Effect of β-endorphin on adherence, chemotaxis and phagocytosis of Candida albicans by peritoneal macrophages

There is growing evidence about the role of neuroendocrine hormones in the regulation of the immune system. In the present study we have examined effects on different stages of phagocytic function of peritoneal macrophages from BALB/c mice induced by β-endorphin. Peritoneal macrophages were incubated (30 min at 37°C) in vitro with 0.22, 0.5 or 2200 ng/ml of this hormone. Adherence capacity was evaluated by means of a substrate adherence technique, chemotaxis in Boyden chambers, and ingestion of Candida albicans on migration inhibitory factor (MIF) dishes. No changes in adherence capacity were found. Chemotaxis, however, increased, and concentration of β-endorphin correlated directly with stimulation. Incubation of macrophages with 0.5 ng/ml of β-endorphin also stimulated phagocytosis of Candida albicans. These results indicate that β-endorphin acts on peritoneal murine macrophages, stimulating some stages of their phagocytic function.     

The in vitro cytotoxicity of native and modified trichloroacetic acid-extracted antigens from Fusobacterium necrophorum for mouse peritoneal macrophages

A Boivin lipopolysaccharide-protein complex (LPS-P), Westphal LPS (W-LPS), Boivin LPS (B-LPS), and a lipid A-associated protein (LAP) were extracted from Fusobacterium necrophorum bov 5 strain and chemically analyzed for protein, total nitrogen, hydrolyzed hexosamine, and nucleic acid content. Dose and time effects on the in vitro cytotoxicity of these preparations for mouse peritoneal macrophages were determined. Macrophage monolayers were exposed to 2.0 ml of 10, 100, 250, 500, or 1000 μg/ml solutions of the individual preparations for 1, 2, 4, or 6 hr at 37°C, and relative toxicities were determined; B-LPS> LPS-P> LAP, W-LPS. The effects of heat, NaOH, and trypsin and pronase on the native antigens were determined. Heat treatment had no effect on the cytotoxicity of B-LPS, but increased the toxicity of LPS-P and LAP for macrophages. Alkaline treatment decreased the cytotoxicity of B-LPS and increased the toxicity of LPS-P and LAP for macrophages. Enzyme treatment had no effect on LPS-P toxicity. The relationship of lipid A and protein content to toxicity for macrophages is discussed. Lipid A appears to be important in the cytotoxicity of B-LPS, and the protein components of LPS-P and LAP may contribute to their toxic activity. The ability of these bacterial cellular antigens to destroy phagocytic cells may facilitate the establishment of necrotic abscesses in susceptible hosts.     

Experimental study of antibiotic-induced immunosuppression in mice—I. Humoral and cell-mediated immune responsiveness related to in vivo antibiotic treatment

By using CBA/J mice as responders, the immunodepressive effect of seven antibacterial chemotherapeutic agents was tested, i.e. penicillin (pen), streptomycin (str), erythromycin (erm), kanamycin (kan), tetracycline (tet), colistin (col) and chloramphenicol (chl). Lymphocytotoxicity power, as well as the ability of each drug to influence secondary humoral (against sheep red blood cells or diphtheria anatoxin) or cell-mediated (against PPD and Coxsackie A₉ virus) immunity were searched.

Erm, col and chl markedly depressed humoral and cell-mediated immune responsiveness in vitro after in vivo treatment with non-cytotoxical amounts. The Th lymphocyte supplementation of B reactive spleen cell population recovered the immune capacity in col and chl in vivo-treated groups, but not in erm-treated group.     

Changes in the phagocytic function of peritoneal macrophages from old mice after strenuous physical exercise

The effect of acute physical exercise (swimming until exhaustion) on the phagocytic function of peritoneal macrophages from old adult BALB/c mice (55 ± 5 weeks old) was studied. Adherence capacity of macrophages to substrate, spontaneous mobility and chemotaxis as well as digestion capacity measured by nitroblue tetrazolium (NBT) reduction in presence of ingested material (latex beads) were not modified with exercise. Adherence to nylon fiber, opsonization and ingestion of Candida albicans (either spontaneous or in presence of serum), ingestion of latex particles as well as nitroblue tetrazolium (NBT) reduction in absence of ingested material (oxidative metabolism measure) were increased in respect to controls with statistically significant differences (P < 0.001) in peritoneal macrophages after the exercise.     

Determination of the oxidative burst chemiluminescent response of avian and murine-derived macrophages versus corresponding cell lines in relation to stimulation with Salmonella serotypes.

In contrast to mammalian systems, avian species lack a resident or harvestable macrophage population in the abdominal exudate. Peritoneal macrophages in the chicken can be elicited if an inflammatory agent such as sephadex is injected. This study examines the kinetics of different macrophage populations, derived by different methods of isolation and from different hosts, with respect to the elicited oxidative burst upon infection with host-adapted Salmonella serotypes.

The nature of the oxidative burst elicited by murine and avian-derived and cell line macrophages was determined after stimulation with phorbol myristate (PMA), zymosan A, and Salmonella serotypes. Both murine and chicken peritoneal macrophages, chicken blood monocytes and corresponding cell lines, J774A.1 and HD-11, were unable to produce a detectable chemiluminescent (CL) response after interaction with Salmonella using the luminescent probe luminol. However, both PMA and zymosan A induced a CL response in all cell types, with PMA eliciting a higher and earlier peak response (pkH) than zymosan A. Lucigenin-enhanced CL in both murine and chicken macrophages was achieved with PMA, zymosan A and Salmonella serotypes. In this case, zymosan A induced higher responses than PMA. In the peritoneal macrophages of both hosts, there were no significant differences in the oxidative burst induced by the different Salmonella serotypes. However, the J774A.1 (murine) cells demonstrated significant differences, with S. enterica serotype Choleraesuis (S. choleraesuis and S. gallinarum producing the highest response. In the HD-11 (chicken) cells, S. choleraesuis and S. dublin elicited the higher CL. With both cell lines, S. abortusovis failed to induce an appreciable CL response.

In these experiments it was demonstrated that oxidative burst was not detectable in monocytes/macrophage populations using luminol, which suggests a link to the lack of a myeloperoxidase system in these cells. Lucigenin-enhanced CL appeared independent from the myeloperoxidase system, indicating production of another oxidative species compared with luminol. No discernable effect of host specificity with regard to Salmonella serotype and respective host was seen in host-derived or cell line macrophages, and cell line macrophages displayed altered functional characteristics with regard to oxidative burst in comparison with their primary counterparts.     

杜仲叶免疫调节机制的网络药理学分析及验证

基于网络药理学及小鼠免疫抑制模型方法探讨杜仲叶免疫调节的作用机制。该研究通过TCMSP数据库筛选出3个杜仲叶活性成分，Uniprot和Swiss Target数据库预测到306个相关联的靶基因，经与OMIM、GeneCards数据库对比，获得105个免疫失调与杜仲叶的交集基因，通过Cytoscape3.8.2软件构建“活性成分-基因”网络。利用STRING数据库进行蛋白质-蛋白质相互作用分析，构建蛋白质-蛋白质相互作用网络(PPI),CytoNCA进行网络拓扑学分析，筛选出21个核心靶点，并对核心靶点进行基因本体(GO)富集分析与KEGG通路分析。结果发现，杜仲叶中的山奈酚、槲皮素、绿原酸等主要化合物通过调节肿瘤坏死因子(TNF)、白细胞介素6(IL-6)、血管内皮生长因子A (VEGFA)、白细胞介素1β(IL-1β)等关键靶点，参与IL-17、肿瘤坏死因子信号通路等，从而发挥免疫调节作用。通过腹腔巨噬细胞体外试验和环磷酰胺免疫抑制体内试验探讨杜仲叶提取物的免疫调节作用，结果显示，1 000～5 000μg·mL^-1浓度的杜仲叶提取物可促进腹腔巨噬细胞增殖和吞噬...     

Absence of lymphokine-enhanced macrophage migration in vitro in the Australian brush-tailed opossum, Trichosurus vulpecula

The supernatants from cultures of either opossum or guinea pig splenic lymphocytes, stimulated with phytohaemagglutinin, significantly enhanced the in vitro migration of guinea pig peritoneal macrophages (P greater than 0.001) but not that of opossum peritoneal macrophages. Failure of opossum macrophages to respond to a putative macrophage chemotactic factor might account for the observed paucity of immune granulomas in these animals and help explain the species' susceptibility to tuberculosis.     

Histological and immunohistochemical characterisation of Mycobacterium bovis induced granulomas in naturally infected Fallow deer (Dama dama)

Mycobacterium bovis infections in fallow deer have been reported in different countries and play an important role in the epidemiology of bovine tuberculosis (bTB), together with other deer species. There is little knowledge of the pathogenesis of bTB in fallow deer. The aim of this study was to perform a histopathological characterisation of the granulomas induced by M. bovis in this species and the immunohistochemical distribution of different cell subsets (CD3+, CD79+, macrophages) and chemical mediators (iNOS, TNF-α, IFN-γ) in the different developmental stages of granulomas. Stage I/II granulomas showed a marked presence of macrophages (MAC387+) expressing high iNOS levels while stage III/IV granulomas showed a decrease in the number of these cells forming a rim surrounding the necrotic foci. This was correlated with the presence of IFN-γ expressing cell counts, much higher in stage I/II than in stage III/IV. The number of B cells increased alongside the developmental stage of the granuloma, and interestingly the expression of TNF-α was very low in all the stages. This characterisation of the lesions and the local immune response may be helpful as basic knowledge in the attempts to increase the vaccine efficacy as well as for disease severity evaluation and for the development of improved diagnostic tools. Immunohistochemical methods using several commercial antibodies in fallow deer tissues are described.     

Experimental study of antibiotic-induced immunosuppression in mice—II. Th, Ts and NC cell involvement

In penicillin(pen), streptomycin(str), kanamycin(kan) and tetracycline(tet)-treated CBA/J adult mice, no difference was noticed as concerned spleen T “helper” (Th) cell activity, as studied by means of response to PHA of X-irradiated/whole T fraction cell mixtures in vitro. On the contrary, in erythromycin(erm), colistin(col) and chloramphenicol(chl)-treated groups, Th cell activity was significantly decreased. On the other hand, spleen T “suppressor” (Ts) cell activity (assayed by response to PHA of mixtures containing pre-incubated with Concanavalin A and whole T cells, respectively) was augmented in samples arising from chl-treated group. These results are also supported by experiments testing Th or Ts soluble factors, induced in spleen T cells belonging to each antibiotic-treated group and purified by affinity chromatography (Concanavalin A-Sepharose 4B columns). Thus, it was confirmed both Th cell deficiency in erm, col or chl-groups, and Ts cell augmentation in chl-group.

As regards spleen “natural cytotoxic” (NC) cell activity, as tested in a xenogeneic “target” cell substrate, a diminished cytotoxic capacity manifested chl-group-derived NC cells, possibly by richness in own “non-specific suppressor” (NSS) cells. NC cell samples in vitro supplemented with NSS cells arising from suckling mouse spleens and a lower cytotoxic activity, in a larger extent in chl-group-derived NC cells, as compared to other groups. The pre-incubation of control-group-NC cells with several antibiotic preparations in vitro was followed by decrease of the cytotoxic values in erm, col and chl-samples, suggesting a drug-induced NC receptor “masking”, that prevented “target” cell recognition in mice—II. In the case of NSS addition in vitro, as strong inhibition of the cytotoximity occurred in chl-treated NC cells derived from the control group, that proves a possible chloramphenicol-induced immunodepression by potentiation of NSS inhibitory effect on NC cells.

Based upon data from the present work, as well as from the previous work, the authors suggest a classification of several mechanisms by which the antibacterial antibiotics can act as immunosuppressive.     

硒对树突状细胞和巨噬细胞功能的调控作用

旨在探讨硒对树突状细胞（dendritic cells,DCs）和巨噬细胞功能的影响,试验将硒分别与髓源性树突状细胞（bone marrow derived dendritic cells,BMDCs）和腹腔巨噬细胞共同培养后,用流式细胞术检测未成熟BMDCs和巨噬细胞的吞噬活性以及BMDCs上的MHCⅡ、CD86、CD80和CD40的表达量,测定经硒处理后的成熟BMDCs对刺激同种异体淋巴细胞增殖和抗原递呈能力,并用ELISA检测BMDCs和巨噬细胞上清液中细胞因子（IL-12、IL-1β、IFN-γ、IL-6、IL-10、TNF-α、NO）水平的变化。结果显示,当硒的质量浓度在0.18~0.09 mg·L^-1时,BMDCs和巨噬细胞的吞噬活性显著增强（P<0.05）,并且BMDCs上的MHCⅡ、CD86和CD80的表达量显著升高（P<0.05）,对刺激同种异体淋巴细胞的增殖和抗原递呈能力也显著增强（P<0.05）。此外,在BMDCs的上清中,IFN-γ、IL-12和IL-10的含量显著升高（P<0.05）;在巨噬细胞的上清中,IFN-γ、TNF-α和NO的含量显著升高（P<0.05）。结果表明,一定质量浓度的硒可以增强树突状细胞和腹腔巨噬细胞的功能,值得进一步探究硒对免疫功能的影响。     

高通量SNP芯片在牛体外早期胚胎染色体质量鉴定中的初步应用

旨在基于高通量SNP芯片建立一种可评估牛早期胚胎染色体质量和生产性能的方法,为胚胎牛育种提供技术支撑。本研究通过胚胎切割技术分别对荷斯坦奶牛体内囊胚（n=27）、体外囊胚（n=21）、体外2细胞胚胎（n=6）和8细胞胚胎（n=5）进行切割取样并统计发育率,其中体内囊胚组分为1/2体内胚组（切取半个胚胎）和体内滋养层（trophoblast cells,TE）组（切取体内胚胎少量TE）,体外囊胚组、体外2和8细胞胚胎分别为体外TE组（切取体外胚胎少量TE）、体外2细胞（切取体外2细胞胚胎的1个卵裂球）和8细胞组（切取体外8细胞胚胎的1个卵裂球）。切割取样后进行全基因组扩增（whole-genome amplification,WGA）,对扩增成功且DNA量大于1 000 ng的样品进行SNP芯片检测,芯片检出率大于90%的进行生产性能评估,低于90%的进行染色体片段缺失分析和数据填充。结果显示：1）切割部分TE后,体内TE组剩余部分和体外TE组剩余部分继续培养24 h后的发育率分别为（94.4±5.6）%和（90.5±6.6）%,而1/2体内胚组剩余部分的发育率显著降低,仅为（22.2±14.7）%。2）1/2体内胚组和体外2细胞组扩增成功率为100%,而体内TE组、体外TE组和体外8细胞组扩增的成功率分别为（94.4±5.6）%、（76.2±9.5）%和（60.0±24.5）%;与1/2体内胚组相比,体内TE组和体外TE组经WGA后DNA量均显著下降（P < 0.05）,且体外TE组扩增后DNA量显著低于体内TE组（P<0.05）。3）相较于1/2体内胚组（91.2±1.6）%,体内TE组（76.7±15.2）%、体外TE组（74.3±9.6）%、体外2细胞组（76.1±6.9）%、体外8细胞组（61.2±19.0）%芯片检出率均显著下降（P<0.05）,且检出率均低于90%。4）以至少连续7个SNPs缺失作为染色体片段缺失筛选标准,体外TE组和体内TE组分别筛选到188个和388个染色体缺失片段,相应的缺失片段各包括46和48个基因;GO和KEGG富集分析结果显示,体外TE组缺失基因主要与细胞骨架和细胞分化相关,而体内TE组缺失基因则主要与细胞物质分泌和运输相关。5）在64 958个SNPs填充位点中52 334个SNPs填充位点的填充准确性（R²）在0.99～1之间且填充准确性为91%,说明填充的准确率较高,育种值估计显示,体外TE组生产性能低于体内TE组。综上,利用胚胎切割、单细胞基因组扩增和基因组芯片可在微创的前提下,实现对植入前早期胚胎染色体质量和生产性能的评估。同时,体内外胚胎染色体缺失片段对比分析发现体外发育过程中细胞骨架等基因异常表达可能是导致体外胚胎质量差的原因之一。     

SPF大白猪和长白猪CIITA基因剪接突变体的鉴定分析及在不同组织中的表达模式

为了鉴定分析SPF大白猪和长白猪CIITA剪接突变体，研究其在猪不同组织中的表达模式。本研究以5周龄的8头SPF大白猪和7头SPF长白猪为研究对象，设计特异性引物对CIITA基因进行扩增、克隆和测序，对获得的序列分析其剪接突变体特征，利用在线软件对不同剪接突变体进行生物信息学分析，同时利用荧光定量PCR方法检测CIITA基因剪接突变体在2头SPF长白猪8种组织中的表达模式。结果显示，在SPF大白猪和长白猪CIITA基因CDS区存在3种不同的剪接突变体（CIITA-A、CIITA-B和CIITA-C），其CDS长分别为939、984和1 698 bp。CIITA-A和CIITA-B突变体由于Exon 11部分核苷酸缺失（40和199 bp）使终止密码子提前，从而致使编码CIITA蛋白的结构域受到了剪接的影响，缺失了末端富含亮氨酸的4个重复结构域，蛋白结构发生了明显的改变。进化树结果表明，针对CIITA，猪与绵羊和牛具有较近的亲缘关系，在不同猪品种中具有较高的保守性。同时，CIITA不同剪接突变体在猪8种组织中均有表达，且表达量趋于一致，均在脾或肺中表达量较高，其次是肾、脊髓、甲状腺、肝、胸腺，在心脏中表达最低。本研究从SPF大白猪和长白猪上共获得了3个CIITA剪接突变体，其中两个蛋白结构缺失了4个富含亮氨酸的重复结构域，且不同剪接突变体在猪不同组织中均有表达，表达量稍有差异，为后续CIITA基因在机体内的免疫调控功能研究奠定了理论基础。     

致病性大肠杆菌HPI对Caspase-1细胞焦亡相关分子表达的影响

为研究云南撒坝猪致病性大肠杆菌高致病性毒力岛（HPI）致猪源巨噬细胞焦亡的分子机制,本试验以云南撒坝猪致病性大肠杆菌HPI阳性株感染猪源巨噬细胞为切入点,从云南楚雄州某规模养殖场采集撒坝猪仔猪黄白痢的粪便,对大肠杆菌进行分离纯化,并通过PCR技术对HPI irp2基因进行检测,分别以HPI阳性株（HPI⁺）和阴性株（HPI^-）感染巨噬细胞,并设立LPS+ATP组和空白对照组,于0.5和6 h收集各组细胞及其上清。应用实时荧光定量PCR法检测不同组Caspase-1、IL-1β和IL-18 mRNA表达水平的变化;应用ELISA检测细胞上清pro-IL-1β、pro-IL-18、IL-1β和IL-18含量的变化。结果显示,试验成功分离得到致病性大肠杆菌,经PCR检测成功获得HPI irp2基因阳性株,经实时荧光定量PCR法检测后发现HPI⁺组、HPI^-组与空白对照组相比,Caspase-1、IL-1β和IL-18 mRNA表达水平均呈上调趋势,且HPI⁺组高于HPI^-组。ELISA检测结果显示,与空白对照组相比,HPI⁺组和HPI^-组pro-IL-1β、pro-IL-18、IL-1β和IL-18的蛋白表达量普遍呈上调趋势,且HPI⁺组均高于HPI^-组。结果表明,云南撒坝猪致病性大肠杆菌HPI可通过上调猪源巨噬细胞中Caspase-1、IL-1β和IL-18 mRNA的表达量促进猪源巨噬细胞炎性因子IL-1β和IL-18的释放,诱发炎症,最终促进猪源巨噬细胞发生细胞焦亡。     

The pathogenesis and immunology of bluetongue virus infection of ruminants

Bluetongue (BLU) virus is transmitted from infected to susceptible ruminants by hematophagous vector midges (Culicoides species). Cattle are important reservoir hosts of the virus because infection typically is asymptomatic and characterized by prolonged cell associated viremia, and because at least some species of insect vector preferentially feed on cattle. Interaction of BLU virus with the cell membrane of erythrocytes in infected cattle likely facilitates both prolonged viremia as well as infection of the insect vector. BLU disease is most common in sheep and some wildlife species. A variety of host, agent and environmental factors clearly can influence expression of disease in these species. The pathogenesis of BLU virus infection of cattle and sheep is remarkably similar, thus the basis for expression of disease in sheep but not cattle remains to be firmly established. Some difference in susceptibility of endothelial cells to infection in the two species is one potential explanation.

Ruminants develop a variety of antiviral responses after BLU virus infection. Antibodies to outer capsid protein VP2 are responsible for virus neutralization, and confer resistance to reinfection with the homologous serotype of BLU virus. Antibodies to epitopes on proteins which are common to all viruses of the BLU serogroup form the basis of current diagnostic serologic tests. Cell mediated responses have been incompletely characterized, in part because BLU virus replicates within dividing lymphocytes and virus-mediated cytolysis inhibits in vitro blastogenesis. Immunological competence of ruminants to BLU virus arises prior to midgestation, and suggestions that persistent immune tolerant BLU virus infection occurs after in utero exposure of cattle have not been substantiated and are not consistent with recent findings.     

Distribution and activation of T-lymphocyte subsets in tuberculous bovine lymph-node granulomas

The immune response against mycobacterial infections is dependant upon a complex interaction between T lymphocytes and macrophages in the context of the granuloma. For this study, we performed the analysis of 18 stage I or II, and 13 stage III or IV granulomas found in lymph nodes from 8 experimentally and 2 naturally infected cattle. T-cell subpopulations (CD3(+), CD4(+), CD8(+), WC1(+), CD25(+)) were investigated by immunohistochemistry. In the majority of stage I/II lesions, CD8(+) and CD25(+) cells were predominantly found in the lymphocytic outer region of the granuloma, suggesting a possible role for activated CD8(+) cells in the initial attempt to restrain the granuloma growth. CD4(+) T cells appeared equally distributed in the lymphocytic mantle and in the internal areas of the granulomas. WC1(+) cells appeared interspersed among the macrophages. We speculated that this could indicate a role for these 2 subsets in the maintenance and the maturation of the granuloma. In stage III/IV lesions, all of the T-cell subsets investigated appeared interspersed among the mononuclear component of the granulomas. In general terms, there was a higher density of CD8(+) cells compared with CD4(+) cells. However, there was no sense of rimming effect for any of the investigated cell populations.     

柠檬苦素对小鼠卵母细胞体外成熟及其体外受精胚胎发育的影响

【目的】研究柠檬苦素(limonin,Lim)对小鼠卵母细胞体外成熟(IVM)及后续体外受精(IVF)胚胎发育潜能的影响,旨在为体外成熟培养系统的优化提供参考。【方法】在小鼠体外成熟培养液中添加不同浓度的Lim(0、10、20、50 μmol/L),成熟培养12 h后统计小鼠卵母细胞第一极体(PBI)排出率,筛选体外成熟培养液中添加Lim的最适浓度;在体外成熟培养液中添加最适浓度的Lim,以0 μmol/L Lim为对照组,成熟培养12 h,通过免疫荧光染色检测活性氧(ROS)、谷胱甘肽(GSH)以及线粒体膜电位(MMP)水平;通过实时荧光定量PCR检测卵母细胞抗氧化及凋亡相关基因的mRNA表达水平。将最适Lim组及对照组卵母细胞体外成熟24 h后进行体外受精,于体外受精24 h和3.5 d分别统计胚胎卵裂率和囊胚率,并用Fluorescein-dUTP和Hoechst 33342染色分别检测囊胚总细胞数及囊胚内凋亡细胞比率。【结果】与0 μmol/L Lim组相比,20 μmol/L Lim组小鼠卵母细胞PBI排出率显著升高(P＜0.05),后续试验均用20 μmol/L Lim进行处理。与对照组组相比,20 μmol/L Lim组小鼠卵母细胞内ROS水平显著降低(P＜0.05),GSH、MMP水平均显著增加(P＜0.05),抗氧化相关基因(GPx3、CAT和Prdx3)、抗凋亡相关基因(Bcl-2、Bcl-xl)表达水平均显著上调(P＜0.05),促凋亡相关基因(Caspase-3)表达水平显著下调(P＜0.05);体外受精胚胎的卵裂率、囊胚率、囊胚总细胞数均显著增加(P＜0.05),囊胚内细胞凋亡比率显著下降(P＜0.05)。【结论】在体外成熟培养液中添加20 μmol/L Lim可以通过抑制氧化应激和细胞凋亡、增加MMP水平提高小鼠卵母细胞质量,从而提高体外受精胚胎的发育潜力。     

Survival of Mycobacterium avium subsp. paratuberculosis in bovine monocyte-derived macrophages is not affected by host infection status but depends on the infecting bacterial genotype

In this study we investigated the ability of different Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) strains to survive in bovine monocyte-derived macrophages (MDMs) of cows naturally infected with M. paratuberculosis and control cows. We tested the hypotheses that infection status of cows affects macrophage killing ability and that survival of M. paratuberculosis in macrophages is dependent on the strain. Peripheral blood mononuclear cells (PBMC) were obtained from Johne's disease-positive (n = 3) and age and stage of lactation matched Johne's disease-negative (n = 3) multiparious cows. Following differentiation, MDMs were challenged in vitro with four M. paratuberculosis strains of different host specificity (cattle and sheep). Two hours and 2, 4, and 7 days after infection, ingestion, and intracellular survival of M. paratuberculosis strains were determined by fluorescence microscopy. There was no effect of the origin of MDMs (Johne's disease-positive or control animals) on phagocytosis, survival of bacteria, or macrophage survival. In contrast, important strain differences were observed. These findings suggest that some M. paratuberculosis strains interfere more successfully than others with the ability of macrophages to kill intracellular pathogens which may make it important to include strain typing when designing control programs.     

Killing of Brucella antigen-sensitized macrophages by T lymphocytes in bovine brucellosis

The present study was an investigation into the role of T lymphocytes in the killing of antigen-sensitized macrophages (MΦ) in bovine brucellosis. Following confirmation of bovine T lymphocyte cell lines derived from Brucella abortus Strain 19 vaccinated steers as antigen-specific in proliferation studies using various antigens, we adapted an apoptosis assay for evaluation of cytotoxicity by these bovine T cells against autologous monocyte-derived macrophages (MDMΦ) as target cells. Various B. abortus antigen preparations were tested including whole γ-irradiated B. abortus bacteria (γBA), a soluble cytosolic protein fraction and a membrane-associated protein fraction. Both polyclonal and cloned T lymphocyte cell lines exhibited cytotoxicity against MDMΦ targets in an antigen-specific fashion. Polyclonal and cloned T lymphocyte cell lines demonstrated cytotoxic responses to varying degrees against B. abortus antigens regardless of whether the antigen used was whole nonviable bacteria, a soluble protein extract or a membrane-associated fraction of extracted bacteria. To further develop correlation of these responses to an in vivo host defense mechanism, cytotoxicity was evaluated using target cells that had been infected with live B. abortus S19 or B. abortus Strain 2308. Cytotoxic responses were also demonstrated consistently against infected targets with either strain of B. abortus although in most cases, cytotoxicity was higher against target cells sensitized with γBA compared to those infected with live bacteria. Cloned T lymphocyte cell lines were all CD4⁺, CD8⁻ cells indicating that the observed cytotoxic responses were most likely due to an inflammatory T_h1 response and may represent an important host defense mechanism induced by vaccination with live attenuated strains of B. abortus in cattle.     

Effects of midazolam on equine innate immune response: a flow cytometric study

Benzodiazepines (BDZ) are among the most frequently used class of psychotropic drugs employed in veterinary medicine in Brazil and worldwide due to their anxiolytic, muscle relaxant and anticonvulsant effects [J. Clin. Pharmacol. 33 (1993) 124]. Peripheral benzodiazepine receptor (PBR) sites were described in peripheral organs, endocrine steroidogenic tissues and immune organs and cells. Midazolam is a mixed-type agonist of PBRs. The present study is focused on the effects of midazolam on equine peripheral blood neutrophils, peritoneal macrophages and cortisol levels in plasma. Adult horses were treated with a single dose of midazolam (0.06 or 0.1 mg/kg) or with 0.9% NaCl. Immune cells were collected 24 h after treatment for flow cytometry analysis of Staphylococcus aureus-induced phagocytosis and oxidative burst. Plasma cortisol concentration was measured 30, 90, 180 and 360 min after midazolam treatment. Midazolam induced a dose-dependent reduction on: (1) peripheral blood neutrophil and peritoneal macrophage oxidative burst; (2) the capacity of both peripheral blood neutrophils and peritoneal macrophages to phagocyte S. aureus. Increments on plasma cortisol concentration were not found after 0.06 and 0.1 mg/kg of midazolam. The effects on oxidative burst of neutrophils and macrophages from horses treated with midazolam were interpreted as a consequence of an impairment of S. aureus-induced phagocytosis. The present data suggest that midazolam, most probably acting on PBRs present on equine macrophage and neutrophil membranes, might have changed some mechanisms related to both phagocytosis and oxidative burst. These results support the use of flow cytometry to identify functional properties and dysfunction of equine immune cells. They also confirm the notion that changes in the functional capacity of the immune system may represent an important hazard of exposure to drugs or chemicals.     

感染鲤浮肿病毒镜鲤的组织病理变化及病毒分布规律研究

【目的】 研究感染鲤浮肿病毒（Carp edema virus,CEV）镜鲤的组织病理特征及CEV在各组织中的分布规律,以期为鲤浮肿病的诊断和防控提供参考。【方法】 利用HE染色观察发病镜鲤心脏、肝脏、皮肤、脾脏、肾脏、脑、鳃组织的病理变化情况;用免疫组织化学法检测CEV在镜鲤各组织的分布和蛋白表达情况;利用CEV P4a基因保守区域设计3条探针,于5'-端标记FAM进行原位杂交,检测CEV在镜鲤各组织的核酸分布情况;实时荧光定量PCR检测感染镜鲤各组织的病毒载量。【结果】 HE染色观察发现,患病镜鲤的鳃丝肿胀、充血,鳃丝间有脱落堆积的红细胞和炎性细胞,肝脏细胞深染萎缩,胆小管出血,脾脏组织出现明显间隙并伴有出血,肾脏组织充血明显,肾小管有明显闭合现象。免疫组织化学结果显示,在患病鱼的鳃和肝脏组织中大量表达CEV P4a蛋白,在其他组织中也有少量表达。原位杂交结果显示,CEV核酸大量存在于鳃组织中,在心脏、脾脏和肾脏中均有少量阳性信号。实时荧光定量PCR结果显示,鳃组织病毒载量极显著高于心脏、肝脏、皮肤、脾脏、肾脏、脑组织（P<0.01）。【结论】 感染CEV后,镜鲤内脏组织出现不同程度充血、出血,病毒主要分布在鳃,说明鳃是CEV增殖的最主要靶器官。