猪sCD2对其天然配体及SRBC培养上清液促淋转效应的影响

以２、４、１６、３２倍稀释猪血清ｓＣＤ２或５、１０、１００、５００倍稀释猪ＰＢＭＣｓＣＤ２，分别替代最优水平组合中的ｓＣＤ２，进行淋转抑制试验。试验结果提示，２、４倍稀释猪血清ｓＣＤ２或５、１０倍稀释猪ＰＢＭＣｓＣＤ２，分别对猪血清ｓＣＤ２配体或ＳＲＢＣｓＣＤ２配体促淋转及协同ＰＨＡ－Ｐ促淋转效应有显著或极显著的掏作用（Ｐ〈０．０５或Ｐ〈０．０１）。在ＳＲＢＣ培养上清液和猪血清中可能存在一定量的ｓ     

雏鸡马立克氏病强毒感染后淋巴细胞化学发光的变化

本研究采用化学发光（ＣＬ）法测定雏鸡马立克氏病强毒（ｖＭＤＶ）人工感染后免疫器官淋巴细胞活性氧自由基（ＡＯＲ）产生水平。结果为：脾脏淋巴细胞ＣＬ值于７、１４和５６日龄显著高于健康对照雏鸡（Ｐ＜０．０５，Ｐ＜０．０１）；胸腺淋巴细胞ＣＬ值４２和５６日龄显著高于健康对照雏鸡（Ｐ＜０．０５）；法氏囊淋巴细胞ＣＬ值１４、２８和４２日龄显著高于健康对照雏鸡（Ｐ＜０．０５）。     

仔猪断奶应激对血液和生化的影响

随机选取 １０ 头健康的４２～４５ 天断奶长白仔猪,分别于断奶时、断奶后７ 天、１４ 天上午采血,检验血液学、血清生化等多项指标,结果如下：仔猪血清 Ａ Ｓ Ｔ、 Ｃ Ｋ、 Ｌ Ｄ Ｈ 活性在断奶后 ７ 天时都升高,其中 Ａ Ｓ Ｔ、 Ｌ Ｄ Ｈ活性与断奶时相比差异极显著（ Ｐ＜ ００１）;血清 Ａ Ｍ Ｙ Ｌ 活性,断奶后均比断奶时高,但无统计学上差异（ Ｐ＞００５）;血清 Ｔ Ｐ、 Ｇ Ｌ Ｏ Ｂ、 Ａ Ｌ Ｂ、 Ｂ Ｕ Ｎ、 Ｃ Ｈ Ｏ Ｌ 含量在断奶后极显著或显著降低（ Ｐ＜ ００１ 或 Ｐ＜ ００５）; Ｇ Ｌ Ｕ、 Ｃ Ｒ Ｅ Ａ、 Ｔ Ｃ Ｏ２ 含量降低,但无统计学上差异（ Ｐ＞ ００５）。血液 Ｒ Ｂ Ｃ、 Ｈ Ｇ Ｂ、 Ｈ Ｃ Ｔ、 Ｍ Ｃ Ｖ 断奶后极显著降低（ Ｐ＜００１）, Ｍ Ｃ Ｈ Ｃ 断奶后极显著升高（ Ｐ＜ ００１）; Ｗ Ｂ Ｃ、 Ｐ Ｌ Ｔ 等断奶后虽有变化,但均无统计学上差异（ Ｐ＞００５）。结果表明,仔猪在断奶后一段时间,处于营养缺乏性单纯小红细胞性贫血状态。     

内毒素休克时山羊血液流变性的变化及山莨菪碱对其影响

本文通过给山羊静注大肠杆菌内毒素诱导内毒素休克,探讨内毒素休克时血液流变性的变化规律,并观察山莨菪碱（６５４－２）对其影响。结果表明,山羊内毒素休克时,低切率全血比粘度（ＬＢＶ）和低切率全血还原比粘度（ＬＢＲＶ）明显升高（Ｐ＜０．０５）,血浆比粘度（ＰＶ）和红细胞聚集指数（ＡＩ）均显著增高（Ｐ＜０．０１,Ｐ＜０．０５）,红细胞变形能力（ＲＣＤ）显著下降（Ｐ＜０．０５,Ｐ＜０．０１）,当静注内毒素前１０ｍｉｎ给予６５４－２（２．５ｍｇ／ｋｇ）山羊的ＬＢＶ和ＬＢＲＶ在１～５ｈ显著高于对照组,５ｈ前ＰＶ和ＡＩ值比对照组有明显升高,第７ｈ后与对照组间无明显差异（Ｐ＞０．０５）,且显著低于休克组（Ｐ＜０．０５）,其ＲＣＤ亦趋于正常。提示山羊内毒素休克时血液流变参数明显改变,血液粘度增加,红细胞聚集加剧,而应用６５４－２具有显著改变血液状态,缓解微循环障碍发生。     

放血对家兔免疫功能的影响

通过测定放血家兔的免疫指标显示：谢血家兔淋巴细胞转化率及ＲＢＣ－ＣＲ１花环率较放血前及对照组有显著提高（Ｐ≤０．０５）；ＲＢＣ－ＩＣ花环率与对照组相比无显著性差异（Ｐ〉０．０５）；血细胞数量和血红蛋白含量，其放血前后均无变化。表明通过谢血确能显著提高淋巴细胞反应能力和细胞膜上Ｃ３ｂ受体活性，增强其免疫吸附作用。     

用免疫组织化学染色法检测猪生殖-呼吸道综合征病毒(PRRSV)抗原的研究

本试验用抗 Ｐ Ｒ Ｒ Ｓ Ｖ 单克隆抗体（ Ｓ Ｄ Ｏ Ｗ １７）建立了检测猪体内 Ｐ Ｒ Ｒ Ｓ Ｖ 抗原的免疫组化染色法,主要步骤：组织块在１００ ｍ Ｌ／ Ｌ中性缓冲液福尔马林中固定２４～４８ ｈ→常规脱水、石蜡包埋、切片４～５μｍ →二甲苯脱蜡→１００％ 酒精→１０ ｍ Ｌ／ Ｌ盐酸酒精 ３０ ｍ ｉｎ（消除内源性过氧化物酶）→９５％ 酒精→８５％ 酒精→７５％ 酒精→蒸馏水→０．０１ ｍ ｏｌ／ Ｌ Ｐ Ｂ Ｓ→１０％ 马血清３７ ℃３０ ｍ ｉｎ 后转４ ℃过夜→０．０１ ｍ ｏｌ／ Ｌ Ｐ Ｂ Ｓ３×５ ｍ ｉｎ→生物素化马抗鼠 Ｉｇ Ｇ（１∶２００） ３７ ℃ ６０ ｍ ｉｎ→０．０１ ｍ ｏｌ／ Ｌ Ｐ Ｂ Ｓ３× ５ ｍ ｉｎ→辣根酶标记链亲和素（１∶２００）３７℃ ６０ ｍ ｉｎ→０．０１ ｍ ｏｌ／ Ｌ Ｐ Ｂ Ｓ３×５ ｍ ｉｎ→新鲜配制的 Ｄ Ａ Ｂ室温下显色 ５～１５ ｍ ｉｎ→０．０１ ｍ ｏｌ／ Ｌ Ｐ Ｂ Ｓ洗→常规脱水、透明、封片。经对８ 例试验感染 Ｐ Ｒ Ｒ Ｓ Ｖ 仔猪各种组织内 Ｐ Ｒ Ｒ Ｓ Ｖ 抗原的检测,本法具有简单、快速、特异性强,结果清晰、稳定及重复性好等特点,是检测组织内 Ｐ Ｒ Ｒ Ｓ Ｖ 抗原的一种好方法。     

鸡马立克氏病毒致鸡淋巴细胞糖皮质激素受体改变的特性

研究证明，鸡感染马立克氏病毒（ＭＤＶ）后，外周血淋巴细胞（ＰＢＬ）糖皮质激素受体（ＧＲ）含量减少，其减少程度随病情的发展而加重，不因攻毒后时间的延伸而改变，提示鸡马立克氏病（ＭＤ）的发生与发展，与病毒所致ＰＢＬ的抗应激能力降低有关；ＭＤ肿瘤组织的ＧＲ含量较正常鸡ＰＢＬ的减少４０％；而含瘤病鸡的ＰＢＬ ＧＲ含量与之基本相同（Ｐ＞０．０５），说明发生肿瘤的ＭＤ鸡，其ＰＢＬ与瘤组织细胞的变化程度相似；Ｍ     

鸡减蛋综合征病毒DNA结合蛋白的基因结构及同源分析

从中国发病鸡群中分离的鸡减蛋综合征病毒（ＥＤＳＶ）ＡＡ－２株，经常规方法提取其病毒核酸后，构建了限制性内切酶ＰｓｔⅠ及ＨｉｎｄⅢ水解片段的基因文库。对其中ＨｉｎｄⅢ－Ｆ片段（５２．９～５８．９ｍｕ）的正反２条链的序列测定发现，其反链存在１个编码容量为３８７个氨基酸（ａａ）的开放读码框架（ｏｐｅｎｒｅａｄｉｎｇｆｒａｍｅ，ＯＲＦ），经Ｇｅｎｅｂａｎｋ／ＥＭＢＬ同源搜寻后证实其编码产物为ＥＤＳＶ的ＤＮＡ结合蛋白（ＤＢＰ）。与其他腺病毒ＤＢＰ的氨基酸序列进行同源比较，其Ｎ端同源性在１９．０％～４６．２％之间，Ｃ端同源性在２７．７％～６０．４％之间。腺病毒ＤＢＰ的３个保守序列ＣＲ１，ＣＲ２，ＣＲ３在ＥＤＳＶＤＢＰ中有较大变化，但ＥＤＳＶＤＢＰ的锌指框架（Ｚｎ２＋ｆｉｎｇｅｒｍｏｔｉｆ）却保留了对其功能必需的残基。     

黄芪多糖和香菇多糖对vMDV感染雏鸡淋巴细胞化学发光的影响

１日龄雏鸡人工感染ｖＭＤＶ，并注射黄芪多糖（ＡＰＳ）和香菇多糖（Ｌｅｎ），分别于７、１６、２８、４２和５６日龄检测雏鸡淋巴细胞化学发光（ＣＬ）的变化。结果表明：（１）ＡＰＳ对７、１４日龄ｖＭＤＶ感染雏鸡胸腺、脾脏和法氏囊淋巴细胞的ＣＬ有显著抑制作用（ｐ〈０．０５，ｐ〈０．０１），在２８、４２日龄则显著增强淋巴细胞ＣＬ（ｐ〈０．０５，ｐ〈０．０１）；（２）Ｌｅｎ对７、１４日龄ｖＭＤＶ感染雏鸡胸腺、脾     

西咪替丁对鸡细胞和体液免疫功能的作用

观察了西咪替丁在左旋咪唑在鸡细胞和体液免疫功能的作用。对２８日龄鸡每天分别按２．５、１０．０、１００．０和４００．０ｍｇ／ｋｇ体重西咪替丁或２．５、１０．０ｍｇ／ｋｇ体重左旋咪唑钦水给药,连用３ｄ后再分别肌肉注射绵羊细胞（ＳＲＢＣ）和牛血清白蛋白（ＢＳＡ）或布鲁氏菌（ＢＡ）抗原。测定外周血淋巴细胞转化率和ＣＤ４＾＋／ＣＤ８＾＋淋巴细胞比值;测定血清ＳＲＢＣ、ＢＳＡ、ＢＡ抗体效价和血清补体总活性。结     

Functional impairment of PRRSV-specific peripheral CD3+CD8high cells

The replication of porcine reproductive and respiratory syndrome virus (PRRSV) in lungs and lymphoid tissues of PRRSV-infected pigs is already strongly reduced before the appearance of neutralizing antibodies, indicating that other immune mechanisms are involved in eliminating PRRSV at those sites. This study aimed to determine whether PRRSV Lelystad virus (LV)-specific cytotoxic T-lymphocytes (CTL) can efficiently eliminate PRRSV-infected alveolar macrophages. Therefore, CTL assays were performed with PRRSV-infected alveolar macrophages as target cells and autologous peripheral blood mononuclear cells (PBMC) from PRRSV-infected pigs as a source of PRRSV-specific CTL. PBMC of 3 PRRSV-infected pigs were used either directly in CTL assays, or following restimulation in vitro. CTL assays with pseudorabies virus (PRV) Begonia-infected alveolar macrophages and autologous PBMC, from 2 PRV Begonia-inoculated pigs, were performed for validation of the assays. In freshly isolated PBMC, derived from PRRSV-infected pigs, CTL activity towards PRRSV-infected macrophages was not detected until the end of the experiment (56 days post infection – dpi). Restimulating the PBMC with PRRSV in vitro resulted in proliferation of CD3⁺CD8^high cells starting from 14 dpi. Although CD3⁺CD8^high cells are generally considered to be CTL, CTL activity was not detected in PRRSV-restimulated PBMC of the 3 pigs until 49 dpi. A weak PRRSV-specific CTL activity was observed only at 56 dpi in PRRSV-restimulated PBMC of one pig. In contrast, a clear CTL activity was observed in PRV Begonia-restimulated PBMC, derived from PRV Begonia-infected pigs, starting from 21 dpi. This study indicates that PBMC of PRRSV-infected pigs contain proliferating CD3⁺CD8^high cells upon restimulation in vitro, but these PBMC fail to exert CTL activity towards PRRSV-infected alveolar macrophages.     

Identification of guinea pig gammadelta T cells and characterization during pulmonary tuberculosis

Guinea pigs are an alternative small animal model for many disease studies. Here we describe a pan-gammadelta monoclonal antibody (anti-TCRdelta1) specific for the constant region of human T cell receptor delta chains that cross-reacts with a subpopulation of guinea pig (Cavia porcellus) lymphocytes. The phenotype and distribution of this subpopulation is consistent with the guinea pig gammadelta T cell subset. FACS analysis of fresh PBMC and splenocytes from na?ve guinea pigs revealed the presence of a subset of cells that stained with the anti-TCRdelta1 mAb. The relative percentage of anti-TCRdelta1 positive cells in PBMC and tissues is similar to that described for gammadelta T cells in other species. Immunohistochemistry of tissues also revealed a distribution of anti-TCRdelta1 positive cells consistent with gammadelta T cells. These data are further supported by staining of a polyclonal guinea pig T cell line that became progressively CD4 and CD8 negative in long-term culture. Analysis of PBMC from guinea pigs following aerosol infection with virulent Mycobacterium tuberculosis revealed no apparent changes in the steady-state percentage of blood gammadelta+ T cells. Taken together, these data suggest that the anti-TCRdelta1 antibody recognizes the gammadelta T cell subset in guinea pigs. This reagent may be useful for examining gammadelta T cells in various disease models where the guinea pig is a more desirable model for study.     

Globotriaosylceramide (Gb(3)/CD77) is synthesized and surface expressed by bovine lymphocytes upon activation in vitro

Neutral glycosphingolipids (GSLs) are considered activation markers on human lymphocytes, which are fundamental for studying the immune system. For cattle, only a limited number of activation markers has yet been identified. We recently showed that Shiga toxin 1, known to use globotriaosylceramide (Gb(3) syn. CD77) as a cellular receptor, depresses proliferation of activated bovine lymphocytes [Infect. Immunol. 67 (1999b) 2209]. In order to confirm the expression of Gb(3)/CD77 on bovine lymphocytes, we flowcytometrically examined a bovine B-lymphoma cell line (BL-3) and bovine peripheral blood mononuclear cells (PBMC) before and after mitogenic stimulation and biochemically characterized neutral GSLs extracted from PBMC. CD77 was detected on the surface of BL-3 cells and cultured PBMC essentially after mitogenic stimulation. Although expressed by all PBMC subpopulations identified, the portion of CD7+ cells was highest for BoCD8+ cells, followed by B-cells and BoCD4+ cells at day 4 of cultivation. Ceramide trihexoside of stimulated PBMC was structurally determined as Gal(alpha1-4)Gal(1-4)Glc(1-1)ceramide (Gb(3)). Biochemically, Gb(3) was also detected within unstimulated PBMC which contained ceramide monohexoside (CMH) and Gb(3) in a ratio of about 4:1. However, stimulation induced an increase of CMH and Gb(3) by a factor of 2.5 and 10, respectively, implicating that bovine lymphocytes regulate surface expression of Gb(3)/CD77 predominantly by quantitative changes in the Gb(3) metabolism. This report presents Gb(3)/CD77 as the first GSL identified on bovine immune cells and highly recommends this activation dependent antigen as a useful tool to investigate lymphocyte activation within the bovine immune system.     

Effect of synthetic agonists of toll-like receptor 9 on canine lymphocyte proliferation and cytokine production in vitro

Synthetic agonists of TLR9 containing novel DNA structures and R'pG (wherein R=1-(2'-deoxy-beta-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine) motifs, referred to as immune modulatory oligonucleotides (IMOs), have been shown to stimulate T(H)-1-type-immune responses and potently reverse allergen-induced T(H)-2 responses to T(H)-1 responses in vitro and in vivo in mice. In order to investigate the immunomodulatory potential of IMOs in dogs, canine peripheral blood mononuclear cells (PBMC) from healthy dogs were stimulated with three different IMOs and a control IMO, alone or in combination with concanavalin A (ConA). Lipopolysaccharide (LPS) was used as a positive control for B lymphocyte activation. Carboxyfluorescein diacetate succinimidyl ester and phenotype staining was used to tag proliferating T and B lymphocytes (CD5(+) and CD21(+)) by flow cytometry. Real-time PCR and ELISA were processed to assay cytokine production of IFN-gamma, IL-10, TGF-beta, IL-6 and IL-10. Like LPS, IMOs alone induced neither proliferation of CD5(+) T cells nor CD21(+) B cells, but both LPS and IMO had the capacity to co-stimulate ConA and induced proliferation of B cells. In combination with ConA, one of the IMOs (IMO1) also induced proliferation of T cells. IMO1 also significantly enhanced the expression of IFN-gamma on the mRNA and protein level in canine PBMC, whereas expression of IL-10, TGF-beta and IL-4 mRNAs was not induced by any of the IMOs. These results indicate that in canine PBMC from healthy dogs, IMO1 was able to induce a T(H)-1 immune response including T- and B-cell proliferation.     

Colostrum induced phenotypic and trafficking changes in maternal mononuclear cells in a peripheral blood leukocyte model for study of leukocyte transfer to the neonatal calf

The relationship between the colostral environment and the function of leukocytes in colostrum is not clearly defined. This study examined the effects of defatted, acellular colostrum (AC) on the phenotype of peripheral blood mononuclear cells (PBMC) and their capacity to enter the circulation of neonatal calves after ingestion as a model of this relationship. Maternal PBMC were exposed to medium alone or medium supplemented with 25% AC. Expression of CD11a, CD11b, CD11c, CD43, CD49d, CD49e, and CD62L was assessed on freshly isolated and treated PBMC. Exposure to AC increased the percentage of cells expressing CD11a, CD11c and CD43, but decreased the percentage of cells expressing CD62L relative to freshly isolated PBMC. The density of expression of CD11b and CD11c was reduced, but increased for CD43 after exposure to AC relative to freshly isolated PBMC. Density of CD62L expression and percentage of cells expressing CD11a and CD43 were significantly different for cells treated with AC relative to medium alone. Further, these changes could not be attributed to occult bacterial contamination of the AC, as treatment of PBMC with LPS in the same medium yielded none of the observed changes. Maternal PBMC (treated as described) were labeled with the fluorescent tracer, PKH26-GL, and fed to neonatal calves within 6 h of birth. The circulation of these cells in the neonate was monitored by flow cytometry. We observed that: (1) cells exposed to AC, but not medium alone, entered the circulation; (2) peak trafficking occurred 12-24 h after ingestion; (3) a large fraction of labeled cells appeared in the neonatal circulation; and (4) labeled cells disappeared from circulation by 36 h after ingestion. This study indicates that exposure to the colostral environment induced phenotypic changes facilitating trafficking of colostral cells into the neonate.     

Molecular cloning and expression analysis of pig CD81

CD81, also known as TAPA-1 (target of antiproliferative antibody 1), is a member of the tetraspanin family of proteins and a component of the B cell co-receptor complex. Several studies have shown that CD81 plays significant roles in a variety of immune responses, including activation of B cells and T cells. In this study, we cloned pig Cd81 cDNA using RT-PCR coupled with rapid amplification of cDNA ends (RACE)-PCR and determined the complete cDNA sequence of pig Cd81. Pig Cd81 cDNA contains an open reading frame (711 bp) encoding 236 amino acids. The identity of pig CD81 with those of human, cattle, rat, and mouse are 90.30%, 92.26%, 86.22%, and 86.22%, respectively. Alignment of the CD81 amino acid sequence with those of mammalian species showed that the large extracellular loop (LEL) is the most divergent, whereas other domains are largely conserved. Pig Cd81 mRNA was detected by RT-PCR in a broad range of tissues, including lymphoid tissues as well as nonlymphoid tissues, indicated variety of cellular functions of CD81 in most pig tissues. Flow cytometry analyses demonstrated that human CD81 antibody recognizes a pig CD81 on the cell surface. Further, immunohistochemistry analysis using human CD81 antibody on pig spleen was revealed that CD81 expression is widely diffused in spleen tissue. Future study will be focused on defining the functional role of CD81 during the course of pig infectious diseases.     

不同品种育成猪基线免疫性状的比较

为了比较不同品种育成猪基线免疫指标的差异,研究选用巴马香猪、本地黑猪和杜长大3个品种育成猪,采用显微镜平板计数法计数红、白细胞总数;血涂片法测定白细胞分类计数;EA花环形成试验检测B淋巴细胞的百分率;流式细胞仪检测T淋巴细胞亚群百分率。结果表明:巴马香猪红细胞总数(RBC)、中性粒细胞百分率(GR%)、嗜酸性粒细胞百分率(EO%)以及T淋巴细胞亚群CD4+、CD8+百分率(CD4+%、CD8+%)等5项指标与本地黑猪、杜长大t检验差异显著(P<0.05),其中RBC和GR%显著高于本地黑猪和杜长大,EO%以及CD3+%、CD4+%、CD8+%显著低于本地黑猪和杜长大(P<0.05)。B淋巴细胞百分率(BLY%)显著低于本地黑猪,嗜碱性粒细胞(BASO%)显著低于杜长大,而白细胞总数(WBC)、淋巴细胞百分率(LY%)、单核细胞百分率(MO%)和CD4+/CD8+等4项指标与本地黑猪、杜长大基本一致。对本地黑猪与杜长大进行比较发现,WBC与BYL%2项差异显著。说明品种是影响基线免疫性状的一个重要因素,但对同一品种猪,各项指标间没有相关性。     

Expression of Recombinant bovine L-selectin in Escherichia coli and insect cells.

Bovine L-selectin was expressed in bacteria using pGEX vector and in insect cells infected with recombinant baculovirus in order to obtain recombinant protein for preparation of specific antiserum and its functional studies. In bacterial expression, L-selectin fusion protein with glutathione S-transferase was detected in the insoluble fraction with the expected molecular weight of 60 kDa by SDS-PAGE and reacted with anti-bovine CD62L monoclonal antibody in immunoblot analysis. In insect cells infected with recombinant baculovirus, a band corresponding to L-selectin was not observed in SDS-PAGE with protein staining, but they apparently reacted with anti-bovine CD62L monoclonal antibody in immunoblot analysis. Furthermore, the indirect immunofluorescence test revealed that bovine L-selectin was efficiently expressed on the surface of Sf9 cells infected with recombinant baculovirus, and flow cytometric analysis showed that the percentage of CD62L positive cells in bovine PBMC was about 66% and that most Sf9 cells infected with recombinant baculovirus had specific immunofluorescence.