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Trauma remains a major cause of death throughout the world, especially for patients younger than 45 years. Due to rapid advances in clinical management, both in the acute and prehospital settings, trauma patients survive devastating injuries at unprecedented rates. However, these patients can often face life threatening complications that stem from the robust innate immune response induced by severe hemorrhage, leading to further tissue injury rather than repair. The complement and coagulation cascades are key mediators in this disordered reaction, which includes the development of trauma‐induced coagulopathy. There is increasing evidence that cross‐talk between these two pathways allows rapid amplification of their otherwise targeted responses and contributes to overwhelming and prolonged systemic inflammation. In this article, we summarize the initial steps of innate immune response to trauma and review the complex complement and coagulation cascades, as well as how they interact with each other. Despite progress in understanding these cascades, effective therapeutic targets have yet to be found and further research is needed both to improve survival rates as well as decrease associated morbidity.

Intestinal ischemia followed by reperfusion leads to local and remote organ injury attributed to inflammatory response during the reperfusion phase. The extent to which ischemia contributes to ischemia/reperfusion injury has not been thoroughly studied. After careful evaluation of intestinal tissue following 30 min of ischemia, we noticed significant local mucosal injury in wild-type mice. This injury was drastically reduced in C3-deficient mice, suggesting C3 involvement. Depletion of circulating complement with cobra venom factor eliminated, as expected, injury recorded at the end of the reperfusion phase but failed to eliminate injury that occurred during the ischemic phase. Immunohistochemical studies showed that tissue damage during ischemia was associated with increased expression of C3/C3 fragments primarily in the intestinal epithelial cells, suggesting local involvement of complement. In vitro studies using Caco2 intestinal epithelial cells showed that in the presence of LPS or exposure to hypoxic conditions the cells produce higher C3 mRNA as well as C3a fragment. Caco2 cells were also noted to produce cathepsins B and L, and inhibition of cathepsins suppressed the release of C3a. Finally, we found that mice treated with a cathepsin inhibitor and cathepsin B–deficient mice suffer limited intestinal injury during the ischemic phase. To our knowledge, our findings demonstrate for the first time that significant intestinal injury occurs during ischemia prior to reperfusion and that this is due to activation of C3 within the intestinal epithelial cells in a cathepsin-dependent manner. Modulation of cathepsin activity may prevent injury of organs exposed to ischemia.

Complement system activation is recognized as a deleterious component of the mammalian physiological response to traumatic injury with severe hemorrhage (TH). Female Yorkshire swine were subjected to a simulated austere prehospital battlefield scenario. Each animal underwent controlled hemorrhage of 22 mL/kg at 100 mL/min rate for approximately 10 min followed by soft tissue injury, femur fracture, and spleen injury. Subsequent blood loss was uncontrolled. Twenty-eight minutes postinjury the animals were randomized into treatment or no treatment with recombinant human C1 esterase inhibitor (C1INH) (500 IU/kg, n = 11) and into receiving or not permissive hypotensive resuscitation (n = 14) with infusion of 45 mL/kg lactated Ringer's solution (2× blood lost). Observation and animal maintenance continued for 6 h at which time the animals had either expired or were euthanized. Heart, lung, and small intestine tissue samples were collected. Pharmacokinetic, hemodynamic, and metabolic parameters as well as survival time, plasma complement activity and tissue deposition, cytokine levels, and tissue injury were determined. We found that administration of C1INH protected tissues from damage, reduced the levels of inflammatory cytokines, and improved blood chemistry. Immunohistochemical analyses revealed that C1INH administration following TH markedly reduced complement activation and deposition in tissues. Importantly, C1INH administration prolonged survival of animals particularly in those which received resuscitation fluid infusion. Our data urge early administration of C1INH to limit organ damage and prolong survival of those injured in the battlefield.

Background Acute trauma coagulopathy (ATC) is defined by an admission prothrombin time ratio (PTr) >1.2 or reduced clot amplitude, on viscoelastic testing. It is associated with increased mortality. Whether ATC occurs after military trauma and whether it is similar to civilian ATC has not been studied. Military trauma differs from civilian trauma with most injuries from explosions and high velocity gun-shot wounds (GSW). This study aimed to characterize military ATC, and to explore possible differences due to the mechanism of injury. Methods: Casualties meeting full trauma team activation on admission to a military field hospital in Afghanistan were eligible for enrollment. After routine blood sampling, waste plasma was prepared, frozen and transported to the UK for further analysis. Findings 77% had ATC as defined by PTr >1.2; only 19% had ATC as defined by a ROTEM A5

C3a is important in the regulation of the immune response as well as in the development of organ inflammation and injury. Furthermore, C3a contributes to liver regeneration but its role in intestinal stem cell function has not been studied. We hypothesized that C3a is important for intestinal repair and regeneration. Intestinal organoid formation, a measure of stem cell capacity, was significantly limited in C3-deficient and C3a receptor (C3aR) 1-deficient mice while C3a promoted the growth of organoids from normal mice by supporting Wnt-signaling but not from C3aR1-deficient mice. Similarly, the presence of C3a in media enhanced the expression of the intestinal stem cell marker leucine-rich repeat G-protein-coupled receptor 5 (Lgr5) and of the cell proliferation marker Ki67 in organoids formed from C3-deficient but not from C3aR1-deficient mice. Using Lgr5.egfp mice we showed significant expression of C3 in Lgr5+ intestinal stem cells whereas C3aR1 was expressed on the surface of various intestinal cells. C3 and C3aR1 expression was induced in intestinal crypts in response to ischemia/reperfusion injury. Finally, C3aR1-deficient mice displayed ischemia/reperfusion injury comparable to control mice. These data suggest that C3a through interaction with C3aR1 enhances stem cell expansion and organoid formation and as such may have a role in intestinal regeneration.

Ischemia-reperfusion (IR) injury to the small intestine following clamping of the superior mesenteric artery results in an intense local inflammatory response that is characterized by villous damage and neutrophil infiltration. IL-17A, a cytokine produced by a variety of cells in response to inflammatory cytokines released following tissue injury, has been implicated in IR injury. Using Il17a−/−, Il23r−/−, and Rorc−/− mice and administration of anti–IL-17A and anti–IL-23 neutralizing Abs to wild-type mice, we demonstrate that intestinal IR injury depends on IL-17A and that IL-17A is downstream of the binding of autoantibody to ischemia-conditioned tissues and subsequent complement activation. Using bone marrow chimeras, we demonstrate that the IL-17A required for intestinal IR injury is derived from hematopoietic cells. Finally, by transferring autoantibody-rich sera into Rag2γc−/− and Rag2−/− mice, we demonstrate that innate lymphoid cells are the main producers of IL-17A in intestinal IR injury. We propose that local production of IL-17A by innate lymphoid cells is crucial for the development of intestinal IR injury and may provide a therapeutic target for clinical exploitation.

Blast-induced ocular trauma is a frequent cause of morbidity for survivors of improvised explosive devices. Blast overpressure (BOP) of 120 ± 7 KPa has been shown to cause damage to lungs, brain, and gut in a rat model; however, the effects of BOP on ocular tissues have not been characterized. To elucidate the pathophysiology of blast-induced ocular trauma, ocular tissues from rats subjected to blast were examined for evidence of apoptosis by the detection of activated caspase 3 and TUNEL assay in their ocular tissues.A compressed air shock tube was used to deliver 120 ± 7 KPa of BOP for duration of 2 msec to the right side of the rats. Rats were then euthanized at specific time points after blast exposure (3 hours, 24 hours, 48 hours). Ocular tissues were processed for immunohistochemistry to detect activated caspase 3 and TUNEL assay. Tissues were evaluated for relative levels of positive signal as compared to nonblast exposed controls.Activated caspase 3 was detected in the optic nerve, ganglion layer, and inner nuclear layer post blast exposure. At 24 and 48 hours, the inner nuclear layer from the right side had more cells with activated caspase 3. In the optic nerve, the highest levels of activated caspase 3 were detected on the right side at 24 hours post blast.BOP of 120 ± 7 KPa induces optic neuropathy and retinal damage. In both the optic nerve and retina, caspase 3 was activated in the right and left sides following blast exposure. The results of this study reveal that blast exposure induces apoptosis in both the optic nerve and retinal tissues.

BackgroundComplementopathy (rapid complement activation and consumption after trauma) has been reported in trauma patients, but the underlying mechanism of these phenomena and their clinical significance remain unclear. This study aimed to determine the complement/complement pathway activation and identify the association of complement activation with clinical outcomes in trauma patients.MethodsWe studied 33 trauma patients with mean Injury Severity Score of 25, and 25 healthy volunteers. Sera were collected on patients’ arrival at the emergency department, as well as 1, 2, 3, 5, and 7 days after trauma, to measure the levels of terminal complement activation product soluble C5b-9 (sC5b-9) by ELISA. In addition, the functional complement activation pathway was evaluated using a commercial complement system screening kit.ResultsSerum concentrations of sC5b-9 (complement terminal pathway activity) were significantly increased in trauma patients throughout the entire observation period except on day 1. Complement terminal activities were significantly higher in 27 of 33 patients with systemic inflammatory response syndrome (SIRS) than non-SIRS patients on day 2, day 5, and day 7. Increased serum levels of sC5b-9 positively correlated with SIRS. Functional complement analysis revealed that the classical pathway was the predominant pathway responsible for complement activation. Burn patients tended to have a greater and prolonged classical pathway activation than non-burn patients, and burn injury and blunt injury were associated with higher blood levels of sC5b-9 than penetrating injury.DiscussionEarly complement activation through the classical pathway after trauma is observed and positively correlated with the development of SIRS. Thus, monitoring of the complement system might be beneficial in the care of critically injured patients.Level of evidenceIII.Study typePrognostic.

Ischemia-reperfusion (IR) injury causes a vigorous immune response that is amplified by complement activation, leading to local and remote tissue damage. Using MRL /lpr mice, which are known to experience accelerated tissue damage after mesenteric IR injury, we sought to evaluate whether complement inhibition mitigates organ damage. We found that complement depletion with cobra venom factor protected mice from local and remote lung tissue damage. Protection from injury was associated with less complement (C3) and membrane attack complex deposition, less neutrophil infiltration, and lower levels of local proinflammatory cytokine production. In addition, complement depletion was able to decrease the level of oxidative stress as measured by glutathione peroxidase 1 mRNA levels and superoxide dismutase activity. Furthermore, blockage of C5a receptor protected MRL/ lpr mice from local tissue damage, but not from remote lung tissue damage. In conclusion, although treatments with cobra venom factor and C5a receptor antagonist were able to protect mice from local tissue damage, treatment with C5a receptor antagonist was not able to protect mice from remote lung tissue damage, implying that more factors contribute to the development of remote tissue damage after IR injury. These data also suggest that complement inhibition at earlier, rather than late, stages can have clinical benefit in conditions that are complicated with IR injury.

Trauma remains the main cause of death for both civilians and those in uniform. Trauma-associated coagulopathy is a complex process involving inflammation, coagulation, and platelet dysfunction. It is unknown whether activation of complement, which occurs invariably in trauma patients, is involved in the expression of trauma-associated coagulopathy. We designed a prospective study in which we enrolled 40 trauma patients and 30 healthy donors upon arrival to the emergency department of BIDMC. Platelets from healthy individuals were incubated with sera from trauma patients and their responsiveness to a thrombin receptor-activating peptide was measured using aggregometry. Complement deposition on platelets from trauma patients was measured by flow cytometry. Normal platelets displayed hypoactivity after incubation with trauma sera even though exposure to trauma sera resulted in increased agonist-induced calcium flux. Depletion of complement from sera further blocked activation of hypoactive platelets. Conversely, complement activation increased aggregation of platelets. Platelets from trauma patients were found to have significantly higher amounts of C3a and C4d on their surface compared with platelets from controls. Depletion of complement (C4d, C3a) reversed the ability of trauma sera to augment agonist-induced calcium flux in donor platelets. Our data indicate that complement enhances platelet aggregation. Despite its complement content, trauma sera render platelets hypoactive and complement depletion further blocks activation of hypoactive platelets. The defect in platelet activation induced by trauma sera is distal to receptor activation since agonist-induced Ca2+ flux is elevated in the presence of trauma sera owing to complement deposition.

Tissue injury following ischemia-reperfusion (I/R) occurs as a consequence of actions of soluble factors and immune cells. Growing evidence supports a role for platelets in the manifestation of tissue damage following I/R. Spleen tyrosine kinase has been well documented to be important in lymphocyte activation and more recently in platelet activation. We performed experiments to evaluate whether inhibition of platelet activation through inhibition of spleen tyrosine kinase prevents tissue damage after mesenteric I/R injury. Platelets isolated from C57BL/6J mice fed with R788 for 10 days were transfused into C57BL/6J mice depleted of platelets 2 days before mesenteric I/R injury. Platelet-depleted mice transfused with platelets from R788-treated mice before mesenteric I/R displayed a significant reduction in the degree of remote lung damage, but with little change in the degree of local intestinal damage compared with control I/R mice. Transfusion of R788-treated platelets also decreased platelet sequestration, C3 deposition, and immunoglobulin deposition in lung, but not in the intestine, compared with control groups. These findings demonstrate that platelet activation is a requisite for sequestration in the pulmonary vasculature to mediate remote tissue injury after mesenteric I/R. The use of small-molecule inhibitors may be valuable to prevent tissue damage in remote organs following I/R injury.

Ischemia/reperfusion (IR) instigates a complex array of inflammatory events which result in damage to the local tissue. IR-related organ damage occurs invariably in several clinical conditions including trauma, organ transplantation, autoimmune diseases and revascularization procedures. We critically review available pre-clinical experimental information on the role of immune response in the expression of tissue damage following IR. Distinct elements of the innate and adaptive immune response are involved in the expression of tissue injury. Interventions such as prevention of binding of natural antibody to antigen expressed on the surface of ischemia-conditioned cells, inhibition of the ensuing complement activation, modulation of Toll-like receptors, B or T cell depletion and blockade of inflammatory cytokines and chemokines limit IR injury in preclinical studies. Clinical trials that will determine the therapeutic value of each approach is needed.

Background C-reactive protein (CRP) is an acute pro-inflammatory mediator that has been demonstrated to enhance ischemia/reperfusion (IR) injury by virtue of activating the complement system. CRP is able to interact with complement proteins such as C1q, complement factor H, and C4b-binding protein. Since complement activation is central in the expression of tissue injury following IR, we have investigated the effects of human decay-accelerating factor (DAF), a complement inhibitor, on CRP-potentiated complement activation and tissue injury in mice subjected to mesenteric IR. Materials and Methods Male C57B1/6 mice were allocated into eight groups: (1) Sham-operated group without IR injury; (2) CRP+Sham group; (3) IR group; (4) CRP+IR group; (5) DAF group; (6) CRP+DAF group; (7) IR+DAF group, and (8) CRP+IR+DAF group. Intestinal and lung injury, neutrophil infiltration, myeloperoxidase (MPO) expression, complement component deposition, and interleukin-6 (IL-6) production were assessed for each treatment group of mice. Results We report that administration of DAF significantly attenuates the CRP-enhanced intestinal injury as well as remote lung damages following acute mesenteric IR in mice, while DAF inhibits complement activation, suppresses neutrophil infiltration, and reduces IL-6 production. Conclusions Our study suggests that inhibition complement activation with DAF may prove useful for the treatment of post-ischemic inflammatory injuries associated with an increased production of CRP.

Multiple elements are known to participate in ischemia/reperfusion (I/R)-mediated tissue injury. Amongst them, B cells have been shown to contribute by the production of antibodies that bind to ischemic cells and fix complement. It is currently unknown whether B cells participate through antibody-independent mechanisms in the pathogenesis of I/R. In a mesenteric I/R model we found that B cells infiltrate the injured intestine of normal and autoimmune mice 2 h after reperfusion is established. B cell depletion protected mice from the development of I/R-mediated intestinal damage. The protection conferred by B cell depletion was significantly greater in MRL/lpr mice. Finally, we show that ischemic tissue expressed the B cell-attractant CXCL13 and infiltrating B cells expressed the corresponding receptor CXCR5. Our data grant B cells an antibody-independent role in the pathogenesis of intestinal I/R and suggest that B cells accumulate in the injured tissue in response to the chemokine CXCL13.

Dietary copper is an essential trace element with roles in both functional and structural aspects of the cardiovascular system. In particular, the vascular response to inflammatory stimuli is known to be significantly augmented in copper-deficient rats. The current study was designed to quantify the extent of injury-induced neointimal proliferation and stenosis in rats fed diets either adequate or deficient in copper. Male, weanling Sprague-Dawley rats were fed purified diets that were either adequate (CuA; 5.6 μg Cu/g) or deficient (CuD; 0.3 μg Cu/g) in copper for 4 weeks. Balloon injury was induced in the left external carotid arteries. Fourteen days after injury, histomorphometric analysis of cross-sections from carotid arteries showed increased neointimal formation in the CuD group compared with the CuA controls (neointima/media ratio: 4.55 ± 0.93 vs 1.45 ± 0.2, respectively). These results correspond with data indicating that the activity of Cu/Zn-superoxide dismutase (SOD) is depressed in rats fed this CuD diet. Because superoxide anion and redox status are known to play a key role in the extent of neointimal formation in response to injury, we propose that the exaggerated neointimal proliferation seen in the CuD group is the result of the diminished Cu/Zn-SOD activity.

Complement system is activated in patients with trauma. Although complement activation is presumed to contribute to organ damage and constitutional symptoms, little is known about the involved mechanisms. Because complement components may deposit on RBCs, we asked whether complement deposits on the surface of RBC in trauma and whether such deposition alters RBC function.A prospective experimental study.Research laboratory.Blood samples collected from 42 trauma patients and 21 healthy donors.None.RBC and sera were collected from trauma patients and control donors. RBCs from trauma patients (n = 40) were found to display significantly higher amounts of C4d on their surface by flow cytometry compared with RBCs from control (n = 17) (p0.01). Increased amounts of iC3b were found in trauma sera (n = 27) (vs 12 controls, p0.01) by enzyme-linked immunosorbent assay. Incubation of RBC from universal donors (type O, Rh negative) with trauma sera (n = 10) promoted C4d deposition on their surface (vs six controls, p0.05). Complement-decorated RBC (n = 6) displayed limited their deformability (vs six controls, p0.05) in two-dimensional microchannel arrays. Incubation of RBC with trauma sera (n = 10) promoted the phosphorylation of band 3, a cytoskeletal protein important for the function of the RBC membrane (vs eight controls, p0.05), and also accelerated calcium influx (n = 9) and enhanced nitric oxide production (n = 12) (vs four and eight controls respectively, p0.05) in flow cytometry.Our study found the presence of extensive complement activation in trauma patients and presents new evidence in support of the hypothesis that complement activation products deposit on the surface of RBC. Such deposition could limit RBC deformability and promote the production of nitric oxide. Our findings suggest that RBC in trauma patients malfunctions, which may explain organ damage and constitutional symptoms that is not accounted for otherwise by previously known pathophysiologic mechanisms.

β-Glucans possess broad immunomodulatory properties, including activation of innate immune functions such as oxidative burst activity. The differential roles of complement receptor type 3 (CR3) and Dectin-1, the known β-glucan receptors, and their associated signaling pathways in the generation of oxidative burst induced by different physical forms of Saccharomyces cerevisiae-derived β-glucan were examined in human peripheral blood mononuclear cells (PBMC). In this study whole glucan particle (WGP) or immobilized soluble β-glucan (ISG) was used to represent the phagocytizable or the nonphagocytizable form of a fungus, respectively. Oxidative burst as measured by the formation of superoxide (SO) was detected in PBMC in response to WGP and ISG. SO induction with WGP was concluded to be Dectin-1-mediated and required Src family kinases, phosphatidylinositol-3 kinase and protein kinase B/Akt. In contrast, the SO induction generated by ISG was CR3-mediated and required focal adhesion kinase, spleen tyrosine kinase, phosphatidylinositol-3 kinase, Akt, p38 mitogen activated protein kinase, phospholipase C and protein kinase C. The study results support the hypothesis that human PBMC, specifically monocytes, utilize distinct receptors and overlapping, but distinct, signaling pathways for the oxidative burst in response to challenge by different physical forms of β-glucan.

Background Blast-induced neurotrauma (BINT) is the signature life threatening injury of current military casualties. Neuroinflammation is a key pathological occurrence of secondary injury contributing to brain damage after blast injury. We have recently demonstrated that blast-triggered complement activation and cytokine release are associated with BINT. Here, we evaluated if administration of the complement inhibitor recombinant human decay-accelerating factor (rhDAF) is beneficial on neuroinflammation and neurodegeneration in a rat model of moderate BINT. Administration of rhDAF after exposure to moderate blast overpressure (BOP, 120 kPa) mitigated brain injury characterized by neuronal degeneration. rhDAF treatment reduced complement hemolytic activity at 3 hours and tissue complement deposition at 3, 24, and 48 hours as well as systemic and local cytokine release at 24 hours post BOP. Furthermore, rhDAF protected blood–brain barrier (BBB) integrity and reduced cytotoxic edema. Interaction between complement cleavage component, C3a and C3a receptor and tau phosphorylation were also attenuated in rhDAF treated animals at 3 and 24 hours after BOP. These novel findings suggest early complement targeted inhibition as a new therapeutic strategy to decrease neuroinflammation and neurodegeneration after blast TBI. Result Administration of rhDAF after exposure to moderate blast overpressure (BOP, 120 kPa) mitigated brain injury characterized by neuronal degeneration. rhDAF treatment reduced complement hemolytic activity at 3 hours and tissue complement deposition at 3, 24, and 48 hours as well as systemic and local cytokine release at 24 hours post BOP. Furthermore, rhDAF protected blood–brain barrier (BBB) integrity and reduced cytotoxic edema. Interaction between complement cleavage component, C3a and C3a receptor and tau phosphorylation were also attenuated in rhDAF treated animals at 3 and 24 hours after BOP. Conclusion These novel findings suggest early complement targeted inhibition as a new therapeutic strategy to decrease neuroinflammation and neurodegeneration after blast TBI.

Inflammation and vascular injury triggered by ischemia/reperfusion (I/R) represent a leading cause of morbidity and mortality in a number of clinical settings. Wnt and its homolog partners R-spondins, in addition to regulating embryonic development have recently been demonstrated to serve as wound-healing agents in inflammation-associated conditions. Here we ask whether R-spondins could prevent inflammation-associated tissue damage in ischemic disorders and thus investigate the role of R-spondin3 (R-spo3) in a mouse model of mesenteric I/R. We demonstrate that R-spo3 ameliorates mesenteric I/R-induced local intestinal as well as remote lung damage by suppressing local and systemic cytokine response and deposition of IgM and complement in intestinal tissues. We also show that decreased inflammatory response is accompanied by tightening of endothelial cell junctions and reduction in vascular leakage. We conclude that R-spo3 stabilizes endothelial junctions and inhibits vascular leakage during I/R and thereby mitigates the inflammatory events and associated tissue damage. Our findings uniquely demonstrate a protective effect of R-spo3 in I/R-related tissue injury and suggest a mechanism by which it may have these effects.

Background The implementation of a human research protection program in Afghanistan and the mobilization of the combat casualty research team have made it possible to design and efficiently conduct multifaceted, multisite, and prospective research studies in a combat environment. Still, to conduct research in such an environment, several unique challenges must be overcome. Methods This article describes the development and conduct of three ongoing trauma-related biomedical research studies in Afghanistan, highlighting the challenges and lessons learned within the context of these studies. Results Key challenges include the process of developing and getting approval for in-theater research protocols, the informed consent process, and logistics of conducting a biomedical research study in an austere environment. Despite these challenges, important lessons learned that can contribute to the success of a protocol include the need for clear operating procedures, judicious selection for which data points must be collected in-theater, and the importance anticipating the "fog and friction" of war. Conclusion As we continue the journey toward more sophisticated research capabilities in combat, this article will help inform the design and conduct of future research performed in a theater of war. Conducting biomedical research in a combat zone is an important but difficult element of military medicine.

Background Complement is invariably activated during trauma and contributes to tissue injury. Recombinant human decay-accelerating factor (DAF), a complement regulatory protein that inhibits both classical and alternative pathways, improves survival and reduces tissue damage in animal models of tissue injury. The extent to which DAF may facilitate resuscitation in hemorrhaged large animals is not known. Methods Male Yorkshire swine assigned to one of six groups were subjected to controlled, isobaric hemorrhage over 15 min to a target mean arterial pressure (MAP) of 35 mm Hg. Hypotension was maintained for 20 min followed by a bolus intravenous injection of DAF or vehicle followed by Hextend resuscitation. Animals were observed for 3 h after hypotensive Hextend resuscitation. Survival, blood chemistry, and physiological parameters were recorded. Additionally, tissue from lung, small intestine, liver, and kidney were subjected to histopathologic evaluation and tissue deposition of complement proteins was determined by immunohistochemistry, dot-blot, and Western blot analyses. Results Administration of DAF (25 μg/kg) to animals subjected to hemorrhage prior to Hextend infusion significantly improved survival (73% versus 27%); protected gut, lung, liver, and kidney tissue from damage; and resulted in reduced resuscitation fluid requirements when compared with animals subjected to hemorrhage and resuscitation with Hextend alone. Animals treated with a higher dose of DAF (50 μg/kg) followed by Hextend fluid resuscitation did not experience the same benefit, suggesting a narrow therapeutic range for use of DAF as adjunct to Hextend fluid. Conclusion DAF improved survival and reduced early Hextend fluid resuscitation requirements in swine subjected to hemorrhagic shock. These benefits are attributed to decreased complement deposition and limited organ damage.

The robust inflammatory response that occurs during ischemia reperfusion (IR) injury recruits factors from both the innate and adaptive immune systems. However the contribution of platelets and their products such as Platelet Factor 4 (PF4; CXCL4), during the pathogenesis of IR injury has not been thoroughly investigated. We show that a deficiency in PF4 protects mice from local and remote tissue damage after 30 minutes of mesenteric ischemia and 3 hours of reperfusion in PF4-/- mice compared to control B6 mice. This protection was independent from Ig or complement deposition in the tissues. However, neutrophil and monocyte infiltration were decreased in the lungs of PF4-/- mice compared with B6 control mice. Platelet-depleted B6 mice transfused with platelets from PF4-/- mice displayed reduced tissue damage compared with controls. In contrast, transfusion of B6 platelets into platelet depleted PF4-/- mice reconstituted damage in both intestine and lung tissues. We also show that PF4 may modulate the release of IgA. Interestingly, we show that PF4 expression on intestinal epithelial cells is increased after IR at both the mRNA and protein levels. In conclusion, these findings demonstrate that may PF4 represent an important mediator of local and remote tissue damage.

Ischemia-reperfusion (I/R) injury is a leading cause of morbidity and mortality. A functional role for platelets in tissue damage after mesenteric I/R is largely unknown. The hypothesis that mesenteric I/R local and remote injury are platelet dependent was tested. Using a murine mesenteric I/R model, we demonstrate that platelets orchestrate remote lung tissue damage that follows mesenteric I/R injury and also contribute, albeit to a lesser degree, to local villi damage. While lung damage is delayed compared with villi damage, it increased over time and was characterized by accumulation of platelets in the pulmonary vasculature early, followed by alveolar capillaries and extravasation into the pulmonary space. Both villi and lung tissues displayed complement deposition. We demonstrate that villi and lung damage are reduced in mice made platelet deficient before I/R injury and that platelet transfusion into previously platelet-depleted mice before I/R increased both villi and lung tissue damage. Increased C3 deposition accompanied platelet sequestration in the lung, which was mostly absent in platelet-depleted mice. In contrast, C3 deposition was only minimally reduced on villi of platelet-depleted mice. Our findings position platelets alongside complement as a significant early upstream component that orchestrates remote lung tissue damage after mesenteric I/R and strongly suggest that reperfusion injury mitigating modalities should consider the contribution of platelets.

Gut commensal bacteria play important roles in the development and homeostasis of intestinal immunity. However, the role of gut commensals in intestinal ischemia/reperfusion (I/R) injury is unclear. To determine the roles of gut commensal bacteria in intestinal IR injury, we depleted gut microbiota with a broad-spectrum antibiotic cocktail and performed mesenteric I/R (M I/R). First, we confirmed that antibiotic treatment completely depleted gut commensal bacteria and diminished the size of secondary lymphoid tissues such as the Peyer's patches. We next found that antibiotic treatment attenuated intestinal injury following M I/R. Depletion of gut commensal bacteria reduced the expression of Toll-like receptor (TLR)2 and TLR4 in the intestine. Both are well-known receptors for gram-positive and -negative bacteria. Decreased expression of TLR2 and TLR4 led to the reduction of inflammatory mediators, such as TNF, IL-6, and cyclooxygenase-2. Intestinal I/R injury is initiated when natural antibodies recognize neo-antigens that are revealed on ischemic cells and activate the complement pathway. Thus we evaluated complement and immunoglobulin (Ig) deposition in the damaged intestine and found that antibiotic treatment decreased the deposition of both C3 and IgM. Interestingly, we also found that the deposition of IgA also increased in the intestine following M I/R compared with control mice and that antibiotic treatment decreased the deposition of IgA in the damaged intestine. These results suggest that depletion of gut commensal bacteria decreases B cells, Igs, and TLR expression in the intestine, inhibits complement activation, and attenuates intestinal inflammation and injury following M I/R.

Blast-induced neurotrauma (BINT) is a major medical concern yet its etiology is largely undefined. Complement activation may play a role in the development of secondary injury following traumatic brain injury; however, its role in BINT is still undefined. The present study was designed to characterize the complement system and adaptive immune-inflammatory responses in a rat model of moderate BINT. Anesthetized rats were exposed to a moderate blast (120 kPa) using an air-driven shock tube. Brain tissue injury, systemic and local complement, cerebral edema, inflammatory cell infiltration, and pro-inflammatory cytokine production were measured at 0.5, 3, 48, 72, 120, and 168 h. Injury to brain tissue was evaluated by histological evaluation. Systemic complement was measured via ELSIA. The remaining measurements were determined by immunohistoflourescent staining. Moderate blast triggers moderate brain injuries, elevated levels of local brain C3/C5b-9 and systemic C5b-9, increased leukocyte infiltration, unregulated tumor necrosis factor alpha (TNFα), and aquaporin-4 in rat brain cortex at 3- and 48-hour post blast. Early immune-inflammatory response to BINT involves complement and TNFα, which correlates with hippocampus and cerebral cortex damage. Complement and TNFα activation may be a novel therapeutic target for reducing the damaging effects of BINT inflammation.

Background Activation of complement system has been associated with tissue injury after hemorrhage and resuscitation in rats and swine. This study investigated whether administration of human recombinant decay-accelerating factor (DAF; a complement regulatory protein that inhibits classical and alternative pathways) reduces tissue damage in a porcine model of hemorrhagic shock. Methods Male Yorkshire swine assigned to four groups were subjected to controlled, isobaric hemorrhage over 15 minutes to a target mean arterial pressure of 35 mmHg. Hypotension was maintained for 20 minutes followed by a bolus intravenous injection of DAF or vehicle then animals were observed for 200 minutes. Blood chemistry and physiological parameters were recorded. Tissue samples from lung and small intestine were subjected to histopathological evaluation and detection of tissue deposition of complement proteins by immunohistochemistry and Western blot analyses. Results Administration of DAF significantly reduced intestinal and lung tissue damage in a dose-dependent manner (5, 25, and 50 g/kg). In addition, DAF treatment improved hemorrhageinduced hyperkalemia. The protective effects of DAF appear to be related to its ability to reduce tissue complement activation and deposition on affected tissues. Conclusions DAF treatment decreased tissue complement activation and deposition in hemorrhaged animals and attenuated tissue damage at 200 minutes post treatment. The observed beneficial effects of DAF treatment on tissue injury after 20 minutes of severe hypotension presents an attractive model of small volume resuscitation, particularly in situations with a restrictive medical logistical footprint such as far-forward access to first responders in the battlefield or in remote rural or mountainous environments.

Background Activated complement system is known to mediate neuroinflammation and neurodegeneration following exposure to hypoxic-ischemic insults. Therefore, inhibition of the complement activation cascade may represent a potential therapeutic strategy for the management of ischemic brain injury. Decay-accelerating factor (DAF, also known as CD55) inhibits complement activation by suppressing the function of C3/C5 convertases, thereby limiting local generation or deposition of C3a/C5a and membrane attack complex (MAC or C5b-9) production. The present study investigates the ability of DAF to protect primary cultured neuronal cells subjected to sodium cyanide (NaCN)-induced hypoxia from degeneration and apoptosis. Methods Cultured primary cortical neurons from embryonic Sprague-Dawley rats were assigned one of four groups: control, DAF treatment alone, hypoxic, or hypoxic treated with DAF. Hypoxic cultures were exposed to NaCN for 1 hour, rinsed, followed by 24 hour exposure to 200 ng/ml of recombinant human DAF in normal medium. Human DAF was used in the present study and it has been shown to effectively regulate complement activation in rats. Neuronal cell function, morphology and viability were investigated by measuring plateau depolarization potential, counting the number dendritic spines, and observing TUNEL and MTT assays. Complement C3, C3a, C3a receptor (R) production, C3a-C3aR interaction and MAC formation were assessed along with the generation of activated caspase-9, activated caspase-3, and activated Src. Results When compared to controls, hypoxic cells had fewer dendritic spines, reduced plateau depolarization accompanied by increased apoptotic activity and accumulation of MAC, as well as up-regulation of C3, C3a and C3aR, enhancement of C3a-C3aR engagement, and elevated caspase and Src activity. Treatment of hypoxic cells with 200 ng/ml of recombinant human DAF resulted in attenuation of neuronal apoptosis and exerted significant protection against neuronal dendritic spine loss and plateau depolarization reduction. Furthermore, treatment with DAF resulted in decreased accumulation of C3a, MAC, C3a-C3aR interaction, caspase-9, activated caspase-3, and pTyr416-Src (activated Src) tyrosine kinase. Conclusion DAF was found to reduce neuronal cell death and apoptosis in NaCN induced hypoxia. This effect is attributed to the ability of DAF to limit complement activation and inhibit the activity of Src and caspases 9 and 3. This study supports the inhibiting of complement as a neuroprotective strategy against CNS ischemia/reperfusion injury.

Pamuk ON, Lapchak PH, Rani P, Pine P, Dalle Lucca JJ, Tsokos GC. Spleen tyrosine kinase inhibition prevents tissue damage after ischemia-reperfusion. Am J Physiol Gastrointest Liver Physiol 299: G391-G399, 2010. First published June 3, 2010; doi: 10.1152/ajpgi.00198.2010.-Reperfusion injury to tissue following an ischemic event occurs as a consequence of an acute inflammatory response that can cause significant morbidity and mortality. Components of both the innate (complement, immunoglobulin, monocytes, and neutrophils) and adaptive (B and T lymphocytes) immune systems have been demonstrated to mediate tissue injury. Spleen tyrosine kinase (Syk) is responsible for membrane-mediated signaling in various cell types including B lymphocytes, macrophages, and T cells. We investigated the ability of a small drug Syk inhibitor, R788, to protect mice against mesenteric ischemia-reperfusion (I/R)-induced local (intestine) and remote lung injury. Mice were fed with chow containing a Syk inhibitor for 6 days before the performance of intestinal I/R, which resulted in silencing of the expression of the active phosphorylated Syk. Syk inhibition significantly suppressed both local and remote lung injury. The beneficial effect was associated with reduced IgM and complement 3 deposition in the tissues and significant reduction of polymorphonuclear cell infiltration. Our data place Syk upstream of events leading to the binding of natural antibodies to the ischemia-conditioned tissues and urge the consideration of the use of Syk inhibitors in the prevention or improvement of tissue injury of organs exposed to ischemia or hypoperfusion.

Complement activation has been demonstrated to contribute significantly to the expression of IR-induced tissue damage. Each of the three complement pathways, classic, alternative, and lectin, has been implicated in the instigation of tissue pathology. In this study, we used a selective inhibitor of the alternative pathway, that is, a soluble form of complement receptor of the immunoglobulin superfamily (CRIg-Fc) to determine whether it can prevent IR tissue injury. We demonstrate that treatment of C57B1/6 mice prior to mesenteric IR prevents local (intestinal) and remote (lung) injury by limiting deposition of complement and entry of polymorphonuclear cells to the sites of injury. Our results show that CRIg-Fc represents a candidate to limit IR injury as it occurs in various clinical conditions.

Ischemia-reperfusion (IR) injury represents a major clinical challenge, which contributes to morbidity and mortality during surgery. The critical role of natural immunoglobulin M (IgM) and complement in tissue injury has been demonstrated. However, cellular mechanisms that result in the deposition of natural IgM and the activation of complement are still unclear. In this report, using a murine intestinal IR injury model, we demonstrated that the β-actin protein in the small intestine was cleaved and actin filaments in the columnar epithelial cells were aggregated after a transient disruption during 30 min of ischemia. Ischemia also led to deposition of natural IgM and complement 3 (C3). A low dose of cytochalasin D, a depolymerization reagent of the actin cytoskeleton, attenuated this deposition and also attenuated intestinal tissue injury in a dose-dependent manner. In contrast, high doses of cytochalasin D failed to worsen the injury. These data indicate that ischemia-mediated aggregation of the actin cytoskeleton, rather than its disruption, results directly in the deposition of natural IgM and C3. We conclude that ischemia-mediated aggregation of the actin cytoskeleton leads to the deposition of natural IgM and the activation of complement, as well as tissue injury.

Elements of the innate and adaptive immune response have been implicated in the development of tissue damage after ischemic reperfusion (I/R). Here we demonstrate that T cells infiltrate the intestine of C57BL/6 mice subjected to intestinal I/R during the first hour of reperfusion. The intensity of the T cell infiltration was higher in B6.MRL/lpr mice subjected to intestinal I/R and reflected more severe tissue damage than that observed in control mice. Depletion of T cells limited I/R damage in B6.MRL/lpr mice, whereas repletion of B6.MRL/lpr lymph node-derived T cells into the I/R-resistant Rag-1−/− mouse reconstituted tissue injury. The tissue-infiltrating T cells were found to produce IL-17. Finally, IL-23 deficient mice, which are known not to produce IL-17, displayed significantly less intestinal damage when subjected to I/R. Our data assign T cells a major role in intestinal I/R damage by virtue of producing the pro-inflammatory cytokine IL-17.

The purpose of this study was to measure the cytotoxicity of six endodontic irrigants on cultured gingival fibroblasts using the CyQuant assay. Human gingival fibroblasts were grown in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum at 37 degrees C and 5% CO(2). At confluence, cells were split, plated in 96-well plates and incubated for 24-h to allow attachment. The following irrigants were tested at various concentrations: Sodium hypochlorite (NaOCl); iodine potassium-iodide (IKI); Betadine scrub (BS); calcium hydroxide [Ca(OH)2]; chlorine dioxide (SCD) and DMEM (positive control). Experimental groups were compared by the logarithmic difference between the clinical and LD50 concentrations of a particular irrigant. The results showed that IKI and Ca(OH)2 were significantly less cytotoxic than SCD, NaOCl, and BS. In conclusion, IKI and Ca(OH)2 are well tolerated by human gingival fibroblasts.

Dietary copper is an essential trace element with roles in both functional and structural aspects of the cardiovascular system. In particular, the vascular response to inflammatory stimuli is known to be significantly augmented in copper-deficient rats. The current study was designed to quantify the extent of injury-induced neointimal proliferation and stenosis in rats fed diets either adequate or deficient in copper. Male, weanling Sprague-Dawley rats were fed purified diets that were either adequate (CuA; 5.6 microg Cu/g) or deficient (CuD; 0.3 microg Cu/g) in copper for 4 weeks. Balloon injury was induced in the left external carotid arteries. Fourteen days after injury, histomorphometric analysis of cross-sections from carotid arteries showed increased neointimal formation in the CuD group compared with the CuA controls (neointima/media ratio: 4.55 +/- 0.93 vs 1.45 +/- 0.2, respectively). These results correspond with data indicating that the activity of Cu/Zn-superoxide dismutase (SOD) is depressed in rats fed this CuD diet. Because superoxide anion and redox status are known to play a key role in the extent of neointimal formation in response to injury, we propose that the exaggerated neointimal proliferation seen in the CuD group is the result of the diminished Cu/Zn-SOD activity.

Based on observations that vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) have altered resting potentials as well as abnormal cell proliferation rates, neointima formation after controlled balloon injury was compared in arteries from SHR and Wistar Kyoto rats (WKY). SHR aortic VSMC showed hyperpolarized resting membrane potentials (-93+/-8 mV) when compared to those from WKY (-61+/-6 mV). Histomorphometric analysis of cross sections from aortic segments submitted to balloon injury showed reduced neointima formation in SHR (neointima/media ratio: 0.04+/-0.03) as compared to WKY (0.2+/-0.1). On the other hand, in injured carotid arteries, neointima formation was more extensive in SHR (neointima/media ratio 5.0+/-0.9) than in WKY (0.8+/-0.7), leading in most cases to luminal occlusion. Measurements of VSMC resting potential showed that carotid artery cells from SHR were depolarized with respect to those from WKY (-46+/-4 vs. -69+/-5 mV, respectively). The results demonstrate an inverse relationship between VSMC membrane polarization and neointima formation in SHR arteries, suggesting that genetic modifications in SHR determine a dysfunctional cellular physiology that may influence cell proliferation subsequent to injury.

Several innate and adaptive immune cell types participate in ischemia/reperfusion induced tissue injury. Amongst them, platelets have received little attention as contributors in the process of tissue damage after ischemia reperfusion (I/R) injury. It is currently unknown whether platelets participate through the immunologically important molecules including, CD40 and when activated, CD154 (CD40L), in the pathogenesis of I/R injury. We hypothesized that constitutive expression of CD40 and activation-induced expression of CD154 on platelets mediate local mesenteric and remote lung tissue damage after I/R injury. Wild type (WT; C57BL/6J), CD40 and CD154 deficient mice underwent mesenteric ischemia for 30 minutes followed by reperfusion for 3 hours. WT mice subjected to mesenteric I/R injury displayed both local intestinal and remote lung damage. In contrast, there was significantly less intestinal damage and no remote lung injury in CD40 and CD154 deficient mice when compared to WT mice. Platelet-depleted WT mice transfused with platelets from CD40 or CD154 deficient mice failed to reconstitute remote lung damage. In contrast, when CD40 or CD154 deficient mice were transfused with WT platelets lung tissue damage was re-established. Together, these findings suggest that multiple mechanisms are involved in local and remote tissue injury and also identify platelet-expressed CD40 and/or CD154 as mediators of remote tissue damage.