Gene expression data from compstatin treated E.coli-Induced primate sepsis model
ABSTRACT: Severe sepsis leads to massive activation of coagulation and complement cascades that could contribute to multiple organ failure (MOF) 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 post E. coli challenge in a baboon model of sepsis-induced MOF. Microarray was used to study the affect of complement pathway on global gene expression pattern in sepsis, aims on exploring and discovering the new target genes as potential drugs for the early treatment and prevention of sepsis. Overall design: Lung and liver tissues were obtained from three normal healthy animals (as Ctl), three animals challenged with sublethal dose of E. coli as SLEC, three animals treated with Compstatin at different sepsis stages after E.coli challenge as SLEC-CST0 and SLEC-CST5. All the animals challenged with E. coli were sacrified at 24 hours post challenge. Total RNAs were isolated from these tissues, hybridized with Affymetrix Human Genome GeneChip U133A 2.0.
INSTRUMENT(S): [HG-U133A_2] Affymetrix Human Genome U133A 2.0 Array
Project description:Severe sepsis leads to massive activation of coagulation and complement cascades that could contribute to multiple organ failure (MOF) 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 post E. coli challenge in a baboon model of sepsis-induced MOF. Microarray was used to study the affect of complement pathway on global gene expression pattern in sepsis, aims on exploring and discovering the new target genes as potential drugs for the early treatment and prevention of sepsis. Lung and liver tissues were obtained from three normal healthy animals (as Ctl), three animals challenged with sublethal dose of E. coli as SLEC, three animals treated with Compstatin at different sepsis stages after E.coli challenge as SLEC-CST0 and SLEC-CST5. All the animals challenged with E. coli were sacrified at 24 hours post challenge. Total RNAs were isolated from these tissues, hybridized with Affymetrix Human Genome GeneChip U133A 2.0.
Project description: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.
Project description:Bacterial sepsis triggers robust activation of the complement system with subsequent generation of anaphylatoxins (C3a, C5a) and the terminal complement complex (TCC) that together contribute to organ failure and death. Here we tested the effect of RA101295, a 2-kDa macrocyclic peptide inhibitor of C5 cleavage, using in vitro whole-blood assays and an in vivo baboon model of Escherichia coli sepsis. RA101295 strongly inhibited E. coli-induced complement activation both in vitro and in vivo by blocking the generation of C5a and the soluble form of TCC, sC5b-9. RA101295 reduced the E. coli-induced "oxidative burst," as well as leukocyte activation, without affecting host phagocytosis of E. coli RA101295 treatment reduced plasma LPS content in E. coli-challenged baboons, implying reduced complement-mediated bacteriolysis, whereas treated animals showed slightly improved bacterial clearance during the bacteremic stage compared with controls. Treatment with RA101295 also improved consumptive coagulopathy and preserved endothelial anticoagulant and vascular barrier functions. RA101295 abolished sepsis-induced surges in proinflammatory cytokines and attenuated systemic circulatory and febrile responses, likely reflecting decreased systemic levels of LPS and C5a. Overall, RA101295 treatment was associated with significant organ protection and markedly reduced mortality compared with nontreated controls (four of five animals survived in a 100% lethal model). We therefore conclude that inhibition of C5 cleavage during the bacteremic stage of sepsis could be an important therapeutic approach to prevent sepsis-induced inflammation, consumptive coagulopathy, and subsequent organ failure and death.
Project description:Sepsis concurrently activates both coagulation and complement systems. Although complement activation by bacteria is well documented, work in mice and in vitro suggests that coagulation proteases can directly cleave complement proteins. We aimed to determine whether generation of coagulation proteases in vivo can activate the complement cascade in 2 highly coagulopathic models. We compared temporal changes in activation biomarkers of coagulation (thrombin-antithrombin [TAT]), fibrinolysis (plasmin-antiplasmin [PAP]), and complement (C3b, C5a, C5b-9) in baboons infused with factor Xa (FXa) and phospholipids (FXa/phosphatidylcholine-phosphatidylserine [PCPS]) vs LD100 Escherichia coli We found that, albeit with different timing, both FXa/PCPS and E coli infusion led to robust thrombin and plasmin generation. Conversely, only E coli challenge activated the complement system, reaching a maximum at 2 hours postchallenge during the peaks of lipopolysaccharide and bacteremia but not of TAT and PAP. Despite inducing a strong burst of thrombin and plasmin, FXa/PCPS infusion did not produce measurable levels of complement activation in vivo. Similarly, ex vivo incubation of baboon serum with thrombin, plasmin, or FXa did not show noticeable complement cleavage unless supraphysiologic amounts of enzymes were used. Our results suggest that in vivo-generated thrombin and plasmin do not directly activate the complement in nonhuman primates.
Project description:Sepsis is a severe infection-induced systemic inflammatory syndrome. Inhibition of downstream inflammatory mediators of sepsis, e.g., TNF-alpha, has failed in clinical trials. The aim of this study was to investigate the effects of inhibiting CD14, a key upstream innate immunity molecule, on the early inflammatory and hemostatic responses in a pig model of gram-negative sepsis. The study comprised two arms, whole live Escherichia coli bacteria and E. coli lipopolysaccharide (LPS) (n=25 and n=9 animals, respectively). The animals were allocated into treatment (anti-CD14) and control (IgG isotype or saline) groups. Inflammatory, hemostatic, physiological, and microbiological parameters were measured. The proinflammatory cytokines TNF-alpha, IL-1beta, IL-6, and IL-8, but not the anti-inflammatory cytokine IL-10, were efficiently inhibited by anti-CD14. Furthermore, anti-CD14 preserved the leukocyte count and significantly reduced granulocyte enzyme matrix metalloproteinase-9 release and expression of the granulocyte membrane activation molecule wCD11R3 (pig CD11b). The hemostatic markers thrombin-antithrombin III complexes and plasminogen activator inhibitor-1 were significantly attenuated. Anti-CD14 did not affect LPS or E. coli DNA levels. This study documents that CD14 inhibition efficiently attenuates the proinflammatory cytokine response and granulocyte activation and reverses the procoagulant state but does not interfere with LPS levels or bacterial counts in E. coli-induced sepsis.-Thorgersen, E. B., Hellerud, B. C., Nielsen, E. W., Barratt-Due, A., Fure, H., Lindstad, J. K., Pharo, A., Fosse, E., Tønnessen, T. I., Johansen, H. T., Castellheim, A., Mollnes, T. E. CD14 inhibition efficiently attenuates early inflammatory and hemostatic responses in Escherichia coli sepsis in pigs.
Project description: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 10(9) 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-?), 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-?, 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.
Project description:Sphingosine 1-phosphate (S1P) is an important regulator of vascular integrity and immune cell migration, carried in plasma by high-density lipoprotein (HDL)-associated apolipoprotein M (apoM) and by albumin. In sepsis, the protein and lipid composition of HDL changes dramatically. The aim of this study was to evaluate changes in S1P and its carrier protein apoM during sepsis. For this purpose, plasma samples from both human sepsis patients and from an experimental Escherichia coli sepsis model in baboons were used. In the human sepsis cohort, previously studied for apoM, plasma demonstrated disease-severity correlated decreased S1P levels, the profile mimicking that of plasma apoM. In the baboons, a similar disease-severity dependent decrease in plasma levels of S1P and apoM was observed. In the lethal E. coli baboon sepsis, S1P decreased already within 6-8 hrs, whereas the apoM decrease was seen later at 12-24 hrs. Gel filtration chromatography of plasma from severe human or baboon sepsis on Superose 6 demonstrated an almost complete loss of S1P and apoM in the HDL fractions. S1P plasma concentrations correlated with the platelet count but not with erythrocytes or white blood cells. The liver mRNA levels of apoM and apoA1 decreased strongly upon sepsis induction and after 12 hr both were almost completely lost. In conclusion, during septic challenge, the plasma levels of S1P drop to very low levels. Moreover, the liver synthesis of apoM decreases severely and the plasma levels of apoM are reduced. Possibly, the decrease in S1P contributes to the decreased endothelial barrier function observed in sepsis.
Project description:Bleeding from traumatic injury is the leading cause of death for young people across the world, but interventions are lacking. While many agents have shown promise in small animal models, translating the work to large animal models has been exceptionally difficult in great part because of infusion-associated complement activation to nanomaterials that leads to cardiopulmonary complications. Unfortunately, this reaction is seen in at least 10% of the population. We developed intravenously infusible hemostatic nanoparticles that were effective in stopping bleeding and improving survival in rodent models of trauma. To translate this work, we developed a porcine liver injury model. Infusion of the first generation of hemostatic nanoparticles and controls 5 min after injury led to massive vasodilation and exsanguination even at extremely low doses. In naïve animals, the physiological changes were consistent with a complement-associated infusion reaction. By tailoring the zeta potential, we were able to engineer a second generation of hemostatic nanoparticles and controls that did not exhibit the complement response at low and moderate doses but did at the highest doses. These second-generation nanoparticles led to cessation of bleeding within 10 min of administration even though some signs of vasodilation were still seen. While the complement response is still a challenge, this work is extremely encouraging in that it demonstrates that when the infusion-associated complement response is managed, hemostatic nanoparticles are capable of rapidly stopping bleeding in a large animal model of trauma.
Project description:The green rice leafhopper Nephotettix cincticeps have two mutualistic symbiotic bacteria (Candidatus Sulcia muelleri and Candidatus Nasuia deltocephalinicola) in its symbiont special organ bacteriome and are also infected to rickettsia. In order to determine immune challenge is induced or not in N. cincticeps, we investigated gene expression of Escherichia coli challenged N. cincticeps. Overall design: One day adult N. cincticeps females were pricked with E. coli with a sterile needle. Control adults were pricked with sterilized water. Total RNA of five females was extracted after 3 h and 12 h incubation respectively. E.coli challenged and control Nephotettix cincticeps samples were prepared 3 replicates for 3h and 4 replicates for 12h.
Project description:Staphylococcus aureus infections can produce systemic bacteremia and inflammation in humans, which may progress to severe sepsis or septic shock, even with appropriate antibiotic treatment. Sepsis may be associated with disseminated intravascular coagulation and consumptive coagulopathy. In some types of mouse infection models, the plasma coagulation protein factor XI (FXI) contributes to the pathogenesis of sepsis. We hypothesize that FXI also contributes to the pathogenesis of sepsis in primates, and that pharmacological interference with FXI will alter the outcome of Staphylococcus aureus-induced lethality in a baboon model. Pretreatment of baboons with the anti-FXI antibody 3G3, a humanized variant of the murine monoclonal 14E11 that blocks FXI activation by FXIIa, substantially reduced the activation of coagulation, as reflected by clotting times and plasma complexes of coagulation proteases (FXIIa, FXIa, FIXa, FXa, FVIIa, and thrombin) with serpins (antithrombin or C1 inhibitor) following infusion of heat-inactivated S aureus 3G3 treatment reduced fibrinogen and platelet consumption, fibrin deposition in tissues, neutrophil activation and accumulation in tissues, cytokine production, kininogen cleavage, cell death, and complement activation. Overall, 3G3 infusion protected the structure and function of multiple vital organs, including lung, heart, liver, and kidney. All treated animals reached the end point survival (7 days), whereas all nontreated animals developed terminal organ failure within 28 hours. We conclude that FXI plays a role in the pathogenesis of S aureus-induced disseminated intravascular coagulation and lethality in baboons. The results provide proof of concept for future therapeutic interventions that may prevent sepsis-induced organ failure and save lives in certain forms of sepsis.