Linomide,a novel immunomodulator that prevents death in four models of septic shock

Intravenous injections of 50 μ.g Staphylococcus aureus enterotoxin B (SEB) or bacterial lipopolysaccharide (LPS) are lethal, provided that mice are simultaneously sensitized with either N-galactosamine (GalN) or the anti-glucocorticoid RU-38486. Similar to the synthetic glucocorticoid (GC) receptor agonist dexamethasone, pharmacological doses of the immunomodulator linomide (quinoline-3-carboxamide) prevent death in all four models of lethal septic shock (LPS + GalN, LPS + RU-38486, SEB + GalN, and SEB + RU-38486) and inhibit the secretion of tumor necrosis factor, one of the major intermediate effector molecules of SEB and LPS toxicity. In this system, cyclosporine A (CsA), although effective in suppressing SEB toxicity, fails to counteract the lethal effect of LPS. This observation, together with the fact that linomide acts in the presence of excess amounts of GC receptor antagonist, indicates that linomide functions in a different way to that of known immunosuppressive agents like CsA and GC.     

抗内毒素Fab'的制备

目的 研究抗内毒素卵黄免疫球蛋白(IgY)的活性片断Fab',探讨防治内毒素血症的新途径.方法 用内毒素(LPS)作为抗原免疫25周龄德国罗曼鸡,改良水溶法提取抗内毒素IgY,胃蛋白酶切后提取Fab'片断,光密度法测抗内毒素Fab'的浓度和含量、ELISA检测抗内毒素Fab'效价、SDS-聚丙烯酰胺凝胶电泳检测其分子量及纯度. 结果 抗内毒素Fab'含量为4.2 mg/mL蛋黄液,效价为1∶51 200,纯度为92%,相对分子质量为44 000. 结论 抗内毒素Fab'产量大、效价高、特异性强.     

LBP对LPS激活巨噬细胞内p38信号通路的影响 

目的:探讨脂多糖结合蛋白(LBP)对脂多糖(LPS)激活肺泡巨噬细胞内p38信号通路的调节作用。方法:经硫酸铵盐析、Bio-Rex70阳离子交换层析和MonoQ阴离子交换层析, 从大鼠急性期血清中分离纯化LBP。分别用0.01mg/L和1mg/L的LPS刺激肺泡巨噬细胞, 并加入不同浓度LBP(0mg/L、0.01mg/L、0.1mg/L、1mg/L和10mg/L), 观察肺泡巨噬细胞中p38蛋白激酶的磷酸化程度。结果:纯化的大鼠LBP在SDS-PAGE的60kD处呈现单一条带, 并可增强LPS与单核细胞的结合。当LPS浓度为0.01mg/L时, 1mg/L以下的LBP可明显增敏LPS对肺泡巨噬细胞内p38信号通路的激活, 并且这种增敏作用随LBP浓度的增加而增强。但LBP为10mg/L时, LBP对LPS的增敏作用反而有所减弱;当LPS为1mg/L时, LBP对LPS激活肺泡巨噬细胞内p38信号通路无调节作用。结论:LBP对低浓度LPS(0.01mg/L)激活肺泡巨噬细胞内p38信号通路有明显的调节作用;而高浓度LPS(1mg/L)不需LBP的增敏作用, 可能通过LBP非依赖途径直接激活p38信号通路。     

Modulation of the transcripts for tumor necrosis factor-α and its receptors in vivo

We investigated the effects of a single bacterial lipopolysaccharide (LPS) injection in vivo on the gene expression of tumor necrosis factor-α (TNF) and its receptors: TNF receptor type I (TNF-R 55 kDa or TNF-R1) and TNF receptor type II (TNF-R 75 kDa or TNF-R2) in various tissues and white blood cells. While TNF mRNA rapidly accumulated in most tissues, TNF-R1 and TNF-R2 mRNA levels were found to be differentially regulated in lung, spleen, lymph nodes and white blood cells. In most cases, TNF-R mRNA levels did not parallel TNF mRNA levels. These observations indicate that TNF-R of both types are capable of modulating the host response to LPS, not only by shedding of their extracellular domains, but also by strict regulation of their gene expression.     

Loss of lipopolysaccharide receptor CD14 from the surface of human macrophage-like cells mediated by Porphyromonas gingivalis outer membrane vesicles

Porphyromonas gingivalis, the major etiologic agent of chronic periodontitis, produces a broad spectrum of virulence factors, including outer membrane vesicles. In this study, we investigated the capacity of P. gingivalis vesicles to promote the shedding or cleavage of the lipopolysaccharide (LPS) receptor CD14 from the surface of human U937 macrophage-like cells. SDS-PAGE/Western immunoblotting analysis of gingival crevicular fluid samples from patients affected by moderate or advanced periodontitis revealed the presence of soluble CD14 and CD14 fragments, thus supporting the hypothesis of an in vivo shedding and cleavage of CD14 receptors. Flow cytometry analysis of macrophage-like cells treated with a vesicle-containing culture supernatant of P. gingivalis showed a significant decrease in the binding of anti-human CD14 to the cell surface. However, no accumulation of soluble CD14 or immunoreactive CD14 fragments in the assay supernatant could be demonstrated by ELISA. Treatment of macrophage-like cells with various concentrations of P. gingivalis vesicles substantially suppressed TNF-alpha production triggered by Escherichia coli LPS. This suppressive effect was much less important using heat-treated vesicles or in the presence of leupeptin, a gingipain inhibitor, during the treatment. Recombinant human CD14 receptors were found to be susceptible to proteolytic degradation by P. gingivalis vesicles. A purified Arg-gingipain preparation produced much more degradation than a Lys-gingipain preparation. This study provides evidence that P. gingivalis outer membrane vesicles contribute to the loss of membrane-bound CD14 receptors and that gingipains degrade this LPS receptor. Such a phenomenon, which results in an hyporesponsiveness of macrophages to LPS stimulation, may contribute to an increased capacity of P. gingivalis, and other periodontopathogens, to evade the host immune system mechanisms.     

LPS对大鼠肺微血管内皮细胞SSeCKS的表达影响

目的研究细菌脂多糖（LPS）对大鼠肺微血管内皮细胞（rat pulmonary microvascular endothelial cell，RPMVEC）Src抑制的蛋白激酶C底物（Src-suppressed C kinase substrate，SSeCKS）表达和细胞内定位的影响，探讨SSeCKS参与细胞骨架结构改变的可能机制。方法用植块培养法体外培养大鼠肺微血管内皮细胞，用抗大鼠CD31抗体进行细胞鉴定。LPS刺激体外培养的RPMVEC，用定量PCR、免疫印迹方法检测LPS刺激RPMVEC不同时间SSeCKS mRNA和蛋白的表达情况；用0．05μmol／L蛋白激酶C（PKC）抑制剂（Calphostin C）预处理RPMVEC30min后再用LPS刺激6h，免疫荧光细胞化学法观察Calphostin C对LPS诱导SSeCKS与纤维状肌动蛋白（filamentous—actin，F-actin）细胞内定位和结构改变的影响。结果定量PCR结果显示LPS刺激RPMVEC1h后SSeCKS表达水平达到最高，Westernblot结果与定量PCR结果相一致，同时，LPS以时间依赖的方式诱导SSeCKS磷酸水平增加。免疫荧光结果显示LPS刺激后，F-actin发生重构，细胞内形成应力纤维，SSeCKS向核周、细胞膜纤维、板状伪足末端聚集；Calphostin C部分抑制LPS对内皮细胞F-actin和SSeCKS细胞内定位改变的影响。结论LPS能够诱导内皮细胞SSeCKS表达增加和细胞内定位改变，PKC参与I娲诱导内皮细胞F-ac，6n的重构和SSeCKS重新分布；提示SSeCKS可能与LPS诱导内皮细胞F-actin的重构有关。     

Pre-exposure of murine macrophages to lipopolysaccharide inhibits the induction of nitric oxide synthase and reduces leishmanicidal activity

Murine macrophages produce nitric oxide (NO) from L-arginine on stimulation with lipopolysaccharide (LPS), alone or with interferon-γ (IFN-γ). The effect of incubation of macrophages with low concentrations of LPS on NO synthesis on subsequent stimulation was investigated, using a murine macrophage cell line, J774, and peritoneal macrophages from CBA mice. Cells which had been incubated with LPS produced significantly lower amounts of NO, and expressed lower levels of NO synthase activity, following stimulation with IFN-γ and LPS, or with a high concentration of LPS. This effect was not reversed by tumor necrosis factor-α. The ability of CBA macrophages to kill the intracellular parasite Leishmania major was markedly reduced by pre-incubation with LPS. Reduced NO production by macrophages previously exposed to LPS is a manifestation of endotoxin tolerance, and may represent an important means of regulation of NO synthesis and thus a survival mechanism for intracellular parasites.     

Divergent effects of prostaglandin receptor signaling on neuronal survival

Induction of cyclooxygenase-2 (COX-2) with production of prostaglandins occurs in a wide spectrum of acute and chronic neurodegenerative diseases and is associated with neuronal death. Inhibition of the COX-2 pathway and downstream production of prostaglandins protect neurons in rodent models of cerebral ischemia and neurodegeneration. Recent studies investigating the functions of selected prostaglandin receptor pathways in mediating COX-2 neurotoxicity have demonstrated both toxic and paradoxically neuroprotective effects of several receptors in models of excitotoxicity. In this study, we investigate the functions of additional prostaglandin receptors not previously characterized in organotypic models of glutamate excitotoxicity. We find that PGD₂, PGI₂, and PGF_2α receptors protect motor neurons in an organotypic spinal cord model of amyotrophic lateral sclerosis (ALS). In addition, PGI₂ and TXA₂ receptors rescue CA1 neurons in an organotypic hippocampal model of N-methyl-d-aspartate excitotoxicity. However, in a model of inflammation induced by lipopolysaccharide, prostaglandin receptors previously found to be protective in excitotoxicity now cause CA1 neuronal death. Taken together, these studies identify novel eicosanoid receptor signaling pathways that mediate neuronal protection in excitotoxic paradigms; these data also support the emerging hypothesis that the toxic/protective effects of eicosanoid signaling on neuronal viability diverge significantly depending on whether excitotoxicity or inflammation predominates as the underlying toxic stimulus.     

脂多糖诱导的小鼠腹腔巨噬细胞基因表达谱分析

目的　利用基因芯片技术分析脂多糖 (LPS)活化的小鼠腹腔巨噬细胞基因表达谱 ,以更全面地了解LPS诱导的巨噬细胞反应。方法　以未刺激的和用 1mg/LLPS刺激的小鼠腹腔巨噬细胞制备3 3 P标记的cDNA探针 ,分别与含有 1176个已知基因的小鼠cDNA芯片杂交。结果　活化组和未刺激组间的 2倍差异表达基因为 118个 ,3倍差异表达基因为 6 9个 ,其中 4 4个上调 ,2 5个下调。转录因子、细胞内信号调节蛋白、炎症细胞因子和细胞凋亡相关基因的转录发生明显的调节变化。结论提供了LPS活化的巨噬细胞综合基因表达信息 ,并筛选出一些新的可能与LPS活化相关的基因。     

Ketamine inhibits LPS-induced calcium elevation and NF-kappa B activation in monocytes

Objective:To investigate whether ketamine could inhibit lipopolysaccharide (LPS)-induced intracellular calcium elevation and NF-kappa B activation in monocytes. Materials and methods:Isolated rat monocytes were challenged with 10 g/ml LPS with or without the presence of various concentrations of ketamine (10, 100, 1000 M). Intracellular calcium was monitored by laser confocal microscopy. NF-kappa B activity of the nuclear extracts of monocytes was analyzed by electrophoretic mobility shift assay (EMSA). Results:LPS provoked a significant calcium elevation and enhanced NF-kappa B activity in monocytes. Ketamine above concentration of 100 M inhibited endotoxin-induced intracellular calcium elevation and NF-kappa B activity. Ketamine itself had no effect on either of them. Conclusions:These findings suggest that ketamine could suppress NF-kappa B in monocytes exposed to endotoxin, and this anti-inflammatory effect might act through attenuating intracellular calcium elevation.Received 31 October 2003; returned for revision 18 December 2003; accepted by I. Ahnfelt-Rønne 26 Januaryy 2004