G. Peer

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.

Complement inhibition decreases the procoagulant response and confers organ protection in a baboon model of Escherichia coli sepsis.

Severe sepsis leads to massive activation of coagulation and complement cascades that could contribute to multiple organ failure and death. To investigate the role of the complement and its crosstalk with the hemostatic system in the pathophysiology and therapeutics of sepsis, we have used a potent inhibitor (compstatin) administered early or late after Escherichia coli challenge in a baboon model of sepsis-induced multiple organ failure. Compstatin infusion inhibited sepsis-induced blood and tissue biomarkers of complement activation, reduced leucopenia and thrombocytopenia, and lowered the accumulation of macrophages and platelets in organs. Compstatin decreased the coagulopathic response by down-regulating tissue factor and PAI-1, diminished global blood coagulation markers (fibrinogen, fibrin-degradation products, APTT), and preserved the endothelial anticoagulant properties. Compstatin treatment also improved cardiac function and the biochemical markers of kidney and liver damage. Histologic analysis of vital organs collected from animals euthanized after 24 hours showed decreased microvascular thrombosis, improved vascular barrier function, and less leukocyte infiltration and cell death, all consistent with attenuated organ injury. We conclude that complement-coagulation interplay contributes to the progression of severe sepsis and blocking the harmful effects of complement activation products, especially during the organ failure stage of severe sepsis is a potentially important therapeutic strategy.

Two-stage response to endotoxin infusion into normal human subjects: Correlation of blood phagocyte luminescence with clinical and laboratory markers of the inflammatory, hemostatic response

ObjectiveTo characterize a two-stage response of normal human subjects to an intravenous bolus infusion of endotoxin by using phagocyte luminescence and molecular markers of hemostatic and inflammatory system, phagocyte, and endothelial cell perturbation. DesignProspective evaluation of the response of normal subjects to endotoxin. SettingMedical intensive care unit, university laboratories. SubjectsNormal healthy male subjects ranging in age from 24 to 37 yrs. MeasurementsLuminescence measurements of phagocyte oxidase, oxidase-myeloperoxidase, opsonin receptor expression, clinical chemical and molecular markers of hemostatic and inflammatory system activation, phagocyte activation, and endothelial cell injury. ResultsResponse to intravenous bolus endotoxin includes two stages. The symptomatic first stage (time [T]-0 to T + 8 hrs) included sharp rises in phagocyte oxidase, oxidase-myeloperoxidase, and opsonin receptor expression with the appearance of morphologic and molecular markers of hemostatic and inflammatory system activation, phagocyte activation, and endothelial cell injury. All variables with the exception of soluble thrombomodulin, soluble fibrin monomer, lactate, white cell count, and opsonin receptor expression returned to baseline by T + 8 hrs, by which time all acute clinical symptoms had disappeared. The asymptomatic second stage (T + 8–48 hrs) included a second peak of oxidase activity at T + 24 hrs with an elevated but declining lactate and opsonin receptor expression. This coincided with a second high peak of soluble fibrin monomer, sustained elevated concentrations of soluble thrombomodulin and plasma soluble tissue factor antigen, a decrease in factor VIIa concentration to 50% of baseline at T + 12–24 hrs, and an increase in C-reactive protein/C3d and C4d complexes, which peaked at T + 24 hrs. ConclusionsThe results suggest that the human response to endotoxin consists of a previously well-characterized, symptomatic, first stage acute inflammatory response followed by an asymptomatic second stage that is marked by a second peak of oxidase activity, a continued increase and sustained elevation of markers of hemostatic and complement system activation, and endothelial cell injury. We conclude that this second stage represents a recovery or reperfusion phase of a compensated response to endotoxin in which contact of the microvasculature perturbed during the first stage is reestablished with blood in a second asymptomatic stage.

Infection-induced Modulation Of M1 And M2 Phenotypes In Circulating Monocytes: Role In Immune Monitoring And Early Prognosis Of Sepsis

To monitor and better understand the immunoinflammatory sequelae in sepsis and septic shock, systemic and monocyte-related cytokine responses were evaluated in baboons with experimental peritonitis induced by an E. coli–laden fibrin clot. Despite similar bacterial inocula, considerable interindividual variability in clinical manifestation and outcome of infection was observed. Because monocytes and macrophages are a key component of innate immunity, we hypothesized that early polarization of distinct activation programs in circulating monocytes that culminates in the emergence of either classically (M1) or alternatively (M2) activated monocytes may underlie the observed susceptibility or resistance to infection. To test our hypothesis, we analyzed infection-induced expression of cytokine mRNAs in monocytes isolated from surviving and dead animals. Our data show that resistance to E. coli sepsis may well be associated with a mixed M1/M2 activation state of circulating monocytes, whereas M1 phenotype appeared to be prevailing in monocytes from animals that died. Together with data on systemic cytokine responses, the latter findings indicate that morbidity and mortality of animals with gram-negative sepsis may well result from an overwhelming proinflammatory response. Collectively, our data contribute to a better understanding of cytokine networking in the immunoinflammatory response to microbial infection and suggest M1/M2 immunophenotypic profiling of readily available circulatory monocytes for early prognosis of severe infections.

Reduced neutrophil CD10 expression in nonhuman primates and humans after in vivo challenge with E. coli or lipopolysaccharide

CD10, also known as neutral endopeptidase or CALLA, is a major metalloproteinase that regulates levels of biologically active peptides that initiate inflammatory, cardiovascular, and neurogenic responses. Relative tissue expression levels of CD10, its peptide substrates, and their receptors constitute the basic regulatory mechanism. Neutrophils contain abundant CD10 and are rapid responders to an inflammatory septic challenge. Expression of neutrophil surface antigens in response to inflammation was studied in the primate model of Escherichia coli-mediated sepsis and in human volunteers injected with lipopolysaccharide (LPS). There was a rapid and profound (up to 95%) reduced baboon neutrophil CD10 expression in response to E. coli injections of 5.71 × 106 CFU/kg to 2.45 × 109 CFU/kg that gradually resolved to preinjection levels. The reduction was both dose and time dependent. Reduced CD10 antigen on mature baboon neutrophils and bands was observed by immunohistochemistry. Human volunteers challenged with 4ng/kg LPS experienced transient chills, nausea, fever, and myalgia. Up to ∼20% of their neutrophils had reduced CD10 expression, peaking at 2 to 8 h after injection. By 24 h, neutrophil CD10 expression resolved to preinjection levels. In contrast, in both the baboon and human studies, other neutrophil surface antigens were only slightly decreased (CD11a) or increased (CD11b, CD18, CD35, CD66b, and CD63). These data present the novel observation that neutrophil CD10 expression decreases significantly in response to in vivo inflammatory challenge. This decrease appears to be unique to CD10 and may contribute to a reduced regulation of bioactive peptides released in response to inflammatory challenge.

PERITONITIS IN THE BABOON: A PRIMATE MODEL WHICH SIMULATES HUMAN SEPSIS

The physiological, hemostatic, and immunological responses of 12 chronically instrumented conscious baboons with sepsis due to Escherichia coli peritonitis were compared with that of similarly instrumented controls. Chronic indwelling cannulae were placed in the aorta and pulmonary artery to monitor pressure, cardiac output, and obtain blood samples. At t = 0 a sterile or E. coli-laden fibrin clot containing 1.9-6.7 x 10(11) CFU/kg was introduced into the peritoneal cavity. The control animals were group 1 (n = 3). The animals with peritonitis were divided into three groups depending on their clinical response. Group 2 animals (n = 3) were clinically well at the time of sacrifice (day 14), group 3 (n = 4) survived but were obviously sick on day 14, and group 4 (n = 5) died of sepsis. Implantation of a sterile fibrin clot was well tolerated with little hemodynamic change and a transient minimal inflammatory response in group 1. Implantation of an E. coli-containing clot elicited a hyperdynamic cardiovascular response and evoked a marked inflammatory reaction and a disseminated intravascular coagulopathy. Five of 12 (42%) E. coli animals died from sepsis. In general, the physiological, hemostatic, and immunological disturbances tended to be greatest in these animals. Autopsy revealed residual peritoneal inflammation and varying degrees of inflammation in the lungs, adrenal, spleen, liver, and kidneys in all the animals that received E. coli with the inflammatory infiltrate increasing in severity from group 2 through group 4. Tissue necrosis was observed only in the latter group. We conclude that the cardiovascular, hemostatic, and immunological responses of baboons with sepsis due to E. coli peritonitis exhibit a variable course that resembles the clinical manifestations of gram-negative sepsis in humans.

Acute Lung Injury and Fibrosis in a Baboon Model of Escherichia coli Sepsis

Sepsis-induced inflammation of the lung leads to acute respiratory distress syndrome (ARDS), which may trigger persistent fibrosis. The pathology of ARDS is complex and poorly understood, and the therapeutic approaches are limited. We used a baboon model of Escherichia coli sepsis that mimics the complexity of human disease to study the pathophysiology of ARDS. We performed extensive biochemical, histological, and functional analyses to characterize the disease progression and the long-term effects of sepsis on the lung structure and function. Similar to humans, sepsis-induced ARDS in baboons displays an early inflammatory exudative phase, with extensive necrosis. This is followed by a regenerative phase dominated by proliferation of type 2 epithelial cells, expression of epithelial-to-mesenchymal transition markers, myofibroblast migration and proliferation, and collagen synthesis. Baboons that survived sepsis showed persistent inflammation and collagen deposition 6-27 months after the acute episodes. Long-term survivors had almost double the amount of collagen in the lung as compared with age-matched control animals. Immunostaining for procollagens showed persistent active collagen synthesis within the fibroblastic foci and interalveolar septa. Fibroblasts expressed markers of transforming growth factor-β and platelet-derived growth factor signaling, suggesting their potential role as mediators of myofibroblast migration and proliferation, and collagen deposition. In parallel, up-regulation of the inhibitors of extracellular proteases supports a deregulated matrix remodeling that may contribute to fibrosis. The primate model of sepsis-induced ARDS mimics the disease progression in humans, including chronic inflammation and long-lasting fibrosis. This model helps our understanding of the pathophysiology of fibrosis and the testing of new therapies.

Comparison of the capacity of rhTNF-alpha and Escherichia coli to induce procoagulant activity by baboon mononuclear cells in vivo and in vitro.

The procoagulant activity of mononuclear cells (MNCs) may play an important role in the disseminated intravascular coagulation seen in septic shock. This study compares the capacity of Escherichia coli (E. coli) and recombinant human TNF-α (rhTNF-α) to induce procoagulant activity by baboon MNCs. In vivo studies showed that MNC procoagulant activity was significantly increased at T + 120 min after LD100 E. coli infusion into baboons. Most of this procoagulant activity was attributable to tissue factor. In contrast, a bolus infusion of rhTNF-α (150 μg/kg) and a monoclonal antibody to activated protein C (2 mg/kg) did not induce any increase of MNC procoagulant activity at T + 120 min even though the plasma TNF-a level was 10 times higher than that seen following infusion of E. coli. In vitro studies showed that E. coli at concentrations comparable to that observed in the in vivo study and LPS at a concentration of 2.5 ng/mL induced more intense tissue factor expression by both human and baboon monocytes than rhTNF-α in the concentrations ranging from 10 to 1,000 ng/mL. These results suggest that TNF-α alone is not sufficient to induce noticeable MNC procoagulant activity, at least, in the early stage of this septic shock model.

Complement inhibition decreases early fibrogenic events in the lung of septic baboons.

Acute respiratory distress syndrome (ARDS) induced by severe sepsis can trigger persistent inflammation and fibrosis. We have shown that experimental sepsis in baboons recapitulates ARDS progression in humans, including chronic inflammation and long‐lasting fibrosis in the lung. Complement activation products may contribute to the fibroproliferative response, suggesting that complement inhibitors are potential therapeutic agents. We have been suggested that treatment of septic baboons with compstatin, a C3 convertase inhibitor protects against ARDS‐induced fibroproliferation. Baboons challenged with 109 cfu/kg (LD50) live E. coli by intravenous infusion were treated or not with compstatin at the time of challenge or 5 hrs thereafter. Changes in the fibroproliferative response at 24 hrs post‐challenge were analysed at both transcript and protein levels. Gene expression analysis showed that sepsis induced fibrotic responses in the lung as early as 24 hrs post‐bacterial challenge. Immunochemical and biochemical analysis revealed enhanced collagen synthesis, induction of profibrotic factors and increased cell recruitment and proliferation. Specific inhibition of complement with compstatin down‐regulated sepsis‐induced fibrosis genes, including transforming growth factor‐beta (TGF‐β), connective tissue growth factor (CTGF), tissue inhibitor of metalloproteinase 1 (TIMP1), various collagens and chemokines responsible for fibrocyte recruitment (e.g. chemokine (C‐C motif) ligand 2 (CCL2) and 12 (CCL12)). Compstatin decreased the accumulation of myofibroblasts and proliferating cells, reduced the production of fibrosis mediators (TGF‐β, phospho‐Smad‐2 and CTGF) and inhibited collagen deposition. Our data demonstrate that complement inhibition effectively attenuates collagen deposition and fibrotic responses in the lung after severe sepsis. Inhibiting complement could prove an attractive strategy for preventing sepsis‐induced fibrosis of the lung.

Sepsis-induced lung fibrosis in baboons is reduced by the treatment with a complement inhibitor

Pulmonary fibrosis is a major and common medical condition, characterized by progressive scaring and decline in lung function. Persistent inflammation and acute lung injury in response to sepsis are potential triggers of the fibrotic response. Recently, we have reported that Escherichia coli sepsis in baboons strongly induces procoagulant responses and affects the integrity of the lung. These effects are diminished by the treatment with compstatin, a C3 convertase complement inhibitor [1].