Ontogeny of IgM-producing cells in the mandarin fish Siniperca chuatsi identified by in situ hybridisation

The ontogeny of IgM-producing cells was studied in juvenile mandarin fish Simperca chuatsi, an important fish in China's aquaculture sector. The IgM-producing cells were localised through in situ hybridisation with a probe complementary to the Ig mu-chain in lymphoid-related tissues, including head kidney, spleen, thymus, intestine and gills. In head kidney, transcripts of Ig mu were first detected at 20 days post-hatching (dph) with a few positive signals. and the number of IgM-producing cells increased obviously from 39 dph onwards. At 136 dph, a large amount of positive cells were observed in the entire organ with clusters of these cells located around the blood vessels. In spleen, IgM-producing cells were found from 26 dph onwards, followed by an increase until 67 dph: clusters of positive cells were also detected around blood vessels at 102 dph. In thymus, IgM-producing cells were first observed at 39 dph; thereafter, no obvious increase was detected until 78 dph. The positive cells in thymus were distributed mainly in the outer zone of thymus. A few IgM-producing cells were still observed in thymus of 1-year-old mandarin fish. IgM-producing cells were not detected in the intestine until 87 dph, with several discrete positively stained cells distributed in the lamina propria. IgM-producing cells, scattered mainly in primary gill filaments around blood vessels, were detected in gills from 90 dph. As in other teleosts, these results indicated that the head kidney appears to be the primary organ for IgM production in mandarin fish, and IgM-producing cells exist in all organs examined in the present study, implying their lymphoid role in fish. In addition, it is suggested that vaccination after 20 dph may be much more effective in mandarin fish. (C) 2009 Elsevier B.V. All rights reserved.

IgM, IgD and IgY and their expression pattern in the Chinese soft-shelled turtle Pelodiscus sinensis

Three Ig isotypes, IgM, IgD, and IgA, were previously known in reptiles. Here, in this report we describe IgM, IgD and a novel immunoglobulin heavy-chain isotype upsilon (IgY) in Chinese soft-shelled turtle (Pelodiscus sinensis). The IgM and IgY constant domains are characteristically similar to their counterparts described in other vertebrates. The expression of IgM and IgD were detected at mRNA level early during embryonic development, and their expression increased during further development. However, the IgY expression was not detected in larval turtles until 90 days after hatching-out. The increase in the transcription of these three Ig molecules was analyzed by using real-time PCR in spleen, kidney and blood following the injection of inactivated Aeromonas hydrophila. The primary increase in the expression of these three Igs was observed I week after the first injection, although not statistically significant, and the second injection 2 weeks after the first injection provoked a significant increase in the expression of these Igs, revealing a pattern of primary and secondary antibody response in the turtle. The present study represents the first report on reptile IgY and the pattern of IgM, IgD and IgY transcription in reptiles. (C) 2009 Elsevier Ltd. All rights reserved.

Distribution of IgM, IgD and IgZ in mandarin fish, Siniperca chuatsi lymphoid tissues and their transcriptional changes after Flavobacterium columnare stimulation

The gene sequences of three different immunoglobulin (Ig) heavy chains, namely IgM, IgD and IgZ, were cloned from mandarin fish (Siniperca chuatsi) recently. In this study the distribution of these three kinds of Ig-producing cells in lymphoid-related tissues as head kidney, spleen, gill and intestine were investigated by using in situ hybridization, and their transcriptional changes were also analyzed by quantitative real-time PCR during 8 weeks after immunization. IgM-producing cells could be detected obviously and abundantly in all the tissues examined. A few numbers of IgD and IgZ positive cells were both detected in head kidney and spleen. IgZ positive cells could be detected in gill moderately while IgD showed negative results, otherwise no IgD or IgZ positive cells could be detected in intestine. After stimulated with bacterial pathogen Flavobacterium columnare G(4), the transcripts of these three Ig genes exhibited quite different kinetics. Significantly increased transcription of IgM gene was observed in almost all the tissues examined especially in boosted group. In contrast with IgM, seldom strong increase was examined for IgD and IgZ genes. For IgD, it seemed that the first injection could stimulate the immune response easier, since in almost all the tissues significant increase was detected at 1 or 2 weeks after injection. For IgZ, boosted injection could not enlarge the up-regulation of gene expression of first injection. This is the first case to report the transcriptional kinetics of three Ig genes in teleost after bacterin immunization. (C) 2008 Elsevier B.V. All rights reserved.

猪-猕猴延迟性异种移植排斥反应的发生机制

　目的:探讨猪2猕猴延迟性异种移植排斥反应(DXR) 的发生机制。方法:建立湖北白猪2云南猕猴的腹腔异位心 脏移植模型,应用中华眼镜蛇毒因子(Y2CVF) 完全清除受者体内补体,并应用环孢素A(CsA) 、环磷酰胺(CTX) 和甲泼尼龙(M. P) 三联免疫抑制治疗。检测血清C3、C4、抗猪内皮细胞天然抗体,免疫组化方法染色检测移植物中C3、C5b29、IgG、IgM、细胞间 黏附分子21 ( ICAM21) 、肿瘤坏死因子2α(TNF2α) 、单核巨噬细胞(CD68) 、NK细胞(CD57) 、CD4 + T 细胞和CD8 + T 细胞的表达。 结果:移植心存活时间分别为8、10、13 和13 天,血清C3 和补体总活性均下降为0 ,抗猪内皮细胞天然抗体水平在移植后则有 一个更为明显的下降,在移植心失功前2～4 天开始天然抗体稍有回升,但较术前正常时仍明显偏低。移植心有程度不等的 C3、C4、C5b29、IgG及IgM 沉积,大量的单核细胞(50 %) ,少量的NK细胞(8 %～10 %) 、CD4 + T 细胞(15 %) 和CD8 + T 细胞 (25 %) 。移植物血管内皮细胞表面出现ICAM21 的表达上调,移植物间质中出现TNF2α的表达增加。结论:体液免疫和细胞免 疫参与猪2猕猴DXR 排斥反应的发生。

猪到猕猴异种心脏移植超急性排斥反应的免疫学及病理学变化

　观察猪到猕猴异种心脏移植超急性排斥反应时的免疫学及病理学变化。方法　 采用猪到猕猴腹腔内异位心脏移植模型,检测发生超急性排斥反应者的血液中补体、天然抗体及T 淋巴细胞亚群的变化,并对移植心脏进行免疫组化(测定C3 、C4 、C5b29 、IgG及IgM 的沉积) 及病理学 分析。结果　发生超急性排斥反应时,血清补体C3 、C4 的含量、总补体活性及抗猪内皮细胞天然抗 体均有一定程度的下降;CD4 + / CD8 + T 淋巴细胞的比率也有所下降;移植心脏中均有补体C3 、C4 、 C5b29 的沉积, IgG及IgM 也均有沉积,但IgG和IgM 沉积强度的差异无统计学意义;病理学改变主 要为心肌间质弥漫性出血、水肿,毛细血管内普遍淤血。结论　补体通过经典途径激活参与猪到猕猴 异种心脏移植超急性排斥反应;超急性排斥反应时受者血中天然抗体水平明显下降;CD4 + T 淋巴细 胞可能参与异种移植超急性排斥反应过程并有所消耗;发生超急性排斥反应的移植物突出病理表现 为间质出血。

人精子抗原决定簇的末端单糖参与小鼠IgA单抗与抗原的结合

对消化道免疫获得的33个抗精子IgA单抗, 12个IgG和35个IgM单抗靶抗原的生化性质及末端单糖做了鉴定。免疫印迹的结果显示, IgA、IgG和IgM类单抗靶抗原的分子量范围分别为10-89、11-75和12-94KDa。有12个单抗的靶抗原为非蛋白类的糖复合物, 一个IgA单抗(A22)的靶抗原为不含糖的蛋白。凝集素封闭和糖苷酶消化试验的结果显示, 98.7%单抗的靶抗原分子末端含一种或几种糖。五种凝集素对IgA类单抗靶抗原的抗原的封闭效应均较强, 表明IgA类单抗靶抗原的抗原决定簇含有岩藻糖、乙酰氨基葡萄糖、乙酰氨基半乳糖、半乳糖和甘露糖等末端单糖者较多。IgG类单抗靶抗原的抗原决定簇则含有带岩藻糖、乙酰氨基半乳糖和#alpha#-甘露糖等末端单糖者较多。内切-#beta#-半乳糖苷酶消化试验的结果表明, 54.4%IgA类单抗的靶抗原为依赖半乳糖苷连接的糖肽化合物。这些结果表明, 经消化道免疫能产生IgA及其它类别抗体的绝大多数人精子抗原为含多种类型末端单糖的膜表面分子。

Effects of testosterone undecanoate as a male contraceptive candidate on rat immunological features

Testosterone undecanoate (TU) is under phase III clinical trial as a hormonal male contraceptive in China. Sex hormones can modulate the immune system. Female hormonal contraceptives may affect SIV/HIV-1 transmission. To evaluate the safety of TU and to understand whether long-term use of TU for a male contraceptive affects users' immunological features, adult male rats were treated for a 32-week TU-treated phase at the dose of 20 mg TU/kg body weight and a 24-week recovery phase. The reproductive and immunological parameters of 4-6 rats in each subgroup were examined at the stated time point. The mean sperm count and viability in the treated rats were significantly suppressed (p < 0.01). In the TU-treated group: the mean blood leukocyte and lymphocyte counts; the proliferation indexes of T cells from peripheral blood mononuclear cells (PBMC) and spleen; and, of B cells from spleen, as well as the mean counts of blood T, NK, and B cells decreased in comparison with those of control group. These decreases were not significant (p > 0.01). Similarly, the mean serum IgM, IgG, and IgA levels and complement activity in TU-treated rats were lower than those in control group (p > 0.01), and the changes in the antibody levels of the examined genital secretions were not significant (p > 0.01). The changes in the thickness of urethra epithelium, and in secretory component (SC) expression in genitals were not observed in the treated group. These results demonstrated that long-term supraphysiological TU injection did not obviously affect the examined rat immunological parameters.

草鱼免疫球蛋白M重链基因的克隆及表达

用RACE-PCR方法扩增出草鱼免疫球蛋白M(immunoglobulin M,IgM)分泌型的重链全长cDNA。其cDNA全长为1 940nt,包含5′非编码区20nt,3′非编码区189nt,开放阅读框1 731nt,编码576个氨基酸。序列比对分析表明草鱼IgM与鲤的相似性高达68%,与斑马鱼、斑点叉尾鱼回、鳜以及大西洋鳕的相似性分别为61%、43%、33%和30%。ClustalX比对结果显示:草鱼IgM中存在半胱氨酸和色氨酸的保守位点。进化分析表明,草鱼IgM与斑马鱼的IgM聚为一枝。荧光定量P

柱状黄杆菌胞外多糖对草鱼的免疫促进作用

将柱状黄杆菌胞外多糖(EPS)溶液经腹腔注射免疫草鱼,以注射灭菌生理盐水作对照。免疫一周后,分别提取受免鱼与对照鱼肝脏、脾脏、体肾和头肾4种组织中的总RNA,并通过RT-PCR半定量的方法检测不同组织中C-反应蛋白(CRP)、白介素1(IL-1)、主要组织相容性复合体I(MHC I)、免疫球蛋白M(IgM)、干扰素(IFN)等5种免疫基因的表达情况。结果显示,CRP在受免鱼肝脏中的表达显著上升;MHC I在受免鱼脾脏、体肾中的表达显著下降;IgM在受免鱼4种组织中的表达皆显著上升;IL-1在受免鱼4种组织

四种方法对鲢血清免疫球蛋白的纯化及分子量测定

采用mannan-binding protein(MBP)、TG及免疫亲和层析法和饱和硫酸铵沉淀法纯化鲢血清免疫球蛋白IgM,并比较了4种纯化方法的纯化效果.SDS-PAGE显示鲢血清免疫球蛋白的重链和轻链分子量分别为76.4 kD和27.2 kD,推算其总分子量约828.8kD.

草鱼免疫应答的初步研究

研究了草鱼在不同水温条件下受抗原刺激后其中和抗体的变化。15℃培养条件下中和抗体上升缓慢,9周内滴度低于1:8;20℃时,3周后抗体可上升到1:256,最高达1:5270,而在25℃时,1周中和抗体即达到1:570,最高可达1:20000以上。并探索了从草鱼血清中提纯抗体的条件,研究其抗体的特性。草鱼血清中的抗体为大分子蛋白,容易解离为抗原性相同,分子量近似于人IgG的较小分子,含有较多的二硫键,具有类似IgM的某些特性。

Effects of cyanobacterial toxin microcystin-LR on the transcription levels of immune-related genes in grass carp Ctenopharyngodon idella

Recent studies in mammals have revealed that the cyanobacterial toxin MC-LR suppresses immune functions. Nevertheless, immunotoxic effects of microcystins have been little studied in fish. In this paper, we present the profiles of the immune modulation of MC-LR in grass carp, and quantitative real-time PCR methodology was developed for the measurement of relative transcription changes of six immune-related genes in the spleen and head kidney of the grass carp Ctenopharyngodon idella, which were intraperitoneally injected with 50 mu g MC-LR center dot kg(-1) body weight in a three-week period. This study was focused exclusively on gene transcription level changes at different time points after MC-LR exposure, so, only one dose was given. The investigated genes were interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), type I interferon (Type I IFN), peptidoglycan recognition protein-L (PGRP-L), immunoglobulin M (IgM) and major histocompatibility complex class I (MHC-I) genes. The results demonstrated that the transcription levels of the TNF-alpha, type I IFN, and PGRP-L genes in the spleen and head kidney were significantly low at all time points, and those of IL-1 beta were significantly low in the head kidney at different time points. In addition, IgM and MHC-I transcription levels were only significantly low in the spleen and head kidney at 21 d postinjection. The changes in the transcription levels of immune-related genes induced by MC-LR confirmed its effect on inhibiting immune function at the transcription level.

Molecular cloning and characterization analysis of immunoglobulin M heavy chain gene in European eel (Anguilla anguilla)

In this study, the immunoglobulin M heavy chain gene of European eel (Anguilla anguilla) was cloned and analyzed. The full-length cDNA of the IgM heavy chain gene (GenBank accession no. EF062515) has 2089 nucleotides encoding a putative protein of 581 amino acids. The IgM heavy chain was composed of leader peptide (L), variable domain (VH), CH1, CH2. Hinge, CH3, CH4, and C-terminus and two novel continuous putative N-glycosylation sites were found close to the second cysteine of CH3 in A. anguilla-H1 and A. anguilla-H2. The deduced amino acid sequence of the European eel IgM heavy chain constant region shared similarities to that of the Ladyfish (Elops saurus). Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), Grass carp (Ctenopharingodon idella), Common carp (Cyprinus carpio), Channel catfish (Ictalurus punctatus), and the orange-spotted grouper (Epinephelus coioides) with the identity of 46.1%, 39.7%, 38.9%, 32.4%, 32.3%, 31.7%, and 30.7%, respectively. The highest level of IgM gene expression was observed in the kidney, followed by the spleen, gills, liver, muscle and heart in the apparently healthy European eels. (C) 2008 Elsevier B.V. All rights reserved.

Generation and characterization of monoclonal antibodies against the flounder Paralichthys olivaceus rhabdovirus

Two MAbs (3C7 and 3C9) against flounder Paralichthys olivaceus rhabdovirus (PORV) were generated with hybridoma cell fusion technology and characterized by an indirect enzyme-linked immunosorbent assay, isotype test, Western blot and immunodot analysis and immunofluorescence assay. Isotyping tests demonstrated that both of the two MAbs belonged to IgM subclass. Western blot analysis showed the MAbs reacted with 42, 30, and 22 kDa viral proteins, which were localized within the cytoplasm of PORV-infected grass carp ovary (GCO) cells analyzed by indirect immunofluorescences tests. The MAb 3C7 was also selected at random for detecting virus antigens in the inoculated grass carp tissues by immunohistochemistry assay. Flow cytometry tests showed that at the 36 h postinfection (0.25 PFU/cell), the 23% PORV-infected GCO cells could be distinguished from the uninfected cells with the MAb 3C7. Such MAbs could be useful for diagnosis and potential treatment of viral infection. (C) 2007 Elsevier B.V. All rights reserved.