Sequence analyses of feline B7 costimulatory molecules

Using RT-PCR amplifications with mRNA from mitogen-stimulated feline peripheral blood mononuclear cells, cDNA of feline B7-1 (CD80) and B7-2 (CD86) were cloned. The cDNA were sequenced and putative translated protein sequences compared with known counterpart sequences. Hydrophilicity patterns of the feline CD80 and CD86 which were only 26.8% identical at the amino acid sequence were very distinct from each other, but similar to the putative human CD80 and CD86 proteins, respectively. The feline CD80 gene encoded a protein of 292 amino acids and the CD86 gene encoded a protein of 329 amino acids. Amino-terminal signal sequences, extracellular Ig V- and Ig C-like domains, transmembrane domains, and carboxyl cytoplasmic domains were identified in both molecules. Although the most conserved domain among the CD80 sequences was the Ig C-like domain, the most conserved domain among the CD86 sequences was the Ig V-like domain. Among the known sequences, the bovine CD80 and the porcine CD86 sequences available for comparisons were identified as most closely related to the feline CD80 (63.3%) and CD86 (67.5%), respectively. The mouse molecules were the least identical (43.6 and 43.6%, respectively) with the feline CD80 and CD86 proteins. The human CD80 and CD86 molecules were 56.3 and 57.0% identical with the feline molecules.     

Molecular cloning of a cDNA encoding the feline CD62L

We cloned a cDNA fragment encoding a feline homologue of L-selectin (CD62L). The extracellular region of the feline CD62L fragment contained a calcium-dependent (C-type) lectin domain, an epidermal growth factor-like domain, and two Sushi/CCP/SCR domains. The flow cytometric analysis confirmed that the feline CD62L molecule, which was expressed 293T cells, retained an epitope recognized by an anti-human CD62L monoclonal antibody (Leu-8).     

猪圆环病毒2型感染对猪CD80和CD86 mRNA表达的影响

通过构建猪共刺激分子CD80和CD86的缺失cDNA竞争分子,用竞争PCR技术定量检测了猪圆环病毒2型(PCV2)感染后猪肺泡巨噬细胞(PAM)中CD80和CD86的mRNA水平,分析了PCV2感染对猪共刺激分子CD80和CD86的mRNA表达的影响.结果显示,PCV2感染后,PAM中CD80和CD86的mRNA水平变化趋势一致,只是CD86的升降幅度更大.在PCV2感染后3d,CD80与CD86的mRNA水平下降至最低,随后迅速上升,至14d达到高峰,尔后快速下降,21d及以后恢复正常.本研究结果表明,PCV2初期可明显抑制猪共刺激分子CD80和CD86的基因转录.     

Molecular cloning and sequencing of the cDNA encoding the feline FcgammaRIIIA (CD16) homologue

We amplified the cDNA encoding the feline FcgammaRIIIA (CD16) homologue from peripheral blood mononuclear cells by polymerase chain reaction and cloned two forms of FCGR3A cDNA. Sequencing analysis revealed that the open reading frame of feline FCGR3A cDNA consists of 750 or 747 base pairs encoding 250 or 249 amino acid residues, respectively. Comparison of the predicted amino acid sequence of feline FCGR3A cDNA with those of other mammalians' homologues revealed that the extracellular domain has a relatively low homology. However, the cytoplasmic domain contained an 8-amino acid motif, Leu-Phe-Val-Val-Asp-Thr-Gly-Leu, which was considered to interact with an accessory molecule such as the gamma chain of Fc receptors for IgE to form heterodimeric complexes.     

牛病毒性腹泻病毒感染牛外周血单核细胞对CD80和CD86 mRNA转录的影响

用非致细胞病变（noncytopathic,NCP）和致细胞病变（cytopathic,CP）型牛病毒性腹泻病毒（BVDV）感染临床健康BVDV检测阴性的荷斯坦奶牛外周血单核细胞（PBMC）,利用实时荧光定量PCR技术对感染后共刺激分子CD80和CD86mRNA转录水平的变化进行定量分析。结果表明,在NCP型BVDV感染牛PBMC后CD80在4h（P〈0.05）和12,24h（P〈0.01）出现2次转录高峰,CD86在6h（P〈0.05）出现转录高峰;CP型BVDV感染后,CD80在24h（P〈0.05）出现转录高峰,CD86在6h（P〈0.05）出现转录高峰。尽管CD80在NCP型BVDV感染后呈现较复杂的动态变化,但结果提示NCP型和CP型BVDV感染均可导致牛PBMC的共刺激分子CD80和CD86基因转录在感染早期明显受到抑制,PBMC的抗原呈递能力受到影响。     

槲皮素和乙酸龙脑酯对小鼠的保胎作用及IL-10和CD80~+/CD86~+细胞的影响

本文旨在研究槲皮素和乙酸龙脑酯对细菌脂多糖(LPS)诱导小鼠流产的保护作用及其对小鼠子宫IL-10水平和脾脏CD80+/CD86+细胞数量的影响。试验选用LPS尾静脉注射(0.10μg/鼠),制造小鼠流产模型,于怀孕第4~7d分别口服不同保胎药物,检测各组(n=10)小鼠的流产率、胚胎吸收率、子宫组织IL-10水平和脾脏CD80+/CD86+表达的变化。发现LPS诱导流产的小鼠IL-10含量降低,CD80+/CD86+比值升高,预先口服不同保胎药物后,不同程度的抑制了LPS的作用,其中以槲皮素和乙酸龙脑酯联合使用组的保胎效果最为明显,并且IL-10含量显著升高,CD80+/CD86+比值显著降低,与LPS流产组比较均差异显著(P0.05)。这些结果表明,LPS诱导流产与机体IL-10和共刺激分子CD80+/CD86+有关。保胎中药成分槲皮素和乙酸龙脑酯有降低小鼠脾脏CD80+/CD86+比值,促进母胎界面Th2细胞因子IL-10分泌的作用。     

Molecular cloning and sequencing of feline CD7

Human CD7 is one of the earliest molecules to appear in T cell development. In this study, putative feline CD7 cDNA was identified based on its similarities with human and mouse CD7 genes. The feline CD7 cDNA contained an open reading frame consisting of 630 nucleotides. The amino acid sequence of feline CD7 had 47.7% identity with that of human CD7, and 52.9% with that of mouse CD7. In addition, the feline CD7 protein fused with histidine tag was expressed in 293T cells. The expression was confirmed by indirect immunofluorescence assay.     

Comparative examination of cats with feline leukemia virus-associated enteritis and other relevant forms of feline enteritis

Cats with feline leukemia virus (FeLV)-associated enteritis (FAE), enteritis of other known viral etiology (parvovirus [PV], enteric coronavirus [CoV]), and enteritis of unknown etiology with histologic features similar to those of FAE and PV enteritis (EUE) and FeLV-negative and FeLV-positive cats without enterocyte alterations were examined. Amount and types of infiltrating leukocytes in the jejunum and activity and cellular constituents of mesenteric lymph nodes, spleen, and bone marrow were determined. PV and CoV infections were confirmed by immunohistologic demonstration of PV and CoV antigen, ultrastructural demonstration of viral particles in the intestinal content, and in situ hybridization for PV genome. FeLV infection was detected by immunohistology for gp70, p27, and p15E. Latent FeLV infection was excluded by polymerase chain reaction methods for exogenous FeLV DNA. Enterocyte lesions involved the crypts in cats with PV enteritis, FAE, and EUE and the villous tips in cats with CoV enteritis. Inflammatory infiltration was generally dominated by mononuclear cells and was moderate in the unaltered intestine and in cats with PV enteritis and marked in cats with FAE, CoV enteritis, and EUE. In cats with EUE, myeloid/histiocyte antigen-positive macrophages were relatively numerous, suggesting recruitment of peripheral blood monocytes. Lymphoid tissues were depleted in cats with PV enteritis and with EUE but were normal or hyperplastic in cats with FAE. Bone marrow activity was decreased in cats with PV enteritis; in cats with FAE or EUE and in FeLV-positive cats without enterocyte alterations, activity was slightly increased. In cats with FAE and PV enteritis, a T-cell-dominated response prevailed. EUE showed some parallels to human inflammatory bowel disease, indicating a potential harmful effect of infiltrating macrophages on the intestinal epithelium.     

黄芩及其成分对RU486诱导小鼠流产的保胎作用及脾脏CD80+、CD86+细胞数量的影响

为了研究共刺激分子CD80(B7-1)、CD86(B7-2)在流产发生机制中的意义,探讨中药黄芩及其成分的安胎作用及机理,本试验用米非司酮(RU486)皮下注射(每鼠90μg)诱导BALB/c小鼠流产,流式细胞仪测定脾脏中CD80 、CD86 细胞的数量。发现RU486诱导流产的小鼠脾脏中CD80 细胞显著升高,CD86 细胞显著减少。预先经口给予保胎中药黄芩及其单体成分黄芩苷和黄芩素,则能显著抑制RU486的流产作用,使母鼠胚胎吸收率降低,脾脏中CD80 细胞数量显著降低,CD86 细胞数量显著升高。这些结果表明,RU486诱导流产与机体共刺激分子CD80 、CD86 有关。保胎中药黄芩及单体成分有调节共刺激分子CD80 、CD86 细胞数量的作用。     

Cloning of cDNAs encoding multiple subtypes of feline interferon-alpha from the feline epitherial cell line

Mammalian interferon (IFN)-alpha consists of a 23-amino acid signal peptide and a 166-amino acid mature protein. Feline (Fe) IFN-alpha has an extra unique molecule consisting of a 171-amino acid mature protein with a 5-amino acid insertion. We cloned eight new subtypes of cDNA encoding FeIFN- alpha from a feline epithelial cell line. Among all the FeIFN-alpha subtypes, including six that have previously been reported, the variations were found to be far less than those of IFN-alphas of other animals.     

PRRSV和PCV2共感染对猪肺泡巨噬细胞CD80-CD86mRNA转录的影响

用猪生殖与呼吸综合征病毒（PRRSV）与猪圆环病毒2型（PCV2）共感染40日龄健康大白仔猪，利用实时荧光定量PCR技术对共感染仔猪肺泡巨噬细胞（PAM）共刺激分子CD80一CD86的mRNA转录水平进行了定量分析。结果表明，在感染后第3d和第7dCD80与CD86mRNA转录显著下调（P〈0．05），感染后第14dCD86mRNA转录水平仍低于未感染对照组。尽管CD80mRNA转录水平在第14d和第28d高于对照组，CD86mRNA转录水平在第28d高于对照组，两者在第42d均高于对照组，但无显著差异。证实，PRRSV和PCV2共感染可导致猪肺泡巨噬细胞的共刺激分子CD80-CD86基因转录在感染早期明显受到抑制，PAM的抗原呈递能力受到影响。     

Comparison of CD34, CD31, and factor VIII-related antigen immunohistochemical expression in feline vascular neoplasms and CD34 expression in feline nonvascular neoplasms

The diagnosis of vascular neoplasms is often facilitated by the use of immunohistochemical markers such as factor VIII-related antigen, CD31, and CD34. However, the relative sensitivity and specificity of these markers have not been compared in cat vascular neoplasms. In this study, these 3 immunohistochemical markers were evaluated in 61 endothelial neoplasms (50 hemangiosarcomas and 11 hemangiomas) in 59 cats. All neoplasms were labeled by all 3 markers. CD34 had the highest average immunolabeling intensity in neoplastic endothelial cells. CD31 had the lowest average background labeling, followed by CD34 and factor VIII-related antigen, respectively. CD34 expression was also examined in 130 nonvascular neoplasms of cats; 14 of 62 epithelial neoplasms, 39 of 43 mesenchymal neoplasms, 8 of 23 leukocytic neoplasms, and 2 of 2 melanomas were positive. Given the broad expression of CD34 in mesenchymal neoplasms, this marker has limited diagnostic relevance for vascular neoplasms of cats.     

Molecular cloning and sequencing of canine T-cell costimulatory molecule (CD28)

T-cells express CD28 and CTLA-4, and through binding to their shared ligands (CD80/CD86) on antigen presenting cells, provide a potent co-stimulatory signal for T-cell activation and proliferation. To investigate the role of CD28 in canine immune system, we hereby report the molecular cloning and sequencing of the full-length complementary DNA (cDNA) coding for canine CD28, from pokeweed mitogen stimulated canine peripheral blood lymphocytes. The cloned cDNA contains an open reading frame of 663 nucleotides, encoding for a polypeptide of 221 amino acids. The amino acid sequence of the canine CD28 showed 91.9, 80, and 79.6% similarities with those of the cat, cattle, and human counterparts, respectively. Five sequence motifs of TATT or ATTTA involved in the regulation of gene expression by influencing mRNA stability are found in the 3' untranslated region. The hexapeptide motif (MYPPPY), five cysteine residues, a potential N-glycosylation site and a cytoplasmic phosphatidylinositol 3-kinase binding site in canine CD28 molecule are completely conserved in canine CTLA-4. The availability of full length canine CD28 will provide a useful molecule for studying its role in dog immune system.     

Chemokine receptors and co-stimulatory molecules: unravelling feline immunodeficiency virus infection

Feline immunodeficiency virus (FIV) infection of the domestic cat induces an immunodeficiency characterised by a gradual depletion of CD4+ T-helper lymphocytes. The virus targets T-helper cells by way of an interaction between its envelope glycoprotein (Env) and the cell surface molecule CD134 (OX40), a member of the nerve growth factor receptor/tumour necrosis factor receptor superfamily. The Env–CD134 interaction is a necessary prerequisite for the subsequent interaction with CXCR4, the only chemokine receptor identified to date to act as a co-receptor for FIV. As T-helper cell expression of CD134 and CXCR4 is restricted to activated cells, FIV targets selectively antigen-specific T-helper cells. With disease progression the cell tropism of the virus expands; this may be the result of changes in the way in which Env interacts with CD134, a less stringent Env–CD134 interaction enabling the Env to interact more readily with CXCR4 and thus broadening the cell tropism of virus. In contrast, viruses that are present in early infection may have a narrower cell tropism, reflecting a more stringent interaction with CD134. Accordingly, “early” viruses may target CD134-expressing cells more efficiently and be more resistant to neutralising antibody. It is these early viruses that may be transmitted and should be considered as candidates for the development of vaccine regimes and novel therapeutic agents.     

Polymorphisms in the feline TNFA and CD209 genes are associated with the outcome of feline coronavirus infection

Feline infectious peritonitis (FIP), caused by feline coronavirus (FCoV) infection, is a highly lethal disease without effective therapy and prevention. With an immune-mediated disease entity, host genetic variant was suggested to influence the occurrence of FIP. This study aimed at evaluating cytokine-associated single nucleotide polymorphisms (SNPs), i.e., tumor necrosis factor alpha (TNF-α), receptor-associated SNPs, i.e., C-type lectin DC-SIGN (CD209), and the five FIP-associated SNPs identified from Birman cats of USA and Denmark origins and their associations with the outcome of FCoV infection in 71 FIP cats and 93 FCoV infected non-FIP cats in a genetically more diverse cat populations. A promoter variant, fTNFA - 421 T, was found to be a disease-resistance allele. One SNP was identified in the extracellular domain (ECD) of fCD209 at position +1900, a G to A substitution, and the A allele was associated with FIP susceptibility. Three SNPs located in the introns of fCD209, at positions +2276, +2392, and +2713, were identified to be associated with the outcome of FCoV infection, with statistical relevance. In contrast, among the five Birman FIP cat-associated SNPs, no genotype or allele showed significant differences between our FIP and non-FIP groups. As disease resistance is multifactorial and several other host genes could involve in the development of FIP, the five genetic traits identified in this study should facilitate in the future breeding of the disease-resistant animal to reduce the occurrence of cats succumbing to FIP.

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