Immunological tools for the assessment of both humoral and cellular immune responses in Foxes (Vulpes vulpes) using ovalbumin and cholera toxin B as an antigenic model

The immune response in the fox (Vulpes vulpes), despite the success of the oral rabies vaccine is not well characterized, and specific immunological tools are needed. To investigate both the humoral and cellular immune response, we used ovalbumin (OVA) and cholera toxin B (CTB) as an antigenic model to set-up ELISA and ELISPOT antibodies secreting cells (ASC) assays in the fox model. Identification of antibodies that cross-react with fox immunoglobulin was performed by Western blot, and their use was adapted for both the ELISA and ELISPOT ASC assay. The humoral and cellular specific immune responses were assessed after intra-muscular or intra-nasal immunization. Intra-muscular immunization resulted in the development of both cellular and humoral anti-OVA and anti-CTB responses in peripheral blood mononuclear cells (PBMCs). Immunization via the intra-nasal route resulted in the development of a cellular and humoral response against CTB in PBMCs. This immune response was confirmed using splenocytes from immunized animals by ELISPOT assay at euthanasia. Females immunized via the intra-nasal route developed specific anti-CTB IgM, IgA and IgG in vaginal fluids after the initial boost (day 26) showing that mucosal immunization produces a vaginal immune response in foxes. These immunological tools developed here are now available to be adapted to other antigenic models to facilitate further immune studies in foxes.     

Human papillomavirus (HPV) type 16-specific CD8+ T cell responses in women with high grade vulvar intraepithelial neoplasia

Human papillomavirus (HPV)-associated vulvar intraepithelial neoplasia (VIN) has serious sequelae for the sufferer. Current treatments are associated with poor response and high relapse rates. The development of HPV-specific T cell immunotherapies offers a new approach to treatment. This will require a detailed understanding of the spectrum of T cell responses induced by HPV antigens, and how effectively viral antigens can be accessed by the immune system. We have investigated the frequency and spectrum of HPV16-specific CD8+ T cell responses to three HPV16 antigens in 9 women with high grade VIN (VIN3). CD4-depleted populations of responder cells were screened against overlapping 30-35mer peptides covering the sequences of HPV16 E6, E7 and E4 using ELISPOT assays of IFN-gamma release. We demonstrated CD8+ T cell reactivity to one or more of the proteins in 6 of 9 patient samples. All 6 of these responders recognised peptides covering the E7 protein, 3 of 9 women responded to E6 peptides, but no reactivity was seen to E4. Our results suggest that HPV16-specific cytotoxic T cells (CTLs) are relatively common in women with persistent VIN3. The HPV-specific CTL response, however, seems to be ineffective. There is some evidence that there are problems associated with the processing and presentation of HPV antigens by the infected vulvar epithelium. It will be crucial to address this in the design of any T cell based therapy for HPV-associated VIN and vulval cancer.     

Prolonged cold-preservation of nerve allografts

The goal of this study was to determine the effect of varying durations of cold-preservation on the immunogenicity of nerve allografts and their subsequent ability to facilitate neuroregeneration across a short nerve gap. Allografts preserved for 1, 4, and 7 weeks were compared to untreated allografts and isografts. There was a shift from an interferon-gamma-producing cellular response (untreated allografts) to an absence of response (7-week cold-preserved allografts and isografts). There were no detectable alloantibodies by flow cytometry. Histomorphometry distal to the graft showed robust regeneration in the isograft and 7-week cold-preserved groups when compared to the untreated allograft group. Increasing duration of cold-preservation diminished the cellular immune response. This cold-preservation does not preclude subsequent nerve regeneration across a short nerve graft. Prolonged cold-preservation of nerve allograft tissue could serve as a means to produce unlimited graft material for use in peripheral nerve reconstruction.     

Rotavirus-specific B cells induced by recent infection in adults and children predominantly express the intestinal homing receptor alpha4beta7

In vivo replication of rotaviruses is generally limited to enterocytes. Because of this restriction, most blood circulating rotavirus-specific B cells are hypothesized to originate in Peyer's patches and should express the intestinal homing receptor alpha4beta7. To test this hypothesis in humans, we used a flow cytometry assay that identifies antigen-activated (IgD-) B cells (CD19+) that express surface rotavirus-specific immunoglobulin. With this assay we could detect rotavirus-specific B cells in both children and adults with an acute rotavirus (RV) infection. Staining with an anti-alpha4beta7 monoclonal antibody, we could determine that B cells that express rotavirus-specific surface immunoglobulin predominantly express alpha4beta7. The response of rotavirus-specific antibody-secreting cells in the peripheral blood of children and adults with acute rotavirus infection was also studied by ELISPOT. The antibody-secreting cells of children were mainly of the IgM isotype, while the antibody-secreting cells of adults were predominantly of the IgA and IgG isotype. alpha4beta7+ and alpha4beta7- subsets of peripheral blood mononuclear cells were purified using paramagnetic beads and then tested in the ELISPOT assay. Rotavirus-specific antibody-secreting cells were predominantly present in the alpha4beta7+ subpopulation. The flow cytometry assay we have described will permit future studies to characterize the phenotype of virus-specific B cells and could be useful in the study of the immunogenicity and protective efficacy of RV vaccines and the identification of markers of protective immunity.     

Accuracy of an immune diagnostic assay based on RD1 selected epitopes for active tuberculosis in a clinical setting: a pilot study

A previous case-control study reported that an in-vitro interferon (IFN)-γ response to early secreted antigenic target (ESAT)-6 selected peptides was associated with active tuberculosis (A-TB). The objective of the present pilot study was to evaluate the diagnostic accuracy of this assay for TB disease in a clinical setting. An IFN-γ ELISPOT assay was performed on samples from patients with suspected A-TB using two peptides selected from ESAT-6 protein and three peptides selected from culture filtrate 10 (CFP-10) proteins. The results were compared with those obtained by two commercially available assays approved for diagnosis of TB infection (T SPOT-TB and QuantiFERON-TB Gold) which use ESAT-6/CFP-10 (RD1) overlapping peptides. Sensitivity to the RD1 selected peptides was 70% (positive for 16 of 23 patients with microbiologically diagnosed A-TB) and specificity was 91% (positive for three of 32 controls). In contrast, the sensitivity and specificity were 91%and 59%, respectively, for T SPOT-TB, and were 83% and 59%, respectively, for QuantiFERON-TB Gold. The RD1 selected peptides assay had the highest diagnostic odds ratio for A-TB. Thus, the results suggest that an assay based on RD1 selected peptides has a higher diagnostic accuracy for A-TB in a clinical setting compared with commercially available assays based on RD1 overlapping peptides.     

Insights into the role of host genetic and T-cell factors in resistance to HIV transmission from studies of highly HIV-exposed Thais

Studies of resistance to HIV-1 transmission are likely to be valuable for the design of vaccines and other efforts to prevent HIV. Here, we review the T-cell and genetic factors associated with resistance to HIV-1 transmission in studies of highly exposed but persistently seronegative (HEPS) women from northern Thailand. Women were enrolled in two sex-worker studies and in a discordant couple study. We performed Cr⁵¹ cytotoxic T lymphocyte (CTL), interferon-γ (IFN-γ) ELISPOT, and proliferation assays as well as genetic studies, including HLA-class I typing. CTL and ELISPOT studies showed a skewing of T-cell responses to conserved HIV-1 proteins in HEPS, but not in HIV-1-seropositive women. T-cell responses were extremely long-lived in some HEPS women. In the two sex-worker studies, HLA-A11 was associated with resistance to HIV-1 transmission. These data provide promise for the ability of CTL to control HIV and emphasize the importance of developing HIV vaccines that stimulate strong, long-lasting T-cell responses.     

酶联免疫斑点方法改进及应用

建立了ELISPOT方法并通过改进 ,应用到抗人CD8杂交瘤细胞及LACA小鼠体外脾细胞分泌IgG量的检测 ,同时观察六味地黄多糖CA4 3在体外对LACA小鼠脾细胞分泌IgG的影响。结果表明 10 μg/mlLPS作用下可明显提高小鼠脾细胞体外产生抗体的量和提高分泌抗体的细胞数。 5 0 μg/mlCA4 3在体外与LPS具有相似的作用 ,可以刺激小鼠脾细胞分泌IgG的量和分泌抗体的细胞数量增多。     

Large numbers of dysfunctional CD8+ T lymphocytes bearing receptors for a single dominant CMV epitope in the very old

Longitudinal studies suggest that a set of immune parameters including high percentages of peripheral CD8⁺, CD28^–, CD57⁺ T lymphocytes, low CD4 and B cell counts, and poor T cell proliferative responses to mitogens is associated with decreased remaining longevity in the free-living very elderly (>85 years). This combination of immune parameters was also significantly associated with an inverted CD4/CD8 ratio and cytomegalovirus seropositivity. Here, using tetramer technology, we show markedly increased numbers of CD8⁺ T cells bearing receptors for one single CMV epitope in the very elderly. Moreover, the fraction of these tetramer-reactive cells secreting interferon- after specific antigenic stimulation was significantly lower in the old than in the young, as was the percentage of CD28-positive cells in this population. Therefore, we conclude that marked expansions of CMV-specific CD8⁺ T cells have occurred and that the obsession of a large fraction of the entire CD8⁺ T cell subset with one single viral epitope may contribute to the increased incidence of infectious disease in the elderly by shrinking the T cell repertoire available for responses to other antigens.     

自身免疫糖尿病细胞免疫学研究进展

自身免疫糖尿病是一种T细胞介导的胰岛 β细胞选择性破坏的自身免疫病。主要组织相容性复合物 (ma jorhistocompabilitycomplex ,MHC)限制性中枢及外周T细胞自身耐受丧失 ,细胞免疫调节失衡 ,胰岛自身抗原反应性T细胞克隆活化 ,Th1细胞及其细胞因子释放占优势 ,将启动、维持和加速自身免疫性胰岛炎 ,导致糖尿病发生。应用ELISPOT和可溶性MHC -肽四聚体技术并解决T细胞分析的标准化问题 ,将显著促进自身免疫糖尿病与细胞免疫学研究的发展。     

Systemic immune response in whiplash injury and ankle sprain: elevated IL-6 and IL-10

Whiplash injury and whiplash-associated disorders (WAD) are significant problems of modern society. Numerous attempts have been made to characterize the nature of whiplash injury. Whether the immune system is involved during the disease process is not known. In a prospective study, using enzyme-linked immunospot (ELISPOT) assays, we examined numbers of blood mononuclear cells (MNC) secreting pro- (IFN-gamma, TNF-alpha, IL-6) and anti-inflammatory (IL-10) cytokines in patients with WAD and, for reference, patients with ankle sprain and multiple sclerosis and healthy subjects. An immune response reflected by elevated numbers of TNF-alpha- and IL-10-secreting blood MNC was observed in patients with WAD examined within 3 days compared to 14 days after the whiplash injury. The patients with WAD examined within 3 days after the injury had also higher numbers of IL-6 and IL-10 secreting blood MNC compared to healthy subjects. The alterations of cytokine profiles observed in WAD were also observed in patients with ankle sprain when examined within 3 days after trauma. In contrast, there were no differences for cytokine profiles between patients with WAD examined 14 days after the whiplash injury and healthy subjects. Relatively minor trauma like WAD and ankle sprain are associated with a systemic dysregulation in numbers of cells secreting pro- as well as anti-inflammatory cytokines.