Influence of cyclooxygenase inhibitors on gut immune cell distribution and apoptosis rate in experimental sepsis

The aim of this study was to determine if cyclooxygenase (COX) inhibitors influence immune cell distribution in the small intestinal mucosa and mesenteric lymph nodes (MLNs), the grade of mucosal damage, and the rate of apoptosis in septic rats. The effects induced by a selective COX-2 inhibitor (SC-236) were compared with those of a nonselective COX-1 and -2 inhibitor (indomethacin). Cecal ligation and puncture (CLP), CLP + SC-236 p.o, and CLP + indomethacin p.o, were evaluated. Animals were harvested 6 and 24 h after CLP, respectively. The concentration of proinflammatory cytokines was higher in ascitic fluid than in blood. CLP + SC-236 attenuated IL-6 in plasma and in ascitic fluid and CLP + indomethacin augmented TNF-alpha in ascitic fluid compared with CLP at 6 h. CLP + SC-236 gave a lesser degree of mucosal damage compared with CLP alone or with indomethacin at 6 and 24 h (P < 0.05). Untreated CLP had significant reductions in the number of T lymphocytes in the villi and increases of macrophages in the mucosa and MLNs compared with controls (P < 0.05). CLP + indomethacin decreased T lymphocytes in the villi and MLNs. CLP caused an enhanced apoptosis in the mucosa compared with controls (P < 0.05), pretreatment with COX inhibitors did not significantly change this. Both COX inhibitors enhanced apoptosis in MLNs and attenuated the increase of macrophages in mucosa and MLNs (P < 0.05). It is proposed that the increased apoptosis and the decrease in T cells in the mucosa may be causally related. Apoptosis of lymphocytes may impair the immunologic defense in sepsis. Furthermore, loss of intestinal epithelial cells may compromise bowel wall integrity and facilitate translocation.     

脓毒症大鼠肠黏膜免疫屏障功能的变化

目的 研究脓毒症对大鼠肠黏膜免疫屏障功能的影响.方法 60只SD大鼠随机(随机数字法)分为对照组(n=15)和脓毒症组(n=45),采用盲肠结扎穿孔术(CLP)建立脓毒症模型.模型建立后3 h、6 h和12 h留取回肠黏膜和全血标本.分别进行肠黏膜形态学观察、肠防御素5(RD-5)及肠三叶因子3(TFF_3)Mrna表达水平检测、肠黏膜淋巴细胞凋亡分析,以及外周血中肠源性细菌DNA定性检测.结果 CLP所致脓毒症导致大鼠回肠黏膜明显损害,主要表现为上皮脱落、固有层分离、毛细血管出血和溃疡形成;脓毒症组模型建立后3 h即出现RD-5和TFF_3 Mrna表达显著性减少(与正常组比较,P<0.05),且6 h和12 h组进行下降(与3 h组比较,P<0.05),肠黏膜淋巴细胞凋亡数亦显著增加(P<0.05);同时,脓毒症组全血肠源性细菌DNA扩增全部阳性.结论 脓毒症时大鼠肠黏膜免疫屏障功能显著减退,且随脓毒症的发展而进行性恶化.     

CD4-expressing cells are early mediators of the innate immune system during sepsis

It is well established that the immune response to sepsis is mediated by leukocytes associated with the innate immune system. However, there is an emerging view that T lymphocytes can also mediate this response. Here, we observed a significant depletion of both CD4 and CD8 T cells in human patients after blunt trauma. To determine what effect the loss of these cells may have during a subsequent infection, we obtained CD4- and CD8-deficient mice and subjected them to cecal ligation and puncture (CLP). We observed that CD4 knockout (KO) mice showed increased CLP-induced mortality compared with CD8-deficient and wild-type (WT) mice especially within the first 30 h of injury. CD4 KO mice also exhibited significantly increased IL-6 concentrations after the CLP. The CD4 KO mice had an increased concentration of bacteremia as compared with WT mice. Antibiotic treatment decreased mortality in the CD4 KO mice as compared with no changes in the wild mice after CLP. Neutrophils isolated from septic CD4 KO mice showed decreased spontaneous oxidative burst compared with neutrophils taken from septic controls. We examined the role of IFN-gamma by using mice deficient in this cytokine and found these mice to have significantly higher mortality as compared with WT mice. Finally, we detected a 2-fold increase in CD11b+ cells that exhibited intracellular IFN-gamma staining in the peritoneum of WT mice after CLP. The data suggest that CD4+ cells may facilitate the early clearance of bacteria by regulating neutrophils function possibly through an IFN-gamma-dependent mechanism.     

儿茶酚胺类药物对脓毒性休克大鼠心肌的影响

目的 :利用生化指标和形态学方法研究儿茶酚胺类药物对脓毒性休克大鼠心脏的影响与机制。方法 :用盲肠结扎穿孔术 (CL P)制成大鼠的脓毒性休克模型。采用多巴酚丁胺 (DB)、去甲肾上腺素 (NE)及两者最小剂量联合应用以纠正血压。动物随机分为假手术对照组、CL P对照组、CL P+DB组、CL P+NE组、CL P+DB+NE组 ,每组各 8只大鼠。心肌损害程度应用血清心肌钙蛋白 I(c Tn I)和肌酸激酶 (CK)浓度来表达 ,用光镜与电镜检查心肌组织形态学变化。结果 :脓毒性休克大鼠 c Tn I升高 (P<0 .0 5 ) ,儿茶酚胺类药物浓度不影响 c Tn I的水平 ;但 CK总量水平在儿茶酚胺治疗组升高 (P均 <0 .0 5 ) ;心肌组织形态学检查与 c Tn I的结果相符合。结论 :脓毒性休克心肌损害由缺血引起 ,没有明确的证据显示儿茶酚胺类药物能加重心肌损害。     

Protective effects of rhizoma paridis total saponins on septic rats

OBJECTIVE: To investigate the protective effect of rhizoma paridis total saponins and its mechanism on septic rats. METHODS: Septic model was reproduced by cecal ligation and puncture (CLP) in Wistar rats. Rhizoma paridis total saponins was administered to observe its protective effects on septic rats. Blood was collected to determine serum tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta(IL-1beta)levels at 2, 6, 12, 24 and 48 hours after operation by means of enzyme-linked immunosorbent assay (ELISA). The pathological changes of lung tissue were observed with light microscope at 72 hours after operation. The peritoneal macrophages (PMZ) in rats were isolated and the release of TNF-alpha and IL-1beta in PMZ after exposure to lipopolysaccharide (LPS, 100 mug/L) were measured by ELISA. RESULTS: Mortality in the rhizoma paridis total saponins group was significantly lower than the CLP group (50.0% vs. 85.0%, P<0.05). The levels of TNF-alphaand IL-1beta in serum were significantly lower than those of the CLP group at the same time (P<0.05 or P<0.01). The degree of inflammatory injury to the lung was much milder than that in the CLP group. In the in vitro experiment, it was shown that rhizoma paridis total saponins in concentrations of 5, 10, 20 and 40 mg/L could inhibit remarkably the release of TNF-alpha and IL-1beta from LPS-stimulated PMZ of rats (all P<0.01). The differences in TNF-alpha levels among the groups showed no statistically significant difference(all P>0.05). The level of IL-1beta in 5 mg/L group was significantly higher than that of the 10 mg/L group (P<0.05), but showed no difference with those of 20 mg/L and 40 mg/L groups (both P>0.05). CONCLUSION: Rhizoma paridis total saponins can protect the CLP rats by inhibiting the activation of rat PMPhi to release cytokines and ameliorating acute lung injury.     

Effect of L-Arginine on intestinal mucosal injury of rats with severe abdominal infection]

OBJECTIVE: To investigate the effect of L-Arginine on intestinal mucosal injury of rats with severe abdominal infection. METHODS: Rats received cecal ligation and puncture (CLP) to reproduce sepsis model. A total of 18 Wistar rats were divided into two groups randomly (each n=9): L-Arginine group and model group. Three hundred mg/kg of L-Arginine was injected into the abdomen in rats of L- Arginine group after CLP. Model group received equal volume of normal saline. Blood sample was harvested and the serum levels of nitric oxide (NO) and inducible nitric oxide synthase (iNOS) were determined at 24 hours after operation in both groups. The histopathological change of intestinal mucosa was observed under light microscope and mucosa damage index was determined. RESULTS: The intestinal mucosal damage was observed both in model group and L- Arginine group after CLP, but the injury was milder in L- Arginine group. There was significant difference in mucosa injury index between L-Arginine group and model group (3.4+/-0.6 vs. 4.1+/-0.5, P<0.05). The serum level of NO [(76.1+/-26.2) micromol/L vs. (87.3+/-16.7) micromol/L, P>0.05] and iNOS [(30.6+/-7.4) U/L vs(44.4+/-6.6) U/L, P<0.01] in L-Arginine group were lower than those in model group. CONCLUSION: L-Arginine could protect against intestinal mucosal injury and depress the serum level of iNOS in severe abdominal infection of rats.     

MHC class II expression restricted to CD8alpha+ and CD11b+ dendritic cells is sufficient for control of Leishmania major

Control of the intracellular protozoan, Leishmania major, requires major histocompatibility complex class II (MHC II)-dependent antigen presentation and CD4+ T cell T helper cell 1 (Th1) differentiation. MHC II-positive macrophages are a primary target of infection and a crucial effector cell controlling parasite growth, yet their function as antigen-presenting cells remains controversial. Similarly, infected Langerhans cells (LCs) can prime interferon (IFN)gamma-producing Th1 CD4+ T cells, but whether they are required for Th1 responses is unknown. We explored the antigen-presenting cell requirement during primary L. major infection using a mouse model in which MHC II, I-Abeta(b), expression is restricted to CD11b+ and CD8alpha+ dendritic cells (DCs). Importantly, B cells, macrophages, and LCs are all MHC II-negative in these mice. We demonstrate that antigen presentation by these DC subsets is sufficient to control a subcutaneous L. major infection. CD4+ T cells undergo complete Th1 differentiation with parasite-specific secretion of IFNgamma. Macrophages produce inducible nitric oxide synthase, accumulate at infected sites, and control parasite numbers in the absence of MHC II expression. Therefore, CD11b+ and CD8alpha+ DCs are not only key initiators of the primary response but also provide all the necessary cognate interactions for CD4+ T cell Th1 effectors to control this protozoan infection.     

The protective effect of N-acetylcysteine on apoptotic lung injury in cecal ligation and puncture-induced sepsis model

Apoptotic loss of parenchymal cells may lead to organ dysfunctions in critically ill patients with septic states. As an antioxidant, the protective effects of N-acetylcysteine (NAC) are documented in many experimental and clinical studies. In this experimental study, we investigated the role of chronically used NAC in septic lung injury on a cecal ligation and puncture (CLP) model. To evaluate this, 30 male Wistar rats were randomly divided into four groups as sham (n = 7), CLP (n = 8), sham + NAC (n = 7) and CLP + NAC (n = 8) groups. NAC was administered 150 mg kg(-1) day through intramuscular route beginning 6 h after the operations and lasting for a period of 1 week. One week later, histopathology and epithelial apoptosis were assessed by hematoxylin-eosin and immunohistochemically by M30 and caspase 3 staining to demonstrate septic lung injury. Additionally, lung tissue myeloperoxidase (MPO) activity, malondialdehyde (MDA), and nitrite/nitrate levels were measured. The MPO activity and MDA levels in lung homogenates were found to be increased in CLP group and the administration of NAC prevented their increase significantly (P < 0.05). However, there were no significant differences among the groups regarding nitrite/nitrate levels. The number of apoptotic cells was significantly lower in CLP+NAC group than CLP group, and this finding was supported by M30 and caspase 3 expression in lung (P < 0.05). Lung histopathology was also protected by NAC in CLP-induced sepsis. In conclusion, the chronic use of NAC inhibited MPO activity and lipid peroxidation, which resulted in reduction of apoptosis in lung in this CLP model. Because lung tissue nitrite/nitrate levels did not change significantly, organs other than the lungs may be responsible for producing the increased nitric oxide during sepsis. The chronic use of NAC needs further investigation for its possible antiapoptotic potential in septic states besides its documented antioxidant and antiinflammatory effects.     

基于STAT3信号通路探讨艾司洛尔对脓毒症大鼠急性肝损伤的保护作用

目的探讨艾司洛尔(ES)对脓毒症大鼠急性肝损伤的保护作用及相关信号通路。方法48只雄性SPF级大鼠随机分为假手术(Sham)组、盲肠结扎穿孔(CLP+NS)组和艾司洛尔干预(CLP+ES)组(每组16只)。Sham组采用盲肠探查术,CLP+NS组、CLP+ES组采用CLP法建立脓毒症大鼠模型。CLP+ES组经颈内静脉微量泵入ES稀释液6 h,Sham组和CLP+NS组给予等质量生理盐水。术后6 h、24 h各组分别处死8只大鼠。采用HE染色,观察脓毒症大鼠肝组织形态学变化,生化分析仪检测血清肝功能指标,酶联免疫吸附法(ELISA)检测肝组织中炎性细胞因子水平,Western blot检测肝组织中STAT3信号通路标志蛋白的表达。结果CLP+NS组脓毒症大鼠肝组织炎性细胞浸润明显,而CLP+ES组炎性细胞减少,肝细胞坏死程度好转。术后6 h、24 h,CLP+NS组血清天冬氨酸转氨酶(AST)、丙氨酸转氨酶(ALT)和肝组织匀浆中高迁移率族蛋白B-1(HMGB-1)、白细胞介素-6(IL-6)均升高(P<0.05);而CLP+ES组较CLP+NS组均降低(P<0.05)。术后6 h,与CLP+NS组比较,CLP+ES组脓毒症大鼠肝组织中磷酸化信号转导和转录激活因子3(p-STAT3)表达水平明显下降(P<0.05),细胞因子信号转导抑制因子3(SOCS3)表达明显上升(P<0.05)。术后24 h,CLP+ES组上述蛋白表达与CLP+NS组比较差异无统计学意义(P>0.05)。结论艾司洛尔通过抑制STAT3信号通路,抑制炎性细胞因子释放,从而发挥对脓毒症大鼠急性肝损伤的保护作用。     

脓毒症大鼠脾脏淋巴细胞凋亡的实验研究

目的 观察脓毒症大鼠脾脏T、B淋巴细胞的数量变化及细胞凋亡情况,探讨脓毒症时免疫失衡的机制.方法 健康雄性Wistar大鼠80只,按随机数字表法分为假手术组(30只)、模型组(50只).采用盲肠结扎穿孔术(CLP)制备脓毒症大鼠模型,于制模后6、12、24、48、96 h活杀动物取脾脏,行苏木素-伊红(HE)染色,光镜下观察脾脏组织病理变化;采用免疫组化法检测脾脏CD4~+、CD8~+T淋巴细胞和B淋巴细胞以及Bax、Bcl-2蛋白表达;采用原位末端缺刻标记法(TUNEL)检测脾脏细胞凋亡指数.结果 光镜下观察模型大鼠脾脏白髓逐渐萎缩,淋巴小结结构破坏.制模6、12、24、48、96 h,模型组大鼠脾脏CD4~+T淋巴细胞数、B淋巴细胞数、Bcl-2蛋白表达均较假手术组明显降低(P均<0.01),CD8~+T淋巴细胞数与假手术组比较差异无统计学意义(P均>0.05),细胞凋亡指数及Bax蛋白表达均较假手术组显著增加(P均<0.01).相关分析表明:Bcl-2蛋白表达与细胞凋亡指数呈负相关(r=0.659,P<0.01),而Bax蛋白表达与细胞凋亡指数呈正相关(r=0.522,P<0.01).结论 脓毒症早期免疫功能处于紊乱状态,表现为脾脏CD4~+T淋巴细胞数、B淋巴细胞数减少,细胞凋亡显著增加;Bax、Bcl-2在脓毒症脾细胞凋亡中发挥了关键作用.     

The anti-inflammatory effect of curcumin in an experimental model of sepsis is mediated by up-regulation of peroxisome proliferator-activated receptor-gamma

OBJECTIVE: Although phytochemical curcumin has been shown to possess anti-inflammatory properties, it remains unknown whether this agent has any beneficial effects in sepsis. The purpose of this study was to demonstrate whether curcumin protects septic animals and, if so, whether activation of peroxisome proliferator-activated receptor (PPAR)-gamma, an anti-inflammatory nuclear receptor, plays any role. DESIGN: Prospective, controlled, and randomized animal study. SETTING: A research institute laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: A bolus injection of 0.2 micromol of curcumin was given intravenously to male adult rats, followed by continuous infusion of curcumin (0.24 micromol/day) for 3 days via a primed 2-mL mini-pump. The rats were then subjected to sepsis by cecal ligation and puncture (CLP). MEASUREMENTS AND MAIN RESULTS: Serum levels of liver enzymes (alanine aminotransferase and aspartate aminotransferase), lactate, albumin, and tumor necrosis factor (TNF)-alpha were measured at 20 hrs after CLP (i.e., late stage of sepsis). In addition, a 10-day survival curve was conducted following CLP and cecal excision with or without curcumin treatment. Furthermore, macrophages cell line RAW 264.7 cells were treated with curcumin followed by stimulation with endotoxin. TNF-alpha and PPAR-gamma expression were then measured. The results indicate that intravenous administration of curcumin before the onset of sepsis attenuated tissue injury, reduced mortality, and decreased the expression of TNF-alpha in septic animals. Similar results were also found when curcumin was administered after the onset of sepsis. Moreover, the down-regulated PPAR-gamma in the liver at 20 hrs after CLP was significantly improved by curcumin treatment. Concurrent administration of curcumin and GW9662, a specific PPAR-gamma antagonist, completely abolished the beneficial effects of curcumin under such conditions. In cultured RAW 264.7 cells, curcumin inhibited endotoxin-induced increases in TNF-alpha expression and markedly up-regulated PPAR-gamma expression without affecting cell viability. Curcumin also prevented morphologic alterations in macrophages induced by endotoxin. CONCLUSIONS: The protective effect of curcumin makes it or its analogues strong candidates as a novel therapy for sepsis. The beneficial effect of curcumin appears to be mediated by up-regulation of nuclear receptor PPAR-gamma.     

丙酮酸乙酯对脓毒症大鼠肠黏膜屏障功能保护作用的研究

目的 观察丙酮酸乙酯(ethyl pyruvate ,EP)干预治疗对脓毒症大鼠生存率和肠黏膜屏障的影响.方法 ①EP对脓毒症大鼠生存率的影响:无特定病原雄性SD大鼠100只随机分为假手术组(A组)、脓毒症组(B组)、EP早期治疗组(C组)及EP延迟治疗组(D组),每组25只,利用盲肠结扎穿孔法(cecal ligation and puncture,CLP)制作大鼠脓毒症模型,各组均于术后6、12、18、24、36、48、60、72 h腹腔内注射给药3 mL,C、D组分别于术后6、12 h开始予EP(40 mg/kg),A、B两组同法予等量林格乳酸钠溶液(ringer lactate solution ,RLS),每隔12 h记录死亡情况,分析比较5 d生存率;②EP对脓毒症大鼠肠黏膜屏障的影响:80只无特定病原雄性SD大鼠随机分为四组,每组20只,分组及给药方法与方法一相同,术后24、48 h各处死10只.测定各时间点血浆D-乳酸、DAO的变化,同时用透射电镜观察术后48 h肠黏膜上皮细胞超微结构的变化.采用Kaplan- Meier生存分析法进行生存分析,多组均数间比较采用单因素方差分析的方法, 多组均数间两两比较采用SNK-q检验,P＜0.05为差异有统计学意义.结果 A、B、C、D四组大鼠5 d生存率分别为100%、24%、68%、56%,与B组相比,C、D组大鼠5 d生存率明显提高(P<0.05),C、D组间差异无统计学意义(P＞0.05);与B组相比,C、D组术后24 h和48 h血浆D-乳酸含量明显下降(P<0.01);与B组相比,C组、D术后24 h和48 h血浆DAO活性明显下降(P<0.01),C、D组术后24、48 h血浆D-乳酸含量、DAO活性差异无统计学意义(P＞0.05);电镜下C、D组肠黏膜上皮细胞损伤较B组明显减轻,细胞间紧密连接较清楚.结论 脓毒症时肠黏膜损伤严重,EP早期与延迟干预治疗能有效保护肠黏膜屏障,提高5 d生存率,具有抗脓毒症作用 .     

Neuraminidase-treated macrophages stimulate allogenic CD8+ T cells in the presence of exogenous interleukin 2

Prior work has shown that purified, resident, and inflammatory peritoneal macrophages are weak stimulators of the allogeneic MLR. We have identified conditions whereby thioglycollate-elicited macrophages become stimulatory, but primarily for the CD8+ T cell subset. The conditions were to treat the macrophages with neuraminidase and to supplement the MLR with rIL-2. These treatments together led to proliferative and cytotoxic responses by isolated CD8+ but not CD4+ T cells. Likewise when MHC-congenic strains were evaluated, an MLR was observed across isolated class I but not class II MHC barriers. Pretreatment of the macrophages with IFN-gamma further enhanced expression of class I MHC products and stimulatory activity, but did not seem essential. While these treatments did not render macrophages stimulatory for an MLR in purified CD4+ cells, blastogenesis of CD4+ cells was observed when the MLR involved bulk T cells. Small allogeneic B lymphocytes behaved similarly to macrophages, in the pretreatment with neuraminidase and supplementation with rIL-2 rendered B cells stimulatory for allogeneic, enriched, CD8+, but not CD4+, T cells. Spleen adherent cells, which are mixtures of macrophages and dendritic cells, stimulated both CD4+ and CD8+ T cells, and neither neuraminidase nor exogenous IL-2 was required. We think that these data suggest that most macrophages and small B cells lack three important functions of dendritic cells: a T cell-binding function that can be remedied by neuraminidase treatment, a T cell growth factor-inducing function that can be bypassed with exogenous IL-2, and an IL-2 responsiveness function that is required by CD4+ lymphocytes.     

Beta-glucan attenuates inflammatory cytokine release and prevents acute lung injury in an experimental model of sepsis

Sepsis is one of the most important risk factors in acute respiratory distress syndrome (ARDS). beta-Glucan is a potent reticuloendothelial modulating agent, the immunobiological activity of which is mediated in part by an increase in the number and function of macrophages. In this study, we investigated the putative protective role of beta-glucan against sepsis-induced lung injury. Sepsis was induced by cecal ligation and puncture (CLP) in Wistar rats. The control group received saline, and the treatment groups received beta-glucan or beta-glucan + beta-1,3-D-glucanase. Five hours thereafter, plasma tumor necrosis factor (TNF) alpha, interleukin (IL) 1beta, and IL-6 levels were determined. Presence of lung injury was determined via lung tissue myeloperoxidase (MPO) activity, intercellular adhesion molecule (ICAM) 1 levels, and histopathological examination at 18 h after CLP. In a separate set of experiments, survival was monitored for 7 days after CLP. beta-Glucan treatment led to a significant increase in survival rate (63% in glucan-treated rats vs 38% in saline-treated rats). Administration of the beta-glucan inhibitor abrogated beta-glucan's survival benefit (50%). After CLP, plasma TNF-alpha, IL-1beta, and IL-6 concentrations were increased in control animals. When beta-glucan was administered, it completely blocked the elevation of TNF-alpha, IL-1beta, and IL-6. Administration of beta-1,3-D-glucanase suppressed glucan-induced decrease in cytokines. Animals treated with beta-glucan showed a significant reduction in lung injury score, a marked decrease in ICAM-1 expression, and a significant decrease in MPO levels. In contrast, beta-1,3-D-glucanase caused a significantly increased MPO and ICAM-1 levels in the lung. These data reveal that beta-glucan treatment improved the course of CLP-induced peritonitis and attenuated the lung injury. Administration of beta-glucanase inhibited the beta-glucan activity and resulted in enhanced lung injury.     

Interferon-gamma and tumor necrosis factor-alpha specifically induce formation of cytomegalovirus-permissive monocyte-derived macrophages that are refractory to the antiviral activity of these cytokines.

Monocytes/macrophages are key cells in the pathogenesis of human cytomegalovirus (HCMV). Although HCMV infection in monocytes is restricted to early events of gene expression, productive infection has been demonstrated in differentiated macrophages in vitro. We examined the cellular and cytokine components that are essential for HCMV replication in Concanavalin A-stimulated monocyte-derived macrophages (MDM). By negative selection, depletion of CD8+ T lymphocytes, but not CD4+ T lymphocytes, CD19+ B cells, or CD56+ NK cells, resulted in a 60-70% reduction in the number of HCMV-infected MDM, and a 4 log decrease in virus production. Neutralization of IFN-gamma and TNF-alpha, but not IL-1, IL-2, or TGF-beta, decreased production of virus by 4 logs and 2 logs, respectively. Subsequently, addition of recombinant IFN-gamma or TNF-alpha to purified monocyte cultures was sufficient to produce HCMV-permissive MDM. While IFN-gamma and TNF-alpha possess antiviral properties, addition of these cytokines to permissive MDM cultures did not affect production of HCMV. Thus, rather than inhibiting replication of HCMV, IFN-gamma and TNF-alpha specifically induce differentiation of monocytes into HCMV-permissive MDM, which are resistant to the antiviral effects of these cytokines.     

Characterization of the tissue macrophage and the interstitial dendritic cell as distinct leukocytes normally resident in the connective tissue of rat heart

Immunohistological studies with a mouse anti-rat macrophage mAb (BMAC-5) demonstrated the presence of numerous positive cells in the interstitial connective tissues of many organs. The pattern resembled that seen with anti-MHC class II antibodies, with the striking exception that BMAC-5+ cells were rare or absent in the portal triad, the islets of Langerhans, and the kidney. Double-labeling fluorescence studies were therefore performed in rat heart using the BMAC-5 mAb in combination with rabbit antisera to pure rat class II MHC antigens and pure rat leukocyte common (CD45) antigens. The tissue macrophages in heart were identified as BMAC-5+, MHC class II-negative, leukocyte common antigen-positive cells. They could be distinguished from the BMAC-5-, MHC class II-positive, leukocyte common antigen-positive interstitial dendritic cells. Moreover, 7 d after lethal irradiation, the class II-positive interstitial dendritic cells had completely disappeared from heart, whereas the BMAC-5+ macrophages were present in undiminished numbers. These studies strongly suggest that the interstitial dendritic cell and the tissue macrophage represent two distinct populations of leukocytes within the connective tissues of antigenically secluded organs such as the heart. They have potentially important implications for the physiology of the immune system, as well as for autoimmunity and transplantation.     

Treatment with recombinant human tumor necrosis factor-alpha protects rats against the lethality, hypotension, and hypothermia of gram-negative sepsis.

Tumor necrosis factor (TNF) is a peptide secreted by macrophages in response to endotoxin that can produce many of the changes seen in septic shock. After cecal ligation and puncture (CLP) rats gradually develop tachycardia, hypotension, tachypnea, and hypothermia. At 5 h post-CLP, rats have a peak in serum levels of endotoxin and 60% of rats have blood cultures that grow Gram-negative rods (Escherichia coli and Klebsiella pneumonia). At 20 h post-CLP all rats develop positive blood cultures. Serum levels of TNF are not reproducibly measurable in rats following CLP. Rats undergoing CLP have a 50-80% mortality with deaths usually occurring 24-72 h postinjury. Repetitive (twice daily x 6 d) i.p. injection of sublethal doses of recombinant human TNF-alpha (100 micrograms/kg) to rats undergoing CLP 1 d after the treatment period resulted in a significant reduction in mortality compared to control rats previously unexposed to rTNF (P less than 0.03). Animals treated with rTNF had no hypotension or hypothermia after CLP and regained normal food intake faster than control rats. 12 h after CLP the gene expression for manganous superoxide dismutase (MnSOD), an inducible mitochondrial metalloenzyme responsible for cellular resistance to injury from toxic reactive oxygen species, was higher in livers of rats treated with rTNF suggesting that the TNF treatment augmented expression of this protective enzyme. Unlike MnSOD, expression of the gene for copper-zinc SOD was not affected by CLP or rTNF treatment. The results suggest that prior treatment with recombinant TNF can ameliorate the lethality, hypotension, hypothermia, and anorexia of Gram-negative sepsis in rats and that the mechanism may be related to enhanced hepatic expression of the gene for MnSOD. Repeated administration of recombinant TNF may be a strategy to minimize mortality and morbidity of Gram-negative sepsis.     

电针足三里穴对腹腔脓毒症大鼠肠缺血及氧自由基损伤的作用研究

目的 探讨电针足三里穴对脓毒症大鼠肠缺血和氧自由基损伤的保护作用.方法 采用盲肠结扎穿孔术(CLP)制备大鼠脓毒症模型.雄性Wistar大鼠32只,随机分为CLP+电针足三里(CLP/EA)组、CLP+假电针(CLP/SEA)组、迷走神经切断+CLP+SEA(VA/CLP/SEA)组、VA+CLP+EA(VA/CLP/EA)组,每组8只.EA组持续针刺双侧足三里穴30 min,刺激强度为2～3 mA,2～100 Hz;SEA组采用相同频率和强度刺激非经非穴(足三里外侧旁开0.5 cm)30 min;VA组于CLP前切断迷走神经.各组大鼠于CLP后6 h测定空肠黏膜血流量(JMBF),处死动物取空肠组织,测定丙二醛(MDA)含量、黄嘌呤氧化酶(XOD)和二胺氧化酶(DAO)活性及肠组织含水量.结果 与CLP/SEA组比较,CLP/EA组JMBF和DAO活性显著增加,XOD和MDA及肠组织含水量显著降低(P均<0.05);VA/CLP/SEA组和VA/CLP/EA组JMBF和DAO活性显著降低,XOD和MDA水平显著升高,且组织含水量明显高于CLP/EA组(P均<0.05).VA/CLP/EA组与VA/CLP/SEA组各指标均无明显差异(P均＞0.05).结论 电针足三里可显著增加CLP大鼠JMBF和DAO活性,减轻肠组织水肿和脂质过氧化损伤;切断腹腔迷走神经能减轻或消除电针的作用,增强小肠组织过氧化反应,加重肠组织水肿和黏膜细胞损害.电针足三里穴对肠缺血和氧自由基损伤的保护机制可能与兴奋胆碱能通路有关.     

氯胺酮对感染性休克大鼠保护作用的研究

目的　观察氯胺酮对感染性休克大鼠血流动力学、血浆肿瘤坏死因子α( TNFα)和白细胞介素 6 ( IL 6 )水平的影响 ,探讨其可能的抗休克机制。方法　取健康成年雄性 ( SD)大鼠 2 0只 ,采用盲肠结扎加穿孔 ( CL P)法复制败血症或感染性休克模型。随机分为假 CL P组、CL P组、氯胺酮 组和氯胺酮 组。假CL P和 CL P组术前 30 m in经股静脉持续输注生理盐水 5 ml· kg- 1· h- 1 ,氯胺酮 和氯胺酮 组分别输注氯胺酮 5 m g· kg- 1· h- 1和 10 m g· kg- 1· h- 1。经股动脉穿刺置管 ,持续监测平均动脉压 ( MAP)、心率 ( HR)及采集血样 ,应用酶联免疫吸附试验 ( EL ISA)检测血浆 TNFα和 IL 6水平。结果　 CL P组术后 MAP进行性下降 ,HR则先加快后减慢 ;血浆 TNFα和 IL 6水平明显升高。两种剂量的氯胺酮处理均能逆转 MAP和 HR下降 ,同时抑制血浆 TNFα和 IL 6水平升高 ,尤以氯胺酮 组作用更加明显。结论　氯胺酮对败血症或感染性休克大鼠具有明显的保护效应 ,其机制可能主要是拮抗促炎性细胞因子的产生