一株植物乳杆菌内化于小鼠回肠派伊尔结及其免疫调节作用的研究

目的 探讨决定Lactobacillus plantarum Lp6 在小鼠小肠派伊尔结 (PP)内化的细菌源因素,并分析该菌株的免疫调节作用.方法 FITC标记不同处理的细菌,分析在含PPs的回肠结扎段内化的情况.给小鼠灌胃活或灭活细菌,研究其对小鼠脾、PP细胞增殖和腹腔巨噬细胞吞噬活性的影响.结果 甘露糖可以强烈的抑制细菌内化,灭活和四甲基脲处理可以较明显的减低细菌内化.活菌和灭活菌可以特异性的调节腹腔巨噬细胞、PP细胞和脾淋巴细胞的活性.结论 细菌表面甘露糖糖凝集素是影响细菌PP内化的最主要因素.细菌活力、表面疏水性也有一定的作用.活菌和灭活可以不同程度的增强小鼠腹腔巨噬细胞的吞噬活性并抑制脾和PP淋巴细胞增殖.     

prtP基因对副干酪乳杆菌调节小鼠肠道免疫作用的影响北大核心CSCD

目的研究副干酪乳杆菌丝氨酸蛋白酶编码基因prtP对小鼠肠道免疫调节作用的影响。方法BALB/c雌性小鼠随机分7组：PBS对照组、副干酪乳杆菌亲本株（Lactobacillus paracasei, Lp）、prtP缺失突变株（Lp△prtP）及回复株ReLp△prtP对应的高、中剂量组（1×109 CFU/ml、5×108 CFU/ml）连续灌胃0～21 d后对免疫器官指数、脾脏淋巴细胞转化试验、巨噬细胞能量代谢水平、腹腔巨噬细胞吞噬能力、细胞因子、脾脏CD11c＋、CD80＋有核细胞数进行检测。 结果与prtP基因缺失突变株相比,副干酪乳杆菌亲本株能显著提高小鼠脾脏淋巴细胞转化率、腹腔巨噬细胞的能量代谢水平以及腹腔巨噬细胞吞噬能力,能显著提高血清中IFN-α、IL-10、IFN-γ的表达,显著提高CD11c＋、CD80＋双阳性细胞数量。结论副干酪乳杆菌对肠道免疫功能的促进作用与prtP基因有关。     

PELA幽门螺杆菌口服疫苗微球黏膜免疫研究

目的：应用复乳挥发法制备PELA泛影葡胺显影微球与缨门螺杆菌超声上清口服疫苗微球，并进行靶向与黏膜免疫研究。方法：采用CT技术，研究显影微球的靶向，PELA幽门曙杆菌超声上清口服疫苗微球口服免疫小鼠后，运用ELISA法检测血清，唾液，肠粘液的抗体改变情况，ELISPOT法分析派伊尔氏结（PP结）抗原特异性抗体形成细胞（ASC）的数量增减，结果；口粒径在10um以下的微球，首先粘附在胃肠黏膜表面，后投递于PP结；H.pylori疫苗微球免疫后可诱导较高的唾液sIgA水平和肠道sIgA反应，PP结抗原特异性抗体形成细胞（ASC）数量与肠道 sIgA水平密切相关，结论：可生物降解的PELA微球可用于靶向口服疫苗的研究。     

水龙骨多糖成分及免疫调节活性的初步研究

目的研究水龙骨多糖(Rhizoma polypodiodis nipponicae polysaccharide,RPNP)的组成及体内外免疫调节活性。方法采用水提醇沉法提取RPNP,HPLC分析单糖组成,MTT法考察RPNP对Raw264.7细胞增殖能力的影响,中性红染色法考察其对Raw264.7细胞吞噬能力的影响,ELISA检测细胞因子的分泌水平。采用环磷酰胺诱导建立免疫抑制小鼠模型,考察RPNP对其脏器指数和小肠派氏结数量的影响。结果 RPNP是由Man、Rha、Glc、Gal、Ara 5种单糖组成的酸性杂化多糖,其总糖、糖醛酸、蛋白质及核酸含量分别为42.98%、25.25%、1.30%、5.87%。适当剂量的RPNP能够提高Raw264.7细胞的增殖及吞噬能力,促进TNF-α及IL-6的分泌,并能够增加免疫抑制小鼠的脏器指数及派氏结个数。结论从水龙骨中分离得到的酸性杂化多糖,在适当剂量下能够活化巨噬细胞,增强免疫抑制小鼠免疫功能。     

嗜酸乳杆菌黏附派伊尔结及抑制病原菌侵袭性的研究

目的 研究嗜酸乳杆菌(Lactobacillus acidophilus)FN001I黏附小鼠派伊尔结的黏附介导物,以及L. acidophilus FN001抑制病原菌黏附及侵袭派伊尔结的效果.方法 通过化学、酶预处理研究黏附因子性质,通过单糖抑制黏附的性质研究黏附特异性.利用3种竞争黏附试验研究L aci-dophilus FN001抑制病原菌黏附的作用,利用小肠结扎段法及电镜观察L. acidophilus FN001抑制大肠杆菌侵袭派伊尔结的效果.结果 蛋白酶处理可以显著降低黏附,高碘酸处理可以显著增强黏附,甘露糖和甲基-α-D-甘露糖可以显著抑制黏附,L.acidophilus FN001可不同程度抑制病原菌黏附派伊尔结,对大肠杆菌效果最佳.结论 L. ncidophilus FN001通过蛋白样物质利用甘露糖特异方式黏附大鼠派伊尔结.L. acidophilus FN001可抑制通过相似方式黏附派伊尔结的病原菌.     

乳杆菌调节小鼠空肠派伊尔结基因表达的通路分析

目的:探索乳杆菌免疫作用机制.方法:BALB/c小鼠灌胃乳杆菌,从小鼠空肠派伊尔结提取RNA,基因芯片分析基因表达情况,基于已知的基因网络数据库利用Onto-Tools分析乳杆菌刺激引起的特异性基因网络.结果:Onto-Tools分析得到的通路中,涉及到免疫的通路有T细胞受体信号通路、B细胞受体信号通路、细胞因子和细胞因子受体通路、抗原加工和呈递等;涉及到粘膜屏障的有粘着斑、粘着连接、紧密连接和细胞粘附分子;还有涉及到细胞周期和凋亡的通路;其中通路中影响因子最高的为MAPK信号通路和粘着斑.结论:乳杆菌刺激引起大量蛋白表达,激活粘膜屏障,引起T细胞受体信号通路、B细胞受体信号通路、细胞因子和细胞因子受体通路等通路,从而造成免疫应答.     

链球菌蛋白对RAW264.7小鼠巨噬细胞免疫学活性的调节作用

目的:探讨链球菌蛋白对RAW264.7小鼠巨噬细胞免疫活性调节及其相关作用机制.方法:不同浓度的链球菌蛋白与RAW264.7小鼠巨噬细胞共同作用后,采用MTT 法检测细胞的增殖活化;吞噬中性红试验观察巨噬细胞的吞噬功能;生物化学法检测巨噬细胞上清液中TNF-α和IL-6 的含量;RT-PCR法检测细胞中TNF-α、IL-6和Toll样受体(Toll-like receptors,TLRs)的mRNA表达情况;流式细胞术检测细胞表面TLR2和TLR4的表达强度.结果:链球菌蛋白对巨噬细胞的生长增殖和吞噬功能有较强的刺激作用(P<0.05),促进TNF-α和IL-6的表达和分泌(P<0.05),并可上调巨噬细胞表面模式识别受体TLR2和TLR4的表达(P<0.05).结论:链球菌蛋白通过刺激RAW264.7小鼠巨噬细胞的增殖,增强细胞吞噬活性以及诱导细胞因子的产生等发挥其免疫调节作用.     

香菇多糖口服和注射给药对DTH小鼠耳后淋巴结和小肠派氏结中T淋巴细胞的影响

目的:通过比较口服和注射香菇多糖对DTH小鼠耳后淋巴结和小肠派氏结中T淋巴细胞的影响,探讨肠道粘膜免疫系统在口服药物发挥免疫调节作用中的重要性。方法:28只6～8周龄NIH小鼠随机分为对照组、环磷酰胺免疫抑制模型组、香菇多糖灌胃组、香菇多糖注射组,各组小鼠用2,4-二硝基氟苯背部致敏,4天后耳廓攻击。攻击30小时后,对比各组小鼠耳肿胀差异,并应用流式细胞术检测被攻击耳廓的耳后淋巴结和小肠派氏结中T淋巴细胞的比例变化和活化水平。结果:与环磷酰胺免疫抑制模型组比较,香菇多糖能有效增强小鼠DTH反应,降低耳后淋巴结和小肠派氏结的T细胞比例,提高T细胞活化水平,且口服组的作用明显优于注射组。结论:对比注射给药,口服香菇多糖可以更有效地调节肠道粘膜免疫系统和整体免疫系统功能,提示肠道粘膜免疫系统在口服药物发挥免疫调节作用中扮演着重要的角色。     

全蝎水提物对巨噬细胞活化作用研究

目的:观察全蝎水提物(Buthus martensii water extract,BMWE)在体外对小鼠巨噬细胞株RAW264.7的活化作用。方法:MTT法测定细胞活性;采用FITC标记的酵母测定吞噬活性;利用Griess试剂和ELISA方法测定细胞上清NO、TNF-α和IL-6释放量。结果:BMWE在不影响细胞活力的剂量范围内可增强正常及免疫抑制的RAW264.7细胞吞噬荧光酵母能力(P0.01),增加RAW264.7细胞NO释放量(P0.01),促进RAW264.7细胞TNF-α和IL-6产生(P0.01),均具有良好的量效关系。结论:BMWE可明显提高巨噬细胞吞噬功能与分泌能力,活化巨噬细胞,此作用与全蝎传统用于治疗疮疡、瘰疬,现代用于肿瘤的临床应用相关。     

乳杆菌肽聚糖调节小鼠免疫细胞基因表达的通路分析

目的:探索乳杆菌肽聚糖免疫调节作用的机制.方法:BALB/c小鼠腹腔注射乳杆菌肽聚糖,从腹腔巨噬细胞和脾淋巴细胞提取RNA,基因芯片分析基因表达情况,基于已知的基因网络数据库利用PathwayExplorer和GeneMAPP分析乳杆菌肽聚糖刺激特异性的基因网络.结果:PathwayEx-plorer分析得到的最显著变化的基因网络是"细胞因子-细胞因子受体相互作用"和"辅助性T细胞表面分子".GeneMAPP分析得到最显著基因网络是核糖体蛋白、炎性反应基因网络和血红素合成基因网络.结论:乳杆菌肽聚糖刺激引起大量蛋白表达,引起保护性炎性反应,激活Th细胞,可能引起Th1免疫反应.     

Susceptibilities of macrophage populations to infection in vitro by Leishmania donovani.

Many studies have demonstrated differences in the resistance of strains of mice to infection by Leishmania donovani, Salmonella typhimurium, and Mycobacterium bovis BCG; this resistance/susceptibility phenotype seems to be controlled by a single gene. The present study investigated the susceptibility of liver, lung, peritoneal, and spleen macrophages to infection by L. donovani promastigotes in vitro; the objective was to determine if the susceptibility of animals was expressed by their macrophages when infected in vitro. This study indicated that the Lsh phenotype was only expressed by liver macrophages. The liver macrophages of the susceptible C57BL/6J strain were significantly more phagocytic than those of the resistant C57L/J strain; infection affected the phagocytic activity of the macrophage population. These results indicated that only liver macrophages can express the Lsh gene. Recognition of expression is in part due to its effect on the phagocytic activity of the macrophages.     

Enhanced mucosal delivery of antigen with cell wall mutants of lactic acid bacteria

The potential of recombinant lactic acid bacteria (LAB) to deliver heterologous antigens to the immune system and to induce protective immunity has been best demonstrated by using the C subunit of tetanus toxin (TTFC) as a model antigen. Two types of LAB carriers have mainly been used, Lactobacillus plantarum and Lactococcus lactis, which differ substantially in their abilities to resist passage through the stomach and to persist in the mouse gastrointestinal tract. Here we analyzed the effect of a deficiency in alanine racemase, an enzyme that participates in cell wall synthesis, in each of these bacterial carriers. Recombinant wild-type and mutant strains of L. plantarum NCIMB8826 and L. lactis MG1363 producing TTFC intracellularly were constructed and used in mouse immunization experiments. Remarkably, we observed that the two cell wall mutant strains were far more immunogenic than their wild-type counterparts when the intragastric route was used. However, intestinal TTFC-specific immunoglobulin A was induced only after immunization with the recombinant L. plantarum mutant strain. Moreover, the alanine racemase mutant of either LAB strain allowed induction of a much stronger serum TTFC-specific immune response after immunization via the vagina, which is a quite different ecosystem than the gastrointestinal tract. The design and use of these mutants thus resulted in a major improvement in the mucosal delivery of antigens exhibiting vaccine properties.     

Adhesive capability of Lactobacillus plantarum 299v is important for preventing bacterial translocation in endotoxemic rats

The preventive effect of the probiotic Lactobacillus plantarum 299v on bacterial translocation (BT) and the role of adhesion were studied in septic rats. Five groups of rats were pretreated as follows: negative and positive control groups received regular drinking water; the oatmeal group received drinking water mixed with oatmeal; the Lp 299v group received drinking water mixed with oatmeal containing 10(9) colony-forming units (CFU) L. plantarum 299v/ml; the Lp 299v-adh(-) group received drinking water with oatmeal containing 10(9) CFU/ml of modified L. plantarum 299v (L. plantarum 299v-adh(-)) lacking adhesive properties to enterocytes. On day 8, all rats except the negative control group were given lipopolysaccharide (LPS) intraperitoneally. After 24 h, mesenteric lymph node (MLN), liver and ileum were harvested for culture. Incidence of BT after LPS challenge was 25% and 88% in MLN and liver, respectively. BT increased to 75% in MLN and 100% in liver of endotoxemic rats pretreated with oatmeal. Pretreatment with L. plantarum 299v reduced BT to 0% and 12% in MLN and liver, respectively. L. plantarum 299v-adh(-) did not prevent BT to MLN. Flow cytometry revealed reduced adherence of these bacteria to intestinal epithelial cells compared to L. plantarum 299v. Thus, L. plantarum 299v prevents BT in septic rats, an effect probably dependent on bacterial adherence to the intestinal mucosa. Further, our findings indicate that oatmeal (prebiotics) without probiotics does not prevent BT during sepsis.     

Selection of potential probiotic lactobacilli from pig feces to be used as additives in pelleted feeding

Thirty-five isolates from pig feces were identified as Lactobacillus reuteri (12 strains), Lactobacillus mucosae (7), Lactobacillus plantarum (6), Lactobacillus kitasatonis (3), Lactobacillus rossiae (2), Lactobacillus ultunensis (2), Lactobacillus crispatus (2), and Lactobacillus intestinalis (1) by partial sequence analysis of the 16S rRNA. All isolates were detected at 8-9 log CFU g(-1). Preliminarily, strains were selected based on resistance to heat treatments (ca. 70 degrees C for 10 s). The decrease in viability for some L. reuteri, L. mucosae, L. plantarum, L. kitasatonis, and L. rossiae strains was lower than 1 log cycle. Selected strains were further characterized for acid and bile salt resistance, and antibacterial activity. Except for L. kitasatonis, tolerance to simulated gastric and intestinal conditions was enhanced for all strains by addition of reconstituted skimmed milk. Antibacterial activity was found against Gram-positive and -negative potential pathogens. L. reuteri 8.1, 3S7, 6.2, and 1.2, L. mucosae 1.1R, L. plantarum 4.1, and L. rossiae 4.4 were freeze-dried and mixed (1%, w/w) into pig feed before pelleting. After pelleting, pig feed contained 10-9 log CFU kg(-1) of lactobacilli. L. plantarum 4.1, and L. reuteri 3S7 were selected based on their bile salt resistance, pH tolerance, antimicrobial activity and heat resistance. The findings in this study provide a strong basis for exploring the potential of porcine lactobacilli isolates to be used in pelleted feeding as probiotic additives.     

Effect of specific colostral antibodies and selected lactobacilli on the adhesion of Helicobacter pylori on AGS cells and the Helicobacter-induced IL-8 production

Helicobacter pylori infection is the most common cause of gastritis, gastric ulcer and adenocarcinoma. It has proven difficult to cure because of its capability to develop strains resistant to antibiotics. The effect of three strains of lactic acid bacteria (LAB) and bovine colostral preparations on the adhesion of H. pylori NCTC 11637 on gastric adenocarcinoma (AGS) cells and on the interleukin (IL)-8 production was studied. Before infection, H. pylori were pretreated with Lactobacillus plantarum MLBPL1, Lactobacillus rhamnosus GG, Lactococcus lactis , or with a colostral preparation with or without specific H. pylori antibodies. The relative number of H. pylori adhered on AGS cells was determined by urease test. IL-8 produced by the cells was studied by enzyme-linked immunosorbent assay. Colostral preparations with and without specific antibodies reduced the adhesion of H. pylori on AGS cells in a dose-dependent manner. Live LAB at a concentration of 10¹⁰ CFU/ml reduced the adhesion by approximately 50% ( P  < 0.05). After the infection of AGS cells by H. pylori, the IL-8 level rose up to about 10-fold (5500 ± 1600 pg/ml). Pretreatment of H. pylori with colostral preparations or high concentrations of LAB prevented this IL-8 rise. Similar effect was seen with live and heat-killed LAB, the live LAB being more effective. Heat-killed LAB at a concentration of 10¹⁰ CFU/ml rose the IL-8 level of non-infected cells significantly. Suppression of IL-8 production by LAB or colostral products could have a suppressive effect on inflammation in Helicobacter infection.     

Role of eicosanoids in regulation of macrophage phagocytic functions by platelet-activating factor during endotoxic shock

We studied the role of eicosanoids in the regulation of macrophage phagocytic functions by products secreted in heterogeneous populations of macrophages and platelet-activating factor during endotoxic shock. Phagocytic activity depended on the metabolism of arachidonic acid in target macrophages and the ratio between its cyclooxygenase and lipoxygenase metabolites produced by heterogeneous populations of macrophages and affecting target cells. The regulatory effect of platelet-activating factor on phagocytosis was related to its interaction with producting of the arachidonic acid cascade. Depending on the quantitative ratio of eicosanoids, platelet-activating factor produced various effects on phagocytic functions of heterogeneous macrophage populations. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 130, No. 9, pp. 309–312, September, 2000     

Role of eicosanoids in regulation of macrophage phagocytic functions by platelet-activating factor during endotoxic shock

We studied the role of eicosanoids in the regulation of macrophage phagocytic functions by products secreted in heterogeneous populations of macrophages and platelet-activating factor during endotoxic shock. Phagocytic activity depended on the metabolism of arachidonic acid in target macrophages and the ratio between its cyclooxygenase and lipoxygenase metabolites produced by heterogeneous populations of macrophages and affecting target cells. The regulatory effect of platelet-activating factor on phagocytosis was related to its interaction with producting of the arachidonic acid cascade. Depending on the quantitative ratio of eicosanoids, platelet-activating factor produced various effects on phagocytic functions of heterogeneous macrophage populations. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 130, No. 9, pp. 309–312, September, 2000     

NK cells mediate increase of phagocytic activity but not of proinflammatory cytokine (interleukin-6 [IL-6], tumor necrosis factor alpha,and IL-12) production elicited in splenic macrophages by tilorone treatment of mice during acute systemic candidiasis

The participation of NK cells in the activation of splenic macrophages or in resistance to systemic candidiasis is still a matter of debate. We had previously reported that there is a correlation between natural killer cell activation and resistance to systemic candidiasis. In those experiments we had used tilorone to boost NK cell activity in mice. Here we show a mechanism elicited by tilorone in splenic macrophages which could explain their effect on mouse survival during acute disseminated Candida albicans infection. The results demonstrate that tilorone treatment elicits, by a direct effect, the production of proinflammatory cytokines (interleukin-6 [IL-6], tumor necrosis factor alpha [TNF-alpha], and IL-12) by splenic macrophages. In addition, it increases the capacity of splenic macrophages to phagocytize C. albicans through activation of NK cells. We also demonstrate that the presence of NK cells is essential for maintaining a basal level of phagocytic activity, which characterizes splenic macrophages of na?ve control mice. The results demonstrate that it is possible to identify two phenotypically and functionally peculiar cell populations among splenic macrophages: (i). cells of the "stimulator/secretor phenotype," which show high levels of major histocompatibility complex (MHC) class II surface expression, are poorly phagocytic, and synthesize the proinflammatory cytokines IL-6, TNF-alpha, and IL-12, and (ii). cells of the "phagocytic phenotype," which express low levels of MHC class II molecules, are highly phagocytic, and do not secrete proinflammatory cytokines.