Inhibition of cyclooxygenase-2 by NS-398 following hemorrhage and subsequent sepsis: no beneficial effects in either gender

BACKGROUND: Inhibition of cyclooxygenase-2 with a reduction of prostaglandin E(2)production by the specific antagonist NS-398 has been shown to have beneficial effects on immune function and survival in a trauma model. Immune function after experimental hemorrhagic shock and subsequent sepsis may be gender-related, with enhanced immunity and better survival in females. However, it remains unclear if the observed effect of NS-398 treatment is gender-related following hemorrhagic shock and subsequent sepsis. METHODS: Male and female CBA/J mice (age: 2-3 months) were subjected to hemorrhagic shock (35 +/- 5 mm Hg for 90 min and fluid resuscitation) or sham operation. At resuscitation and after 20 and 40 h each received either NS-398 10 mg/kg or placebo i.p. At 48 h after resuscitation, either splenocytes and peritoneal macrophages (pM phi) were harvested (n = 8 per group), or polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Following CLP, either 10-day survival (n = 15 per group) was determined or pM phi and splenocytes were harvested 4 h after CLP (n = 8 per group). Cytokine release of pM phi, and splenocyte proliferation and responsiveness in vitro were assessed. RESULTS: Treatment with NS-398 led to lower PGE(2) levels as compared to placebo-treated animals, reaching significance (p < 0.05) in males. Placebo-treated males had significantly depressed proinflammatory immune response (IL-1, IL-6, IL-2, IFN-gamma) after hemorrhagic shock and experienced further suppression by CLP (all, p < 0.05). In contrast, young females displayed unchanged cytokine release after hemorrhagic shock, but a comparable suppression following CLP. Treatment with NS-398 did not influence cytokine release nor survival. CONCLUSIONS: Despite a significant reduction of PGE(2) concentration, NS-398 treatment has no beneficial effects on cytokine release and survival in this model of hemorrhage and subsequent sepsis.     

What is the role of interleukin 10 in polymicrobial sepsis: anti-inflammatory agent or immunosuppressant?

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 micrograms/mL). To assess the in vivo effects of IL-10 neutralization on survival after CLP, C3H/HeN mice (16 per group) were given 250 micrograms 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.     

Mechanism of immune dysfunction in sepsis: inducible nitric oxide-meditated alterations in p38 MAPK activation

BACKGROUND: After the onset of sepsis, there is a marked dysfunction in cell-mediated immunity that contributes to the morbidity and mortality seen in this condition. Although both nitric oxide (NO) from inducible NO synthase (iNOS) and the activation of p38 mitogen-activated protein kinase (p38 MAPK) appear to contribute to this immune dysfunction, the extent to which NO regulates p38 MAPK activity in sepsis remains unknown. METHODS: To examine this, we induced sepsis by cecal ligation and puncture (CLP) in iNOS knockout (iNOS -/-) or C57BL/6 control mice. Twenty-four hours after CLP or sham operation, splenic T cells and macrophages were isolated and then stimulated with monoclonal antibody against the T-cell marker CD3 (anti-CD3) or lipopolysaccharide. At 4 or 24 hours after stimulation, cytokine release was determined by enzyme-linked immunosorbent assay, and p38 MAPK phosphorylation (activation) was determined by immunoblotting with antibody specific to phosphorylated p38 MAPK. RESULTS: Splenic T-cell p38 MAPK activation and interleukin (IL)-10 release was increased by CLP, whereas Th1 cytokine (IL-2, interferon-gamma) release was depressed. iNOS gene deficiency inhibited p38 MAPK activation in splenic T cells taken from septic mice, and also suppressed IL-10 release in both sham and septic mice. Interestingly, although deficiency of iNOS restored IL-2 release after CLP, both sham and CLP T cells remained depressed in their ability to release interferon-gamma. Septic insult markedly suppressed C57BL/6 splenic macrophage release of proinflammatory agents tumor necrosis factor, IL-12, and IL-1, while augmenting the release of IL-10. However, although deficiency of iNOS concomitantly restored the ability to produce tumor necrosis factor while suppressing the rise in IL-10 release and p38 MAPK activation, it only partially restored IL-1 release and had no effect on IL-12 production seen after CLP. CONCLUSION: These data suggest that NO release from iNOS regulates aspects of sepsis-induced immune dysfunction by the activation of p38 MAPK.     

Simvastatin treatment improves survival in a murine model of burn sepsis: Role of interleukin 6

Infection is the most common and most serious complication of a major burn related to burn size. Recent studies have demonstrated that statin treatment can decrease mortality in murine or human sepsis. In the current study mice were anesthetized and subjected to a dorsal 30% TBSA scald burn. Simvastatin or placebo were administered by intraperitoneal injection once daily or every 12 h. On post burn day 7 cecal ligation and puncture with a 21-gauge needle (CLP) was performed under ketamine/xylazine anesthesia, the two different dosing schedules were continued and survival was monitored. In other groups of mice, interleukin-6 (IL-6) levels in blood were measured in mice at 7 days after injury. A simvastatin dependent improvement in survival was observed in the burn sepsis model. This protection was found to be dose and time dependent. In addition, statin treatment reduced the elevation in IL-6 levels of mice burned 7 days previously. However, IL-6 levels in burned mice with or without statin treatment were elevated by CLP to the same degree. The results of these studies suggest that statin treatment reduces mortality in mice with burns and CLP and that this effect may not be mediated via IL-6 levels.     

Preferable inotropic action of procaterol, a potent bronchodilator, on impaired diaphragmatic contractility in an intraabdominal septic model

Intraabdominal sepsis can lead to acute respiratory failure, and concomitant diaphragmatic dysfunction may be aggravated by sepsis-induced airway hyperreactivity. We previously reported that isoproterenol, a nonselective β-adrenoceptor agonist, increased diaphragmatic contractility and accelerated recovery from fatigue during sepsis. The purpose of this study was to demonstrate the direct inotropic effect of a potent bronchodilator and β₂-selective adrenoceptor agonist, procaterol, on fatigued diaphragmatic contractility in an intraabdominal septic model. Rats were divided into two groups: a cecal ligation and perforation (CLP) group and a sham group. CLP was performed in the CLP group whereas laparotomy alone was performed in the sham group. The left hemidiaphragm was removed at 16 h after the operation. The diaphragmatic tissues were exposed to procaterol (10⁻⁸–10⁻⁶ M), and muscle contractility was assessed. Intracellular cyclic AMP levels were also measured in the CLP model. Procaterol caused an upward shift in the force–frequency curves in the CLP group whereas it had no effect on the curves in the sham group. Procaterol significantly increased cyclic AMP levels in the CLP model. We conclude that the potent bronchodilator procaterol had a direct and positive inotropic effect on the diaphragm in an intraabdominal septic model.     

右美托咪啶对脓毒症大鼠血清炎性因子和氧化应激反应的影响

目的 探讨右美托咪啶对脓毒症大鼠血清炎性因子和氧化应激反应的影响.方法 健康雄性SD大鼠30只,10～ 14周龄,体重250～300 g,采用随机数字表法,将其随机分为3组(n=10):假手术组(S组)、脓毒症组(CLP组)和脓毒症+右美托咪啶组(CLP+D组).CLP组和CLP+D组采用盲肠结扎穿孔术制备脓毒症模型,CLP+D组术毕静脉输注右美托咪啶10 μg·kg-1·h-1至大鼠死亡或术后12 h,S组和CLP组给予等容量生理盐水1ml·kg-1·h-1.每组取5只大鼠,分别于术前(基础状态)、术后1、6、12 h时采集股动脉血样,测定血清IL-6、IL-10、SOD及MDA的水平.记录术后12 h内大鼠生存情况.结果 与S组比较,CLP组术后1、6、12 h时血清IL-6、IL-10和MDA的浓度升高,血清SOD活性和生存率降低(P＜0.05);与CLP组比较,CLP+D组术后6、12 h时血清IL-6和MDA的浓度降低,术后12 h时血清SOD活性和生存率升高(P＜0.05).结论 右美托咪啶可以提高脓毒症大鼠的生存率,其机制与抑制炎性因子释放及氧化应激反应有关.     

Depressed T cell-derived IFN-γ following trauma-hemorrhage: a potential mechanism for diminished APC responses

Introduction Prolonged immunosuppression has been demonstrated after trauma-hemorrhage resulting in an increased susceptibility to sepsis. The contribution of antigen-presenting cells (APC) vs T cells to this diminished immune response, however, remains unknown. Materials and methods To study this, male mice were trauma-hemorrhaged (35 ± 5 mmHg for 90 min and resuscitation) or sham operated. At 24 h thereafter, spleens were harvested and T cells (via Microbeads) and APC (via adherence) were isolated. Cocultures of combined T cells and APC were established for 48 h, stimulated with ConA and LPS. The T cell-derived cytokine IFN-γ and IL-12 for APC responses were measured in the supernatants by the multiplex assay. Results The release of IFN-γ was suppressed by T cells after trauma-hemorrhage irrespective of whether sham or trauma-hemorrhage APC were added. Trauma-hemorrhaged APC did not affect T cells-derived IFN-γ release by sham T cells. In contrast, trauma-hemorrhaged T cells depressed the release of IL-12 by APC. The release of IL-12 by trauma-hemorrhaged APC was not altered when sham T cells were cocultured. Conclusion Prolonged immunosuppression after trauma-hemorrhage appears to be predominantly due to diminished T cell function. Thus, attempts to prevent immunodysfunction should be directed towards T cells. Best abstracts – Surgical Forum 2007 Presented at the 29th Annual Conference on Shock, Broomfield, CO, 3–6 June 2006.Awarded with the travel award of the Shock Society 2006.     

Effects of Adiponectin on Mortality and Its Mechanism in a Sepsis Mouse Model

ABSTRACT

The mortality of sepsis is increasing and conventional therapies for it have no better therapeutic effects. We investigated the effects of adiponectin (APN) on mortality and high mobility group box 1 (HMGB1) in polymicrobial sepsis mouse models. Sepsis models were established by cecal ligation and puncture (CLP) in BALB/c mice. Animals were randomly divided into four groups including control group (C group), model group (CLP group), early APN treatment group (APN + CLP group), and late APN treatment group (CLP + APN group). Mice in each group were killed at 6, 12, 24, and 48 hr after CLP, respectively, to collect samples for determining the levels of serum interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), high mobility group protein-1 (HMGB1), and the expression of lung tissue HMGB1 mRNA. The survival curves in the four groups were drawn. The mortality rates were significantly lower in APN + CLP (30%) and CLP + APN (40%) groups than in CLP group (80%) seven days after CLP. Serum levels of cytokines (IL-6 and TNF-α) and HMGB1 were significantly reduced (p < .05) in APN + CLP and CLP + APN groups compared with those of CLP group. There was a significant correlation between serum HMGB1 and lung HMGB1 mRNA (r = 0.891). The levels of HMGB1 and HMGB1 mRNA were higher in CLP, APN + CLP, and CLP + APN groups than in C group (p < .01), but were lower in APN + CLP and CLP + APN groups than in CLP group (p < .01). APN can reduce the mortality rate and plays an anti-inflammatory role in polymicrobial sepsis mouse models through inhibiting HMGB1.     

Sepsis-induced changes in macrophage co-stimulatory molecule expression: CD86 as a regulator of anti-inflammatory IL-10 response

BACKGROUND: Sepsis remains a substantial risk after surgery or other trauma. Macrophage dysfunction, as a component of immune suppression seen during trauma and sepsis, appears to be one of the contributing factors to morbidity and mortality. However, whereas it is known that the ability of macrophages to present antigen and express major histocompatibility complex MHC class II molecules is decreased during sepsis, it is not known to what extent this is associated with the loss of co-stimulatory receptor expression. Our objectives in this study were, therefore, to determine if the expression of co-stimulatory molecules, such as CD40, CD80, or CD86, on peritoneal/splenic/liver macrophages were altered by sepsis (cecal ligation [CL] and puncture [CLP] or necrotic tissue injury (CL) alone; and to establish the contribution of such changes to the response to septic challenge using mice that are deficient in these receptors. METHODS: To address our first objective, male C3H/HeN mice were subjected to CLP, CL, or sham (n = four to six mice/group), and the adherent macrophages were isolated from the peritoneum, spleen, or liver at 24 h post-insult. The macrophages were then analyzed by flow cytometry for their ex vivo expression of CD40, CD80, CD86, and/or MHC II. RESULTS: The expression of CD86 and MHC II, but not CD40 or CD80, were significantly decreased on peritoneal macrophages after the onset of sepsis or CL alone. In addition, CD40 expression was significantly increased in Kupffer cells after sepsis. Alternatively, splenic macrophages from septic or CL mice did not show changes in the expression of CD80, CD86, or CD40. To the degree that the loss of CD86 expression might contribute to the changes reported in macrophage function in septic mice, we subsequently examined the effects of CLP on CD86 -/- mice. Interestingly, we found that, unlike the background controls, neither the serum IL-10 concentrations nor the IL-10 release capacity of peritoneal macrophages from septic CD86 -/- mice were increased. CONCLUSION: Together, these data suggest a potential role for the co-stimulatory receptor CD86/B7-2 beyond that of simply promoting competent antigen presentation to T-cells, but also as a regulator of the anti-inflammatory IL-10 response. Such a role may implicate the latter response in the development of sepsis-induced immune dysfunction.     

Intraabdominal bacterial infections significantly alter regeneration and function of the liver in a rat model of major hepatectomy

Background No systematic investigations of interactions of postoperative infections and liver regeneration after resection are available. Materials and methods Male Sprague–Dawley rats underwent sham operation, 70% partial hepatectomy (PH), cecal ligation and puncture (CLP), or synchronous PH + CLP and were killed at regular intervals. Liver regeneration and function were measured by the mitotic index, Bromo-deoxy-uridine labeling, and Ki-67 as well as bilirubin, albumin, and indocyanine green plasma disappearance rate. The inflammatory response was evaluated by determination of IL-1β and myeloperoxidase (MPO) activity. Bacterial concentrations in different organs were quantified. Results Simultaneous CLP + PH resulted in a significantly delayed regeneration kinetic, which was most pronounced at 24 h. This was preceded by hyperinflammation with increased liberation of pro-inflammatory cytokines in the PH + CLP group at 6 h. After 48 h, the pro-inflammatory response declined, and regeneration proceeded also in the PH + CLP group. Liver function was found impaired in both groups; however, it was significantly worse in the PH + CLP group. Especially after 48 h, when regeneration peaked in this group, liver function significantly declined. At 96 h, only minor differences were seen, but the persistently elevated proliferative activity indicated the delay of regeneration after PH + CLP. Conclusion The present analysis shows that infectious conditions delay liver regeneration. Our data suggest a cross-linkage of both conditions via the functional liver capacity. A direct role of microorganisms seems unlikely; however, the inhibitory effect of the pro-inflammatory cytokines may be involved. Best abstracts — Surgical Forum 2007.     

Major injury induces increased production of interleukin-10 by cells of the immune system with a negative impact on resistance to infection.

OBJECTIVE: The purpose of this study was to compare the production of interleukin-10 (IL-10) by peripheral blood mononuclear cells (PBMC) from injured patients and control subjects to determine the responsible cell types and to relate IL-10 production to the occurrence of sepsis. A mouse model of burn injury was used to confirm the human findings and to assess the importance of IL-10 in the lowered resistance to infection after injury. SUMMARY BACKGROUND DATA: Severe injury is associated with depressed immune responses. Although IL-10 is known to inhibit several aspects of immune reactivity, the role of IL-10 in postinjury immune suppression remains controversial. METHODS: Peripheral blood mononuclear cells from 14 burn and 12 trauma patients and 16 healthy individuals were studied at serial intervals for IL-10 production stimulated by a T-cell mitogen, phytohemagglutinin, and by bacterial lipopolysaccharide. To determine the source of IL-10, CD4+ and CD8+ lymphocyte subsets were obtained by selective depletion of PBMC with antibody-coated magnetic beads and were stimulated by anti-CD3 antibody to induce IL-10 secretion. In addition, IL-10 production by patients' PBMC in the first 10 days after injury was assessed for correlation with subsequent septic events. Anti-CD3-stimulated IL-10 production also was determined for CD4- and CD8-enriched lymphocyte subsets obtained by antibody and complement depletion of splenocytes harvested from groups of burn and sham burn mice at day 10 after injury, the time of maximal susceptibility to a septic challenge, cecal ligation and puncture (CLP). Finally, to test the importance of IL-10 in immune suppression in vivo, groups of burn and sham burn mice were treated with anti-IL-10 monoclonal antibody or control immunoglobulin G (IgG) on days 1 and 3 postinjury and were observed for survival after CLP on day 10. RESULTS: Patients' PBMC produced significantly more IL-10 than did controls' PBMC 7 to 14 days after injury. Patients' CD4+ (T-helper) but not CD8+ (T-cytotoxic) lymphocytes also showed increased IL-10 production versus those of control subjects early after injury. Increased PBMC IL-10 production in the first 10 days postinjury correlated significantly (p < 0.05) with subsequent septic events. Burn mouse CD4-enriched but not CD8-enriched splenocytes produced more IL-10 than did sham burn splenocyte subsets on day 10 after injury. Burn mice treated with anti-IL-10 antibody but not with control IgG had significantly increased survival after CLP. CONCLUSION: Serious injury in humans and in a mouse burn model is followed by increased stimulated production of IL-10 by cells of the immune system. The CD4+ T-helper cells appear to be a major source of IL-10 after injury. In injured patients, increased IL-10 production is correlated with subsequent septic events, and in the burn mouse, IL-10 appears to induce decreased resistance to infection.     

Interleukin-12 and -18 induce severe liver injury in mice recovered from peritonitis after sublethal endotoxin challenge

BACKGROUND: Postoperative intraabdominal abscess is the major complication after abdominal surgery, and additional infection is often observed and becomes the leading cause of death in septic patients who survive initial resuscitation. Sepsis is initiated and perpetuated by the overzealous systemic production of proinflammatory cytokines-such as tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-12, and IL-18-sometimes resulting in excessive tissue injury and death. The purpose of this study was to assess the correlation between liver and spleen innate cytokine responses and organ dysfunction in sepsis syndrome. METHODS: Peritonitis was induced by cecal ligation and puncture (CLP). All CLP mice survived more than 7 days after the procedure, and serum cytokine (TNF-alpha, IL-12, IL-18, and IL-10) levels peaked 12 hours after CLP; thereafter, they returned to basal levels 7 days after CLP. The mice were injected with a sublethal dose of lipopolysaccharide (LPS) 7 days after CLP. Survival rates, tissue damage, serum cytokine levels, and cytokine production of liver or spleen mononuclear cells (MNCs) were evaluated. RESULTS: All CLP mice died within 6 hours from liver injury 7 days after LPS challenge, but all sham mice survived. IL-12, IL-18, and IFN-gamma levels in supernatants of the liver MNCs stimulated with LPS in CLP mice were significantly higher than those in sham mice 7 days after the procedure. Furthermore, serum IL-12 and IL-18 levels and liver MNCs IL-12, IL-18, and IFN-gamma production were significantly increased in CLP mice compared with sham mice after LPS challenge. Thereafter, effects of anti-IL-12 and/or anti-IL-18 antibody were evaluated in LPS-injected CLP mice. The survival rate of LPS-injected CLP mice treated with both anti-IL-12 and anti-IL-18 antibody was significantly better than that of untreated mice. Furthermore, liver damage was improved. CONCLUSION: Mice recovered from mild peritonitis died of severe liver injury by subsequent injection of a sublethal dose of LPS, and this liver injury was related to the collaborating production of IL-12 and IL-18 by liver MNCs.     

Prolactin modulates survival and cellular immune functions in septic mice

BACKGROUND: The immunomodulatory properties of the pituitary hormone prolactin have been demonstrated. It was proposed that prolactin is important in maintaining normal immune response in several pathological states. We investigated the effect of prolactin administration on the survival and cellular immune functions during systemic inflammation. MATERIALS AND METHODS: Male NMRI mice were subjected to laparotomy (LAP) or sepsis induced by cecal ligation and puncture (CLP). Mice were treated with either saline (LAP/saline; CLP/saline) or prolactin (LAP/PRL, CLP/RPL; 4 mg/kg s.c.). Survival of septic mice was determined 24 and 48 h after CLP. Forty-eight hours after the septic challenge, the proliferative capacity, cytokine release (IL-2, IL-6, IFN-gamma) and apoptosis of splenocytes were determined. Additionally, monitoring of circulating leukocyte distribution was performed (WBC; CD3+, CD4+, CD8+, B220+, NK1.1+, F4/80+ cells by FASCan). RESULTS: CLP was accompanied by a mortality of 47% and induced a decrease in splenocyte proliferation and apoptosis rate. Administration of prolactin significantly increased the mortality of septic mice (81%). This was paralleled by a further decrease of splenocyte proliferation and an increased splenocyte apoptosis. In addition, administration of prolactin augmented the sepsis-induced inhibition of IL-2 release, attenuated the sepsis-induced inhibition of IFN-gamma release, and did not affect the release of IL-6. However, prolactin did not affect the sepsis-induced changes of circulating leukocyte subpopulations. CONCLUSIONS: We conclude that prolactin has profound immunomodulatory properties and that administration of prolactin in pharmacological doses is associated with a decreased survival and an inhibition of cellular immune functions in septic mice.     

Splenic immune suppression in sepsis: A role for IL-10-induced changes in P38 MAPK signaling

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-gamma 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 microg concanavalin A (ConA)/ml in the presence or absence of either monoclonal antibody (Mab) to IL-10 (4 microg/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-gamma) 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.     

Genetic background determines susceptibility during murine septic peritonitis.

BACKGROUND: The tolerance of mouse strains to cecal ligation and puncture (CLP), a clinically relevant model of sepsis, can vary greatly. We compared the immune response and bacterial eradication during CLP in two mouse strains with different susceptibilities to the lethal effects in an effort to understand alterations in tolerance. MATERIALS AND METHODS: CLP of increasing severity was performed on Swiss Webster mice. Interleukin (IL)-12 levels, bacterial counts, and myeloperoxidase were determined. We then compared the same parameters in Swiss Webster and in BALB/c mice and determined survival for both mouse strains after CLP. RESULTS: Bacterial counts locally and systemically as well as serum IL-12 correlated with the severity of CLP in Swiss Webster mice. Lung myeloperoxidase increased with increasing severity CLP, while peritoneal myeloperoxidase decreased. Following CLP, one-half of the Swiss Webster mice survived versus none of the BALB/c mice. Despite worsened survival, BALB/c mice had lower bacterial counts and similar IL-12 levels compared to Swiss Webster mice. Myeloperoxidase and IL-6 levels were similar between experimental groups. CONCLUSIONS: Swiss Webster and BALB/c mice have significantly different susceptibilities to the lethal effects of CLP, and this difference may be related to IL-12 responsiveness.     

An exaggerated inflammatory response after CLP correlates with a negative outcome

BACKGROUND: Sepsis is the leading cause of morbidity and mortality in the surgical intensive care unit. We postulate that the variable clinical profile of septic patients is the product of multiple factors, including initiating insult, environment, and genetic make-up. This hypothesis was tested by changing the severity of the insult and the genetic background in an experimental murine model of sepsis. MATERIALS AND METHODS: Eight-week-old, male A/J and C57BL/6J (B6) mice underwent cecal ligation and puncture (CLP). The cecum was ligated just below the ileocecal valve (>1-cm ligation) or 1 cm from the end of the cecum (1-cm ligation) and single punctured using a 16- or 25-gauge needle (CLP16 or CLP25) or double punctured with a 25-gauge needle (CLP25 x 2). Cytokines were measured in plasma samples by ELISA at different time points after CLP. RESULTS: Elevated TNF-alpha and IL-6 plasma levels were observed in A/J as compared to B6 mice at 10 and 20 h after CLP16 (1-cm ligation). In contrast, IL-10 levels were decreased in A/J versus B6 mice at 6 h but increased at 10 h. After CLP25 and CLP25 x 2 (1-cm ligation), TNF-alpha was significantly increased at 10 h, but there was no difference in IL-10 and IL-6. CLP with >1-cm ligation resulted in increased cytokine expression after CLP25, CLP25 x 2, and CLP16 versus 1-cm ligation. Mortality after CLP16 was significantly higher in B6 mice with >1-cm versus 1-cm ligation. A/J mortality did not differ between the two procedures. CONCLUSION: Mortality rate and cytokine profiles after CLP vary depending on the insult severity and the genetic make-up.     

δ阿片受体激动剂对脓毒症大鼠细胞免疫功能的影响

目的 探讨δ阿片受体激动剂对脓毒症大鼠细胞免疫功能的影响.方法 健康成年雄性SD大鼠150只,体重154～198g,随机分为3组(n=50):假手术组(S组)仅穿线,不进行盲肠结扎穿孔;脓毒症组(SEP组)采用盲肠结扎穿孔的方法制备脓毒症模型;δ阿片受体激动剂组(DADLE组)于盲肠结扎穿孔后立即腹腔注射δ阿片受体激动剂DALDE 10 ml/kg,浓度为0.5 mg/ml.于盲肠结扎穿孔后4、8、12 h(T1～3)时各组随机取10只大鼠,抽取下腔静脉血样,采用ELISA法检测血清TNF-α和IL-10的浓度,采用流式细胞仪检测T淋巴细胞亚群变化.记录盲肠结扎穿孔后7 d内大鼠的生存情况.结果 与S组比较,SEP组和DADLE组各时点血清TNF-α和IL-10的浓度、TNF-α/lL-10比值升高,T3时CD4+T细胞水平和CD4+/CD8+降低,CD8+T细胞水平升高(P＜0.05或0.01);与SEP组比较,DADLE组T2.3时血清TNF-α和IL-10的浓度、TNF-α/IL-10比值降低,T3时CD4+T细胞水平和CD4+/CD8+升高,CD8+T细胞水平降低,盲肠结扎穿孔后7 d内大鼠生存率升高(P＜0.05).结论 δ阿片受体激动剂可改善细胞免疫功能,从而减轻炎性反应,有利于脓毒症大鼠的预后.     

瑞芬太尼对腹腔感染脓毒症小鼠肝肺组织诱导型一氧化氮合酶的影响

目的 观察瑞芬太尼对腹腔感染脓毒症小鼠肺组织和肝组织诱导型一氧化氮合酶(iNOS)的影响.方法 采用小鼠盲肠结扎穿孔术制备脓毒症模型,将40只雄性昆明小鼠随机分为假手术组(Sham组)、脓毒症组(CLP组)、瑞芬太尼治疗组(R1组)、瑞芬太尼对照组(R2组).Western blot方法检测肺组织和肝组织iNOS蛋白表达,双抗体夹心酶联免疫吸附法测定肝肺组织白细胞介素(IL)-10和IL-6水平,观察PaO_2、肺组织髓过氧化物酶(MPO)活性、血清ALT和AST活性及电镜下组织超微结构改变.结果 与Sham组比较,CLP组PaO_2显著降低,肺组织和肝组织中iNOS蛋白表达、肺组织MPO活性、ALT和AST活性显著增强(P<0.01),肺组织IL-6和IL-10水平显著增加为649.74±90.47和501.06±57.67(P<0.01),肝组织IL-6和IL-10水平显著增加为341.05±28.73和267.50±41.82,R2组以上指标均无明显变化;与CLP组比较,R1组PaO_2明显增加,iNOS蛋白表达,IL-6和IL-10水平、MPO活性、ALT和AST活性显著降低(P<0.01),电镜显示肺组织和肝组织损伤程度较CLP组略减轻.结论 瑞芬太尼对脓毒症感染致急性肺损伤和肝损伤有保护作用,其机制可能通过抑制iNOS蛋白的表达,降低炎性因子水平有关.