoxemia

P38 mitogen-activated protein kinase (MAPK) is an important component of intracellular signaling cascades that initiate various inflammatory cellular responses. To determine the role of p38 MAPK in the procoagulant response to lipopolysaccharide (LPS), 24 healthy subjects were exposed to an intravenous dose of LPS (4 ng/kg), preceded 3 hours earlier by orally administered 600 or 50 mg BIRB 796 BS (a specific p38 MAPK inhibitor), or placebo. The 600-mg dose of BIRB 796 BS strongly inhibited LPS-induced coagulation activation, as measured by plasma concentrations of the prothrombin fragment F1 + 2. BIRB 796 BS also dose dependently attenuated the activation and subsequent inhibition of the fibrinolytic system (plasma tissue-type plasminogen activator, plasmin-alpha2-antiplasmin complexes, and plasminogen activator inhibitor type 1) and endothelial cell activation (plasma soluble E-selectin and von Willebrand factor). Activation of p38 MAPK plays an important role in the procoagulant and endothelial cell response after in vivo exposure to LPS.     

Effect of recombinant hirudin, a specific inhibitor of thrombin, on endotoxin-induced intravascular coagulation and acute lung injury in pigs

We hypothesized that thrombin activation may play a prominent role in endotoxin-induced secondary organ failure, such as acute lung injury. To test this hypothesis, we administered a thrombin-specific inhibitor, recombinant hirudin, in endotoxemic pigs. The pigs were anesthetized, mechanically ventilated, and prepared with Swan-Ganz and extravascular lung water (EVLW) catheters. A total of 18 randomly selected animals received a pretreatment of 1,000 U/kg of hirudin, followed by a continuous infusion over 6 h of 500 U/kg/h given simultaneously with the infusion of 10 micrograms/kg/h of Salmonella abortus equi endotoxin. Another 18 animals received a continuous infusion over 6 h of endotoxin but did not receive hirudin. All animals were fluid resuscitated with 17 ml/kg/h of saline for the duration of the experiment. Data are expressed as the mean (95% confidence interval). Hirudin reduced the endotoxin-induced consumption of plasma fibrinogen from -110 (-138 to -82) mg/100 ml to -39 (-67 to -12) mg/100 ml (p = 0.0001) and endotoxin-induced increases in the soluble fibrin in plasma from 434 (369 to 499) ng/ml to 236 (171 to 300) ng/ml (p = 0.0002). These data suggest an effective inhibition of the endotoxin-generated thrombin by hirudin. Furthermore, hirudin significantly reduced endotoxin-induced increases in pulmonary vascular resistance from 32 (27 to 37) kdyn x s x cm-5 x kg to 20 (15 to 25) kdyn x s x cm-5 x kg (p = 0.0015) and increases in EVLW from 15.4 (13.2 to 17.6) ml/kg to 12.2 (10.0 to 14.4) ml/kg (p = 0.0299).(ABSTRACT TRUNCATED AT 250 WORDS)     

Antigenicity of a recombinant Ro (SS-A) fusion protein

The antigenicity of the 60-kd human Ro (SS-A) synthesized in vitro from its complementary DNA as a β-galactosidase fusion protein (β-gal—Ro) was evaluated by Western blotting. In this analysis, almost all the anti-Ro (SS-A)-positive sera that bound β-gal-Ro also bound affinity-purified 60-kd human Ro (SS-A) (P > 0.005). Three of the 27 anti-Ro (SS-A) precipitinpositive sera, however, did not show reactivity on Western blot analysis, which suggests that in some sera, antigenicity to Ro (SS-A) is destroyed by denaturation. Of the 22 sera that were reactive with β-gal-Ro, 2 were not reactive with affinity-purified human Ro (SS-A). Two serum samples that did not react with β-gal-Ro were also reactive with affinity-purified human Ro (SS-A). Nevertheless, except for a small percentage of Ro (SS-A) precipitin-positive sera, the frequency of antibody binding to the fusion protein was similar to the frequency of binding to the purified antigen in Western blots. Recombinant Ro (SS-A) antigen may therefore be valuable in the serologic evaluation of anti-Ro (SS-A) autoantibodies.     

Safety and pharmacokinetics of a novel recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in hemophilia B patients

A recombinant fusion protein linking coagulation factor IX (FIX) with human albumin (rIX-FP) has been developed to facilitate hemophilia B treatment by less frequent FIX dosing. This first-in-human dose-escalation trial in 25 previously treated subjects with hemophilia B (FIX ≤ 2 IU/dL) examined the safety and pharmacokinetics of 25, 50, and 75 IU/kg rIX-FP. Patients in the 50-IU/kg cohort underwent a comparative pharmacokinetics assessment with their previous FIX product (plasma-derived or recombinant). No allergic reactions or inhibitors were observed. Four mild, possibly treatment-related adverse events were reported. In the 50-IU/kg cohort (13 subjects), the mean half-life of rIX-FP was 92 hours, more than 5 times longer than the subjects' previous FIX product. After 25 or 50 IU/kg rIX-FP administration, the baseline-corrected mean FIX activity remained elevated at day 7 (7.4 IU/dL and 13.4 IU/dL, respectively) and day 14 (2.5 IU/dL and 5.5 IU/dL, respectively). The incremental recovery of rIX-FP was higher than both recombinant and plasma-derived FIX (1.4 vs 0.95 and 1.1 IU/dL per IU/kg, respectively). These results demonstrated both the safety and improved pharmacokinetics of rIX-FP, thus indicating this new product with extended half-life as possibly able to control and prevent bleeding with less frequent injection. The trial was registered at www.clinicaltrials.gov as no. NCT01233440.     

Inhibition of complement activation by a secreted Staphylococcus aureus protein

Staphylococcus aureus can cause a variety of acute and chronic diseases. The ability of S. aureus to cause persistent infections has been linked to its ability to evade or inactivate host immune responses. We have identified a secreted 19-kDa protein produced by S. aureus that binds to the complement protein C3. N-terminal sequencing of this protein identified it as the extracellular fibrinogen-binding protein (Efb). In this study, we demonstrate that Efb can bind to the alpha -chain of C3 and inhibit both the classical and alternative pathways of complement activation. In addition, we show that Efb can inhibit complement-mediated opsonophagocytosis in a dose-dependent manner and that Efb inhibits complement activity by blocking deposition of C3 or by preventing further complement activation beyond C3b. These data suggest that Efb is a virulence factor involved in facilitating persistent S. aureus infections by interfering with complement activity in vivo.     

Human coagulation factor Va is a cofactor for the activation of protein C.

During blood clotting in vitro, protein C is converted in part to protein Ca, Protein Ca, in turn, inactivates factor Va. This is evidenced by the rapid inactivation of factor Va coagulant activity after clot formation which is associated with the cleavage of the Mr 110,000 peptide of factor Va. When exogenous factor Va is added to serum, it is inactivated only after a lag of 10-20 min. Using purified coagulation factors in the presence of EDTA, we demonstrated that factor Va enhances the rate of protein C activation by thrombin by 50-fold. The Km for factor Va in the reaction is 14 nM, 100 times higher than its Km for accelerating platelet surface prothrombin activation by factor Xa. By this mechanism, factor Va can act as a procoagulant as well as limit dissemination of the coagulation process through the activation of protein C and the subsequent inactivation of both factor Va and factor VIIIa.     

Tissue factor pathway inhibitor dose-dependently inhibits coagulation activation without influencing the fibrinolytic and cytokine response during human endotoxemia

Inhibition of the tissue factor pathway has been shown to attenuate the activation of coagulation and to prevent death in a gram-negative bacteremia primate model of sepsis. It has been suggested that tissue factor influences inflammatory cascades other than the coagulation system. The authors sought to determine the effects of 2 different doses of recombinant tissue factor pathway inhibitor (TFPI) on endotoxin-induced coagulant, fibrinolytic, and cytokine responses in healthy humans. Two groups, each consisting of 8 healthy men, were studied in a double-blind, randomized, placebo-controlled crossover study. Subjects were studied on 2 different occasions. They received a bolus intravenous injection of 4 ng/kg endotoxin, which was followed by a 6-hour continuous infusion of TFPI or placebo. Eight subjects received 0.05 mg/kg per hour TFPI after a bolus of 0.0125 mg/kg (low-dose group), and 8 subjects received 0.2 mg/kg per hour after a bolus of 0.05 mg/kg (high-dose group). Endotoxin injection induced the activation of coagulation, the activation and subsequent inhibition of fibrinolysis, and the release of proinflammatory and antiinflammatory cytokines. TFPI infusion induced a dose-dependent attenuation of thrombin generation, as measured by plasma F1 + 2 and thrombin-antithrombin complexes, with a complete blockade of coagulation activation after high-dose TFPI. Endotoxin-induced changes in the fibrinolytic system and cytokine levels were not altered by either low-dose or high-dose TFPI. The authors concluded that TFPI effectively and dose-dependently attenuates the endotoxin-induced coagulation activation in humans without influencing the fibrinolytic and cytokine response. (Blood. 2000;95:1124-1129)     

Antigenicity of a recombinant Ro (SS-A) fusion protein.

The antigenicity of the 60-kd human Ro (SS-A) synthesized in vitro from its complementary DNA as a beta-galactosidase fusion protein (beta-gal-Ro) was evaluated by Western blotting. In this analysis, almost all the anti-Ro (SS-A)-positive sera that bound beta-gal-Ro also bound affinity-purified 60-kd human Ro (SS-A) (P less than 0.005). Three of the 27 anti-Ro (SS-A) precipitin-positive sera, however, did not show reactivity on Western blot analysis, which suggests that in some sera, antigenicity to Ro (SS-A) is destroyed by denaturation. Of the 22 sera that were reactive with beta-gal-Ro, 2 were not reactive with affinity-purified human Ro (SS-A). Two serum samples that did not react with beta-gal-Ro were also reactive with affinity-purified human Ro (SS-A). Nevertheless, except for a small percentage of Ro (SS-A) precipitin-positive sera, the frequency of antibody binding to the fusion protein was similar to the frequency of binding to the purified antigen in Western blots. Recombinant Ro (SS-A) antigen may therefore be valuable in the serologic evaluation of anti-Ro (SS-A) autoantibodies.