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The contribution of individual genetic determinants of aging to the adverse clinical outcomes and altered inflammation mediator networks characteristic of aged trauma patients is unknown. The AA genotype of the aging-related single-nucleotide polymorphism (SNP) rs2075650 in TOMM40 has been associated with longevity, while the AG and GG genotypes are associated with an increased risk of Alzheimer’s disease. Here, we studied the effect of rs2075650 on clinical outcomes and dynamic biomarker patterns after traumatic injury. Genomic DNA was obtained from blunt trauma patients admitted to the ICU and examined for 551,839 SNPs using an Illumina microarray kit. Plasma was sampled from each patient three times within the first 24 hours and daily from day 1 to 7 then assayed for 31 biomarkers using Luminex. Aged patients (65–90 years) were segregated into AA (n = 77) and AG/GG (n = 17) genotypes. Additional comparisons were made with matched groups of young patients (18–30 years), controlling for injury severity score (ISS) and sex ratio, and also segregated into AA (n = 56) and AG/GG (n = 19) genotypes. Aged patients with the AA genotype had a significantly lower requirement for ventilation and fewer days on mechanical ventilation, as well as significantly higher levels of one mediator and lower levels of two mediators. Dynamic Bayesian Network inference revealed IL-23 as a central node in each network regardless of age or genotype, with MIG and IP-10 also as key mediators in the networks of the aged patients. These findings suggest that an aging-related SNP, rs2075650, may influence clinical outcomes and inflammation networks in aged patients following blunt trauma, and thus may serve as a predictive outcome biomarker in the setting of polytrauma

Experimental pharmacotherapies for the acute respiratory distress syndrome (ARDS) have not met with success in the clinical realm. We hypothesized that chemically modified tetracycline 3 (CMT-3), an anti-inflammatory agent that blocks multiple proteases and cytokines, would prevent ARDS and injury in other organs in a clinically applicable, porcine model of inflammation-induced lung injury. Pigs (n = 15) were anesthetized and instrumented for monitoring. A "2-hit" injury was induced: (a) peritoneal sepsis-by placement of a fecal clot in the peritoneum, and (b) ischemia/reperfusion-by clamping the superior mesenteric artery for 30 min. Animals were randomized into two groups: CMT-3 group (n = 7) received CMT-3 (200 mg/kg); placebo group (n = 9) received the same dose of a CMT-3 vehicle (carboxymethylcellulose). Experiment duration was 48 h or until early mortality. Animals in both groups developed polymicrobial bacteremia. Chemically modified tetracycline 3 treatment prevented ARDS as indicated by PaO(2)/FIO(2) ratio, static compliance, and plateau airway pressure (P < 0.05 vs. placebo). It improved all histological lesions of ARDS (P < 0.05 vs. placebo). The placebo group developed severe ARDS, coagulopathy, and histological injury to the bowel. Chemically modified tetracycline 3 treatment prevented coagulopathy and protected against bowel injury. It significantly lowered plasma concentrations of interleukin 1β (IL-1β), tumor necrosis factor α, IL-6, IL-8, and IL-10. This study presents a clinically relevant model of lung injury in which CMT-3 treatment prevented the development of ARDS due in part to reduction of multiple plasma cytokines. Treatment of sepsis patients with CMT-3 could significantly reduce progression from sepsis into ARDS.

Remote and systemic inflammatory responses after long bone fractures have been well described, but the mechanisms underlying these changes remain unexplained. We hypothesized that bone components locally exposed to injured soft tissue are capable of inducing a systemic inflammatory response associated with acute lung injury, and that this inflammatory cascade requires Toll-like receptor 4 (TLR-4) signaling. Accordingly, male C3H/HeOuJ (TLR-4-competent) and C3H/HeJ (TLR-4-mutant) mice were injected with various bone components (bone marrow cells, bone marrow supernatant, and bone suspension, respectively) in bilaterally injured thigh muscles and euthanized after 6 h. Serum TNF-alpha, IL-6, and IL-10 levels, and pulmonary myeloperoxidase activity was measured using specific enzyme-linked immunosorbent assay kits. Pulmonary permeability changes were assessed with bronchoalveolar lavage. Local exposure of bone components to injured soft tissue induced systemic inflammation and acute lung injury in TLR-4-competent, but not in TLR-4-mutant, animals. These findings suggest that bone components contribute to systemic inflammation and acute lung injury after long bone fractures via TLR-4 signaling and support the notion of a central role for TLR-4 in sensing tissue damage.

Bacillus anthracis (anthrax) can trigger an acute inflammatory response that results in multisystem organ failure and death. Previously, we developed a mathematical model of acute inflammation after gram-negative infection that had been matched qualitatively to literature data. We modified the properties of the invading bacteria in that model to those specific to B. anthracis and simulated the host response to anthrax infection. We simulated treatment strategies against anthrax in a genetically diverse population including the following: (1) antibiotic treatment initiated at various time points, (2) antiprotective antigen vaccine, and (3) a combination of antibiotics and vaccine. In agreement with studies in mice, our simulations showed that antibiotics only improve survival if administered early in the course of anthrax infection. Vaccination that leads to the formation of antibodies to protective antigen is anti-inflammatory and beneficial in averting shock and improving survival. However, antibodies to protective antigen alone are predicted not to be universally protective against anthrax infection. Rather, our simulations suggest that an optimal strategy would require both vaccination and antibiotic administration.

Hemorrhagic shock (HS) is a major cause of morbidity and mortality in trauma patients. The early growth response 1 (Egr-1) transcription factor is induced by a variety of cellular stresses, including hypoxia, and may function as a master switch to trigger the expression of numerous key inflammatory mediators. We hypothesized that HS would induce hepatic expression of Egr-1 and that Egr-1 upregulates the inflammatory response after HS. The Egr-1 mice and wild-type (WT) controls (n>or=5 for all groups) were subjected to HS alone or HS followed by resuscitation (HS/R). Other mice were subjected to a sham procedure which included general anesthesia and vessel cannulation but no shock (sham). After the HS, HS/R, or sham procedures, mice were euthanized for determination of serum concentrations of interleukin (IL) 6, IL-10, and alanine aminotransferase. Northern blot analysis was performed to evaluate Egr-1 messenger RNA (mRNA) expression. Liver whole cell lysates were evaluated for Egr-1 protein expression by Western blot analysis. Hepatic expression of IL-6, granulocyte colony-stimulating factor, and intracellular adhesion molecule 1 mRNA was determined by semiquantitative reverse transcriptase-polymerase chain reaction. The Egr-1 DNA binding was assessed using the electrophoretic mobility shift assay. Hemorrhagic shock results in a rapid and transient hepatic expression of Egr-1 mRNA in WT mice by 1 h, whereas protein and DNA binding activity was evident by 2.5 h. The Egr-1 mRNA expression diminished after 4 h of resuscitation, whereas Egr-1 protein expression and DNA binding activity persisted through resuscitation. The Egr-1 mice exhibited decreased levels of hepatic inflammatory mediators compared with WT controls with a decrease in hepatic mRNA levels of IL-6 by 42%, granulocyte colony-stimulating factor by 39%, and intracellular adhesion molecule 1 by 43%. Similarly, Egr-1 mice demonstrated a decreased systemic inflammatory response and hepatic injury after HS/R compared with their WT counterparts. Early growth response 1 is rapidly upregulated in the liver during and after resuscitation from HS. Our results showing a blunted inflammatory response in Egr-1 mice provides evidence that Egr-1 functions as a proximal signal transduction mechanism responding to shock by amplifying the systemic inflammatory response.

A poorly controlled acute inflammatory response can lead to organ dysfunction and death. Severe systemic inflammation can be induced and perpetuated by diverse insults such as the administration of toxic bacterial products (e.g., endotoxin), traumatic injury, and hemorrhage. Here, we probe whether these varied shock states can be explained by a universal inflammatory system that is initiated through different means and, once initiated, follows a course specified by the cellular and molecular mechanisms of the immune and endocrine systems. To examine this question, we developed a mathematical model incorporating major elements of the acute inflammatory response in C57Bl/6 mice, using input from experimental data. We found that a single model with different initiators including the autonomic system could describe the response to various insults. This model was able to predict a dose range of endotoxin at which mice would die despite having been calibrated only in nonlethal inflammatory paradigms. These results show that the complex biology of inflammation can be modeled and supports the hypothesis that shock states induced by a range of physiologic challenges could arise from a universal response that is differently initiated and modulated.

Hemorrhagic shock (HS) is a complex process that initiates a global stress response. However, the earliest signaling pathways responsible for initiating this response remain unidentified. We have investigated the involvement of the extracellular signal-regulated kinases (ERK 1/2; also known as p42/44) and their activation in the liver by angiotensin II in the early signal transduction after HS. Hemorrhage of mice to 25 mmHg for 30 min was associated with the activation of ERK 1/2 in the liver, and this was accompanied by a 6.7-fold elevation of circulating angiotensin II levels. Similar results were obtained in rats. Both the angiotensin II levels and ERK 1/2 phosphorylation were suppressed by administration of an angiotensin-converting enzyme inhibitor peptide. Plasma from shocked rats, but not shocked rats treated with the angiotensin-converting enzyme inhibitor, increased ERK 1/2 phosphorylation in cultured hepatocytes. Together, these data suggest that angiotensin II is an important stimulus for ERK 1/2 activation in the liver during HS.

It has been previously shown that the inducible nitric oxide (NO) synthase (iNOS; NOS-2) is elevated after hemorrhage, and that iNOS-derived NO participates in the upregulation of inflammation as well as lung and liver injury postresuscitation from shock. The purpose of this study was to elucidate the time course of iNOS mRNA expression, as well as the cellular and subcellular localization of iNOS protein in the liver posthemorrhage in rats subjected to varying durations of hemorrhagic shock (HS; mean arterial blood pressure [MAP] = 40 mmHg) with or without resuscitation. Expression of iNOS mRNA in rat liver by real-time reverse transcriptase (RT)-PCR demonstrated iNOS upregulation in shocked animals as compared with their sham counterparts as early as 60 min after the initiation of hemorrhage. By 1 h of HS, iNOS protein was detectable in rat liver by immunofluorescence, and this expression increased with time. Immunofluorescence localized iNOS primarily to the hepatocytes, and in particular to hepatocytes in the centrilobular regions. This analysis, confirmed by immunoelectron microscopy, revealed that iNOS colocalizes with catalase, a peroxisomal marker. Furthermore, we determined that iNOS mRNA is detectable by RT-PCR in liver biopsies from human subjects with HS (MAP90 mmHg) associated with trauma (n = 18). In contrast, none of the seven nontrauma surgical patients studied had detectable iNOS mRNA in their livers. Collectively, these results suggest that hepatic iNOS expression, associated with peroxisomal localization, is an early molecular response to HS in experimental animals and possibly in human patients with trauma with HS.

We and others have demonstrated previously that cytokines, including interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNFalpha), regulate LPS recognition proteins such as CD14 in the liver and on hepatocytes. Based on recent findings that the mammalian homologue of Drosophila Toll participates in LPS signaling, we examined the regulation of Toll-Like Receptor (TLR) gene expression by cytokines in vitro and its distribution in vivo with a focus on the liver as a site of host-microbe interaction. Our results show that IL-1beta and/or TNFalpha participate in the upregulation of TLR2 mRNA levels in hepatocytes. Rats treated concurrently with LPS and antagonists of the IL-1 or TNFalpha receptor demonstrated significantly reduced LPS-induced hepatic expression of TLR2 compared to animals treated with LPS alone. The increase in hepatic TLR2 mRNA expression was associated with enhanced transcription as determined by nuclear run-on analysis. LPS treatment in vivo caused a marked TLR2 mRNA up-regulation in all of the tissues examined, with liver showing the highest expression. The high level of TLR2 expression in the liver may have important implications for pathogen-host interactions or microbial signaling.