Type I IFNs drive hematopoietic stem and progenitor cell collapse via impaired proliferation and increased RIPK1-dependent cell death during shock-like ehrlichial infection.

Type I interferons (IFNα/ß) regulate diverse aspects of host defense, but their impact on hematopoietic stem and progenitor cells (HSC/HSPCs) during infection remains unclear. Hematologic impairment can occur in severe infections, thus we sought to investigate the impact of type I IFNs on hematopoiesis in a tick-borne infection with a virulent ehrlichial pathogen that causes shock-like disease. During infection, IFNα/ß induced severe bone marrow (BM) loss, blunted infection-induced emergency myelopoiesis, and reduced phenotypic HSPCs and HSCs. In the absence of type I IFN signaling, BM and splenic hematopoiesis were increased, and HSCs derived from Ifnar1-deficient mice were functionally superior in competitive BM transplants. Type I IFNs impaired hematopoiesis during infection by both limiting HSC/HSPC proliferation and increasing HSPC death. Using mixed BM chimeras we determined that type I IFNs restricted proliferation indirectly, whereas HSPC death occurred via direct IFNαR -mediated signaling. IFNαR-dependent signals resulted in reduced caspase 8 expression and activity, and reduced cleavage of RIPK1 and RIPK3, relative to Ifnar1-deficient mice. RIPK1 antagonism with Necrostatin-1s rescued HSPC and HSC numbers during infection. Early antibiotic treatment is required for mouse survival, however antibiotic-treated survivors had severely reduced HSPCs and HSCs. Combination therapy with antibiotics and Necrostatin-1s improved HSPC and HSC numbers in surviving mice, compared to antibiotic treatment alone. We reveal two mechanisms whereby type I IFNs drive hematopoietic collapse during severe infection: direct sensitization of HSPCs to undergo cell death and enhanced HSC quiescence. Our studies reveal a strategy to ameliorate the type I IFN-dependent loss of HSCs and HSPCs during infection, which may be relevant to other infections wherein type I IFNs cause hematopoietic dysfunction.

The deubiquitinase activity of A20 is dispensable for NF-κB signaling.

A20 has been suggested to limit NF-κB activation by removing regulatory ubiquitin chains from ubiquitinated substrates. A20 is a ubiquitin-editing enzyme that removes K63-linked ubiquitin chains from adaptor proteins, such as RIP1, and then conjugates them to K48-linked polyubiquitin chains to trigger proteasomal degradation. To determine the role of the deubiquitinase function of A20 in downregulating NF-κB signaling, we have generated a knock-in mouse that lacks the deubiquitinase function of A20 (A20-OTU mice). These mice are normal and have no signs of inflammation, have normal proportions of B, T, dendritic, and myeloid cells, respond normally to LPS and TNF, and undergo normal NF-κB activation. Our results thus indicate that the deubiquitinase activity of A20 is dispensable for normal NF-κB signaling.

Developmental defects in zebrafish for classification of EGF pathway inhibitors.

One of the major challenges when testing drug candidates targeted at a specific pathway in whole animals is the discrimination between specific effects and unwanted, off-target effects. Here we used the zebrafish to define several developmental defects caused by impairment of Egf signaling, a major pathway of interest in tumor biology. We inactivated Egf signaling by genetically blocking Egf expression or using specific inhibitors of the Egf receptor function. We show that the combined occurrence of defects in cartilage formation, disturbance of blood flow in the trunk and a decrease of myelin basic protein expression represent good indicators for impairment of Egf signaling. Finally, we present a classification of known tyrosine kinase inhibitors according to their specificity for the Egf pathway. In conclusion, we show that developmental indicators can help to discriminate between specific effects on the target pathway from off-target effects in molecularly targeted drug screening experiments in whole animal systems.

Roles of peroxiredoxin II in the regulation of proinflammatory responses to LPS and protection against endotoxin-induced lethal shock.

Mammalian 2-Cys peroxiredoxin II (Prx II) is a cellular peroxidase that eliminates endogenous H(2)O(2). The involvement of Prx II in the regulation of lipopolysaccharide (LPS) signaling is poorly understood. In this report, we show that LPS induces substantially enhanced inflammatory events, which include the signaling molecules nuclear factor kappaB and mitogen-activated protein kinase (MAPK), in Prx II-deficient macrophages. This effect of LPS was mediated by the robust up-regulation of the reactive oxygen species (ROS)-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and the phosphorylation of p47(phox). Furthermore, challenge with LPS induced greater sensitivity to LPS-induced lethal shock in Prx II-deficient mice than in wild-type mice. Intravenous injection of Prx II-deficient mice with the adenovirus-encoding Prx II gene significantly rescued mice from LPS-induced lethal shock as compared with the injection of a control virus. The administration of catalase mimicked the reversal effects of Prx II on LPS-induced inflammatory responses in Prx II-deficient cells, which suggests that intracellular H(2)O(2) is attributable, at least in part, to the enhanced sensitivity to LPS. These results indicate that Prx II is an essential negative regulator of LPS-induced inflammatory signaling through modulation of ROS synthesis via NADPH oxidase activities and, therefore, is crucial for the prevention of excessive host responses to microbial products.

Tumor necrosis factor inhibits glucocorticoid receptor function in mice: a strong signal toward lethal shock.

As glucocorticoid resistance (GCR) and the concomitant burden pose a worldwide problem, there is an urgent need for a more effective glucocorticoid therapy, for which insights into the molecular mechanisms of GCR are essential. In this study, we addressed the hypothesis that TNFα, a strong pro-inflammatory mediator in numerous inflammatory diseases, compromises the protective function of the glucocorticoid receptor (GR) against TNFα-induced lethal inflammation. Indeed, protection of mice by dexamethasone against TNFα lethality was completely abolished when it was administered after TNFα stimulation, indicating compromised GR function upon TNFα challenge. TNFα-induced GCR was further demonstrated by impaired GR-dependent gene expression in the liver. Furthermore, TNFα down-regulates the levels of both GR mRNA and protein. However, this down-regulation seems to occur independently of GC production, as TNFα also resulted in down-regulation of GR levels in adrenalectomized mice. These findings suggest that the decreased amount of GR determines the GR response and outcome of TNFα-induced shock, as supported by our studies with GR heterozygous mice. We propose that by inducing GCR, TNFα inhibits a major brake on inflammation and thereby amplifies the pro-inflammatory response. Our findings might prove helpful in understanding GCR in inflammatory diseases in which TNFα is intimately involved.

Dichotomy between RIP1- and RIP3-mediated necroptosis in tumor necrosis factor-α-induced shock.

Tumor necrosis factor receptor (TNFR) signaling may result in survival, apoptosis or programmed necrosis. The latter is called necroptosis if the receptor-interacting protein 1 (RIP1) inhibitor necrostatin-1 (Nec-1) or genetic knockout of RIP3 prevents it. In the lethal mouse model of TNFα-mediated shock, addition of the pan-caspase inhibitor zVAD-fmk (zVAD) accelerates time to death. Here, we demonstrate that RIP3-deficient mice are protected markedly from TNFα-mediated shock in the presence and absence of caspase inhibition. We further show that the fusion protein TAT-crmA, previously demonstrated to inhibit apoptosis, also prevents necroptosis in L929, HT29 and FADD-deficient Jurkat cells. In contrast to RIP3-deficient mice, blocking necroptosis by Nec-1 or TAT-crmA did not protect from TNFα/zVAD-mediated shock, but further accelerated time to death. Even in the absence of caspase inhibition, Nec-1 application led to similar kinetics. Depletion of macrophages, natural killer (NK) cells, granulocytes or genetic deficiency for T lymphocytes did not influence this model. Because RIP3-deficient mice are known to be protected from cerulein-induced pancreatitis (CIP), we applied Nec-1 and TAT-crmA in this model and demonstrated the deterioration of pancreatic damage upon addition of these substances. These data highlight the importance of separating genetic RIP3 deficiency from RIP1 inhibition by Nec-1 application in vivo and challenge the current definition of necroptosis.

C-type lectin SIGN-R1 has a role in experimental colitis and responsiveness to lipopolysaccharide.

Pathogen recognition receptors (PRRs) function to maintain the balance between controlled responses to pathogens and uncontrolled innate immune activation leading to inflammation. In the context of commensal bacteria and the etiology of inflammatory bowel disease, although a role for the TLRs is known, there is a less defined function for C-type lectin receptors (CLRs). We demonstrate that mice deficient ((-/-)) in the CLR specific intracellular adhesion molecule-3 grabbing nonintegrin homolog-related 1 (SIGN-R1) (CD209b) have reduced susceptibility to experimental colitis, with a reduction in the disease severity, colon damage, and levels of the proinflammatory cytokines IL-1beta, TNF-alpha, and IL-6. To determine whether SIGN-R1(-/-) mice had a systemic defect in innate activation, we examined the responsiveness of macrophages from SIGN-R1(-/-) mice to TLR ligands. SIGN-R1(-/-) peritoneal macrophages, but not bone marrow-derived macrophages, have a specific defect in IL-1beta and IL-18 production, but not other cytokines, in response to the TLR4 ligand LPS. In vivo SIGN-R1(-/-) mice had significantly reduced susceptibility to LPS-induced shock. To address the synergistic relationship between SIGN-R1 and TLR4 in the context of experimental colitis, SIGN-R1/TLR4(-/-) mice were generated. SIGN-R1/TLR4(-/-) mice displayed reduced susceptibility to experimental colitis relative to severity of disease observed in wild-type or TLR4(-/-) mice. The in vivo use of a blocking mAb confirmed a functional role for SIGN-R1 in LPS-induced shock and experimental colitis. These data indicate a role for SIGN-R1 in the regulation of inflammation in a model of experimental colitis and illustrate that SIGN-R1 is a critical innate factor in response to LPS.

Hypoxia-inducible factor (HIF1α) gene expression in human shock states.

INTRODUCTION: Hypoxia-inducible factor-1 (HIF1) controls the expression of genes involved in the cellular response to hypoxia. No information is available on its expression in critically ill patients. Thus, we designed the first clinical study in order to evaluate the role of HIF1α as a prognosis marker in patients with shock. METHODS: 50 consecutive adult patients with shock and 11 healthy volunteers were prospectively included. RNA was extracted from whole blood samples and expression of HIF1α was assessed over the first 4 hours of shock. The primary objective was to assess HIF1α as a prognostic marker in shock. Secondary objectives were to evaluate the role of HIF1α as a diagnostic and follow-up marker. Patient survival was evaluated at day 28. RESULTS: The causes of shock were sepsis (78%), hemorrhage (18%), and cardiac dysfunction (4%). The HIF1α expression was significantly higher in the shock patients than in the healthy volunteers (121 [72-168] vs. 48 [38-54] normalized copies, p < 0.01), whatever the measured isoforms. It was similar in non-survivors and survivors (108 [range 84-183] vs. 121 [range 72-185] normalized copies, p = 0.92), and did not significantly change within the study period. CONCLUSIONS: The present study is the first to demonstrate the increased expression of HIF1α in patients with shock. Further studies are needed to clarify the potential association with outcome. Our findings reinforce the value of monitoring plasma lactate levels to guide the treatment of shock.

Gene Expression Scoring of Immune Activity Levels for Precision Use of Hydrocortisone in Vasodilatory Shock.

PURPOSE: Among patients with vasodilatory shock, gene expression scores may identify different immune states. We aimed to test whether such scores are robust in identifying patients' immune state and predicting response to hydrocortisone treatment in vasodilatory shock. MATERIALS AND METHODS: We selected genes to generate continuous scores to define previously established subclasses of sepsis. We used these scores to identify a patient's immune state. We evaluated the potential for these states to assess the differential effect of hydrocortisone in two randomized clinical trials of hydrocortisone versus placebo in vasodilatory shock. RESULTS: We initially identified genes associated with immune-adaptive, immune-innate, immune-coagulant functions. From these genes, 15 were most relevant to generate expression scores related to each of the functions. These scores were used to identify patients as immune-adaptive prevalent (IA-P) and immune-innate prevalent (IN-P). In IA-P patients, hydrocortisone therapy increased 28-day mortality in both trials (43.3% vs 14.7%, P = 0.028) and (57.1% vs 0.0%, P = 0.99). In IN-P patients, this effect was numerically reversed. CONCLUSIONS: Gene expression scores identified the immune state of vasodilatory shock patients, one of which (IA-P) identified those who may be harmed by hydrocortisone. Gene expression scores may help advance the field of personalized medicine.

Targeted disruption of the gene encoding the murine small subunit of carboxypeptidase N (CPN1) causes susceptibility to C5a anaphylatoxin-mediated shock.

Carboxypeptidase N (CPN) is a plasma zinc metalloprotease, which consists of two enzymatically active small subunits (CPN1) and two large subunits (CPN2) that protect the protein from degradation. Historically, CPN has been implicated as a major regulator of inflammation by its enzymatic cleavage of functionally important arginine and lysine amino acids from potent phlogistic molecules, such as the complement anaphylatoxins C3a and C5a. Because of no known complete CPN deficiencies, the biological impact of CPN in vivo has been difficult to evaluate. Here, we report the generation of a mouse with complete CPN deficiency by targeted disruption of the CPN1 gene. CPN1(-/-) mice were hypersensitive to lethal anaphylactic shock due to acute complement activation by cobra venom factor. This hypersensitivity was completely resolved in CPN1(-/-)/C5aR(-/-) but not in CPN1(-/-)/C3aR(-/-) mice. Moreover, CPN1(-/-) mice given C5a i.v., but not C3a, experienced 100% mortality. This C5a-induced mortality was reduced to 20% when CPN1(-/-) mice were treated with an antihistamine before C5a challenge. These studies describe for the first time a complete deficiency of CPN and demonstrate 1) that CPN plays a requisite role in regulating the lethal effects of anaphylatoxin-mediated shock, 2) that these lethal effects are mediated predominantly by C5a-induced histamine release, and 3) that C3a does not contribute significantly to shock following acute complement activation.

SCN5A mutation is associated with early and frequent recurrence of ventricular fibrillation in patients with Brugada syndrome.

BACKGROUND: Mutations in SCN5A are reportedly linked to Brugada syndrome (BS), but recent observations suggest that they are not necessarily associated with ventricular fibrillation (VF) in BS patients. Therefore, the clinical importance of SCN5A mutations in BS patients was examined in the present study. METHODS AND RESULTS: The 108 BS patients were examined for SCN5A mutations and various parameters were compared between patients with and without mutations. An implantable cardioverter defibrillator (ICD) was implanted in 49 patients and a predictor of appropriate ICD shock was investigated. The existence of a SCN5A mutation was not associated with initial VF episodes (21.7% vs 20.0%, P=0.373). In the secondary prevention group, appropriate shock-free survival rate was significantly lower in patients with spontaneous type 1 ECG than in those without (41.1% vs 85.7% at 2 years, P=0.014). The appropriate shock-free survival rate was also significantly lower in patients with SCN5A mutations than in those without (28.6% vs 83.3% at 1 year, P=0.040). Appropriate shock was more frequent in patients with SCN5A mutations than in those without (6.6±6.2 vs 1.7±3.0, P=0.007). CONCLUSIONS: SCN5A mutations are associated with early and frequent VF recurrence, but not with initial VF episodes. This is the first report on the genotype-phenotype interaction and clinical significance of this mutation.

Variation in the TLR4 gene influences the risk of organ failure and shock posttrauma: a cohort study.

BACKGROUND: Genetic variation contributes to risk and outcomes of sepsis. We sought to determine whether variation in inflammation related genes is associated with severity of sepsis in trauma patients. METHODS: A cohort of severely injured Caucasian patients was studied and genotyped for candidate single nucleotide polymorphisms (SNPs). These were toll-like receptor 4 (TLR4) A896G, tumor necrosis factor-alpha G-308A, interleukin-6 G-174C, interleukin-1beta C-31T, and cluster of differentiation marker 14C-159T. SNP genotypes among patients with sepsis and complicated sepsis were analyzed by chi2 and logistic regression. Six haplotype-tagging SNPs in the TLR4 gene were subsequently examined to analyze their influence on TLR4 A896G SNPs relationship to sepsis severity. RESULTS: We enrolled 598 patients. Complicated sepsis developed in 147 (25%). Adjusting for independent risk factors, carriage of the variant TLR4 896 G allele was associated with decreased risk of complicated sepsis (odds ratio = 0.3, 95% confidence interval, 0.1-0.7, p = 0.008). Furthermore, two haplotypes seemed to better characterize this risk than the variant TLR4 896G allele. The variant TLR4 896G allele is linked to one common haplotype, which seems to confer a considerably reduced risk of complicated sepsis. (aOR = 0.2 95% confidence interval, 0.05-0.7, p = 0.01). CONCLUSIONS: Variation within TLR4 gene is associated with severity of posttraumatic sepsis. This risk may not be solely related to TLR4 A896G SNP. It is likely that other, uncharacterized variations in the TLR4 gene contribute to sepsis severity. A thorough evaluation of variability within the TLR4 gene is needed to characterize sepsis risk.

A Genetic Approach to the Association Between PCSK9 and Sepsis.

Importance: Whether the PCSK9 gene is associated with the progress from infection to sepsis is unknown to date. Objective: To test the associations between PCSK9 genetic variants, a PCSK9 genetic risk score (GRS), or genetically estimated PCSK9 expression levels and the risk of sepsis among patients admitted to a hospital with infection. Design, Setting, and Participants: This retrospective cohort study used deidentified electronic health records to identify patients admitted to Vanderbilt University Medical Center, Nashville, Tennessee, with infection. Patients were white adults, had a code indicating infection from the International Classification of Diseases, Ninth Revision, Clinical Modification, or the International Statistical Classification of Diseases, Tenth Revision, Clinical Modification, and received an antibiotic within 1 day of hospital admission (N = 61 502). Data were collected from January 1, 1993, through December 31, 2017, and analyzed from April 1, 2018, to March 16, 2019. Exposures: Four known PCSK9 functional variants, a GRS for PCSK9, and genetically estimated PCSK9 expression. Main Outcomes and Measures: The primary outcome was sepsis; secondary outcomes included cardiovascular failure and in-hospital death. Results: Of patients with infection, genotype information was available in 10 922 white patients for PCSK9 functional variants (5628 men [51.5%]; mean [SD] age, 60.1 [15.7] years), including 7624 patients with PCSK9 GRS and 6033 patients with estimated PCSK9 expression. Of these, 3391 developed sepsis, 835 developed cardiovascular failure, and 366 died during hospitalization. None of the 4 functional PCSK9 variants were significantly associated with sepsis, cardiovascular failure, or in-hospital death, with or without adjustment for (1) age and sex or (2) age, sex, and Charlson-Deyo comorbidities (in model adjusted for age, sex, and comorbidities, odds ratios for any loss-of function variant were 0.96 [95% CI, 0.88-1.04] for sepsis, 1.05 [95% CI, 0.90-1.22] for cardiovascular failure, and 0.89 [95% CI, 0.72-1.11] for death). Similarly, neither the PCSK9 GRS nor genetically estimated PCSK9 expression were significantly associated with sepsis, cardiovascular failure, or in-hospital death in any of the analysis models. For GRS, in the full model adjusted for age, sex, and comorbidities, the odds ratios were 1.01 for sepsis (95% CI, 0.96-1.06; P = .70), 1.03 for cardiovascular failure (95% CI, 0.95-1.12; P = .48), and 1.05 for in-hospital death (95% CI, 0.92-1.19; P = .50). For genetically estimated PCSK9 expression, in the full model adjusted for age, sex, and comorbidities, the odds ratios were 1.01 for sepsis (95% CI, 0.95-1.06; P = .86), 0.96 for cardiovascular failure (95% CI, 0.88-1.05; P = .41), and 0.99 for in-hospital death (95% CI, 0.87-1.14; P = .94). Conclusions and Relevance: In this study, PCSK9 genetic variants were not significantly associated with risk of sepsis or the outcomes of sepsis in patients hospitalized with infection.

GITR-GITRL system, a novel player in shock and inflammation.

Glucocorticoid-induced TNFR-Related (GITR) protein is a member of the tumor necrosis factor receptor superfamily that modulates acquired and natural immune response. It is expressed in several cells and tissues, including T cells, natural killer cells, and, at lower levels, in cells of innate immunity. GITR is activated by its ligand, GITRL, mainly expressed on antigen presenting and endothelial cells. Recent evidence suggests that the GITR/GITRL system participates in the development of inflammatory responses, including shock, either due to early response of neutrophils and macrophages, or together with autoimmune/allergic pathogenesis. The pro-inflammatory role of the GITR/GITRL system is due to: 1) modulation of the extravasation process, 2) activation of innate immunity cells, 3) activation of effector T cells also favored by partial inhibition of suppressor T cells and modulation of dendritic function. This review summarizes the in vivo role of the GITR/GITRL system in inflammation and shock, explaining the mechanisms responsible for their effects, considering the interplay among the different cells of the immune system and transduction pathways activated by GITR and GITRL triggering. The hidden aspects about GITR/GITRL function, crucial for treatment planning of inflammatory diseases and shock by modulation of this system is stressed.

Absence of peroxisome proliferators-activated receptors (PPAR)alpha enhanced the multiple organ failure induced by zymosan.

The peroxisome proliferator-activated receptor (PPAR) alpha is a member of the nuclear receptor superfamily of ligand-dependent transcription factors related to retinoid, steroid, and thyroid hormone receptors. The aim of the present study is to evaluate the role of PPAR-alpha receptor on the development of multiple-organ dysfunction syndrome (MODS) induced by zymosan. MODS was induced by peritoneal injection of zymosan (dose, 500 mg/kg i.p. as a suspension in saline) in PPAR-alpha wild-type (PPAR-alphaWT) and PPAR-alpha knockout (PPAR-alphaKO) mice, was assessed 18 h after the administration of zymosan, and was monitored for 12 days (for loss of body weight and mortality). A severe inflammatory process, induced by zymosan administration in wild-type mice, coincided with the damage of liver, kidney, pancreas, and small intestine. Myeloperoxidase activity, indicative of neutrophil infiltration, and lipid peroxidation were significantly increased in zymosan-treated wild-type mice. Zymosan in the wild-type mice also induced a significant increase in the plasma levels of nitrite/nitrate. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine and Fas ligand in the intestine of zymosan-treated wild-type mice. In contrast, the degree of (1) peritoneal inflammation and tissue injury, (2) nitrotyrosine formation and Fas ligand expression, and (3) neutrophil infiltration were markedly enhanced in intestinal tissue obtained from zymosan-treated PPAR-alphaKO mice. Zymosan-treated PPAR-alphaKO mice also showed a significantly increased mortality. Taken together, the present study clearly demonstrates that PPAR-alpha pathway modulates the degree of MODS associated with zymosan-induced nonseptic shock.

Target deletion of complement component 9 attenuates antibody-mediated hemolysis and lipopolysaccharide (LPS)-induced acute shock in mice.

Terminal complement membrane attack complex (MAC) formation is induced initially by C5b, followed by the sequential condensation of the C6, C7, C8. Polymerization of C9 to the C5b-8 complex forms the C5b-9 (or MAC). The C5b-9 forms lytic or non lytic pores in the cell membrane destroys membrane integrity. The biological functionalities of MAC has been previously investigated by using either the mice deficient in C5 and C6, or MAC's regulator CD59. However, there is no available C9 deficient mice (mC9(-/-)) for directly dissecting the role of C5b-9 in the pathogenesis of human diseases. Further, since C5b-7 and C5b-8 complexes form non lytic pore, it may also plays biological functionality. To better understand the role of terminal complement cascades, here we report a successful generation of mC9(-/-). We demonstrated that lack of C9 attenuates anti-erythrocyte antibody-mediated hemolysis or LPS-induced acute shock. Further, the rescuing effect on the acute shock correlates with the less release of IL-1ß in mC9(-/-), which is associated with suppression of MAC-mediated inflammasome activation in mC9(-/-). Taken together, these results not only confirm the critical role of C5b-9 in complement-mediated hemolysis and but also highlight the critical role of C5b-9 in inflammasome activation.

Developmental expression of an amn(+) transgene rescues the mutant memory defect of amnesiac adults.

The Drosophila memory gene amnesiac (amn) has been proposed to encode a neuropeptide protein, which includes regions homologous to vertebrate pituitary adenylyl cyclase-activating peptide (PACAP; Feany and Quinn, 1995). Definitive experiments to link this gene to memory formation, however, have not yet been accomplished (Kandel and Abel, 1995). The experiments described here demonstrate that the putative amn transcript is involved in adult memory formation. With the use of a UAS-amn(+) transgene, we show complete rescue of memory defects in amn(28A), a mutant allele caused by the insertion of a GAL4 enhancer trap transposon (Moore et al., 1998). Study of the amn(28A) reporter reveals widespread expression in the adult brain but also enriched expression in the embryonic and larval nervous systems. To begin addressing the temporal requirement of amn in memory, we asked whether the memory defects could be rescued by restricting transgenic expression to the adult stage. A heat-shock regimen shown previously to rescue fully the amn ethanol sensitivity defect (Moore et al., 1998) failed to rescue the memory defect. These results, coupled with previous genetic and anatomical studies, suggest that adult memory formation and ethanol sensitivity have different temporal and spatial requirements for amn.

Glucocorticoid-induced TNF receptor family gene (GITR) knockout mice exhibit a resistance to splanchnic artery occlusion (SAO) shock.

In the present study, we used glucocorticoid-induced tumor necrosis factor (TNF) receptor family gene knockout (GITR-KO) mice to evaluate a possible role of GITR on the pathogenesis of splanchnic artery occlusion (SAO) shock, which was induced in mice by clamping the superior mesenteric artery and the celiac artery for 30 min, followed thereafter by release of the clamp (reperfusion). At 60 min after reperfusion, animals were killed for histological examination and biochemical studies. There was a marked increase in the lipid peroxidation in the ileum of the SAO-shocked, GITR wild-type (WT) mice after reperfusion. The absence of GITR significantly reduced the lipid peroxidation in the intestine. SAO-shocked WT mice developed a significant increase of ileum tissue, TNF-alpha, and myeloperoxidase activity and marked histological injury. SAO shock was also associated with a significant mortality (5% survival at 24 h after reperfusion). Reperfused ileum tissue sections from SAO-shocked WT mice showed positive staining for P-selectin, intercellular adhesion molecule 1 (ICAM-1), and E-selectin. The intensity and degree of P-selectin, E-selectin, and ICAM-1 were markedly reduced in tissue section from SAO-shocked, GITR-KO mice. SAO-shocked, GITR-KO mice also showed a significant reduction of the TNF-alpha production and neutrophil infiltration into the reperfused intestine, an improved histological status of the reperfused tissues, and an improved survival. Taken together, our results clearly demonstrate that GITR plays an important role in the ischemia and reperfusion injury and put forward the hypothesis that modulation of GITR expression may represent a novel and possible strategy.

The ERRor of Our Ways: Estrogen-Related Receptors are About Energy, Not Hormones, and are Potential New Targets for Trauma and Shock.

As with sharks and horseshoe crabs, some designs of nature need only minor evolutionary adjustments during the millennia to remain superbly adapted. Such is the case at the molecular level for the nuclear receptors (NRs), which seem to have originated concomitantly with the earliest metazoan lineage of animals. A wide array of NRs persists today throughout all animal phyla with many different functions, yet they share a highly conserved protein structure, a testament to their having evolved through numerous gene duplications. Of particular interest for this readership are the estrogen-related receptors (ERRs), which have significant supportive roles in energy creation and regulation, mitochondrial function and biogenesis, development, tissue repair, hypoxia, and cancer. Thus, placed at the nexus of energetics and homeostasis, ERR (in association with the coregulatory molecules peroxisome proliferator-activated receptor-γ coactivator-1α and -ß) can facilitate repair from injury and adaptations to stressful environments. Whereas it is curious that ERRs and some other NRs exist as "orphans" by virtue of having no known cognate ligand, increasing interest in the estrogen receptor has led to the development of synthetic ligands and screening for naturally occurring molecules, either capable of modulating ERR activity. Thus, what is needed now is a nomenclature update for the ERR to focus the mind on energetics and metabolism, the most compromised and crucial systems after trauma and shock.