Study of septic shock in the non-human primate: relationship of pathophysiological response to therapy with anti-TNF antibody.

Therapy with anti-TNF antibody is reported to be effective in preventing morbidity and mortality in baboons given lethal infusions of Escherichia coli. Treated animals survived, and organ histopathology was absent when antibody was administered early after lethal infusions of E. coli. The present study explored the relationship between antibody dosage, pathophysiology, and survivability from shock. When antibody dose was decreased lungs, kidneys, adrenals, spleen, and liver were injured as shown by increased vascular congestion, hemorrhage, edema, and necrosis of tissues. Survival was also affected. All animals treated with 15 mg/kg antibody survived as reported earlier; less than 60% survived with 7.5 mg/kg; 9% survived with 5.0 mg/kg, and all died with 1.5 mg/kg. Serum concentrations of interleukin-6 (IL-6) increased markedly as dose of antibody decreased. The increases in concentrations of IL-6 were associated with increases in morbidity and mortality following E. coli administration.

Antithrombin III replacement in animal models of acquired antithrombin III deficiency.

Plasma antithrombin III (AT III) levels decrease early during Gram-negative septicaemia, and even a moderate decrease in this major inhibitor of the coagulation system is associated with serious disseminated intravascular coagulation (DIC). This study reports the efficacy of high dose (at least 250 units/kg) AT III replacement in three animal models of Escherichia coli endotoxaemia or bacteraemia. Highlights of our findings are: 1. Endotoxaemic rat model: DIC occurs early, before the appearance of deleterious cardiovascular abnormalities; AT III prophylaxis attenuates DIC; and AT III prophylaxis increases permanent survival. 2. Endotoxaemic sheep pulmonary dysfunction model: AT III prophylaxis prevents the typical decrease in arterial oxygen partial pressure and AT III prophylaxis combined with alpha 1-proteinase inhibitor significantly attenuates indices of pulmonary dysfunction. 3. E. coli bacteraemic baboon model: AT III prophylaxis and treatment significantly attenuates indices of DIC and organ damage, and prevents death in an otherwise 100% lethal bacterial challenge. In conclusion, prophylactic treatment with high doses of AT III may be efficacious in disease states of impending DIC such as Gram-negative septicaemia. Data presented herein demonstrate that plasma levels of AT III must be maintained at levels markedly higher than normal to be efficacious in animal models of Gram-negative endotoxaemia or bacteraemia.

Efficacy of monoclonal antibody against tumor necrosis factor alpha in an endotoxemic baboon model.

Tumor necrosis factor alpha (TNF) has been described as a primary mediator of the pathophysiology associated with bacterial sepsis/endotoxemia. We tested the efficacy and possible mechanisms of protection of a monoclonal antibody against TNF (TNF Mab) in a low mortality (29%), endotoxemic baboon model. A number of parameters were monitored at days 0, 1, 2 and 5-7 after challenge with 2 mg E. coli endotoxin/kg. TNF Mab pretreatment (15 mg/kg) prevented the increase in detectable serum TNF and the early perturbations in cardiovascular function which occurred in the control group. Early metabolic dysfunction was delayed in the TNF MAb group and was attenuated thereafter. Dysfunction of the kidney, liver, and coagulation systems and the increased IL-6 levels were significantly attenuated in the TNF MAb group; neutrophil activation was not affected by TNF MAb. No deaths occurred in the TNF MAb group. These results support the hypothesis that TNF plays a central role in the pathophysiology of endotoxic shock.

Lethal Staphylococcus aureus-induced shock in primates: prevention of death with anti-TNF antibody.

A successful experimental treatment for gram-positive sepsis to our knowledge has not been achieved. The objectives of this study were to develop a nonhuman primate model of lethal gram-positive sepsis employing the micro-organism Staphylococcus aureus and to determine the efficacy of treatment using monoclonal antibody (MAb) to tumor necrosis factor alpha (TNF). The antibody was administered intravenously, 15 mg/kg, 30 minutes after the beginning of a 2-hour infusion of S. aureus, 4 x 10(10) colony forming units/kilogram. The baboons infused with S. aureus demonstrated the release of the cytokines TNF and interleukin-6 (IL-6), but endotoxin was not observed in the plasma at any time. Treatment with antibody to TNF abolished the rise in serum TNF levels and reduced the increased levels of IL-6. Treatment with MAb to TNF prevented multiple organ failure and achieved permanent (> 7 day) survival of all baboons.

Characterization of an endotoxemic baboon model of metabolic and organ dysfunction.

An anesthetized endotoxemic baboon model has been developed by infusing 2.0 mg E. coli endotoxin/kg i.v. over 1 hr (n = 7). Animals were monitored for 5-7 days with analyses of: cardiovascular, metabolic, and organ dysfunction; acid base, hemostatic, and hematological alterations; as well as tumor necrosis factor (TNF) and interleukin-6 (IL-6) levels. Pathophysiologies detected at 2 hr included transient decreases in vascular resistance and blood pressure, a 157% increase in blood lactate, and a 90% decrease in circulating neutrophils. Organ dysfunction was not observed until 24 hr and, although thrombocytopenia was prevalent (-72% at 48 hr), disseminated intravascular coagulation (DIC) was not a major pathology. Hematocrit fell 21% by 24 hr and was -41% at 5-7 days. Serum TNF peaked at 90 min (7.8 +/- 0.2 ng/mL) and was undetectable after 3 hr. IL-6 also increased early, peaked at 3 hr (3872 +/- 846 U/mL) and was still detectable at 24 hr. A low mortality primate model of gram-negative sepsis has been developed that is characterized by early cardiovascular and metabolic dysfunction (2-6 hr), late organ dysfunction (24-48 hr), sub-clinical DIC, a prolonged anemia, and a 29% mortality between 48 and 72 hr.

Survival of primates in LD100 septic shock following therapy with antibody to tumor necrosis factor (TNF alpha).

The purpose of this study was to determine the efficacy of treatment with anti-TNF monoclonal antibody in preventing the deleterious effects of sepsis in a nonhuman primate. Experiments were carried out on anesthetized baboons intravenously infused with a lethal dose of Escherichia coli (E. coli). Twelve baboons (six control and six experimental) received 2 hr infusions of E. coli. The experimental group was administered a bolus of anti-TNF antibody, 15 mg/kg, 30 min after beginning the E. coli infusion. Control baboons lived an average of 19 hr (12-34 hr). All antibody-treated baboons survived more than 7 days with a significantly improved quality of life compared to the control group. Although some adverse changes occurred during the monitoring period in surviving baboons, they maintained nearly normal arterial pressures, and serum urea nitrogen and creatinine concentrations. The severe histopathologic changes in lungs, liver, adrenals, kidneys, and spleen documented at death in baboons receiving E. coli only were absent after 7 days in baboons given E. coli and early post-treatment with antibody to TNF.

Efficacy of antithrombin III supplementation in animal models of fulminant Escherichia coli endotoxemia or bacteremia.

Plasma antithrombin III (ATIII) levels decrease early during gram-negative septicemia, and even a moderate decrease in this major inhibitor of the coagulation system is associated with serious disseminated intravascular coagulation (DIC). Herein the efficacy of high-dose (at least 250 units/kg) ATIII supplementation in animal models of Escherichia coli endotoxemia or bacteremia is reported. An endotoxemic rat model demonstrated that: (1) DIC occurs very early, before the appearance of deleterious cardiovascular abnormalities; (2) ATIII prophylaxis attenuates DIC, metabolic dysfunction, and organ damage; (3) ATIII prophylaxis increases permanent survival; (4) ATIII treatment one hour after endotoxin challenge attenuates DIC, metabolic dysfunction, and organ damage, although not as well as when given prophylactically, and survival is not increased. An endotoxemic sheep pulmonary dysfunction model demonstrated that: (1) ATIII prophylaxis prevents the typical decrease in arterial oxygen partial pressure; (2) ATIII prophylaxis combined with alpha-1-proteinase inhibitor significantly attenuates indices of pulmonary dysfunction. An E. coli bacteremic baboon model demonstrated that ATIII prophylaxis and treatment significantly attenuate indices of DIC and organ damage and prevent death in an otherwise completely lethal dose bacterial challenge. In conclusion, prophylactic treatment with high doses of ATIII may be efficacious in disease states of impending disseminated intravascular coagulation, such as primary or secondary gram-negative septicemia.

Antithrombin-III treatment limits disseminated intravascular coagulation in endotoxemia.

Gram-negative septicemia/endotoxemia remains a serious clinical disorder that is often complicated by disseminated intravascular coagulation (DIC). Plasma antithrombin-III (AT-III) levels usually decrease during gram-negative septicemia/endotoxemia, and even moderate decreases in this major inhibitor of the coagulation system are associated with serious DIC. We demonstrated in an earlier study that prophylactic treatment of rats with 250 U/kg of AT-III followed by endotoxin challenge markedly attenuates DIC, indices of organ damage, and metabolic dysfunction. The present study was to determine whether treatment with 250 U/kg AT-III 1 hr after endotoxin challenge would be similarly efficacious. Rats treated with 250 U/kg of AT-III inactivated by human sputum elastase (ATX) served as protein controls. Blood samples for analysis were obtained 4 hr after AT-III or ATX treatment (5 hr after endotoxin challenge). Rats in the ATX treatment group exhibited abnormalities characteristic of endotoxemia, i.e., decreased fibrinogen levels and platelet counts, increases in prothrombin time and activated partial thromboplastin time, elevated serum glutamic oxaloacetic transaminase (SGOT) and alkaline phosphatase (AKP), and hypoglycemia. Treatment with AT-III markedly and significantly (P less than .05) attenuated all of these abnormalities, although survival was not increased. This study strongly suggests that supplementation of plasma AT-III is efficacious after the development of sepsis, although not as efficacious as prophylactic treatment.

Antithrombin-III prevents the lethal effects of Escherichia coli infusion in baboons.

Infusion of Escherichia coli (LD100) was followed by coagulopathic and cell injury responses, cardiovascular collapse, and death in 18 to 32 hr in four out of four baboons. Infusion of AT-III in sufficient amounts to achieve AT-III levels of more than 4 units/ml of plasma before and during the infusion of E. coli reduced the intensity of the coagulopathic and cell injury response and prevented vascular collapse and death in four out of four baboons. Failure to achieve AT-III levels of more than six units/ml at T +60 min during the infusion of E. coli resulted in failure to prevent its lethal effects in three out of three baboons even though levels as high as 10 units/ml were achieved later in the course of the experiment. These studies suggest that thrombin and/or its products can contribute to the inflammatory response to E. coli and that AT-III is of potential value as a prophylactic but not as a therapeutic agent in the treatment of patients at high risk of developing gram negative sepsis.

Synergistic protection from lung damage by combining antithrombin-III and alpha 1-proteinase inhibitor in the E. coli endotoxemic sheep pulmonary dysfunction model.

Septicemic/endotoxic-induced adult respiratory distress syndrome (ARDS) remains a major clinical problem. The present study was to determine in the E. coli endotoxemic sheep ARDS model the efficacy of combination prophylaxis with antithrombin-III (AT-III) and alpha 1-proteinase inhibitor (alpha 1-PI). We reasoned that 1) AT-III supplementation would ameliorate the endotoxin-induced coagulopathy, 2) alpha 1-PI supplementation would attenuate pulmonary damage caused by neutrophil elastase and inactivation of AT-III by neutrophil elastase, and 3) the therapeutic effects of this combination would be additive or synergistic. The typical increases in lung lymph flow microvascular permeability to protein, transvascular protein flow and transvascular protein clearance, and decrease in systemic arterial PO2 were prevented or significantly attenuated during 5 hours of endotoxemia by the AT-III/alpha 1-PI combination pretreatment. Limited efficacy was observed with AT-III pretreatment, and none was seen with alpha 1-PI alone. Results of this study demonstrate that combining AT-III and alpha 1-PI prophylaxis prevents or attenuates indices of ARDS during gram-negative endotoxemia and that this efficacy is due to a statistically significant synergism between AT-III and alpha 1-PI.

Efficacy of antithrombin III in endotoxin-induced disseminated intravascular coagulation.

Antithrombin III (AT III) is a major modulator of the clotting cascade and is decreased in disseminated intravascular coagulation (DIC). AT III was given as a pretreatment to dogs with endotoxin-induced DIC. Significant improvement in clotting parameters (prothrombin time, fibrinogen, fibrin degradation products) was noted. There was no effect on platelets. Mean arterial blood pressure was improved, while there were no other significant changes in other measured hemodynamic, acid-base, or biochemical variables. It was concluded that AT III was effective in ameliorating endotoxin-induced changes in the clotting profile. AT III may prove to be a beneficial therapy in acquired DIC.

Effects of purified fibronectin alone and combined with immunoglobulin G or antithrombin-III on survival during gram-negative peritonitis or endotoxemia in rats.

The present study was performed to determine the effects of pretreatment with purified human plasma fibronectin (FN) on survival in rats challenged with Salmonella typhimurium peritonitis or E. coli endotoxemia. The effects on survival of combining FN with immunoglobulin G (IgG) or antithrombin-III (AT-III) were also determined during S. typhimurium peritonitis and E. coli endotoxemia. Permanent survival was increased 15% in the peritonitis group (p less than 0.05) and 15% in the endotoxemic group (p greater than 0.05). There was no enhancement in survival by combining FN with a subprotective dose of IgG preparation. AT-III alone increased survival by 50% over survival in the control group. Combining FN with AT-III increased survival 10% greater than with AT-III alone, which is suggestive of an additive effect. Results from this study suggest that FN provides modest protection during Gram-negative peritonitis or endotoxemia in the rat. Combining FN with AT-III may augment in an additive manner the marked increase in survival observed with AT-III alone in this and previous studies.

Protection against disseminated intravascular coagulation and death by antithrombin-III in the Escherichia coli endotoxemic rat.

Gram-negative septic shock remains a major clinical problem. One frequently encountered complication of sepsis is disseminated intravascular coagulation (DIC). The present study was to determine in an Escherichia coli endotoxemia awake rat model the efficacy of antithrombin-III (AT-III) prophylaxis and to explore the role of DIC in the pathogenesis of endotoxemia. We demonstrated that DIC occurs very early, before the appearance of detectable serious abnormalities in cardiovascular, metabolic, and biochemical variables indicative of organ damage or dysfunction; AT-III prophylaxis significantly ameliorates DIC, as evidenced by completely preventing the fall in plasma fibrinogen concentration and significantly limiting the increases in prothrombin time and activated partial thromboplastin time after 4 hours of endotoxemia; and AT-III prophylaxis dramatically increases permanent survival. Results of this study suggest that AT-III prophylaxis is very protective above a threshold dosage in an endotoxemic rat model and that protection is in part due to ameliorating DIC. Our data also suggest that DIC occurs very early during endotoxemia and may in part be responsible for the pathogenesis of endotoxemia in the rat. We conclude that AT-III prophylaxis may be efficacious in conditions of impending DIC, such as gram-negative septicemia/endotoxemia.

Effect of fibronectin supplementation in endotoxic shock in the dog.

An earlier study by our group demonstrated significant amelioration of hypotension, hypoglycemia, and acidosis in dogs treated with purified human plasma fibronectin prior to induction of endotoxic shock. The present study was completed to determine whether treatment with purified human plasma fibronectin 1 hr after induction of endotoxic shock would provide similar benefits. To this end, selected hemodynamic, pulmonary, acid-base, metabolic, hematological, and serum chemistry parameters were monitored for 6 hours in two groups of anesthetized dogs in Escherichia coli endotoxic shock. One group was given an intravenous injection of purified human plasma fibronectin, and the other received an equal volume of saline 1 hr after shock induction. Between-group analysis of the data revealed no significant differences between any parameter excepting modest differences in plasma glucose, albumin, alkaline phosphatase, and BUN concentrations. However, even these differences, although statistically significant, were sporadic and unimpressive. This study suggested that treatment of dogs with fibronectin during gram-negative endotoxic shock was not efficacious.

Use of a new, low-pH immunoglobulin G preparation during episodes of bacteremia in the rat.

A rapidly expanding role for immunoglobulin G preparations in conditions other than the classical immunodeficiency syndromes is evident. This relatively new concept of treatment with polyclonal antibody has been tested in the rat with severe Salmonella typhimurium bacteremia with use of a newly developed, native immunoglobulin G preparation for intravenous use (IGIV pH 4.25). IGIV pH 4.25 increased survival time and decreased absolute mortality, prevented hypotension and acidosis, and ameliorated or prevented changes in variables indicative of organ damage during S. typhimurium bacteremia in the rat. Intravenous infusion of IGIV pH 4.25 at high rates did not cause further deterioration in the arterial blood pH in the acid-base-compromised rat and hence should not cause clinically significant decreases in pH in patients with compromised acid-base regulating systems.

Effects of fibronectin pretreatment on cardiovascular, acid-base, metabolic, and organ function indices during endotoxin shock in the dog.

Selected hemodynamic, pulmonary, acid-base, metabolic, hematological, and serum chemistry parameters were monitored for 6 hr in two groups of anesthetized dogs given an intravenous injection of Escherichia coli endotoxin. One group of animals was pretreated with purified human plasma fibronectin, and the other group received an equal volume of saline or dextrose. Between-group analysis showed that the fibronectin-treated group had significantly higher arterial blood pressure and glucose levels, and lower hemoglobin levels, at 4, 5, and 6 hr postendotoxin administration. This group also had higher arterial pH and base excess values at 5 and 6 hr postendotoxin administration. This study, along with others from our laboratory, suggests that exogenous administration of purified plasma fibronectin can be beneficial in the prophylactic treatment of impending sepsis or endotoxemia. However, the amount of benefit appears to be moderate. Whether combining fibronectin with other therapeutic modalities would be additive or synergistic cannot be ruled out and merits investigation.

Skeletal muscle insulin unresponsiveness during chronic hyperdynamic sepsis in the dog.

Recent reports from our laboratory and others have documented changes in insulin unresponsiveness and electrolyte and hormonal changes characteristic of hypodynamic shock states in anesthetized animals. Since most acute shock protocols do not adequately mimic the clinical profile of sepsis, the present study was undertaken to document the hemodynamic and metabolic changes associated with chronic hyperdynamic peritonitis in dogs. Mongrel dogs of either sex weighing 20 +/- 2 kg were surgically instrumented with an electromagnetic aortic flow probe for monitoring cardiac output determinations, and aortic and central venous catheters for withdrawing blood for blood pressure and chemical analyses. Following a recovery period (7-10 days) control hemodynamic and metabolic measurements were made. Sepsis was induced (peritoneal abscess) by implanting a 4" X 4" gauze sponge, previously inoculated with human fecal bacteria, amid the intestines. Experimental (N = 18) and pair-fed control (N = 6) animals were monitored daily for 21 days or until death. During the septic protocol, cardiac index increased from a control value of 3.4 L/min/m2 to 5.5 L/min/m2 by the end of the experimental period. Mean arterial blood pressure, total peripheral resistance index, body weight, and plasma Ca++ fell below control values during the experimental period. Body temperature, plasma glucose, insulin, glucagon, and Mg++ were all elevated with sepsis. At the end of the chronic experimental period, skeletal muscle insulin responsiveness was assessed in the isolated, innervated, constantly perfused gracilis muscle preparation. Pair-fed control animals responded to various concentrations of local insulin infusion by increasing glucose uptake by the gracilis muscle. However, septic animals had a blunted response to local insulin infusion resulting in a decrease in the maximum dose response effect. These data demonstrate that: chronic, hyperdynamic peritonitis in the dog more closely mimics the human clinical profile of sepsis; and hyperdynamic sepsis is associated with a state of skeletal muscle insulin unresponsiveness which results from a post-receptor defect.

Efficaciousness of immunoglobulin G prophylaxis on mortality and physiological variables in gram-negative peritonitis in rodents.

A rapidly expanding role involving the use of immunoglobulin preparations in conditions other than the classical immunodeficiency syndromes is evident in recent years. Treatment with immunoglobulin may be especially important during infection in which specific antibody to pathogens has been consumed, degraded, or not produced and in cases in which antibiotic treatment is ineffective. Recent studies in animals and humans support this concept, although further studies are needed to fully develop it. This study was completed to explore possible beneficial effects of prophylactic treatment of rodents during severe Salmonella typhimurium peritonitis with a newly developed native immunoglobulin G preparation for intravenous use (IGIV pH 4.25). This study demonstrates that IGIV pH 4.25 increases survival time and decreases absolute mortality, prevents hypotension and acidosis, and ameliorates or prevents changes in variables indicative of organ damage during S. typhimurium bacteremia in the rat. Studies in mice indicate that protection is mediated by antibody to cell surface antigen (s) of S. typhimurium.