Early 2-mercaptoethanol-resistant antibody production in endotoxin-treated rats

Experiments are reported concerning both natural and specific antibody production in endotoxin-treated rats.     

Effect of quinolones on TNF-alpha production by human monocytes

Previous studies have shown that in lipopolysaccharide (LPS)--stimulated human monocytes, interleukin-1 (IL-1) production is altered by quinoline derivative antibiotics (quinolones), in a way which depends both on the dose and on the agents used. Given that IL-1 and tumor necrosis factor alpha (TNF) are produced in response to LPS and have some overlapping and synergistic activities, we sought to determine if TNF production was altered under the above-mentioned conditions. We investigated the effects of three quinolones: ciprofloxacin (Cip), pefloxacin (Pef) and ofloxacin (Ofl). These quinolones were found to decrease extracellular TNF production in a dose-dependent manner at concentrations higher than 25 micrograms/ml as previously described by our laboratory with regard to IL-1 production. Moreover, the order of the extracellular decrease in TNF and IL-1 induced by each drug was similar. However, in contrast to IL-1 activity, the quinolones studied also reduced cell-associated TNF. The kinetics of TNF production suggested that the quinolones affected TNF production at a very early step, probably during TNF synthesis rather than during its secretion into the extracellular medium. Furthermore, the quinolone-induced accumulation of intracellular cAMP could explain the extracellular decrease in both IL-1 and TNF production.     

Physiologic responses of cross-linked hemoglobin in endotoxin-treated rats

Purified human cross-linked hemoglobin (alpha alpha Hb) as well as recombinant human hemoglobin is undergoing clinical trials in the setting of acute blood loss and perioperative hemodilution. We have previously demonstrated that in rabbits with circulating plasma Hb, such as alpha alpha Hb, infusion of endotoxin (LPS) impairs myocardial contractility which results in hypotension, tissue hypoperfusion and increased mortality. The untoward cardiovascular effects occurring after the combined infusion of LPS and alpha alpha Hb in this model are similar to those reported for other agents that inhibit nitric oxide (NO) availability. To determine if the deleterious effects of alpha alpha Hb and LPS were species specific, we performed similar studies in rats. Anesthetized Sprague-Dawley rats received LPS (4 mg/kg or 40 mg/kg) alone or in combination with alpha alpha Hb (0.7 g/kg). Mean arterial blood pressures (MAP) increased in the group that received alpha alpha Hb alone (105 +/- 8 to 120 +/- 7 mm Hg, p = 0.2) and a decrease was noted in the groups that received low dose LPS (4 mg/kg, p = 0.5) and high dose LPS (40 mg/kg, p = 0.016). MAP in rats treated with the LPS at either dose combined with alpha alpha Hb remained unchanged. Levels of urine nitrite, which was measured as a surrogate marker for plasma NO, were significantly decreased at 2 hr in groups that received the combination of alpha alpha Hb and LPS at 4 mg/kg (p = 0.022) and 40 mg/kg (p = 0.003). No significant decrease was observed in animals treated only with alpha alpha Hb (p = 0.21) or LPS (4 mg/kg; p = 0.78 and 40 mg/kg; p = 0.65). Survival was evaluated during 72 hr in animals that were infused with high dose LPS (40 mg/kg) alone or in combination with alpha alpha Hb and then allowed to recover. The survival of rats treated with LPS alone or the combination was 29% at the end of 24 hr and was 100% for rats receiving only alpha alpha Hb. The data suggest that the toxicity of alpha alpha Hb appears to be a species specific phenomenon.     

穿心莲内酯通过抑制TNF-α活化的NF-KB信号途径保护和促进成骨分化

目的探索穿心莲内酯是否能够抵抗TNF-α激活的NF-κB信号通路活化,从而保护和促进骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)的成骨分化作用。方法碱性磷酸酶染色和茜素红染色观察穿心莲内酯对BMSCs在有或无TNF-α环境下对成骨分化的保护和促进作用。通过real time PCR分析穿心莲内酯对BMSCs在有或无TNF-α环境下Runx2、Opn和Col1的调节作用。通过免疫荧光染色分析穿心莲内酯对TNF-α激活下NF-κB信号通路活化的影响,从而确定穿心莲内酯保护TNF-α对BMSCs细胞成骨分化影响的机制。结果与对照组相比,穿心莲内酯通过促进BMSCs中Runx2、Opn和Col1的表达,从而促进BMSCs成骨分化。同时,穿心莲内酯能够抑制TNF-α对NF-κB信号途径的激活作用,并能保护TNF-α对BMSCs成骨分化的抑制作用。结论穿心莲内酯通过抑制TNF-α激活的NF-κB信号途径保护和促进BMSCs成骨分化。     

Increased NF-kappa B activity in B cells and bone marrow-derived dendritic cells from NOD mice

Type 1 diabetes results from the breakdown of peripheral tolerance. As regulators of T cell activation, antigen-presenting cells (APC) modulate peripheral tolerance and hence contribute to the immune dysregulation characteristic of insulin-dependent diabetes mellitus (IDDM). We initially observed an increased importance of NOD B cell APC function in a T cell priming assay as compared to non-autoimmune strains. Consistent with this increased APC function, we found that NF-kappa B nuclear translocation is increased in unmanipulated NOD and NOD.B10Sn-H2(b) B cells and that, in addition, NOD B cells are more sensitive to NF-kappa B-activating stimuli. We obtained similar results using NOD bone marrow-derived dendritic cell (BMDC) cultures. As costimulatory molecules have been shown to be NF-kappa B responsive, we examined the expression of these markers on NOD APC. Both B cells and BMDC expressed elevated levels of CD80 and CD40. Finally, NOD B cells provided better allostimulation than B cells from non-autoimmune strains. Therefore, hyperactivation of NF-kappa B and increased expression of CD80 and CD40 by NOD B cells and BMDC may be a contributing factor in the selection of effector T cells observed in IDDM.     

离子硅对成骨细胞NF-kappa B核内转录因子活性的影响

目的　研究离子硅对体外培养的成骨细胞NF-kappa B核转录因子活性的影响。 方法　分别用终浓度为1 mmol/L和2 mmol/L离子硅（SiO32-）处理MC3T3-E1成骨细胞,处理时间分别为6、12、24和48 h,设置对照组（不加处理因素）;采用流式细胞术检测细胞周期,计算细胞的增殖指数;Western blotting方法检测NF-kappa B信号通路的相关蛋白表达量及其变化。 结果　流式细胞术结果显示,与对照组相比,1 mmol/L浓度的离子硅处理24 h组和48 h组,MC3T3-E1细胞增殖明显;Western blot结果显示,1 mmol/L浓度的离子硅促进成骨细胞增殖与p-NF-kappa B表达上升密切相关。 结论　骨材料中释放的微量的硅不会引起成骨细胞损伤,相反,微量的硅酸盐可能通过激活NF-kappa B诱导成骨细胞增殖。     

Effects of ketamine on renal nerve activity, arterial pressure and heart rate in rats

We recorded renal nerve activity (RNA) together with arterial pressure (AP) and heart rate (HR) in 24 Wister rats anesthetized with nitrous oxide to investigate the effects of ketamine on the sympathetic nerve activity and the cardiovascular dynamics. The magnitude and time course of the responses to four graded doses of ketamine (1, 5, 10, 25 mg/kg) were studied in 19 rats. RNA responded biphasically, initially decreasing dose-dependently to minimal values of 89 +/- 4.4, 77 +/- 8.2, 54 +/- 5.2, and 17 +/- 3.7% of control for 1, 5, 10, and 25 mg/kg, respectively, and then increasing above control dose-independently. AP showed a biphasic response. HR first decreased dose-dependently but then increased slightly. In the remaining five rats, we compared the effects of ketamine 5 mg/kg on RNA, AP, and HR before surgical baroceptor denervation with those after the denervation. The denervation abruptly increased RNA, AP, and HR. Ketamine decreased RNA, AP, and HR in the denervated state and returned them to pre-ketamine values without overshoot. The finding that in the nerve intact state ketamine produced the characteristically biphasic response of RNA could be explained by the following mechanisms: (1) ketamine depresses the vasomotor center causing the initial decrease in RNA; (2) ketamine depresses the inhibitory effects of baroreflex causing the successive increase in RNA. The biphasic change in AP could be partly attributed to biphasic responses of RNA to ketamine.     

Verapamil modulates LPS-induced cytokine production via inhibition of NF-kappa B activation in the liver

Objective: To investigate the effect of verapamil on Lipopolysaccharide (LPS)-induced cytokines [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10)] and nuclear factor kappa B (NF-κ B) in the liver. Methods and Materials: Adult male Sprague-Dawley rats were randomly divided into seven groups of eight rats each: control rats treated with saline (0.9 % NaCl); rats treated with saline and then challenged intraperitoneally with LPS (10 mg/kg); rats treated intraperitoneally with different levels of verapamil (1, 2.5, 5, 10 mg/kg) and then challenged with LPS (10 mg/kg); and rats treated only with verapamil (10 mg/kg). TNF-α, IL-6, IL-10 and NF-κ B in the liver tissues were investigated as well as the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) one hour after LPS injection. Results: LPS alone stimulated production of TNF-α, IL-6 and IL-10, and activated NF-κ B in the liver. Pretreatment with verapamil before LPS challenge reduced acute liver injury, down-regulated production of LPS-induced pro-inflammatory cytokines (TNF-α and IL-6), up-regulated production of anti-inflammatory cytokines (IL-10) and inhibited NF-κ B activation in the liver in a dose-dependent manner. Conclusion: Verapamil can attenuate acute liver injury by down-regulating the production of TNF-α and IL-6 and up-regulating IL-10 in the liver, possibly via inhibition of NF-κ B. Received 8 August 2005; returned for revision 25 September 2005; returned for final revision 18 November 2005; accepted by M. Katori 6 December 2005     

PKC/Raf-1/NF-κB信号通路在低氧诱导大鼠单核细胞表达TNF-α中的作用

目的：研究PKC/Raf-1/NF-κB信号通路在低氧诱导大鼠外周血单核细胞（PBMCs）肿瘤坏死因子α（TNF-α）表达中的作用，探讨低氧与全身炎症反应综合征(SIRS)的关系，为进一步研究老年多器官功能障碍综合征(MODSE)的肺启动机制奠定基础。方法:采用明胶法分离大鼠外周血单核细胞，分为chelerythrine+低氧组、forskolin+低氧组和单纯低氧组。Chelerythrine+低氧组和forskolin+低氧组细胞在低氧前分别予10 μmol/L chelerythrine和50 μmol/L forskolin预处理。然后各组均于低氧条件下（3 % O2, 5 % CO2, 92 % N2）培养0、1、3、6、9、12、24 h后，收集细胞及培养液上清，分别采用PKC、Raf活性检测试剂盒、电泳迁移分析法（EMSA）、逆转录PCR（RT-PCR）和酶联免疫吸附试验（ELISA）检测PKC、Raf-1、NF-κB活性及TNF-α表达量。结果:低氧1-9 h，PKC、Raf-1活性、NF-κB结合活性及TNF-α表达量显著高于正常对照组（P<0.01）。低氧后1-24 h，PKC、Raf-1活性、NF-κB结合活性与TNF-α mRNA和蛋白表达水平间呈显著正相关（P<0.01，P<0.05）。10 μmol/L chelerythrine可显著抑制低氧诱导的PKC、Raf-1、NF-κB活性升高和TNF-α表达。50 μmol/L forskolin可显著抑制低氧诱导的Raf-1、NF-κB活性升高及TNF-α表达。结论:低氧可显著增强大鼠外周血单核细胞的PKC、Raf-1活性及NF-κB结合活性,并可诱导其产生大量的促炎症因子TNF-α，这些变化可能与急性呼吸窘迫综合征（ARDS）时血浆中大量促炎症因子持续存在密切相关。     

富氢液对大鼠肾缺血再灌注肾功能及TLR4/NF-κB信号通路表达的影响

目的探讨富氢液(HRS)对大鼠肾缺血再灌注肾功能及TLR4/NF-κB信号通路表达的影响。方法SPF级雄性SD大鼠30只,随机分为假手术组(S组)、缺血再灌注组(I/R组)、富氢液组(HRS组),每组10只。S组麻醉开腹后右肾切除;I/R组行右肾切除,左肾缺血45min,再灌注60min,造肾缺血再灌注模型;HRS组于术前口服40 ml/kg富氢液,连续3d后建立I/R模型。每组于I/R后6h取取血样,分离血浆,多功能生化仪检测肌酐(Scr)和尿素氮(BUN)含量;ELISA检测IL-1β和TNF-α含量;另取肾脏,HE染色观察病理组织学变化,Western-blot检测TLR4、Myd88、NF-κB表达变化。结果 HRS可减轻由I/R造成的肾损伤,减低血浆中Scr、BUN、IL-1β和TNF-α含量,抑制肾脏TLR4、Myd88、NF-κB表达(P0.05)。结论 HRS减轻由I/R引起的肾功能障碍,抑制炎性反应,对肾脏有一定的保护作用,其机制可能与TLR4/NF-κB信号通路有关。     

Effects of anti-rheumatic gold salts on NF-kappa B mobilization and tumour necrosis factor-alpha (TNF-alpha)-induced neutrophil-dependent cytotoxicity for human endothelial cells

We have previously shown that the gold-containing disease-modifying anti-rheumatic drugs, auranofin (AF) and gold sodium aurothiomalate (GSTM) reduce human umbilical vein endothelial cell (HUVEC) adhesion molecule expression and neutrophil (PMN) adherence. AF diminishes E-selectin and intercellular adhesion molecule-1 (ICAM-1) on cytokine-activated HUVEC, while GSTM decreases only E-selectin. Since tight adhesion is critical for PMN to damage EC, we tested whether these drugs modulated human PMN-mediated injury to TNF-alpha-activated HUVEC in vitro (as measured by 51Cr release). Here we show that TNF-alpha caused a prominent PMN-mediated cytotoxicity that was dose-dependently reduced when AF and GSTM were added to the assay system. We also found that a potent inhibitor of NF-kappaB, pyrrolidine dithiocarbamate (PDTC) in a dose-dependent manner impaired TNF-alpha-induced cytotoxicity, indicating a role of NF-kappaB activation in cytokine-induced endothelial injury. To examine the effects of AF and GSTM on TNF-alpha-induced NF-kappaB activation this was measured in HUVEC nuclear extracts by an electrophoretic mobility shift assay. AF, but not GSTM, decreased TNF-alpha-induced NF-kappaB activation in HUVEC. Thus, in this in vitro model of vasculitis, AF and GSTM dose dependently reduced TNF-alpha-mediated neutrophil-dependent cytotoxicity for HUVEC, and AF, but not GSTM, inhibited NF-kappaB mobilization, thereby providing possible mechanisms for effects of AF and GSTM.     

Variable coupling between olfactory system activity and respiration in ketamine/xylazine anesthetized rats

In this study, we have characterized slow and fast oscillations at several stages of olfactory processing under light and deep ketamine/xylazine anesthesia in the albino rat. While monitoring the animal's respiration, we also obtained field potentials from the olfactory bulb and piriform (olfactory) cortex and simultaneously recorded membrane potentials in piriform cortex pyramidal cells. Our results demonstrate that oscillations are generally found at higher frequencies under lighter and lower frequencies under deeper anesthesia. In previous studies of cerebral cortex, similar results in ketamine/xylazine anesthetized animals have been interpreted to correspond with the higher frequencies found during waking and lower frequencies found in the sleep state. Correlation and coherence analysis between data obtained in the bulb and cortex reveals a clear difference in coupling depending on the anesthetic state of the animal. Specifically, activity recorded in the whole system is highly correlated with respiration during deep anesthesia, whereas only the olfactory bulb, and not the cortex, is correlated with respiration during light anesthesia. These data suggest that global activity in the piriform cortex is actually more directly tied to peripheral slow respiratory input during slow wave than fast wave states and that the coupling between olfactory structures can be dynamically modulated by the level of anesthesia and therefore presumably by different brain states as well.