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Traumatic brain injury (TBI) and severe blood loss (SBL) frequently co-occur in human trauma, resulting in high levels of mortality and morbidity. Importantly, each of the individual post-injury cascades is characterized by complex and potentially opposing pathophysiological responses, complicating optimal resuscitation and therapeutic approaches. Large animal models of poly-neurotrauma closely mimic human physiology, but a systematic literature review of published models has been lacking. The current review suggests a relative paucity of large animal poly-neurotrauma studies (N = 52), with meta-statistics revealing trends for animal species (exclusively swine), characteristics (use of single biological sex, use of juveniles) and TBI models. Although most studies have targeted blood loss volumes of 35–45%, the associated mortality rates are much lower relative to Class III/IV human trauma. This discrepancy may result from potentially mitigating experimental factors (e.g., mechanical ventilation prior to or during injury, pausing/resuming blood loss based on physiological parameters, administration of small volume fluid resuscitation) that are rarely associated with human trauma, highlighting the need for additional work in this area.

Background Post-exercise recovery phase is associated with clustering of various cardiovascular events and, therefore, monitoring of cardiac autonomic control via heart rate variability (HRV) during this phase may allow identification of autonomic alterations that are not evident under resting conditions in type 2 diabetes mellitus (T2DM) patients. Purpose To investigate and compare the diagnostic performance of resting and post-exercise HRV for detecting cardiac autonomic neuropathy (CAN) in T2DM patients. Methods Forty-two T2DM patients were categorized as CAN-positive and CAN-negative based on standard cardiovascular autonomic reflex tests (CARTs). Short-term resting and post-exercise HRV after a graded exercise test were evaluated for each participant. Diagnostic performance of both resting and post-exercise HRV measures was computed using standard statistical procedures. Results Diagnostic testing yielded superior diagnostic performance of post-exercise HRV than resting HRV measures. Root mean square of successive differences (RMSSD) between adjacent R-R intervals (p = 0.01), percentage of consecutive N-N intervals that vary by >50 ms (pNN50) (p = 0.03) and total power (TP) (p = 0.01) were significantly better diagnostic indicators of CAN under post-exercise conditions than at rest. Predictive ability of these post-exercise HRV measures for CAN was maintained after adjusting various clinical confounders to cardiac autonomic function. Conclusion Post-exercise HRV measures such as TP, RMSSD and pNN50 were found to be more accurate diagnostic tests for detecting CAN than resting HRV. Hence, monitoring of the HRV measures proposed here during exercise testing protocols may provide important diagnostic information regarding CAN in T2DM.

Respiratory syncytial virus (RSV) is a potent pathogen having global distribution. The main purpose of this study was to gain an insight into distribution pattern of the NA1 genotype of group A RSV across the globe together with its evolutionary dynamics. We focused on the second hypervariable region of the G protein gene and used the same for Phylogenetic, Bayesian and Network analyses. Eighteen percent of the samples collected from 500 symptomatic pediatric patients with acute respiratory tract infection (ARI) were found to be positive for RSV during 2011–15 from New Delhi, India. Of these, group B RSV was predominant and clustered into two different genotypes (BA and SAB4). Similarly, group A viruses clustered into two genotypes (NA1 and ON1). The data set from the group A viruses included 543 sequences from 23 different countries including 67 strains from India. The local evolutionary dynamics suggested consistent virus population of NA1 genotype in India during 2009 to 2014. The molecular clock analysis suggested that most recent common ancestor of group A and NA1 genotype have emerged in during the years 1953 and 2000, respectively. The global evolutionary rates of group A viruses and NA1 genotype were estimated to be 3.49 × 10−3 (95% HPD, 2.90–4.17 × 10−3) and 3.56 × 10−3 (95% HPD, 2.91 × 10−3–4.18 × 10−3) substitution/site/year, respectively. Analysis of the NA1 genotype of group A RSV reported during 11 years i.e. from 2004 to 2014 showed its dominance in 21 different countries across the globe reflecting its evolutionary dynamics. The Network analysis showed highly intricate but an inconsistent pattern of haplotypes of NA1 genotype circulating in the world. Present study seems to be first comprehensive attempt on global distribution and evolution of NA1 genotype augmenting the optimism towards the vaccine development.

Background The gastrointestinal mucosa is an important route of entry for microbial pathogens. The immune cells of Peyer's patch (PP) compartments contribute to the active immune response against infection. Although local anesthetics are widely used in clinical practice, it remains unclear whether local anesthetics such as lidocaine affect PP T cell functions. Methods The aim of this study was to examine if lidocaine has any effects on mouse PP T cell functions. To test this, freshly isolated mouse Peyer's patch T cells were incubated with lidocaine. The effects of lidocaine on concanavalin A-stimulated PP T cell proliferation and cytokine production were assessed. The effect of lidocaine on PP T cell mitogen-activated protein kinase (MAPK) activation was also assessed. Results The results indicate that lidocaine suppresses cell proliferation, cytokine production, and MAPK activation in PP T cells. Furthermore, we found that the chronic in vivo exposure to lidocaine increases bacterial accumulation in PP. Conclusion The enhanced immunosuppressive effects of lidocaine on PP T cell functions could contribute to the host's enhanced susceptibility to infection.

Reduced immune function is frequently a consequence of serious injury such as trauma-hemorrhage (T-H). Injury may lead to reduced T-cell activation, resulting in decreased engagement of costimulatory molecules after antigen recognition and in subsequent immunological compromise and anergy. We hypothesized that inhibition of CD28 expression is one possible mechanism by which immune functions are suppressed after T-H. Male C3H/HeN mice (with or without ovalbumin immunization) were subjected to sham operation or T-H and sacrificed after 24 hours. Splenic T cells were then stimulated with concanavalin A or ovalbumin in vivo or in vitro, and CD28, cytotoxic T-lymphocyte antigen 4 (CTLA-4), CD69, and phospho-Akt expression was determined. T-cell proliferation/cytokine production was measured in vitro. Stimulation-induced CD69, CD28, and phospho-Akt up-regulation were significantly impaired after T-H compared with sham-operated animals; however, CTLA-4 expression was significantly higher in the T-H group. Over a 3-day span, stimulated T cells from sham-operated animals showed significantly higher proliferation compared with the T-H group. IL-2 and IFN-γ were elevated in sham-operated animals, whereas IL-4 and IL-5 rose in the T-H group, revealing a shift from TH1 to TH2 type cytokine production after T-H. Dysregulation of the T-cell costimulatory pathway is therefore likely to be a significant contributor to post-traumatic immune suppression.

Although 17beta-estradiol administration following trauma-hemorrhage attenuates Kupffer cell, splenic and peritoneal macrophage functions, it remains unknown whether 17beta-estradiol has any salutary effects on splenic dendritic cell (DC) functions and if so, whether such effects are mediated via the estrogen receptors (ER). We hypothesized that 17beta-estradiol administration following trauma-hemorrhage has salutary effects on splenic DC functions. Male C3H/HeN (6-8 weeks) mice were randomly assigned to sham operation or trauma-hemorrhage. Trauma-hemorrhage was induced by midline laparotomy and approximately 90 min of hemorrhagic shock (blood pressure [BP] 35 mmHg), followed by fluid resuscitation (4x the shed blood volume in the form of Ringer's lactate). Estrogen receptor (ER)-alpha agonist propyl pyrazole triol (PPT; 5microg/kg), ER-beta agonist diarylpropionitrile (DPN; 5microg/kg), 17beta-estradiol (50microg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. Two hours later, the mice were sacrificed, splenic DCs were isolated and the changes in their apoptosis, co-stimulating factors and MHC class II expression, ability to produce cytokines, and antigen presentation capacity were measured. Apoptosis of splenic DC increased following trauma-hemorrhage; however, 17beta-estradiol administration after trauma-hemorrhage normalized the rate of apoptosis. Moreover, splenic DC cytokines production, co-stimulating factors and MHC class II expression, and antigen presentation capacity were significantly decreased following trauma-hemorrhage; however, 17beta-estradiol as well as PPT also prevented these depressions. In contrast, DPN did not attenuate splenic DC functions following trauma-hemorrhage. Since PPT administration following trauma-hemorrhage was more effective in normalizing splenic DC functions than DPN, the salutary effects of 17beta-estradiol on splenic DC functions are mediated predominantly via ER-alpha.

Background/Aims The aim of this study was to determine which of the estrogen receptor (ER) subtypes plays a predominant role in ameliorating hepatic damage following trauma-hemorrhage. Methods Adult male rats were subjected to hemorrhagic shock (40mmHg for 90min) and resuscitation. ER-α agonist (PPT) or ER-β agonist (DPN) was administered during resuscitation; rats were sacrificed 24h thereafter. Results PPT or DPN decreased elevated plasma α-glutathione S -transferase levels; however, PPT was more effective. PPT or DPN increased hepatic heat shock protein 32 (Hsp32) mRNA/protein expressions above levels observed after trauma-hemorrhage. PPT reduced hepatic NF-κB and AP-1 activity and iNOS expression. Although DPN reduced hepatic NF-κB activity, AP-1 activity remained higher than in shams; hepatic iNOS induction remained elevated. PPT/DPN reduced nitrate/nitrite production and iNOS mRNA in Kupffer cells following trauma-hemorrhage; however, these levels in DPN-treated animals remained higher than sham. Conclusions Although both PPT and DPN decreased hepatic injury following trauma-hemorrhage, ER-α agonist PPT appears to be more effective in downregulating NF-κB and AP-1 activity, and iNOS induction. Thus, ER-α appears to play a predominant role in mediating the salutary effects of E2 in ameliorating hepatic damage following trauma-hemorrhage.

Although nongenomic effects of 17beta-estradiol (E2) are mediated via the estrogen receptor alpha (ER-alpha), the existence of another novel ER, G protein-coupled receptor 30 (GPR30), has been suggested as a candidate for triggering a broad range of E2-mediated signaling. GPR30 also acts independently of the ER to promote activation of the protein kinase A (PKA) pathway, which protects cells from apoptosis through Bcl-2. In this study, we examined whether the salutary effects of E2 in attenuating hepatic injury after trauma-hemorrhage are mediated via GPR30- or ER-alpha-regulated activation of PKA-dependent signaling. At 2 hours after trauma-hemorrhage, administration of E2-conjugated to bovine serum albumin (E2-BSA, membrane impermeable) or E2 induced the up-regulation of ER-alpha and GPR30 and attenuated hepatic injury. This was accompanied by increases in PKA activity and Bcl-2 expression. Inhibition of PKA in E2-BSA-treated trauma-hemorrhage rats by PKA inhibitor H89 prevented the E2-BSA attenuation of hepatic injury. Isolated hepatocytes were transfected with small interfering RNA to suppress GPR30 or ER. We found that suppression of GPR30 but not ER-alpha prevented E2-BSA- or E2-induced PKA activation and Bcl-2 expression. These results suggest that the nongenomic salutary effect of E2 in reducing hepatic injury after trauma-hemorrhage is mediated through the PKA-dependent pathway via GPR30 but not ER-alpha.

Background This study examined whether there is any difference in the lung cytokine and cytokeratin 19 levels between young and middle-aged mice after bone fracture and soft-tissue trauma hemorrhage (Fx-TH). Methods Young (6–8 weeks) and middle-aged (12 months) C3H/HeN male mice were subjected to right lower leg fracture, trauma hemorrhage (mean arterial blood pressure to 35 ± 5 mm Hg for 90 minutes), and resuscitation. Results The tumor necrosis factor α level in the lung increased significantly at 2 hours after Fx-TH in both young and middle-aged mice, whereas at 24 hours the levels remained significantly higher in middle-aged mice. Interleukin-6 levels increased significantly 24 hours after Fx-TH in both groups, whereas interleukin-10 levels increased only in middle-aged mice at 24 hours under those conditions. Monocyte chemoattractant protein-1 levels increased significantly 2 hours after Fx-TH. The protein and messenger RNA levels of cytokeratin 19 were significantly higher in middle-aged mice compared with young mice after Fx-TH. Conclusion These results suggest that age influences the lung inflammatory response after Fx-TH.

Since splenic immune functions are depressed in metestrus females following trauma-hemorrhage, we hypothesized that administration of the androgen receptor antagonist flutamide at the onset of resuscitation will maintain the immune function of the spleen following trauma-hemorrhage. Female C57BL6/J mice (metestrus state, 8-12 weeks old), underwent laparotomy and hemorrhagic shock (35.0+/-5.0 mm Hg for 90 min) and received 17beta-estradiol (50 microg/25 g), flutamide (625 microg/25 g) or 17beta-estradiol+flutamide. Four hours after resuscitation, the in vitro productive capacity of different cytokines (TNF-alpha, IL-6, IL-10, and IFN-gamma) by splenic MPhi and splenocytes were determined by flow cytometry. A significantly decreased cytokine production by both splenocytes and splenic MPhi was observed following trauma-hemorrhage compared to shams. Administration of 17beta-estradiol, flutamide and 17beta-estradiol+flutamide following trauma-hemorrhage resulted in a significant increase in the in vitro IL-6 release by splenic MPhi. The TNF-alpha productive capacity, however, was only restored by 17beta-estradiol and 17beta-estradiol+flutamide administration following trauma-hemorrhage. No significant effect of either treatment was observed with regard to the suppressed splenic MPhi IL-10 release. Anti-CD3 stimulation, administration of 17beta-estradiol and 17beta-estradiol+flutamide, but not the administration of flutamide alone resulted in a significant increased release of TNF-alpha, IL-6 and IFN-gamma compared to vehicle-treated animals. No significant effect of either treatment was found on IL-10 productive capacity. These results collectively suggest that flutamide administration following trauma-hemorrhage in females has beneficial effects on splenic immune function. However, flutamide administration in combination with estrogen does not provide any significant, additional effects over 17beta-estradiol administration alone.

Posttraumatic activation of macrophages enhances development of systemic inflammation/immunosuppression and organ dysfunction. We hypothesized that Kupffer cells are the main source of monocyte chemoattractant protein-1 (MCP-1) production after trauma-hemorrhage, that administration of 17β-estradiol (E2) after trauma-hemorrhage modulates MCP-1 release and reduces remote organ damage, and that salutary effects of E2 are mediated via estrogen receptor (ER)-α. To test these hypotheses, female B57BL/J6 mice received E2 (50 μg/25 g) or vehicle after trauma-hemorrhage and female 129 Sve ER-β −/− transgenic mice and ovariectomized wild-type mice received E2 or ER-α agonist propyl pyrazole triol (50 μg/25 g) after trauma-hemorrhage. Systemic MCP-1 and interleukin-6 and their release by liver, spleen, and lung macrophages were determined by flow cytometry 4 hours after trauma-hemorrhage. Prior Kupffer cell depletion with gadolinium chloride significantly decreased systemic MCP-1 and interleukin-6 after trauma-hemorrhage and was associated with decreased edema/neutrophil infiltration in lung and liver. Kupffer cells were the only macrophages showing significant MCP-1 release, which was markedly reduced by E2 or propyl pyrazole triol in wild-type and in ER-β −/− mice. Pretreatment of mice with anti-MCP-1 antiserum prevented an increase in myeloperoxidase and edema in lung and liver. These findings suggest that Kupffer cell-derived MCP-1 plays a major role in remote organ dysfunction after trauma-hemorrhage.

Although 17beta-estradiol (E2) administration following trauma-hemorrhage (T-H) improves cardiac function in male rodents, it is not known whether the salutary effects of E2 are mediated via estrogen receptor (ER)-alpha or ER-beta, and whether cardiac heat shock proteins (Hsp) are affected by E2 administration. Male Sprague-Dawley rats underwent T-H (mean BP 40 mmHg for 90 min, then resuscitation). ER-alpha agonist propyl pyrazole triol (PPT) (5 microg/kg), ER-beta agonist diarylpropiolnitrile (DPN) (5 microg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. At 24 h after T-H or sham operation, cardiac output (CO), stroke volume (SV), mean blood pressure, and +/- dP/dt max were measured (n=6 rats per group). Cardiac Hsp32, 60, 70, and 90 mRNA/protein expressions and heat shock factor (HSF)-1 DNA binding activity were determined. One-way ANOVA and Tukey's test were used for statistical analysis. CO, SV and +/- dP/dt(max) decreased significantly after T-H, however, administration of ER-beta agonist DPN after T-H restored the above parameters. Moreover, DPN treatment prevented T-H-mediated decrease in Hsp60 mRNA/protein and Hsp90 protein expressions in the heart. Hsp32 and Hsp70 mRNA/protein expression and HSF-1 DNA binding activity in the hearts were increased even above the shams in DPN treated T-H rats. In contrast, no significant change in the above parameters was observed in T-H rats treated with ER-alpha agonist PPT. Thus, the salutary effects of E2 on cardiac function are mediated via ER-beta and ER-beta-induced up-regulation of Hsp likely plays a significant role in the E2-mediated cardioprotection after T-H.

Background Immune dysfunction and post-injury infections are complications associated with thermal injury. Opiates, the analgesic of choice for the treatment of post-burn pain, can also induce similar immune complications. However, the impact of therapeutic opiates on post-burn immune dysfunction is unknown. Materials and Methods C57BL/6 mice were subjected to a small 6.25% total body surface area (TBSA) burn or sham procedure. The mice were left untreated or treated with morphine sulfate by subcutaneous implantation of an Alzet pump that administered morphine sulfate at a rate of 2 mg/kg body weight/day. Plasma, splenocytes and splenic macrophages were isolated for in vitro analysis 1, 4, or 7 days later. Results Neither burn injury nor morphine treatment alone significantly altered splenic T-cell proliferation at 1, 4, or 7 days post-injury/treatment. In contrast, morphine treatment of injured mice suppressed splenic T-cell proliferation at 4 and 7 days post-injury/treatment. The suppressed proliferation of T-cells correlated with increased levels of the nitric oxide and an immunosuppressive Th-2 type phenotype. In contrast morphine treatment did not accentuate the suppressed T-cell proliferative responses associated with larger injuries covering 12.5% and 25% TBSA. Splenic macrophage function was unaffected with the exception that LPS-induced nitric oxide production was elevated in the injured mice treated with morphine. Conclusions These findings demonstrate that those mice treated with a clinically relevant dose of morphine sulfate after an “immunologically insignificant” burn displayed immunosuppression and a Th-2 cytokine profile. Thus, the therapeutic administration of exogenous opiates appears to contribute to the development of post-burn immune dysfunction.

Studies have shown gender dimorphic response of the liver for various hepatic stresses including ischemia/reperfusion, hemorrhagic shock-resuscitation, hepatectomy, liver cirrhosis, endotoxemia, and chronic alcoholic consumption. The mechanisms responsible for the gender dimorphic response include differences in pro-inflammatory cytokine release, production of reactive oxygen species, and alteration in hepatic vasoregulatory action. These effects were shown to be modulated by circulating sex steroid levels. In this regard, modulation of sex steroid levels by agents/drugs has been proposed as a therapeutic option for preventing hepatic damage in various hepatic stress models. Further elucidation of precise mechanisms responsible for the gender-related differences in the hepatic pathophysiology is essential for the potential clinical application of sex hormone modulation therapy. In this article, current progress in our understanding the gender difference in the hepatic pathophysiology under the condition of hepatic stress is reviewed and discussed.

Background Although flutamide (FTM), an androgen-receptor antagonist, normalizes the depressed immune and cardiac function in males after trauma hemorrhage (T-H), the mechanism responsible for its salutary effects remains unknown. We hypothesized that the salutary effects of FTM are mediated via upregulation of estrogen receptors (ERs). Methods Male Sprague-Dawley rats underwent T-H (laparotomy and 90 minutes of hemorrhage (35-40 mm Hg) and then resuscitated with 4× the volume of shed blood in the form of Ringer's lactate). FTM (25 mg/kg) or vehicle (propanediol) was injected subcutaneously 30 minutes before the end of resuscitation. At 2 hours after T-H or sham operation, cardiac output, stroke volume, heart rate, mean arterial pressure, ± dp/dt, and total peripheral resistance were measured (n = 6 rats per group). Immediately after the measurement of cardiac function, cardiomyocytes were isolated, RNA was extracted, and expression of ER-α, ER-β, and androgen-receptor (AR) mRNA in cardiomyocytes was determined by quantitative real-time polymerase chain reaction. ER-α, ER-β, and AR protein levels in cardiomyocytes were also measured by Western blot analysis. Results The depressed cardiac output, stroke volume, and ± dp/dt after T-H were significantly improved in the FTM-treated T-H group. Moreover, the decrease in expression of ER-α and ER-β mRNA and protein in cardiomyocytes in the T-H group was prevented with FTM treatment after T-H. However, expression of cardiomyocytes AR mRNA and protein were not significantly different between the T-H or sham group with or without FTM treatment. Conclusions These findings collectively suggest that, in addition to blockade of androgen receptors, flutamide-mediated ER upregulation is likely to play a role in mediating the salutary effect of flutamide on cardiac function after trauma hemorrhage.

Studies have shown that following bacterial infection or endotoxin administration, immune functions are regulated differently in mice of different genetic background. Since the susceptibility to sepsis following trauma-hemorrhage is dependant on the severity of injury, it is important to determine whether genetic background of the animal influence immune functions after trauma-hemorrhage. The aim of our studies, therefore, was to assess differences in the immune functions in genetically different strains of age-matched C3H/HeN and C57BL/6 male mice following trauma-hemorrhage. The analysis for immune functions included: proliferation of splenocyte and bone-marrow cells, IL-2 and IFN-gamma release by splenocytes, and TNF-alpha and IL-10 release by splenic, peritoneal, liver (Kupffer cell), and bone-marrow macrophages. The results show significant differences in splenocyte and bone-marrow functions, and in the release of the mediators of immune function by immune competent cells: (a) between the two genetic strains, and (b) in each mouse strain following trauma-hemorrhage. Thus, genetic background appears to significantly influence the severity of immune responses in males following trauma-hemorrhage.

Identification of novel diagnostic or therapeutic biomarkers from human blood plasma would benefit significantly from quantitative measurements of the proteome constituents over a range of physiological conditions. Herein we describe an initial demonstration of proteome-wide quantitative analysis of human plasma. The approach utilizes post-digestion trypsin-catalyzed 16O/18O peptide labeling, two-dimensional liquid chromatography (LC)-Fourier transform ion cyclotron resonance ((FTICR) mass spectrometry, and the accurate mass and time (AMT) tag strategy to identify and quantify peptides/proteins from complex samples. A peptide accurate mass and LC-elution time AMT tag database was initially generated using tandem mass spectrometry (MS/MS) following extensive multidimensional LC separations to provide the basis for subsequent peptide identifications. The AMT tag database contains >8,000 putative identified peptides, providing 938 confident plasma protein identifications. The quantitative approach was applied without depletion for high abundant proteins for comparative analyses of plasma samples from an individual prior to and 9 h after lipopolysaccharide (LPS) administration. Accurate quantification of changes in protein abundance was demonstrated by both 1:1 labeling of control plasma and the comparison between the plasma samples following LPS administration. A total of 429 distinct plasma proteins were quantified from the comparative analyses and the protein abundances for 25 proteins, including several known inflammatory response mediators, were observed to change significantly following LPS administration.

Patients without spleens have an increased risk of infection. Previous studies have shown that splenectomy (Spx) influences Kupffer cells (KC), peritoneal macrophages (pMphi) and alveolar macrophages (aMphi) phagocytosis and bactericidal activity. This study examined the effect of Spx on the cytokine production by peripheral blood monocular cells (PBMC), KC, aMphi, pMphi and cells from mesenteric lymph nodes (MLN). We also determined if Spx influences survival following sepsis induced by cecal ligation and puncture (CLP). C57BL/6J male mice (8-10 weeks old) underwent sham operation or Spx. Two weeks after sham or Spx, KC, pMphi, aMphi, PBMC and cells from MLN were isolated and stimulated with LPS. Cell-free supernatants were analyzed for TNF-alpha, IL-6, and IL-10. A significant increase in KC, pMphi and aMphi TNF-alpha and IL-6 release was observed following Spx. The production of IL-10 was also significantly higher in KC under those conditions. In contrast, the release of TNF-alpha, IL-6 and IL-10 was significantly decreased in PBMC after Spx. Similarly, TNF-alpha and IL-6 was also decreased in MLN after Spx. Overall survival after CLP was not different in mice subjected to either sham or Spx. However, the mean time to death was significantly decreased in mice subjected to Spx compared to sham injured mice. These findings suggest that Spx modulates immune cell functions in various tissue compartments of the body and results in early deaths following sepsis.

Background. Current isolation techniques recover only 20% to 50% of the pancreatic islets. Brain death (BI)) is characterized by activation of proinflammatory cytokines (PICs) with reduced islet yields and functionality. We previously reported that 17β-estradiol (E2) induces cytoprotection to human islets exposed to PICs. Furthermore, inhibition of PIC release has been demonstrated after E2 treatment. In the present study, we evaluated if E2 treatment to BD donors would improve pancreatic islet recovery and functionality. Methods. BD was induced in male, 250- to 350-g Lewis rats by inflation of a Fogarty catheter placed intracranially. Rats were mechanically ventilated for 6 hours. Only rats with mean arterial blood pressure >75 mm Hg were used. Animals (n = 6) received E2 (1mg/kg/iv immediately after BD induction), vehicle (V), or the combination of 17β-estradiol and a selective estrogen receptor antagonist ICI 182, 780 (ICI, 3mg/kg/ip/1 hour before BD induction). Islet viability was determined by ethidium bromide-acridine orange. PICs were assessed by ELISA. Islet functionality was determined by static incubation and glucose disposal rate (Kg) after intraportal transplantation (3000 islet equivalen-t[IEQ]/syngeneic steptozotocin-induced diabetic rat). Results. A 2- to 3-fold reduction in TNF-α, IL-1β, and IL-6 was demonstrated in BD donors given-E2; this effect reversed by ICI 182, 780. Pancreatic sections from control BD donors presented 26. 5% ± 4 % TUNEL-positive β-cells compared with 15.1% ± 3% in 17β-estradio-treated animals. Islet recovery was enhanced in E2-treated donors (1233.4 ± 123 IEQ/pancreas) compared with controls (725 ± 224 IEQ P

Several studies demonstrated a sex-specific cytokine secretion by macrophages following trauma-hemorrhage (T-H) and incubation with lipopolysaccharide A (LPS). Although LPS is known to act via the receptors CD14 and TLR4 on macrophages, it remains unknown whether differences in LPS receptor expression in males and females may be responsible for the gender-specific LPS induced cytokine response following (T-H). To study this, male and proestrus female mice (C3H/HeN) were subjected to trauma (laparotomy) followed by hemorrhage or sham operation. At 2 h thereafter, SMphi and PMphi were harvested and cultured for 2 h. The expression of CD14 and TLR4 was measured by flow cytometry on unstimulated SMphi and PMphi as well as after LPS stimulation. The results indicate that the expression of CD14 and TLR4 on SMphi and PMphi from female and male mice was similar in sham-operated animals and after (T-H). Incubation of macrophages with LPS did not alter CD14 and TLR4 expression in the study groups. Thus, the sex specific LPS induced cytokine secretion after (T-H) is not caused by differences in LPS receptor expression on Mphi of male and female mice.

Although immune responses following soft-tissue trauma-hemorrhage are markedly different in young (6-8 weeks) and aged (18-20 months) mice, it remains unknown if there are any differences in immune responses in middle-aged and young mice following bone fracture, soft-tissue trauma-hemorrhage (Fx-TH). To study this, young (6-8 weeks) and middle-aged (approximately 12 months) C3H/HeN male mice were subjected to sham operation or Fx-TH followed by resuscitation with Ringer's lactate. The mice were sacrificed 2 h thereafter and splenocytes, bone marrow cells (BM) and Kupffer cells (KC) were harvested, purified and stimulated with ConA (for splenocytes) or LPS (for BM and KC) in vitro. Splenocyte release of Th1 (IL-2 and IFN-gamma) cytokines was decreased and Th2 (IL-4 and IL-10) cytokine release was increased following Fx-TH in both young and middle-aged mice. However, the decrease in IL-2 and increase in IL-10 were significantly more in middle-aged mice compared to young mice (p0.05). Furthermore, splenocyte proliferation was decreased more in middle-aged mice compared to young mice following Fx-TH (p0.05). Additionally, TNF-alpha production was more in BM from middle-aged compared to BM from young mice after Fx-TH (p0.05). The production of IL-6 and IL-10 was also significantly higher in KC from middle-aged mice compared to young ones following Fx-TH. These results suggest that at middle age, the immune responses to Fx-TH are significantly different from those observed in young mice in different compartments of the body. Although the mechanism of the difference in various compartments in middle-aged vs. young mice following Fx-TH remains unknown, the decreased IL-2 production along with other altered T cell and macrophage functions may contribute to an increased susceptibility to sepsis in middle-aged vs. young individuals.

Studies have shown that 17beta-estradiol has salutary effects on immune functions after trauma-hemorrhage (TH). It remains unknown, however, whether 17beta-estradiol has a similar effect in a double-hit model of TH and subsequent sepsis. It is also unknown if under those conditions the circulating immune cells accurately represent immunological responses occurring in fixed tissues, such as the spleen. To study this, pre-castrated mice were hormonally treated and then subjected to soft-tissue trauma (i.e. midline laporatomy), hemorrhagic shock (MAP 35+/-5mmHg for 90 min followed by resuscitation) and 24 h later sepsis was induced by cecal ligation and puncture (CLP). Splenic macrophages (SMphi) and peripheral blood mononuclear cells (PBMC) were isolated and cultured with LPS. 5alpha-Dihydrotestosterone-treated mice showed a depressed pro-inflammatory cytokine production after TH-sepsis in both SMphi and PBMC. In contrast, the 17beta-estradiol treated groups showed suppressed pro-inflammatory cytokine production in the PBMC population under those conditions. In summary, 17beta-estradiol was able to prevent immune dysfunction after TH and subsequent sepsis. However, the beneficial effects of 17beta-estradiol were limited to tissue-fixed Mphi, suggesting compartmentalization of the response. Thus, events occurring in the tissue-fixed cells are not necessarily reflected in the circulating PBMC population.

Trauma-hemorrhage initiates inflammation in males that leads to a systemic inflammatory response syndrome (SIRS) and immunosuppression, which in turn may develop into multiple organ dysfunction syndrome (MODS). In contrast, females, in the proestrus state (which is associated with elevated estrogen levels), do not show immune depression following trauma-hemorrhage. Studies in experimental models have demonstrated that the underlying basis for this gender-dimorphic immune response following trauma-hemorrhage is due to the marked effects of sex hormones. Sex hormones influence the onset and severity of immune-mediated pathophysiological conditions primarily by modulating immune cell function. Nonetheless, despite extensive studies, the mechanisms of sex hormone action are yet to be precisely defined. Earlier studies had suggested that the sex hormone-mediated modulation of immune activity was primarily via the thymus. In recent years, however, it has become apparent that sex hormones can also influence the immune system by acting on several nonthymic targets such as monocytes, macrophages, lymphoid cells, endothelial cells and the central nervous system. However, more research is needed to define the mechanism responsible for gender-specific immune response to trauma-hemorrhage. These studies will likely contribute to the therapeutic modalities that include manipulation of sex hormones, and/or their receptor agonist/antagonists in the treatment of traumatized patients.

Immune responses in proestrus females are not altered after trauma-hemorrhage, whereas they are markedly depressed in males. Elevated levels of female sex steroids appear to be responsible for maintaining immune responses but it remains unknown, whether estrogen per se is responsible. To study this, proestrus female C3H/HeN mice were subjected to laparotomy (i.e., soft tissue trauma) and hemorrhagic shock (35+/-5 mmHg for 90 min, then resuscitated) or sham operation and received the estrogen receptor antagonist EM-800 or vehicle during resuscitation. Two hours following trauma-hemorrhage, splenocyte proliferation, IL-2, IL-3, IFN-gamma release, and splenic macrophage IL-6 release was maintained in vehicle-treated females. In EM-800-treated females, however, these immune parameters were significantly depressed. Following trauma-hemorrhage, Kupffer cell TNF-alpha release and circulating TNF-alpha were increased only in EM-800-treated females. These findings indicate that the ability of proestrus females to maintain immune function following trauma-hemorrhage is estrogen-dependent and mediated via estrogen receptors.

Kupffer cells have been reported as a major source of proinflammatory cytokines (i.e. IL-6, TNF-alpha), which have been implicated in the pathogenesis of trauma-hemorrhage. Previous studies have shown a protective effect of 17beta-estradiol on immune function and physiological responses following trauma-hemorrhage. In this study, we investigated whether 17beta-estradiol has a direct effect on Kupffer cell cytokine production following trauma-hemorrhage. Male Sprague-Dawley rats were subjected to trauma (midline laparotomy) and hemorrhage (35-40 mmHg for 90 min followed by fluid resuscitation) or sham operation. Two hours later, Kupffer cells were isolated and cultured with 17beta-estradiol in the presence and absence of lipopolysaccharide stimulation. Kupffer cell IL-6 and TNF-alpha production increased following trauma-hemorrhage. Incubation with 17beta-estradiol attenuated the production of IL-6 by cells from both sham and trauma-hemorrhage animals in a dose-dependent manner. The suppression of IL-6 production by 17beta-estradiol was paralleled by a decrease in mRNA levels. In contrast to IL-6, the effects of 17beta-estradiol on TNF-alpha production were minimal. In conclusion, these results indicate the direct downregulation of Kupffer cell IL-6 production by 17beta-estradiol at a molecular level, which might explain in part the previously observed salutary effects of estradiol treatment following trauma-hemorrhage.

Burn injury induces immune dysfunction and alters numerous physiological parameters. While clinical studies indicate that burn injury size profoundly impacts patient immune status, only limited experimental studies have systematically addressed its impact on immune functional parameters. In the present study, mice were subjected to burn injuries of varying sizes and splenic immune cells (splenocytes and macrophages) were isolated 7 days thereafter. Burn injury suppressed splenic T-cell proliferation in an injury size-dependent manner that correlated with the release of the immunosuppressive mediators PGE(2) and nitric oxide. In addition, a shift towards an immunosuppressive Th-2 cytokine profile and a hyperactive macrophage phenotype (increased release of inflammatory mediators) was observed post-injury, however, this effect was in part independent of burn size. Thus, unlike patient survival data, burn injury-induced changes in immune function do not necessarily correlate with the size of the injury.

Fas/Fas ligand (FasL) system is one of the major pathways triggering apoptosis that has been shown to play an important role in development and pathogenesis of various diseases including liver and gastrointestinal diseases. Studies indicate that FasL deficiency provides a survival advantage in mice subjected to polymicrobial sepsis. However, the extent to which Fas/FasL contributes to organ injury during sepsis is unclear. Thus, the aim of this study was to determine whether in vivo administration of a Fas-signaling inhibitor during sepsis preserves organ function.Male adult C3H/HeN mice were subjected to cecal ligation and puncture (CLP) or sham CLP (sham). Twelve hours after CLP, mice received either Fas-receptor fusion protein (FasFP) (200 microg/kg body weight) or the saline vehicle. Twenty-four hours after the onset of sepsis, cardiac output and organ blood flow were measured with radioactive microspheres. Plasma levels of alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase were assessed as indexes of liver damage. Changes in systemic cytokines were measured by enzyme-linked immunosorbent assay. RESULTS. The data indicate that although cardiac output and organ blood flow in the liver, intestine, kidneys, spleen, and heart decreased markedly at 24 hours after CLP, treatment with FasFP maintained the measured hemodynamic parameters and improved hepatic, intestinal, and heart blood flow (P.05) and partially restored spleen and renal blood flow. Moreover, FasFP treatment markedly attenuated the systemic rise in alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and interleukin 10 (P.05).These results not only indicate that there is a role for Fas/FasL-mediated processes in the induction of organ injury but suggest that inhibition of Fas/FasL pathway may represent a novel therapeutic modality for maintaining organ perfusion and preventing liver injury during sepsis.

Introduction. Recent studies have shown that administration of the sex steroid dehydroepiandrosterone (DHEA) in males following trauma-hemorrhagic shock has salutary effects on the depressed cardiovascular and immunological functions under those conditions. Since the effects of sex steroids are gender specific, we examined whether administration of DHEA has any beneficial effects on hepatocellular function in female rats with low estrogen levels following trauma–hemorrhage. Methods. Ovariectomy was performed in female Sprague-Dawley rats 14 days prior to the experiments. The animals then underwent a 5-cm midline laparotomy and were subjected to hemorrhagic shock (40 mm Hg for 90 min). This was followed by fluid resuscitation (Ringer's lactate over 60 min) and administration of DHEA (30 mg/kg BW) or vehicle subcutaneously at the end of resuscitation. At 24 h after resuscitation hepatocellular function, i.e., clearance of indocyanine green (ICG), and hepatocyte damage (serum alanine aminotransferase) were measured. Plasma levels of DHEA and 17β-estradiol were also assayed. Results. Vehicle-treated rats had significantly reduced hepatocellular function, increased ALT activity, and decreased levels of 17β-estradiol following trauma–hemorrhage compared to sham-operated animals (P < 0.05, ANOVA and Student-Newman-Keuls test). In animals receiving DHEA following trauma–hemorrhage, hepatocellular function and ALT activity were similar to those of shams. However, administration of DHEA did not influence the plasma levels of 17β-estradiol. Conclusions. Administration of DHEA following trauma–hemorrhage restored hepatocellular function and reduced hepatic damage that was observed in ovariectomized female rats under such conditions. This salutary effect of DHEA did not appear to be due to elevated levels of plasma 17β-estradiol. We therefore propose that DHEA should be considered a novel, safe, and useful adjunct in the treatment of trauma-induced hepatocellular dysfunction in ovariectomized and postmenopausal females.

Although the hemodynamic response to polymicrobial sepsis is characterized by an early, hyperdynamic phase followed by a late, hypodynamic phase, the factors responsible for producing the transition from the hyperdynamic to the hypodynamic stage are not fully understood. The failure to recognize or prevent this transition may lead to progressive deteriorations in cell and organ functions and ultimately result in multiple organ failure. Despite the fact that several vasoactive mediators (i.e., nitric oxide, prostacyclin, calcitonin gene-related peptide) have been implicated in producing cardiovascular alterations during sepsis, recent studies have indicated that adrenomedullin (AM), a novel vasodilatory peptide, plays an important role in initiating the hyperdynamic response during the early stage of polymicrobial sepsis. In addition, the reduced vascular responsiveness appears to be responsible for producing the transition from the early, hyperdynamic phase to the late, hypodynamic phase of sepsis. Moreover, modulation of AM vascular responsiveness reduces sepsis-induced mortality. In this review the physiological effects of AM, mechanisms of its action, and regulation of its production under various pathophysiological conditions will be discussed. Furthermore, the role of AM in producing the biphasic hemodynamic responses observed during polymicrobial sepsis and approaches for pharmacologically modulating vascular responsiveness and hemodynamic stability under such conditions will be described.

Background: Although studies have shown that there is a marked depression in cell-mediated (T H 1 ) immunity after the onset of sepsis, the mechanism by which this occurs remains unknown. In this regard, the T H2 cytokine IL-4 is known to regulate T H1 and T H2 cell responsiveness primarily through the activation of the signal transducer and activation of transcription factor-6 (Stat6) pathway. Methods: We hypothesized that IL-4 may contribute to the suppression of cell-mediated immunity and to death seen in sepsis and that IL-4 may be acting through the Stat6 pathway. To determine this, we induced cecal ligation and puncture (CLP) or sham-CLP in male BALB/c mice. Mice received 2 mg of monoclonal antibody against IL-4 or IgG control at 12 hours after CLP (ie, at the onset of immune suppression). Splenic T cells were then isolated 24 hours after CLP and stimulated with monoclonal antibody to CD3. Cytokine release and Stat6 phosphorylation (activation) were determined. In a separate group of animals, survival was assessed over 10 days. Results: The results indicate that after CLP, T cells are suppressed in their ability to release the T H1 cytokines, IL-2 and IFN-γ. Alternatively, the release of T H2 cytokines IL-10 and IL-4 is markedly increased after CLP. This was associated with an increase in phosphorylated Stat6 protein. In vivo treatment of mice with monoclonal antibody to IL-4 at 12 hours after CLP restores T H1 responsiveness while preventing the increase in T H2 cytokine release and Stat6 phosphorylation. Furthermore, neutralization of IL-4 markedly increased the survival rates in septic animals. Conclusions: Taken together, these data indicate that the T H2 cytokine IL-4 contributes to the suppression of cell-mediated immunity and death associated with polymicrobial sepsis and suggest that IL-4 may be acting through the Stat6 pathway in septic animals. (Surgery 2000;128:133-8.)

Background. Although it is known that pentoxifylline (PTX) produces various beneficial effects during sepsis, it remains unknown whether this agent has any salutary effects on the depressed vascular responsiveness to adrenomedullin (ADM), a novel potent vasodilatory peptide, under such conditions. Materials and methods. Adult male Sprague–Dawley rats were subjected to polymicrobial sepsis by cecal ligation and puncture (CLP). One hour after CLP, PTX (50 mg/kg body wt) or vehicle (normal saline) was infused intravenously over 90 min. Twenty hours after CLP (i.e., the late, hypodynamic stage of sepsis), the thoracic aorta and small intestine were isolated and preconstricted by norepinephrine. Rat ADM (10−7 M) was applied, and the percentage of ADM-induced relaxation in the aortic rings and resistance vessels in the small intestine was determined. In addition, plasma ADM was determined by radioimmunoassay and tumor necrosis factor α (TNF-α), interleukin (IL)-1β, and IL-6 levels were measured by enzyme-linked immunosorbent assay. Results. The percentage of ADM-induced vascular relaxation in the aortic rings and resistance vessels of the isolated gut was significantly reduced 20 h after CLP. Administration of PTX early after the onset of sepsis, however, prevented the decrease in vascular ADM responsiveness at the macro- and microcirculatory levels. Plasma ADM levels increased after CLP, irrespective of PTX infusion, indicating that the effect of PTX was not mediated by altering ADM release. The upregulated TNF-α, IL-1β, and IL-6 during late sepsis were, however, attenuated by PTX administration, suggesting that maintenance of ADM responsiveness by this agent appears to be due to downregulation of these cytokines. Conclusions. Since early administration of PTX maintains vascular ADM responsiveness even during the late stage of sepsis, this agent appears to be a useful adjunct in preventing the deterioration in hemodynamics and cardiovascular function during the progression of polymicrobial sepsis.

Background. Studies have indicated that a shift from a Th1 to a Th2 response occurs that contributes to the late immunosuppression seen during sepsis. However, the mechanism by which this occurs is unknown. In this regard, mediators released in response to sepsis are thought to upregulate a family of stress-induced mitogen-activated protein kinases (MAPKs), such as JNK, ERK, and p38 MAPK, which may play a role in this process. Materials and methods. To determine the role of MAPK in immune suppression, we induced polymicrobial sepsis in C3H/HeN male mice using cecal ligation and puncture (CLP). Splenic lymphocytes were harvested 24 h post-CLP and stimulated with the T-cell mitogen concanavalinA, and the expression and activation of these MAPKs were assessed by Western analysis. To determine the extent to which these MAPKs may have an impact on splenic immune function, cells were pretreated with a 10 μM concentration of the p38 MAPK inhibitor SB203580 or the MEK inhibitor PD98059 or with DMSO vehicle. The cells were then stimulated with 2.5 μg/ml of the T-cell mitogen concanavalin A, and cytokine release was then determined (by ELISA). Results. In the lymphocytes from CLP mice no JNK signal was detected, however, p38 expression and activation were markedly ( P n = 6) increased. In contrast, the expression of activated ERK markedly decreased following septic challenge. The results indicate that p38 MAPK inhibition with SB203580 suppressed the sepsis-induced augmentation of interleukin-10 release while restoring the suppressed Th1 cytokine interleukin-2 release typically encountered following sepsis. Inhibition of ERK had no effect on cytokine release. Neither PD98059 nor SB203580 had an effect on interferon γ release or on proliferative capacity. Conclusion. This would indicate that the induction of p38 MAPK activation in splenocytes contributes to the immunosuppression seen in late sepsis.

Background. Although the initial response to sepsis includes a hyperdynamic phase and although the increased hepatic perfusion in early sepsis is due solely to the increased portal blood flow, it remains unknown whether the gut plays an important role in producing such a response. Materials and methods. Adult male Sprague–Dawley rats underwent a complete enterectomy (ER) before being subjected to sepsis by cecal ligation and puncture (CLP; the cecum was excised from the removed gut and stitched to the posterior peritoneum in ER groups) or sham operation. At 2 h after CLP (i.e., the early, hyperdynamic phase of sepsis), cardiac output and heart performance (±dP/dtmax), as well as hepatic and renal blood flow, were measured. Systemic and regional oxygen delivery (DO2) and oxygen consumption (VO2) were also determined. Results. Cardiac output, heart performance, organ blood flow, as well as DO2 and VO2, increased significantly 2 h after CLP. ER prior to the onset of sepsis, however, prevented the elevation of those parameters. ER in sham animals did not alter the measured parameters with the exception that portal blood flow decreased by 85% and hepatic arterial blood flow increased by 368%, resulting in no significant reduction in hepatic DO2 and VO2. There were no changes in circulating blood volume among groups, indicating that the effect of ER on hemodynamics after CLP was not due to alterations in blood volume. Conclusion. Since ER immediately before the onset of sepsis prevents the increase in cardiac output and regional hemodynamics, the gut appears to play an important role in producing the hyperdynamic response during the early stage of polymicrobial sepsis.

Background: Studies indicate impaired wound healing after trauma. The underlying mechanism remains unknown. Methods: Mice were subjected to midline laparotomy, and polyvinyl alcohol sponges were implanted subcutaneously before hemorrhage (35 ± 5 mmHg for 90 minutes, resuscitated) or sham operation. Wound exudate cells from the sponges were harvested on the first, third, and fifth postoperative day and cultured for 24 hours. Interleukin (IL)-1β, IL-6, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2), and transforming growth factor (TGF)-β were determined in the supernatants. IL-1β and IL-6 were measured in the wound fluid. Results: Hemorrhage decreased collagen deposition in the wound. TGF-β release was significantly decreased on the first and third postoperative days after hemorrhage, whereas IL-1β and IL-6 release was increased at 3 and 5 days after hemorrhage. Similarly, IL-1β and IL-6 in the wound fluid were significantly increased at 3 days after hemorrhage. Conclusions: Because increased levels of pro-inflammatory cytokines and decreased amounts of TGF-β have been reported to impair the process of wound healing, the increased release of IL-1β and IL-6 and the decreased release of TGF-β after hemorrhage might contribute to the decreased collagen production in those animals. Thus, attempts to locally change the ratio of those cytokines in trauma victims might be useful for improving wound healing in those patients. (Surgery 1999;126:279-85.)

Background: Controversy exists concerning the role of interleukin 10 (IL-10) in sepsis. When IL-10 is used in models of endotoxemia, it appears to protect (by anti-inflammatory effects), whereas in models of polymicrobial sepsis it seems to be deleterious (by immunosuppression?). However, little direct evidence for such an immunosuppressive role is available for polymicrobial sepsis. Thus the aim of this study was to determine whether IL-10 contributes to lymphocyte immunosuppression in a model of cecal ligation and puncture (CLP) and whether neutralization of IL-10 has any salutary effects on survival after sepsis. Methods: To assess the former, polymicrobial sepsis was induced in male C57BL/6J wild-type (+/+) and C57BL/6J-IL-10 knockout(–/–) mice by CLP. Splenocytes were harvested 24 hours later and stimulated with concanavalin A to assess their proliferative capacity and their ability to release the Th1 lymphokines interleukin 2 and interferon gamma (by enzyme-linked immunosorbent assay, nanograms/millilter). To further verify the immunosuppressive role of IL-10, splenocytes were obtained from male C3H/HeN mice 24 hours after CLP and then stimulated in the presence or absence of anti-IL-10 monoclonal antibody (Mab, 4 μg/mL). To assess the in vivo effects of IL-10 neutralization on survival after CLP, C3H/HeN mice (16 per group) were given 250 μg of anti-IL-10 Mab (intraperitoneally) either immediately after CLP (before the initiation of the hyperdynamic phase) or 12 hours after CLP (the beginning of the hypodynamic state). Control mice were given nonspecific rat immunoglobulin G. Results: These data indicate that IL-10 deficiency (–/–) prevents the depression of the proliferative capacity and Th1 lymphokine production after sepsis. Analysis of the interleukin 2–interferon gamma production patterns and proliferative capacity in lymphocytes treated with anti-IL-10 Mab confirmed the role of IL-10 in suppressing lymphocyte responsiveness in CLP. Interestingly, however, only delayed administration (12 hours after CLP) of anti-IL-10 markedly increased survival of mice (Fisher's exact test, P < .05). Conclusion: The results not only illustrate IL-10's role in septic immune dysfunction but document that anti-IL-10 administration beyond the initial proinflammatory hyperdynamic state of polymicrobial sepsis improves survival of animals subjected to sepsis. (Surgery 1999;126:378-83.)

Although a profound depression in immune function occurs following injury, the mechanism responsible for this is not fully understood. Furthermore, steroid hormones are known to be important mediators in the regulation of immune function. Although dehydroepiandrosterone (DHEA), the most plentiful steroid hormone, has been shown to stimulate immune function in normal animals, it is unknown whether DHEA has any salutary or detrimental effects on immune responses after trauma and haemorrhage. To study this, male mice were subjected to trauma, haemorrhage and resuscitation, following which they received either DHEA or vehicle subcutaneously. DHEA administration restored the normally depressed splenocyte proliferation as well as interleukin 2, interleukin 3, and interferon gamma elaboration following trauma and haemorrhage. In an attempt to determine the mechanisms mediating this effect, T cells were stimulated in vitro in the presence of DHEA and a variety of hormone antagonists. The stimulatory effect of DHEA on splenocyte proliferation was unaltered by the testosterone receptor antagonist flutamide, while the oestrogen antagonist tamoxifen completely abrogated its effect. In addition, DHEA administration normalized the elevated serum corticosterone level typically seen following injury. These results indicate, therefore, that DHEA improves splenocyte function after trauma and haemorrhage by directly stimulating T cells and also by preventing a rise in serum corticosterone.

Background.Although hepatocellular dysfunction occurs early after the onset of sepsis, the mechanism responsible for this remains unknown. We tested the hypothesis that the reduction in Kupffer cell (KC) numbers prior to the onset of sepsis prevents the occurrence of hepatocellular dysfunction and reduces levels of the proinflammatory cytokines IL-1β and IL-6 during the early stage of polymicrobial sepsis. Materials and methods.The number of KC in male adult rats was reducedin vivoby intravenous injection of gadolinium chloride 48 h before the induction of sepsis. KC-reduced and KC-normal rats were then subjected to cecal ligation and puncture (CLP, i.e., a model of polymicrobial sepsis) or sham operation followed by administration of normal saline solution. At 5 h after CLP (the early stage of sepsis), hepatocellular function [i.e., the maximal velocity of clearance (Vmax) and efficiency of active transport (Km) of indocyanine green] was measured using a fiber-optic catheter andin vivohemoreflectometer. Whole blood was drawn to measure plasma levels of IL-1β and IL-6 using enzyme-linked immunosorbent assays. Results.Hepatocellular function was depressed and the circulating levels of IL-1β and IL-6 were increased significantly at 5 h after CLP. KC reduction by prior administration of gadolinium chloride, however, prevented the occurrence of hepatocellular dysfunction and the upregulation of IL-1β and IL-6. Conclusions.The KC-derived proinflammatory cytokines IL-1β and IL-6 appear to be directly or indirectly responsible for producing hepatocellular dysfunction during early sepsis. Thus, pharmacologic agents that downregulate the production of one or both of these proinflammatory cytokines in the liver may be useful for maintaining hepatocellular function during the early stage of sepsis.

Background. Studies have indicated that following the induction of sepsis, there is a late (24 h) generalized suppression of the immune response which is associated with increased anti-inflammatory mediator release (e.g., IL-10). However, the mechanisms by which this occurs are unknown. In this regard, recent studies indicate that p38 mitogen-activated protein kinase (p38 MAPK) may play a central role in transducing the signals from immunosuppressive agents which in turn may alter lymphoid cytokine release. The aim of this study, therefore, was to determine whether the anti-inflammatory mediator IL-10 alters splenocyte IL-2 and IFN-γ release, as well as the expression and activation of p38 MAPK in septic animals. Materials and methods. Splenocytes (SPL) (or for some experiments purified T cells) were harvested from mice subjected 24 h earlier to either sepsis by cecal ligation and puncture (CLP) or Sham-CLP and stimulated with 2.5 μg concanavalin A (ConA)/ml in the presence or absence of either monoclonal antibody (Mab) to IL-10 (4 μg/ml) or IgG control. In subsequent studies, sepsis was induced in C57BL/6J and C57BL/6 IL-10 knockout mice, and SPL harvested and stimulated with ConA. SPL cytokine release was measured by ELISA, and the expression and phosphorylation of p38 MAPK were measured by Western analysis. Results. The results indicate that Th1 cytokine (IL-2, IFN-γ) release was depressed by sepsis, while p38 MAPK expression and activity were increased in SPL as well as in T-cells. Neutralization of IL-10 by in vitro use of anti-IL-10 Mab and in the IL-10 knockout animal restored the Th1 response and caused a downregulation of p38 MAPK expression and activity after CLP. Thus, IL-10 appears to contribute to the increase in p38 MAPK activity and expression and the corresponding suppression of Th1 response seen in late sepsis.

Although circulating levels of adrenomedullin (ADM), a newly reported vasodilatory peptide with 52 amino acid residues in the human and 50 amino acid residues in the rat, are elevated during the early and late stages of sepsis, ADM levels in cardiovascular tissues and its precise localization remain to be determined. To study this, rats were subjected to sepsis by cecal ligation and puncture (CLP), followed by administration of 3 ml/100 g b.wt. normal saline to these and sham-operated animals. The heart and thoracic aorta were harvested at 5 h (i.e. the early stage of sepsis) and 20 h (late sepsis) after CLP. Tissue levels of ADM were determined by radioimmunoassay. The localization of ADM in the left ventricle and thoracic aorta was examined by using immunohistochemistry and electron microscopy techniques. The results indicated that ADM levels in the heart and thoracic aorta increased significantly at 5 h after CLP and remained elevated at 20 h after the onset of sepsis. Immunohistochemistry findings showed that ADM immunoreaction products were localized in the cytoplasm of the cardiac myocytes and aortic endothelial cells. Using electron microscopy, ADM immunoreaction products were found in the cytoplasmic matrixes. The immunostainings were also associated with the outer membranes of mitochondria and vesicles of the myocytes as well as vascular endothelial cells. It appears that the cardiovascular tissues, among other organ systems, contribute to the increased levels of plasma ADM under those conditions. Since ADM is localized in different cell populations in the heart and the large blood vessel (i.e. myocytes versus vascular endothelial cells), this peptide may play a differential role in regulating cardiac and vascular functions during sepsis as an autocrine and/or paracrine mediator.

Although hepatocellular dysfunction occurs following trauma and hemorrhagic shock, whether severe hypotension even in the absence of blood loss depresses hepatocellular function remains unknown. The aim of this study, therefore, was to determine whether chemically induced severe hypotension causes hepatocellular dysfunction and, if so, whether IL-6 and PGE 2 are associated with this dysfunction. To study this, hypotension was induced in adult male rats by intravenous infusion of a high dosage of ATP–MgCl 2 solution (3.2 ± 0.45 μmol/min/kg body wt) for 60 min. Blood pressure decreased from 108 ± 6 mm Hg to an average of 43 mm Hg during the infusion period and returned to normal levels immediately after the completion of ATP–MgCl 2 infusion. At 0 and 4 h after hypotension, hepatocellular function [i.e., maximum velocity of indocyanine green clearance ( V max ) and its efficiency ( K m )] was measured using a fiberoptic catheter and in vivo hemoreflectometer. Cardiac output was determined by dye dilution. Microvascular blood flow was assessed by laser Doppler flowmetry. Plasma levels of PGE 2 and IL-6 were measured by radioimmunoassay and bioassay, respectively. The results indicate that severe hypotension in the absence of any blood loss depresses hepatocellular function (i.e., decreased V max and K m ) despite stable cardiac output and hepatic perfusion at 0 and 4 h after the completion of hypotension. Moreover, severe hypotension resulted in significantly increased plasma levels of PGE 2 (only at 0 h) and IL-6. Thus, chemically induced severe hypotension in the absence of any blood loss, which does not significantly reduce cardiac output and hepatic perfusion, depresses hepatocellular function and upregulates IL-6 and PGE 2 production.