Infection of a chimeric simian and human immunodeficiency virus with CCR5-specific HIV-1 envelope to Rhesus macaques

Human immunodeficiency virus (HIV) infects lymphocytes and macrophages via CD4 and chemokine receptors. In this study, the infectivity of a chimeric simian and human immunodeficiency virus (SHIV) having a CCR5-specific HIV-1 envelope gene was examined. A SHIV strain termed SHIV-JRFL could enter cells via CD4 with a chemokine receptor CCR5, not CXCR4, and the viral replication was suppressed by recombinant human RANTES, one of beta-chemokines. The intravenous inoculation of SHIV-JRFL into two rhesus macaques resulted in a systemic infection, though it was rather weak. During the early infection, the production of RANTES from Con A-stimulated PBMCs of the infected monkeys increased. These results suggested that beta-chemokine has the potential to limit the infectivity of an R5-type SHIV.     

猪圆环病毒2型感染猪皮肤源树突状细胞炎性反应能力的变化

用PCV2 B1株经鼻腔接种40日龄SPF仔猪,于接种后3、7、14 d宰杀,收集皮肤源树突状细胞(DC).利用实时荧光定量PCR技术对感染仔猪皮肤源DC的IL-10、TNF-α、IFN-α、IL-8、趋化因子受体1(CCR1)、CCR5在mRNA转录水平的变化进行定量分析.结果表明,IFN-α在接种后3 d(3DPI)显著下调(P＜0.05),TNF-α、IL-10在7DPI时显著上调(P＜0.05);趋化因子IL-8在3、7、14 DPI时均下调,差异接近显著;MCP-1在感染后3、14DPI下调,7DPI均上调,但不显著;MIP-1β在3、7DPI明显上调,14DPI恢复正常;趋化因子受体CCR1、CCR5在3、7和14DPI均上调,且7DPI显著上调(P＜0.05).以上结果表明PCV2在感染早期可抑制DC炎性反应的能力,免疫应答失调,影响了动物机体的细胞和体液免疫功能的发挥.     

Bovine Immunodeficiency Virus Expression in Vitro is Reduced in the Presence of β-Chemokines,MIP-1α, MIP-1β and RANTES

The inhibition of HIV expression in vitro by a cocktail of the -chemokines MIP-1, MIP-1 and RANTES provided the initial evidence that HIV utilizes chemokine receptors as co-receptors for infection of cells. Bovine immunodeficiency virus (BIV), a lentivirus, infects a wide variety of leukocyte populations, but the cellular receptor(s) utilized by this virus for infection of cells is not known. The purpose of this study was to determine whether MIP-1, MIP-1 and RANTES affect BIV expression in vitro, as a prelude to identifying the cellular receptors utilized by this virus. Fetal bovine lung (FBL) cells were pretreated with serial dilutions of a cocktail of the chemokines, and then the cells were infected with BIV. Virus expression in these cells was determined by counting the syncytia that had developed in the cultures by five days after infection. A significant decrease in syncytium formation, corresponding to increasing concentrations of the chemokines, was the result. Reacting the chemokines with chemokine-specific neutralizing antibodies prior to treatment of the cells neutralized the effect of the chemokines on virus replication in a dose-dependent manner, restoring viral expression to a level similar to that of untreated cells. The presence of a CCR5 homologue on the surface of FBL cells was confirmed using an anti-CCR5 monoclonal antibody and FACS analysis. Collectively, these data provide preliminary evidence that BIV may utilize the CCR5 receptor for infection of cells in vitro, but additional studies are necessary to confirm this.     

Production of a monoclonal antibody to canine thymus and activation-regulated chemokine (TARC) and detection of TARC in lesional skin from dogs with atopic dermatitis

A monoclonal antibody to canine thymus and activation-regulated chemokine (TARC/CCL17) was developed to examine the association of TARC with the immunopathogenesis of canine atopic dermatitis (AD). Recombinant canine TARC was prepared using an E. coli expression system. Results of transwell chemotaxis assay demonstrated that the recombinant canine TARC showed chemotactic activity for canine lymphoid cells expressing CC chemokine receptor 4 (CCR4). Mice were then immunized with the recombinant canine TARC to obtain monoclonal antibodies. Among the monoclonal antibodies thereby obtained, one monoclonal antibody (CTA-1) was found to react with both recombinant and authentic canine TARC in ELISA and flowcytometric assays, respectively. Immunohistochemical analysis using the monoclonal antibody CTA-1 demonstrated that keratinocytes were major TARC producing cells in lesional skin of dogs with AD.     

Molecular cloning of canine thymus and activation-regulated chemokine (TARC) gene and its expression in various tissues

Thymus and activation-regulated chemokine (TARC) is known as a functional ligand for CC chemokine receptor 4 (CCR4), which is selectively expressed on Th2 lymphocytes and induces selective migration of the cells to allergic lesions. In this study, we cloned canine TARC cDNA from canine thymus by RT-PCR with rapid amplification of cDNA ends (RACE) method. The canine TARC clone contained a full-length open reading frame encoding 99 amino acids and included four cysteine residues characteristic to CC chemokine family. The canine TARC cDNA showed 77.5%, 67.4%, and 68.5% amino acid sequence similarity with human, mouse and rat homologues, respectively. Expression of TARC mRNA was detected not only in thymus but also in spleen, lymph node, lung and heart of the various normal dog tissues examined. TARC cDNA clone obtained in this study will be useful for further investigation on allergic diseases in dogs.     

Expression of CC chemokine receptor 4 (CCR4) mRNA in canine atopic skin lesion

CC chemokine receptor 4 (CCR4) is a G protein-coupled seven transmembrane receptor that is selectively expressed on Th2 cells and plays an important role in the trafficking of Th2 cells into inflammatory sites. In this study, a full-length canine CCR4 cDNA was cloned and characterized in order to examine the potential role of CCR4 in allergic responses that produce skin lesions in canine atopic dermatitis (AD). The canine CCR4 cDNA reported in this study contained an open reading frame of 1083 nucleotides encoding 360 amino acids. The predicted amino acid sequence of canine CCR4 showed 91.9, 85.3 and 84.5% similarity with those of the human, mouse and guinea pig counterparts, respectively. Expression of CCR4 mRNA was detected in various tissues including thymus, spleen, heart, small intestine and lymph node. Furthermore, it was found that CCR4 mRNA was preferentially expressed in lesional skin of dogs with AD, together with the mRNA of thymus and activation-regulated chemokine (TARC), which is a ligand for CCR4. The present study demonstrates that CCR4 contributes strongly to the immunopathogenesis of canine AD.     

Differential expression of inducible nitric oxide synthase and cytokine mRNA in chicken lines divergent for cutaneous hypersensitivity response

Phytohemagglutinin (PHA)-induced delayed-type hypersensitivity is an immunocompetent trait considered an indicator of cell-mediated immune or T-cell responses. Divergent selection was performed to generate high and low lines for response to PHA-P. Extreme-responder birds of the F2 generation in each line were used to study possible differences in macrophage activity and the associated functional genes. To evaluate macrophage activity, nitric oxide (NO) was estimated both systemically in serum and in in vitro monocyte culture. Semi-quantitative RT-PCR was used to detect the differential mRNA expression patterns of iNOS and MIP-1beta in monocyte culture, whereas T(H)1 cytokines (IL-2 and IFN-gamma) were studied in peripheral blood mononuclear cells (PBMC) at different time intervals after lipopolysaccharide (LPS) induction. The high line showed strong systemic, as well as in vitro NO production, compared to the low line, upon stimulation with NDV and LPS, similar to early and high iNOS mRNA expression. Following the pattern of iNOS gene expression, an early strong expression of cytokines with powerful iNOS-inducing action, such as IFN-gamma and the chemokine MIP-1beta, was observed in the high line. In contrast, for response to PHA-P, low expression of IL-2 was observed in the high compared to the low line. In conclusion, the study revealed that divergent selection for response to PHA-P resulted in a divergent effect on T(H)1 cell activity, resulting in altered macrophage function in chickens. Selection, based on response to PHA-P, could lead to more resistant birds or birds with an enhanced immune response.     

Molecular cloning and expression analysis of the canine chemokine receptor CCR9

The chemokine receptor CCR9, which interacts with the thymus-expressed chemokine TECK/CCL25, contributes to the localization of lymphocytes to the small intestine, and is implicated in the development of human inflammatory bowel disease (IBD); however, their role in canine IBD is unknown. The objective of this study was to isolate cDNA encoding CCR9 and to investigate CCR9 expression in normal canine tissues and lymphoid cell lines. The complete open reading frame contained 1104 bp, encoding 367 amino acids, with 85% and 81% identity to human and mouse homologs, respectively. CCR9 mRNA was detected in all tissues investigated with the highest expression level in the small intestine. CCR9 mRNA was also expressed in GL-1, a canine B cell leukemia cell line, but not in CLBL-1, a canine B cell lymphoma cell line. Immunoblot and flow cytometry analyses of these cell lines using an anti-human CCR9 monoclonal antibody revealed that CCR9 protein expression was detected only in GL-1, indicating the cross-reactivity of the antibody. Using the antibody, flow cytometry showed that the proportions of CCR9(+) cells were small (mean, 4.88%; SD, 2.15%) in the normal canine PBMCs. This study will be useful in understanding canine intestinal immunity and the immunopathogenesis of canine IBD.     

Expression and functional analysis of chemokine receptor 7 in canine lymphoma cell lines

C-C chemokine receptor 7 (CCR7) contributes to cell homing to lymph nodes (LNs). Recent studies reported that CCR7 is also expressed in tumor cells, which correlates with LN metastasis in various cancers. However, the expression of CCR7 in tumor cells is unknown in dogs due to the lack of appropriate antibodies. In the present study, a fusion protein of C-C chemokine ligand 19 (CCL19) was employed as an alternative method to CCR7 antibodies. The fusion CCL19 protein specifically detected CCR7 expressed in canine lymphoma cell lines, which showed active chemotaxis to both canine and mouse ligands. The present study will help further research on the involvement of canine CCR7 in LN metastasis.     

Differential gene regulation of interleukin-1 ligands and receptors in bovine peripheral blood neutrophils and mononuclear cells in response to E. coli lipopolysaccharide (LPS)

The proinflammatory cytokine, interleukin-1, plays a prominent role in the inflammatory reactions that characterize numerous diseases. In this study, we examined the gene expression for bovine IL-1 ligands and receptors by bovine peripheral blood mononuclear cells (MNCs) and neutrophils (PMNs) in response to E. coli lipopolysaccharide (LPS) in vitro. Gene expression of mRNA for IL-1alpha, IL-1beta, IL-1 receptor antagonist (IL-1ra), type 1 IL-1 receptor, type 2 IL-1 receptor, and IL-1 beta converting enzyme (ICE), were measured by a semi-quantitative RT-PCR technique. LPS had little effect on type 1 IL-1R expression in MNC, whereas, it strongly up-regulated type 1 IL-1R expression in PMNs. Co-incubation of PMNs with LPS and bovine recombinant IL-1beta had little additional effect on type 1 IL-1R expression. Incubation of MNCs with LPS resulted in up-regulation of IL-1beta, IL-1ra, and type 2 IL-1R, no change in IL-1alpha, and a decrease in ICE gene expression. Incubation of PMNs with LPS up-regulated IL-1beta gene expression, whereas, IL-1alpha, IL-1ra, type 2 IL-1R and ICE were unchanged. This study provides evidence for differential regulation of gene products of the bovine IL-1 family by peripheral blood mononuclear cells (MNC) and neutrophils (PMNs) in response to E. coli LPS.     

中国荷斯坦奶牛CCR5基因克隆、生物信息学及表达分析

试验旨在克隆中国荷斯坦奶牛CC趋化因子受体5(C-C chemokine receptor type 5,CCR5)基因,对其进行生物信息学分析,并探究CCR5基因在奶牛炎性和健康组织中的表达水平。采用PCR技术扩增并克隆荷斯坦奶牛CCR5基因CDS区全长序列,连接pMD18-T载体并转化大肠杆菌DH5α感受态细胞,通过蓝白斑方法筛选阳性克隆并测序,对序列进行相似性比对及系统进化树构建;应用多种在线生物信息学软件对其编码蛋白进行分析,并利用实时荧光定量PCR方法检测CCR5基因在健康和炎症奶牛乳腺组织中的表达情况。结果显示,中国荷斯坦奶牛CCR5基因CDS区全长1 059 bp,编码352个氨基酸。相似性和遗传进化分析结果显示,奶牛CCR5基因与绵羊的遗传距离最近,高达96.0%,与鸡遗传关系最远,为61.0%,且在不同物种之间CCR5基因高度保守。中国荷斯坦奶牛CCR5蛋白分子质量为40.235 ku,理论等电点(pI)为9.30,为疏水性蛋白但不是分泌蛋白,主要存在于细胞质内;在CCR5蛋白二级结构中α-螺旋和无规则卷曲分别占51.14%和32.95%,三级结构模型预测结果与二级结构一致。实时荧光定量PCR结果显示,CCR5基因在健康奶牛乳腺组织中的表达量极显著低于炎性奶牛乳腺组织(P<0.01),提示其可能参与奶牛乳腺炎的发生过程。本试验结果为进一步研究奶牛CCR5蛋白的功能提供了理论依据,对探究奶牛CCR5基因在奶牛乳腺炎中的调控功能等具有重要意义。     

Cytokine and chemokine gene expression of IL-1beta stimulated equine articular chondrocytes

OBJECTIVE: To evaluate mRNA expression of several proinflammatory and anti-inflammatory cytokines and chemokines in equine unstimulated and interleukin-1beta (IL-1beta)-stimulated chondrocytes. STUDY DESIGN: In vitro experiment using equine chondrocyte cultures. SAMPLE POPULATION: Whole articular cartilage from metacarpophalangeal joints (n=5 horses; 10 fetlocks). METHODS: Chondrocyte monolayer cultures were established from digested adult equine articular cartilage and stimulated with 5 ng/mL of recombinant human IL-1beta. RNA was extracted from the cells 24 hours after stimulation. IL-1beta, IL-4, IL-6, IL-8, tumor necrosis factor-alpha (TNF-alpha), and ubiquitin (house keeping gene) mRNA expression were investigated by real-time RT-PCR. RESULTS: IL-1beta, IL-6, and IL-8 mRNA were expressed in unstimulated chondrocytes from macroscopically normal joints and were significantly up-regulated after stimulation (5/5 horses). IL-4 mRNA was not detected in any samples (0/5 horses). TNF-alpha mRNA, by comparison, was expressed in 2/5 unstimulated samples and in all stimulated samples but a considerable sample variation in response to IL-1beta stimulation was observed. CONCLUSIONS: Equine chondrocytes express mRNA for several proinflammatory cytokines and chemokines and IL-1beta modulates their expression. CLINICAL RELEVANCE: Chondrocytes express proinflammatory cytokines and chemokines capable of modulating a local inflammatory cascade in articular cartilage, which could potentially lead to focal degradation and osteoarthritis.     

Babesia gibsoni rhoptry-associated protein 1 and its potential use as a diagnostic antigen

A cDNA encoding the rhoptry-associated protein 1 (RAP-1) homologue was obtained by immunoscreening an expression library prepared from Babesia gibsoni merozoite mRNA. The complete nucleotide sequence of the gene was 1740bp. Computer analysis suggested that the sequence contains an open reading frame of 1425bp encoding an expected protein with a molecular weight of 52kDa. Based on the sequence similarity, this putative protein was designated as the B. gibsoni RAP-1 (BgRAP-1). The BgRAP-1 gene was expressed in the Escherichia coli BL21 strain, and the recombinant BgRAP-1 was used as the antigen in the enzyme-linked immunosorbent assay (ELISA). The results can differentiate between the B. gibsoni-infected dog sera and the Babesia canis infected dog sera or the normal dog sera. Furthermore, the antibody response against the recombinant protein was maintained during the chronic stage of infection, indicating that the recombinant BgRAP-1 protein might be a useful diagnostic antigen for the detection of antibodies to B. gibsoni infection in dogs.     

反转录病毒介导的稳定表达HIV-1Gag蛋白和增强型绿色荧光蛋白细胞系的建立

本研究旨在建立中国流行株人免疫缺陷病毒(HIV-1)衣壳蛋白(Gag)哺乳动物稳定表达细胞系。将HIV-1核心蛋白基因gag和增强型绿色荧光蛋白基因EGFP依次串联插入反转录病毒载体pFB-neo,构建重组反转录病毒载体pFB-gag-EGFP,并与含有辅助病毒gag-pol和env基因的质粒pVPack-GP、pVPack-10A1共转染HEK293T细胞,包装出的反转录病毒感染小鼠骨髓瘤细胞SP2/0。荧光显微镜下观察绿色荧光蛋白EGFP表达,验证HIV-1核心蛋白Gag表达,G418抗性筛选阳性细胞。结果表明,HIV-1核心蛋白Gag和增强型绿色荧光蛋白可在SP2/0细胞中稳定表达,HIV-1核心蛋白gag基因稳定表达细胞系成功建立,为抗AIDS治疗用基因工程制剂及靶向药物的活性检测提供了理想方法。     

Porcine insulin-like growth factor (IGF)-binding protein-3 elicits bi-phasic effects on IGF-I stimulated DNA synthesis in neonatal porcine skin fibroblasts

The present study was undertaken to investigate the effects of porcine IGFBP-3 on IGF-I stimulated DNA synthesis in neonatal porcine skin fibroblasts. IGF-1 stimulated DNA synthesis in skin fibroblasts in a concentration dependent manner. DNA synthesis was maximally stimulated by 5 to 20 fold at 5 nM IGF-I; half-maximal stimulation was observed at approximately 1 nM IGF-I. Co-incubation of IGFBP-3 with a maximally effective dose of IGF-I (10 nM) did not inhibit the stimulatory effects of IGF-I on DNA synthesis. In contrast, when IGFBP-3 at concentrations of 0 to 20 nM was co-incubated with 1 nM IGF-I, a bi-phasic dose response was observed with IGFBP-3 being inhibitory only at a 10 to 20 fold molar excess to IGF-I. Based on the approximately equal molar ratio of IGFBP-3:IGF-I present in the circulation of control and pST-treated pigs our results suggest that IGFBP-3 does not inhibit the mitogenic effects of IGF-I. In summary, these results indicate that the combination of IGFBP-3 with IGF-I optimizes mitogenic signalling via the type I IGF receptor and suggest that IGFBP-3 does not inhibit the effects of ST that are mediated by IGF-I.     

CXCL8 attenuates chemoattractant-induced equine neutrophil migration

The chemokine, CXCL8, is a potent chemoattractant but it has also been shown to attenuate the migratory response of human neutrophils to the bacterial peptide, FMLP; this could lead to retention of cells in infected tissue and, potentially, to enhanced clearance of bacteria. This study has examined the effect of CXCL8 on equine neutrophil migration and adherence in response to PAF and LTB(4), chemoattractants that may play a role in non-infectious inflammatory conditions of the horse associated with neutrophil recruitment to the target tissue. The effects of CXCL8 on PAF- and LTB(4)-induced responses were determined using a ChemoTx plate migration assay and by measuring adhesion to protein-coated plastic. The CXCR1/2 antagonist, SB225002, was used to investigate whether the observed effects were receptor mediated and the role of cAMP was examined by measuring intracellular cAMP following exposure to agonists alone and in combination and by establishing the effect of dibutyryl cAMP on neutrophil migration. CXCL8, LTB(4) and PAF each induced migration and adhesion. Exposure of neutrophils to a combination of CXCL8 and PAF reduced the magnitude of the responses to that of unstimulated cells. In contrast, although the effect was less than additive, the response to co-stimulation with CXCL8 and LTB(4) were not nearly as pronounced. CXCL8 acted in a receptor mediated manner, the attenuation of PAF-induced responses being reversed by SB225002 at a concentration that blocks CXCR2. CXCL8, PAF and LTB(4) alone increased intracellular cAMP. In co-incubation studies, combination of CXCL8 with PAF led to an additive increase in cAMP whereas no increase above that obtained in response to LTB(4) alone was seen. Dibutyryl cAMP significantly reduced neutrophil migration in response to either CXCL8 or PAF alone. These results demonstrate that CXCL8, in addition to being a potent chemoattractant and pro-adhesive molecule for equine neutrophils, is able to attenuate responses to PAF and, to a much lesser extent, LTB(4). This effect, which appears to be CXCR2-mediated and cAMP dependent, could lead in vivo to trapping of cells at sites of inflammation resulting potentially in either enhanced clearance of injurious stimuli or increased local tissue damage by activated cells.