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The original guidelines of cardiopulmonary resuscitation focused on the establishment of an airway and rescue breathing before restoration of circulation through cardiopulmonary resuscitation. As a result, the airway-breathing-circulation approach became the central guiding principle of resuscitation. Despite new guidelines by the American Heart Association for a circulation-first approach, Advanced Trauma Life Support guidelines continue to advocate for the airway-breathing-circulation sequence. Although definitive airway management is often necessary for severely injured patients, endotracheal intubation (ETI) before resuscitation in patients with hemorrhagic shock may worsen hypotension and precipitate cardiac arrest. In severely injured patients, a paradigm shift should be considered, which prioritizes restoration of circulation before ETI and positive pressure ventilation while maintaining a focus on basic airway assessment and noninvasive airway interventions. For this patient population, the most reasonable current strategy may be to target a simultaneous resuscitation approach, with immediate efforts to control hemorrhage and provide basic airway interventions while prioritizing volume resuscitation with blood products and deferring ETI until adequate systemic perfusion has been attained. We believe that a circulation-first sequence will improve both survival and neurologic outcomes for a traumatically injured patient and will continue to advocate this approach, as additional clinical evidence is generated to inform how to best tailor circulation-first resuscitation for varied injury patterns and patient populations.

Sepsis is a leading cause of death in the United States, claiming more than 215,000 lives every year. A primary condition observed in septic patients is the incidence of acute respiratory distress syndrome, which is characterized by the infiltration of neutrophils into the lung. Prior studies have shown differences in pulmonary neutrophil accumulation in C57BL6J (B6) and AJ mice after endotoxic and septic shock. However, the mechanism by which neutrophils accumulate in the lung after polymicrobial sepsis induced by cecal ligation and puncture still remains to be fully elucidated. We show in this study that lung inflammation, characterized by neutrophil infiltration and expression of inflammatory cytokines, was aggravated in B6 as compared with AJ mice and correlated with a high expression of p19, the interleukin 23 (IL-23)–specific subunit. Furthermore, lipopolysaccharide stimulation of B6- and AJ-derived macrophages, one of the main producers of IL-23 and IL-12, revealed that B6 mice favored the production of IL-23, whereas AJ-derived macrophages expressed higher levels of IL-12. In addition, expression of IL-17, known to be upregulated by IL-23, was also more elevated in the lung of B6 mice when compared with that in the lung of AJ mice. In contrast, pulmonary expression of interferon- was much more pronounced in AJ than that in B6 mice, which was most likely a result of a higher production of IL-12. The expression of the IL-17–dependent neutrophil recruitment factors chemokine (CXC motif) ligand 2 and granulocyte colony-stimulating factor was also higher in B6 mice. Altogether, these results suggest that increased activation of the IL-23IL-17 pathway has detrimental effects on sepsis-induced lung inflammation, whereas activation of the IL-12interferon- pathway may lead, in contrast, to less pronounced inflammatory events. These two pathways may become possible therapeutic targets for the treatment of sepsis-induced acute respiratory distress syndrome.

Itraconazole (ICZ) is commonly used for the treatment of fungal infections, particularly in immunocompromised patients. In addition, ICZ has been recently found to have antiangiogenic effects and is currently being tested as a new chemotherapeutic agent in several cancer clinical trials. We have previously shown that ICZ impaired complex N-linked glycosylation processing, leading to the accumulation of high-mannose glycoproteins on the surface of macrophages (Møs). This investigation was directed at determining the effects of ICZ on phagocytosis as a major function of Møs. We found a significant decrease in the phagocytosis of opsonized bacterial particles in ICZ-treated murine Møs in comparison with nontreated Møs. Furthermore, the impairment of phagocytosis was associated with a decrease in cell surface expression of Fc receptors (FcRs) as well as alteration of their glycosylation pattern. Concomitantly, a reduction in all three isoforms of the FcR family (i.e., Fcgr1, Fcgr2, and Fcgr3) mRNA levels was observed after incubation with ICZ. The effect of ICZ on phagocytosis and FcR expression was reversed by addition of low-density lipoprotein. These studies indicate that ICZ treatment certainly has a dramatic effect on macrophage function, which could result in a potential impairment of the immune system’;s ability to respond to pathogens and may lead to an elevated incidence of infections.

The expression of heat shock proteins (HSPs) is a basic and well-conserved cellular response to an array of stresses. These proteins are involved in the repair of cellular damage induced by the stress, which is necessary for the salutary resolution from the insult. Moreover, they confer protection from subsequent insults, which has been coined stress tolerance. Because these proteins are expressed in subcellular compartments, it was thought that their function during stress conditions was circumscribed to the intracellular environment. However, it is now well established that HSPs can also be present outside cells where they appear to display a function different than the well-understood chaperone role. Extracellular HSPs act as alert stress signals priming other cells, particularly of the immune system, to avoid the propagation of the insult and favor resolution. Because the majority of HSPs do not possess a secretory peptide signal, they are likely to be exported by a nonclassic secretory pathway. Different mechanisms have been proposed to explain the export of HSPs, including translocation across the plasma membrane and release associated with lipid vesicles, as well as the passive release after cell death by necrosis. Extracellular HSPs appear in various flavors, including membrane-bound and membrane-free forms. All of these variants of extracellular HSPs suggest that their interactions with cells may be quite diverse, both in target cell types and the activation signaling pathways. This review addresses some of our current knowledge about the release and relevance of extracellular HSPs.

Large surface area burn injuries lead to activation of the innate immune system, which can be blocked by parasympathetic inputs mediated by the vagus nerve. We hypothesized that vagal nerve stimulation (VNS) would alter the inflammatory response of peritoneal macrophages after severe burn injury. Male BALBc mice underwent right cervical VNS before 30 total body surface area steam burn and were compared with animals subjected to burn alone. Peritoneal macrophages were harvested at several time points following injury and exposed to lipopolysaccharide (LPS) in culture conditions. The inflammatory response of peritoneal macrophages was measured by analyzing changes in nuclear factor B p65Ser536phosphorylation using flow cytometry. We found that peritoneal macrophages isolated from mice subjected to burn injury were hyperresponsive to LPS challenge, suggesting burn-induced macrophage activation. We identified a protective role for VNS in blocking peritoneal macrophage activation. Analysis of the phosphorylation state of nuclear factor B pathway mediator, p65 Rel A, revealed a VNS-mediated reduction in p65Ser536phosphorylation levels after exposure to LPS compared with burn alone. In combination, these studies suggest VNS mediates the inflammatory response in peritoneal macrophages by affecting the set point of LPS responsiveness.

Interactions of toll-like receptors (TLRs) with nonmicrobial factors play a major role in the pathogenesis of early trauma-hemorrhagic shock (THS)–induced organ injury and inflammation. Thus, we tested the hypothesis that TLR4 mutant (TLR4mut) mice would be more resistant to THS-induced gut injury and polymorphonuclear neutrophil (PMN) priming than their wild-type littermates and found that both were significantly reduced in the TLR4mutmice. In addition, the in vivoand ex vivoPMN priming effect of THS intestinal lymph observed in the wild-type mice was abrogated in TLR4mutmice as well the TRIFmut-deficient mice and partially attenuated in Myd88−−mice, suggesting that TRIF activation played a more predominant role than MyD88 in THS lymph–induced PMN priming. Polymorphonuclear neutrophil depletion studies showed that THS lymph–induced acute lung injury was PMN dependent, because lung injury was totally abrogated in PMN-depleted animals. Because the lymph samples were sterile and devoid of endotoxin or bacterial DNA, we investigated whether the effects of THS lymph was related to endogenous nonmicrobial TLR4 ligands. High-mobility group box 1 protein 1, heat shock protein 70, heat shock protein 27, and hyaluronic acid all have been implicated in ischemia-reperfusion-induced tissue injury. None of these “danger” proteins appeared to be involved, because their levels were similar between the sham and shock lymph samples. In conclusion, TLR4 activation is important in THS-induced gut injury and in THS lymph–induced PMN priming and lung injury. However, the THS-associated effects of TLR4 on gut barrier dysfunction can be uncoupled from the THS lymph–associated effects of TLR4 on PMN priming.

Previous studies have shown that treatment with erythropoietin (EPO) exerts important cytoprotective and antiapoptotic effects. Donor organs recovered after cardiac death (DCD) can alleviate the shortage of organs required for transplantation. However, organs obtained subsequent to cardiac death demonstrate an increased incidence of delayed graft function and primary nonfunction. The aim of this study was to determine the effects of EPO administration to the donor in a porcine model of kidney transplantation under DCD conditions. Landrace pigs received 1,000 IU/kg i.v. EPO 30 min before cardiac arrest. Kidneys were then subjected to 30 min of warm ischemia and were transplanted after 24 h of cold storage. Renal dysfunction, injury, and inflammation were evaluated 4 h after transplantation. Transplantation of kidneys from DCD resulted in significant renal dysfunction, injury, and inflammation. This study provides the first evidence that pretreatment of the donor with a single pharmacologically relevant dose of EPO causes substantial attenuation of the dysfunction and injury associated with the transplantation of kidneys recovered after cardiac death.

Common bile duct ligation (CBDL) compromises the hepatic reticuloendothelial system by impairing the clearing of endotoxin and triggering an overwhelming inflammatory response. The response to endotoxin at the level of cytokine release and subsequent mortality depends on the genetic background in experimental mouse models. We hypothesized that the genetic make-up modulates the inflammatory responses after CBDL. The CBD was ligated in male A/J and B6 mice (8 weeks old). At 7 days post-CBDL, the presence of ascites was observed in 80% of B6 mice but in none of the A/J mice (P< 0.001). B6 mice showed higher mortality than A/J mice (P< 0.05). Both strains had marked cholestatic injury documented histologically. Liver chemistries were markedly elevated in both strains after injury. Plasma levels of the anti-inflammatory cytokine IL-10 were significantly higher in A/J than B6 mice at the 4- and 12-h time points (P< 0.05), whereas proinflammatory cytokine TNF-α levels were significantly higher in B6 than A/J mice at 2 h (P< 0.05). Both strains displayed activation of NF-κB after CBDL. In conclusion, the contrasting response observed after CBDL between A/J and B6 mice is largely attributable to genetic differences. Survival after CBDL was correlated with an increase in anti-inflammatory cytokines.

Men are considered more susceptible to sepsis after severe injury than are women, which has been attributed to a suppressing effect of male sex steroids on the inflammatory response. Moreover, the effect of sex steroids on the inflammatory process depends on the genetic background. The present study examined the genetic contribution to survival after endotoxic shock in mice depleted of testosterone by surgical castration. Six-week-old male mice, from strains A/J, AKR/J, C57BL/6J (B6), BALBc/J, DBA/2J, and C3H/HeN, were castrated (CX) or nonoperated (NoOp). Two weeks after surgery, mice were injected intraperitoneally with Escherichia colilipopolysaccharide (15 mg/kg) and the frequency of mortality was monitored. CX A/J mice showed a significantly higher survival rate than NoOp mice, but this protective effect was not observed in the other strains. Administration of 5-α-dihydrotestosterone to CX A/J mice reverted the protection by CX. The protective effect of CX was also observed in crosses of female A/J and male B6 (AXB), but not female B6 and male A/J (BXA), suggesting that protection is linked to the A/J X chromosome. This possibility was corroborated by using consomic mice containing A/J chromosome X and the remaining chromosomes from B6. These results suggest that testosterone is a negative factor in the recovery from endotoxic shock, depending on the genetic background.

The genetic background has recently been recognized as an important element in the response to injury, contributing to the variability in the clinical outcome of critically ill patients. The traditional approach to studying the genetic contribution requires the availability of families with multiple members who have experienced similar disease conditions, a situation that is nearly impossible to find in the case of trauma. Association studies looking at unrelated individuals across populations require large economic and labor-intensive efforts. Thus, a candidate gene approach has been the sole methodology used to correlate genetic variability with clinical outcome. However, this approach cannot provide a comprehensive description of a multigenic condition. Animal models are an alternative for studying the genetic contributions to variability in the response to injury. A murine model is ideal because a large set of inbred strains are available; congenic, consomic, transgenic, and recombinant strains can also be used. Employing this paradigm, we have demonstrated that the response to several stressors, such as injection of E. colilipopolysaccharide (LPS) and polymicrobial sepsis induced by cecal ligation and puncture (CLP), is modified by the genetic background. The inflammatory response in mice has also been shown to be affected by sex, age, and other, nongenetic components such as diet. We have exploited the differences in response among various inbred mouse strains to map loci contributing to the inflammatory response. Fine mapping strategies allow the refinement of sets of candidate genes, which can be identified by positional cloning. Detection of genetic variation affecting the inflammatory response in murine models provides a basis for determining whether polymorphisms in orthologous human genes correlate with particular clinical outcomes from injury. Thus, discovery of these genes could impact patient care by acting as markers of a specific predisposition in humans.

Aging is the result of several detrimental changes that lead to a decrease in homeostasis, an increase in the incidence of degenerative diseases, and death. A caloric-restricted diet (CR), which consists of a significant reduction in calorie intake (40%) without malnutrition, has been shown to delay the onset of age-related diseases and pathologies and to extend life span. The aims of this study were to assess the effects of aging and CR on lipopolysaccharide (LPS)-dependant cytokine production by peritoneal macrophages (PMs). Resident naïve PMs were isolated from 2- to 24-month-old male C57BL/6 mice and were stimulated with Escherichia coliLPS (100 ng/mL) for 1 to 5 h in culture conditions. A linear decrease in the production of LPS-induced tumor necrosis factor α (TNF-α) and interleukin (IL) 10 was observed with age. LPS-induced IL-6 and IL-1β levels were also reduced with age, but in a nonlinear fashion. Expression of CD14, the major receptor for LPS, on the PM surface was also observed to decline with age. Moreover, TNF-α production by PMs was reduced in mice undergoing the two different CR diets of limited daily feeding and intermittent fasting, as compared with ad libitum-fed mice. The results of this study add the new variables age and diet to the paradigm proposing that the response to LPS is modulated by multiple components, including genetic background and sex.

The expression of heat shock or stress proteins (hsps) is a widespread response to stress that results in the protection of cells from subsequent insults, coined stress tolerance. Stress tolerance is apparently due to the preservation of several cellular structures and processes, such as translation. Protection of protein synthesis has been correlated with the presence of Hsp70. In the present study, Hsp70 was found to interact with translating ribosomes. This interaction is due to the preferential binding of Hsp70 to the 40S ribosomal subunit. Additionally, Hsp70 seems to interact weakly with nascent polypeptides within the 60S subunit. The interaction between Hsp70 and ribosomal subunits could also be observed in vitroconditions. Binding of Hsp70 to ribosomes was salt resistant, suggesting that this protein is not bound to transiently associated translational factors. Moreover, protection of protein synthesis requires new gene expression. We speculate that the binding of Hsp70 to ribosomes is part of a mechanism to guarantee the rapid and abundant protein synthesis during stress, particularly the translation of mRNAs encoding for hsps.

The response to injury is dependent on several factors, including the type and extent of the injury, genetics, and the environment. In the present study, the genetic contribution to sepsis was evaluated in a mouse model. Sepsis was induced in two inbred mouse strains, C57BL/6J (B6) and A/J, by cecal ligation and single puncture (CLP). Frequency of mortality was significantly higher in B6 than A/J mice from 36 to 132 h after CLP. Plasma TNF-, IL-1, and IL-6 levels were similar in both strains after CLP. IL-10 plasma levels were significantly higher in B6 mice as opposed to A/J mice after 24 h of CLP. Similarly, hepatic myeloperoxidase activity, an index of polymorphonuclear leukocytes, was elevated in B6 mice as compared with A/J mice after 24 h of CLP. On the contrary, metallothionein mRNA levels were higher in A/J mice compared with B6 mice. Finally, leptin levels were also higher in A/J than B6 mice within 19 h of CLP. This study demonstrates a genetic contribution in the response to sepsis.

Gap junctional cellular communication is important in the propagation of signals that coordinate hepatic metabolism. Hepatocytes express two different connexin Cx genes, Cx32and Cx26, which encode for the subunit component of gap junction channels. Previous studies have shown that the expression of hepatic Cx32 is reduced during inflammatory conditions. The objective of this study was to evaluate whether this decrease in Cx32 expression results in a decrease in hepatic gap junctional communication. Transfer of the dye Lucifer Yellow between hepatocytes was measured after microinjection of single cells in an isolated perfused liver. Livers were harvested from rats subjected to an inflammatory condition induced by administration of bacterial lipopolysaccharide LPS. A decrease in gap junctional cellular communication was observed within 6 h of the LPS treatment. This decrease in dye coupling was reversible, because gap junctional communication returned to control levels within 48 h of the LPS injection. The inhibition of hepatic gap junctional communication was associated with the disappearance of Cx32 and Cx26 from the hepatocyte plasma membrane as detected by indirect immunostaining. Cx32 mRNA levels were also reduced during inflammation as previously reported. However, Cx26 mRNA levels were unaffected or even transiently increased after the injection of LPS without significant increase in the polypeptide level. Thus, the downregulation of Cx32 and Cx26 from the hepatocyte surface is apparently due to a rapid degradation of the polypeptide from the cell surface. We hypothesize that this loss of gap junctional cellular communication within the liver may contribute to the disordered hepatic metabolic that occurs during inflammatory states.

Although the expression of heat shock or stress proteins hsps is a well conserved response to stress, the accumulation of these proteins is different between various celltypes. Particularly, cells of neuronal origin show a reduced expression of Hsp70 after stress. The possible mechanism of this reduced Hsp70 expression was studied in thermally stressed murine neuroblastoma cells N18. These cells showed no detectable levels of Hsp70 or Hsp70 mRNA after heat shock. Hsp70transcription was not detectable after stress. However, heat shock transcription factor 1 HSF1 is active in these cells under stress conditions. Cells transiently transfected with the chloramphenicol acetyltransferaseCAT gene under control of the human heat shock promoter showed a stressdependent expression of CAT, suggesting that the cells contain the factors necessary for the expression of Hsp70.Integration of the foreign human heat shock promoter into genomic DNA did not affect its transcriptional inducibility. These results suggest that the impairment of Hsp70 expression in N18 cells is due to the environment chromatin structure, methylation pattern of the Hsp70locus.

The most primitive mechanism of cellular protection involves the expression of a polypeptide family named heat shock or stress proteins (hsps). Some of these hsps are present in unstressed cells and play an important role in the folding and translocation of polypeptides across membranes. Thus, they have been termed molecular chaperones. Hsps are expressed in response to an array of stresses, including hyperthermia, oxygen radicals, heavy metals, ethanol, and amino acid analogues. In addition, the heat shock response is induced during clinically relevant situations such as ischemia/reperfusion and circulatory and hemorrhagic shock. All of the above stresses have in common that they disturb the tertiary structure of proteins and have adverse effects on cellular metabolism. Pretreatment of cells with a mild stress, sufficient to induce the expression of hsps, results in protection to subsequent insults. This phenomenon has been coined “stress tolerance” and is apparently caused by the resolubilization of proteins that were denatured during the stress. In addition, cellular structures (microfilaments and centrosomes) and processes (transcription, splicing, and translation) are stabilized or repaired during a second stress in stress tolerant cells and organisms. There is a great body of evidence indicating a direct role of hsps in the stabilization of these events. The intrinsic capacity of hsps to protect cells has potential relevance as a natural mechanism of organ protection during harmful environmental conditions and operative procedures, and in the combat against pathogens.

At the molecular level, the inflammatory response is characterized by changes in gene expression of various organ systems. One gene by which expression has been observed to be altered in the liver during inflammation is connexin (Cx) 32. Cx genes encode the polypeptide subunits of the hemichannels that comprise gap junctions. In the present study, an increase in the expression of a different Cx gene, Cx43, was observed in the kidney and lung of rats injected with a sublethal dose (1 mg/kg) of bacterial lipolysaccharide (LPS). To elucidate the possible mechanism by which the Cx43 expression is increased during inflammation, the 5′ flanking region of the gene was cloned and coupled to a reporter gene (human growth hormone). This construct was transfected into cells of renal origin (NRK), which express Cx43 constitutively. The Cx43 promoter activity was indeed found in the cloned region, which contained 725 base pairs upstream of the transcriptional initiation site of the Cx43 gene. The Cx43 promoter activity was found to be increased by incubation of the transfected cells with serum obtained from LPS-treated rats. Moreover, direct incubation of the transfected cells with LPS or interleukin 1β, but not with other cytokines, was observed to increase the Cx43 promoter activity. These results suggest the expression of Cx43 after administration of LPS is part of the inflammatory response. Moreover, the expression of this gene seems to be mediated by proinflammatory mediators.

Multiple organ dysfunction syndrome (MODS) appears to be the result of a complex program influenced by multiple factors, including environmental, physiological, and immunological conditions. Thus, an uncontrolled inflammatory response following a stochastic event, the initial injury, is believed to be the cause for the development of this syndrome. Several lines of evidence suggest that a genetic component could contribute to the regulation of the inflammatory response, as well, but no direct evidence demonstrates a heritable predisposition to MODS. In the present study, a genetic contribution was demonstrated for the inflammatory response induced by the administration of bacterial lipopolysaccharide (LPS) in different, genetically distinct strains of inbred mice. A survey of five inbred strains showed that mortality following administration of Escherichia coliLPS (20 mg/kg) was highest in C57BLV6J (B6) mice, while A/J mice were the most resistant. Accordingly, B6 and A/J mice were examined further for differences in the inflammatory response elicited by LPS. B6 mice showed higher levels of circulating interleukin-1β and interleukin-6, as well as higher mRNA levels of hepatic β-fibrinogen (an acute-phase gene) and metallothionein. Surprisingly, the circulating levels of tumor necrosis factor-α were significantly higher in A/J than in B6 mice after LPS administration. Since B6 and A/J mice were bred and raised in identical environments and received the same LPS challenge, the contrasting inflammatory response that was observed is largely attributable to genetic differences between these two strains. These data illustrate that the response to injury could be modulated by the genetic background of the individual. This information may be pertinent for the care of critically ill patients.

Part of the hepatic response to injury is mediated by the expression of a gene family known as acute phase genes. Expression of acute phase genes is induced by the presence of cytokines in the circulation such as tumor necrosis factor α, interferon γ, and interleukins-1 and −6. Simultaneous with the transcription of acute phase genes, there is a decrease in the expression of other genes such as albumin. In the present study, we report the decrease in transcription of the gene coding for phosphoenolpyruvate carboxykinase (PEPCK), a key regulatory enzyme in the gluconeogenic pathway, in rat liver during an acute inflammatory state induced by bacterial endotoxin. Steady-state mRNA levels of PEPCK decrease 3 h after endotoxin injection, returning to 70 of normal levels after 24 h. This decrease in PEPCK mRNA levels was due to a decline in the rate of transcription of the gene, most likely mediated by an increase in the circulating levels of insulin. However, expression of the PEPCK gene was not induced by an increase in the cellular levels of cAMP during inflammation. Such a lack of response to cAMP may be due to the inactivation of, or decrease in, the levels of the transcriptional factor(s) responsible for PEPCK transcription.