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Sepsis remains one of the leading causes of death in burn patients who survive the initial insult of injury. Disruption of the intestinal epithelial barrier has been shown after burn injury; this can lead to the translocation of bacteria or their products (e.g., endotoxin) from the intestinal lumen to the circulation, thereby increasing the risk for sepsis in immunocompromised individuals. Since the maintenance of the epithelial barrier is largely dependent on the intestinal microbiota, we examined the diversity of the intestinal microbiome of severely burned patients and a controlled mouse model of burn injury. We show that burn injury induces a dramatic dysbiosis of the intestinal microbiome of both humans and mice and allows for similar overgrowths of Gram-negative aerobic bacteria. Furthermore, we show that the bacteria increasing in abundance have the potential to translocate to extra-intestinal sites. This study provides an insight into how the diversity of the intestinal microbiome changes after burn injury and some of the consequences these gut bacteria can have in the host.

Apoptotic injury participates in hepatic fibrosis, but the molecular mechanisms are not well understood. The present study aimed to investigate the role of inducible TIMP1 in the pathogenesis of hepatic apoptosis-fibrosis. Apoptosis was induced with GCDC, LPS, and alcohol in precision-cut liver slices or bile duct ligation (BDL) in rats, as reflected by caspase-3 activity, TUNEL assay, and apoptosis-related gene profiles. The hepatic fibrosis was detected with Picrosirius staining, hydroxyproline determination, and expression profiling of fibrosis-related genes. Levels of TIMP1 were upregulated by the hepatic apoptosis, but downregulated by caspase inhibitor. The inducible TIMP1 was apoptosis-dependent. Once TIMP1 was inhibited with treatment of TIMP1-siRNA, the fibrotic response was reduced as demonstrated by hydroxyproline assay. In addition, the expression of fibrosis-related genes aSMA, CTGF, and TGFb2r were down-regulated subsequent to the treatment of TIMP1-siRNA. TIMP1 could mediate the expression of fibrosis-related genes. TIMP1 was transcriptionally regulated by nuclear factor c-Jun as demonstrated by EMSA and ChIP assay. The treatment of c-Jun siRNA could significantly decrease the expression of TIMP1 induced by alcohol, GCDC, or LPS treatment. Hepatic apoptosis induces the expression of TIMP1. Inducible TIMP1 can modulate the expression of fibrosis-related genes in liver. TIMP1 pathway is a potential target for therapeutic intervention of fibrotic liver diseases.

Studies have shown that burn patients who are intoxicated at the time of injury are more susceptible to infection and have a higher incidence of mortality. A major cause of death in burn and trauma patients regardless of their alcohol (EtOH) exposure is multiple organ dysfunction, which is driven in part by the systemic inflammatory response and activated neutrophils. Neutrophils are short lived and undergo apoptosis to maintain homeostasis and resolution of inflammation. A delay in apoptosis of neutrophils is one important mechanism which allows for their prolonged presence and the release of potentially harmful enzymes. The purpose of this study was to examine whether EtOH intoxication combined with burn injury influences neutrophil apoptosis and whether IL-18 plays any role in this setting. To accomplish this investigation, rats were gavaged with EtOH (3.2 g/kg) 4 h before being subjected to sham or burn injury of ~12.5% of the total body surface area, and then killed on d 1 after injury. Peripheral blood neutrophils were isolated and lysed. The lysates were analyzed for pro-and antiapoptotic proteins. We found that EtOH combined with burn injury prolonged neutrophil survival. This prolonged neutrophil survival was accompanied by a decrease in the levels of the neutrophil proapoptotic protein Bax, and an increase in antiapoptotic proteins Mcl-1 and Bcl-xl. Administration of IL-18 antibody following burn injury normalized the levels of Bax, Mcl-1 and Bcl-xl. The decrease in caspase-3 and DNA fragmentation observed following EtOH and burn injury was also normalized in rats treated with anti-IL-18 antibody. These findings suggest that IL-18 delays neutrophil apoptosis following EtOH and burn injury by modulating the pro-and antiapoptotic proteins.

The immediate appearance of platelets in wounds and the ability of platelets to release growth factors suggest that platelets are an important trigger of the tissue repair process. To examine the effect of systemic thrombocytopenia on both the inflammatory and proliferative aspects of wound healing, adult mice were rendered thrombocytopenic by intraperitoneal administration of a rabbit antimouse platelet serum. Full-thickness excisional dermal wounds were prepared and analyzed for inflammatory cell content, growth factor production, reepithelialization, collagen synthesis, and angiogenesis at multiple time points after injury. Compared to control mice, thrombocytopenic mice exhibited significantly altered wound inflammation. Wounds of thrombocytopenic mice contained significantly more macrophages and T cells, yet exhibited neutrophil content similar to wounds from control mice. Surprisingly, thrombocytopenic mice exhibited no delay in the reparative aspects of wound healing. The rate of wound reepithelialization, collagen synthesis, and angiogenesis was nearly identical for thrombocytopenic and control mice. Analysis of vascular endothelial growth factor, fibroblast growth factor 2, transforming growth factor beta1, keratinocyte growth factor, and epidermal growth factor revealed no difference in the levels of these growth factors in the wounds of control and thrombocytopenic mice. Taken together, the results suggest that the presence of platelets may influence wound inflammation, but that platelets do not significantly affect the proliferative aspects of repair, including wound closure, angiogenesis, and collagen synthesis.The immediate appearance of platelets in wounds and the ability of platelets to release growth factors suggest that platelets are an important trigger of the tissue repair process. To examine the effect of systemic thrombocytopenia on both the inflammatory and proliferative aspects of wound healing, adult mice were rendered thrombocytopenic by intraperitoneal administration of a rabbit antimouse platelet serum. Full-thickness excisional dermal wounds were prepared and analyzed for inflammatory cell content, growth factor production, reepithelialization, collagen synthesis, and angiogenesis at multiple time points after injury. Compared to control mice, thrombocytopenic mice exhibited significantly altered wound inflammation. Wounds of thrombocytopenic mice contained significantly more macrophages and T cells, yet exhibited neutrophil content similar to wounds from control mice. Surprisingly, thrombocytopenic mice exhibited no delay in the reparative aspects of wound healing. The rate of wound reepithelialization, collagen synthesis, and angiogenesis was nearly identical for thrombocytopenic and control mice. Analysis of vascular endothelial growth factor, fibroblast growth factor 2, transforming growth factor beta1, keratinocyte growth factor, and epidermal growth factor revealed no difference in the levels of these growth factors in the wounds of control and thrombocytopenic mice. Taken together, the results suggest that the presence of platelets may influence wound inflammation, but that platelets do not significantly affect the proliferative aspects of repair, including wound closure, angiogenesis, and collagen synthesis.

Standard methods of neutrophil isolation require skilled personnel, are time consuming and use large blood volumes. Kotz and his colleagues have developed a rapid microfluidic chip-based approach for rapidly isolating neutrophils directly from whole blood with 'on-chip' processing for mRNA and protein isolation. The device, which yields sufficient quantities and purities for downstream genomic or proteomic analysis, was validated in a multicenter clinical study of the immune response to severe trauma and burn injury. Neutrophils have key roles in modulating the immune response. We present a robust methodology for rapidly isolating neutrophils directly from whole blood with 'on-chip' processing for mRNA and protein isolation for genomics and proteomics. We validate this device with an ex vivo stimulation experiment and by comparison with standard bulk isolation methodologies. Last, we implement this tool as part of a near-patient blood processing system within a multi-center clinical study of the immune response to severe trauma and burn injury. The preliminary results from a small cohort of subjects in our study and healthy controls show a unique time-dependent gene expression pattern clearly demonstrating the ability of this tool to discriminate temporal transcriptional events of neutrophils within a clinical setting.

Chemokine (C-X-C motif) receptor (CXCR) 4 and atypical chemokine receptor (ACKR) 3 ligands have been reported to modulate cardiovascular function in various disease models. The underlying mechanisms, however, remain unknown. Thus, it was the aim of the present study to determine how pharmacological modulation of CXCR4 and ACKR3 regulate cardiovascular function. In vivo administration of TC14012, a CXCR4 antagonist and ACKR3 agonist, caused cardiovascular collapse in normal animals. During the cardiovascular stress response to hemorrhagic shock, ubiquitin, a CXCR4 agonist, stabilized blood pressure, whereas coactivation of CXCR4 and ACKR3 with CXC chemokine ligand 12 (CXCL12), or blockade of CXCR4 with AMD3100 showed opposite effects. While CXCR4 and ACKR3 ligands did not affect myocardial function, they selectively altered vascular reactivity upon α 1 -adrenergic receptor (AR) activation in pressure myography experiments. CXCR4 activation with ubiquitin enhanced α 1 -AR-mediated vasoconstriction, whereas ACKR3 activation with various natural and synthetic ligands antagonized α 1 -AR-mediated vasoconstriction. The opposing effects of CXCR4 and ACKR3 activation by CXCL12 could be dissected pharmacologically. CXCR4 and ACKR3 ligands did not affect vasoconstriction upon activation of voltage-operated Ca 2+ channels or endothelin receptors. Effects of CXCR4 and ACKR3 agonists on vascular α 1 -AR responsiveness were independent of the endothelium. These findings suggest that CXCR4 and ACKR3 modulate α 1 -AR reactivity in vascular smooth muscle and regulate hemodynamics in normal and pathological conditions. Our observations point toward CXCR4 and ACKR3 as new pharmacological targets to control vasore-activity and blood pressure.

The immediate appearance of platelets in wounds and the ability of platelets to release growth factors suggest that platelets are an important trigger of the tissue repair process. To examine the effect of systemic thrombocytopenia on both the inflammatory and proliferative aspects of wound healing, adult mice were rendered thrombocytopenic by intraperitoneal administration of a rabbit antimouse platelet serum. Full-thickness excisional dermal wounds were prepared and analyzed for inflammatory cell content, growth factor production, reepithelialization, collagen synthesis, and angiogenesis at multiple time points after injury. Compared to control mice, thrombocytopenic mice exhibited significantly altered wound inflammation. Wounds of thrombocytopenic mice contained significantly more macrophages and T cells, yet exhibited neutrophil content similar to wounds from control mice. Surprisingly, thrombocytopenic mice exhibited no delay in the reparative aspects of wound healing. The rate of wound reepithelialization, collagen synthesis, and angiogenesis was nearly identical for thrombocytopenic and control mice. Analysis of vascular endothelial growth factor, fibroblast growth factor 2, transforming growth factor pi, keratinocyte growth factor, and epidermal growth factor revealed no difference in the levels of these growth factors in the wounds of control and thrombocytopenic mice. Taken together, the results suggest that the presence of platelets may influence wound inflammation, but that platelets do not significantly affect the proliferative aspects of repair, including wound closure, angiogenesis, and collagen synthesis.

CXC chemokine receptor (CXCR)-4 agonists have been shown to attenuate inflammation and organ injury in various disease models, including trauma/hemorrhage. The pathophysiological role of CXCR4 during the early response to tissue injury, however, remains unknown. Therefore, we investigated the effects of AMD3100, a drug that antagonizes binding of stromal cell-derived factor (SDF)-1α and ubiquitin to CXCR4 during the initial response to polytrauma in pigs. Fifteen minutes before polytrauma (femur fractures/lung contusion; control: sham), 350 nmol/kg AMD3100, equimolar AMD3100 and ubiquitin (350 nmol/kg each) or vehicle were administered intravenously. After a 60-min shock period, fluid resuscitation was performed for 360 min. Ubiquitin binding to peripheral blood mononuclear cells was significantly reduced after intravenous AMD3100. SDF-1α plasma levels increased transiently >10-fold with AMD3100 in all animals. In injured animals, AMD3100 increased fluid requirements to maintain hemodynamics and enhanced increases in peripheral blood granulocytes, lymphocytes and monocytes, compared with its effects in uninjured animals. Cytokine release from leukocytes in response to Toll-like receptor (TLR)-2 and TLR-4 activation was increased after in vitro AMD3100 treatment of normal whole blood and after in vivo AMD3100 administration in animals subjected to polytrauma. Coadministration of AMD3100/ubiquitin reduced lactate levels, prevented AMD3100-induced increases in fluid requirements and sensitization of the tumor necrosis factor (TNF)-α and interleukin (IL)-6 release upon TLR-2/4 activation, but did not attenuate increases in leukocyte counts and SDF-1α plasma levels. Our findings suggest that CXCR4 controls leukocyte mobilization after trauma, regulates leukocyte reactivity toward inflammatory stimuli and mediates protective effects during the early phase of trauma-induced inflammation.

The purpose of this study was to determine if statistical models for prediction of chest injuries would outperform the clinician's (MD) ability to identify injured patients at risk for a thoracic injury diagnosed by chest radiograph (CXR). A prospective observational study was done during a 12-month period. The study was conducted in a level I trauma center. Injured patients meeting trauma team activation criteria were enrolled to the study. Physical examination findings by a clinician were interpreted and CXR was performed. The accuracy of 2 mathematical models is compared against the accuracy of clinician's clinical judgment in predicting an injury by CXR. Two newly constructed multivariate models, binary logistic regression (LR) and classification and regression tree (CaRT) analysis, are compared to previously published data of clinician clinical assessment of probability of thoracic injury identified by CXR. Data for 757 patients were analyzed. Classification and regression tree analysis developed a stepwise decision tree to determine which signs/symptoms were indicative of an abnormal CXR finding. The sensitivity (CaRT, 36.6%; LR, 36.3%; MD, 58.7%), specificity (CaRT, 98.3%; LR, 98.2%; MD, 96.4%), and error rates (CaRT, 0.93; LR, 0.94; MD, 0.82) show that the mathematical decision aids are less sensitive and risk more misclassification compared to clinician judgment in predicting an injury by CXR. Clinician judgment was superior to mathematical decision aids for predicting an abnormal CXR finding in injured patients with chest trauma.

Despite blood-conservation techniques, hemorrhage during burn excision remains substantial. It is difficult to predict the blood loss that will occur per operation and how many units the patient will require during surgery. This may result in high cross-match-to-transfusion ratios (CMTRs). A retrospective chart review from 2001 to 2003 was performed. All adult patients with >20% total body surface area burns who underwent surgery were included in the study. Variables examined were centimeters excised, estimated blood loss, packed red blood cells transfused during surgery, preoperative and postoperative hematocrit, and CMTR. There were 273 operations. The average estimated blood loss was 820 mL, and; the median posttransfusion hematocrit was 27. Based on the area excised and units transfused, a ratio of packed red blood cells/cm excised was determined. A total of 1.78 U blood were transfused/1000 cm 2 excised to keep hematocrit between 25 and 31; P = 0.02. Estimation of excision area can predict transfusion need, which at our institution yields a low CMTR.