BACKGROUND: The immune responses mediated by CD8+T cells are known to be significant in controlling M. tuberculosis infections. In order to determine the role of cytotoxic CD8+T cells in the protective immune mechanism in latently infected subjects, this study examined whether or not the cytotoxic immune responses of CD8+T cells specific to the M. tuberculosis somatic antigens are induced in BCG vaccinated healthy subjects. METHODS: Cytotoxicity and IFN-gamma elispot assays were used to investigate the activities of CD8+T cells specific for the thyA30-38 peptide epitope in circulating peripheral blood mononuclear cells (PBMC) from BCG-vaccinated HLA-A*0201 and A*0206 subjects. RESULTS: The results indicate the cytotoxic and IFN-gamma immune responses of CD8+T cells specific for thyA30-38 were induced in BCG vaccinated healthy subjects. CONCLUSION: The cytotoxic and IFN-gamma responses by CD8+T cells specific for the M. tuberculosis somatic antigens are induced in BCG-vaccinated subjects, and appear to be involved in the protective immune mechanism in latently infected people against a M. tuberculosis infection.
Enzyme-Linked Immunospot Assay ; Mycobacterium bovis ; Tuberculosis*

OBJECTIVETo compare enzyme-linked immunospot assay (ELISPOT) and tuberculin skin test (TST) and explore their roles in the auxiliary diagnosis of initial pulmonary tuberculosis.

METHODSTotally 123 patients with initial pulmonary tuberculosis (tuberculosis group) and 102 patients with non-tuberculosis pulmonary disease (control group) were enrolled. The peripheral blood mononuclear cells of all participants were co-cultured with early secretiny antigen target-6/culture filtrate protein-10 fusion protein (ESAT-6/CFP-10), and spot forming cells (SFCs) were enumerated by ELISPOT (ESAT-6/CFP-10-ELISPOT). TST was also performed simultaneously.

RESULTSESAT-6/CFP-10-ELISPOT showed significantly higher numbers of SFCs after stimulation in tuberculosis group than in control group (P = 0.000). The sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, positive predictive value, and negative predictive value of ESAT-6/CFP-10-ELISPOT were 91.1% (111/123), 81.4% (82/102), 4.60, 0.12, 0.85, and 0.87 respectively, while the above values of TST were 65.6% (59/90), 45.1% (46/102), 1.31, 0.76, 0.51, and 0.60, respectively. The sensitivity and specificity of ESAT-6/CFP-10-ELISPOT were significantly higher than those of TST (all P = 0.000). The number of SFCs were not significantly different between smear-positive tuberculosis subgroup and smear-negative tuberculosis subgroup (P = 0.166). The sensitivities were 91.8% (67/73) and 88.0% (44/50) in these two subgroups, respectively, (P = 0.448).

CONCLUSIONSESAT-6/CFP-10-ELISPOT may be a more accurate approach for the auxiliary diagnosis of initial pulmonary tuberculosis; meanwhile, it offers certain diagnostic evidences for smear-negative tuberculosis. However, its specificity may be affected by latent tuberculosis infection. On the contrary, TST has poor value in the auxiliary diagnosis of initial pulmonary tuberculosis.

OBJECTIVETo establish a method of detecting spinal tuberculosis (TB) infection by enzyme-linked immunospot (ELlSPOT) assay and evaluate the value of CFP10/ESAT6 fusion protein for diagnosis of spinal TB.

METHODSSuspected spinal TB patients were prospectively recruited in two hospitals (First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine; Nanfang Hospital, Southern Medical University) from May 2012 to December 2013. Data on clinical characteristics of the patients and conventional laboratory results were collected. Compare and analyze the positive detection rate in spinal TB diagnosis by different methods including ELISPOT detection and conventional detection methods.

RESULTS47 patients with spinal TB had available biopsy or surgical specimens for histopathological examination and 41 specimens had pathological features consistent with a diagnosis of TB infection. Among the spinal TB patients and non-TB disease patients,the overall sensitivity, specificity, positive predictive value, and negative predictive value of the ELISPOT assay in spinal TB diagnosis were 82.7%,87.2%,89.6%, and 79.1%,respectively; the 4 indexes of the PPD skin test were 61.5%, 46.2%, 60.4%, and 47.4%, respectively;those of the antibody detection were 55.8%, 61.5%, 65.9%, and 51.1%. The positive rate of ELISPOT was significantly higher than those of PPD skin test and antibody detection test (82.7% vs. 61.5%, Χ² =5.786, P=0.016; 82.7% vs. 55.8%, Χ² =8.847, P=0.003), but not significantly different from the positive rate of pathological examination (82.7% vs. 87.2%, Χ² =0.396, P=0.529). Moderate agreement was found between pathological examination and the ELISPOT assay (87.2%, Κ=0.498, P=0.001).

CONCLUSIONWith high sensitivity and specificity, the ELISPOT assay using CFP10/ESAT6 fusion protein as antigen is an effective technique for auxiliary diagnosis of spinal TB.

Aluminum hydroxide (alum) is the most widely used adjuvant in human vaccines. Nevertheless, it is virtually unknown whether alum acts on B cells. In the present study, we explored the direct effect of alum on Ig expression by murine B cells in vitro. LPS-activated mouse spleen B cells were cultured with alum, and the level of isotype-specific Ig secretion, IgG1 secreting cell numbers, and Ig germ-line transcripts (GLT) were measured using ELISA, ELISPOT, and RT-PCR, respectively. Alum consistently enhanced total IgG1 production, numbers of IgG1 secreting cells, and GLTgamma1 expression. These results demonstrate that alum can directly cause IgG1 isotype switching leading to IgG1 production.
Alum Compounds ; Aluminum Hydroxide ; Animals ; B-Lymphocytes ; Cell Count ; Enzyme-Linked Immunosorbent Assay ; Enzyme-Linked Immunospot Assay ; Humans ; Hydroxides ; Immunoglobulin Class Switching ; Immunoglobulin G ; Mice ; Spleen ; Vaccines

BACKGROUND: Zoster vaccination is recommended for people with a history of herpes zoster (HZ), but the most effective timing of vaccine administration after zoster illness is unresolved. This prospective observational study compared the immunogenicity and safety of administering HZ vaccine at 6-12 months and 1-5 years after zoster illness. MATERIALS AND METHODS: Blood samples were collected before the administration of live zoster vaccine and 6 weeks after vaccination. Varicella-zoster virus (VZV) IgG concentrations and T-cell responses were assessed by glycoprotein enzyme-linked immunosorbent assay and interferon-γ enzyme-linked immunospot assay (ELISPOT), respectively. RESULTS: The baseline geometric mean value (GMV) of VZV IgG was higher in the 6-12 months group than in the 1-5 years group (245.5 IU/mL vs. 125.9 IU/mL; P = 0.021). However, the GMV increased significantly in both groups (P = 0.002 in the 6-12 months group; P <0.001 in the 1-5 years group). The results of the ELISPOT assay were not significant for differences of the GMV between baseline and 6-week post-vaccination groups, while the GMV increased significantly in both groups (P = 0.001 in the 6-12 months group; P <0.001 in the 1-5 years group). CONCLUSION: The immunogenicity of zoster vaccine may be similar whether administered 6-12 months, or >1 year after zoster illness. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02704572
Enzyme-Linked Immunosorbent Assay ; Enzyme-Linked Immunospot Assay ; Glycoproteins ; Herpes Zoster Vaccine* ; Herpes Zoster* ; Herpesvirus 3, Human ; Immunoglobulin G ; Observational Study ; Prospective Studies* ; T-Lymphocytes ; Vaccination*

BACKGROUND: Zoster vaccination is recommended for people with a history of herpes zoster (HZ), but the most effective timing of vaccine administration after zoster illness is unresolved. This prospective observational study compared the immunogenicity and safety of administering HZ vaccine at 6-12 months and 1-5 years after zoster illness. MATERIALS AND METHODS: Blood samples were collected before the administration of live zoster vaccine and 6 weeks after vaccination. Varicella-zoster virus (VZV) IgG concentrations and T-cell responses were assessed by glycoprotein enzyme-linked immunosorbent assay and interferon-γ enzyme-linked immunospot assay (ELISPOT), respectively. RESULTS: The baseline geometric mean value (GMV) of VZV IgG was higher in the 6-12 months group than in the 1-5 years group (245.5 IU/mL vs. 125.9 IU/mL; P = 0.021). However, the GMV increased significantly in both groups (P = 0.002 in the 6-12 months group; P <0.001 in the 1-5 years group). The results of the ELISPOT assay were not significant for differences of the GMV between baseline and 6-week post-vaccination groups, while the GMV increased significantly in both groups (P = 0.001 in the 6-12 months group; P <0.001 in the 1-5 years group). CONCLUSION: The immunogenicity of zoster vaccine may be similar whether administered 6-12 months, or >1 year after zoster illness. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02704572
Enzyme-Linked Immunosorbent Assay ; Enzyme-Linked Immunospot Assay ; Glycoproteins ; Herpes Zoster Vaccine* ; Herpes Zoster* ; Herpesvirus 3, Human ; Immunoglobulin G ; Observational Study ; Prospective Studies* ; T-Lymphocytes ; Vaccination*

OBJECTIVETo highlight the clinical features and diagnosis of chronic disseminated tuberculosis, with emphasizing the usefulness of several recently available diagnostic technologies in this setting.

METHODWe presented a case of chronic disseminated tuberculosis diagnosed with the combined application of interferon-gamma release assay T-SPOT. TB, 18F-fluorodeoxyglucose (FDG)-positron emission tomography (PET), and gene chip assay.

RESULTSA 53-year-old gentleman who had chronic cough for 7 years and fever for 2 weeks was referred to our hospital for further evaluation. 18F-FDG-PET/CT scan showed increased FDG uptake in multiple lesions involving bilateral lungs, supraclavicular, mediastinal and intro-abdominal lymph nodes and bones, mimicking metastatic malignancy. T-SPOT. TB assay revealed significant responses [ early secreting antigen target 6 (ESAT-6): 3 908 spot forming cells (SFCs)/10(6) peripheral blood mononuclear cells (PBMCs), culture filtrate protein (CFP-10): 3 400 SFCs/10(6) PBMCs]. Subsequent biopsy of supraclavicular lymph node, lung, and ilium revealed granulomas, while culture of the obtained tissue yeilded mycobacteria. Gene chip testing identified M. tuberculosis sensitive to isoniazid and rifampin. After 10 weeks of treatment for tuberculosis, the patient's condition was improved and a second T-SPOT. TB assay showed significantly reduced responses (ESAT-6: 1528 SFCs/10(6) PBMCs; CFP-10: 1460 SFCs/10(6) PBMCs).

CONCLUSIONSTimely diagnosis of chronic disseminated tuberculosis requires high index of suspicion. T-SPOT. TB assay, PET/CT, and gene chip assay may provide valuable information that facilitates further diagnostic procedures and treatment decision.

We established an ELISPOT for bovine interferon-gamma (BoIFN-γ), and applied it in the diagnosis of bovine tuberculosis (bTB). Monoclonal antibodies that can bind with native BoIFN-γ were screened as the coating antibody and detecting antibody. After optimization of detecting conditions including coating antibody concentration, cell number, and detecting antibody concentration, the ELISPOT assay was established. Peripheral mononuclear cells (PBMCs) isolated from 30 cows were co-cultured with PPD, and detected with the ELISPOT assay. The optimal conditions of ELISPOT assay were 2.5 μg/mL coating antibody 2G5, 2.5 x 10(5) cells/well, and 1 μg/mL detecting antibody Bio-5E11. In these 30 cows tested both with the ELISPOT assay and the BOVIGAM kit, 11 cows were proved to be positive in ELISOPT assay with the sensitivity of 78.6%, and 12 cows were proved to be negative in ELISOPT assay with the specificity of 75%. The ELISPOT assay for BoIFN-γ could be used to detect bTB efficiently and it might be an alternative method for the diagnosis of bTB.
Animals ; Antibodies, Monoclonal ; Cattle ; Enzyme-Linked Immunospot Assay ; veterinary ; Female ; Interferon-gamma ; isolation & purification ; Sensitivity and Specificity ; Tuberculosis, Bovine ; diagnosis

PURPOSE: To investigate the toxicity of a short-term application of timolol maleate, dorzolamide, and benzalkonium chloride (BAC) on human conjunctival epithelial cells in vitro. METHODS: Chang's conjunctival epithelial cell line was treated for 5 min, 15 min, 30 min, and 60 min with various concentrations of timolol, dorzloamide, or BAC, and then examined 4 hrs or 24 hrs later. Cell viabilities were assessed by MTT assay. The expressions of various cytokines by timolol maleate, dorzolamide, and BAC treatment in human conjunctival epithelial cells were evaluated using ELISPOT. RESULTS: BAC significantly decreased survival of conjunctival epithelial cells in a dose and time dependent manner compared with timolol and dorzolamide. Inflammatory cytokines, IL-6 and IL-8, were highly expressed in conjunctival epithelial cells treated with timolol, dorzolamide, and BAC. CONCLUSIONS: The present study suggests that increased expression of inflammatory markers, IL-6 and IL-8 might explain the ocular surface disorder in patients receiving antiglaucoma medication.
Benzalkonium Compounds ; Cell Survival ; Cytokines ; Enzyme-Linked Immunospot Assay ; Epithelial Cells* ; Humans* ; Interleukin-6 ; Interleukin-8 ; Timolol

In organ transplantation, achieving an operational tolerance is the ultimate goal. However, inducing this tolerance with a minimal dose of anti-rejection drugs can only be safely achieved when guided by biomarkers reflecting the immune reactivity in patients. We review recently described biomarkers and assays for the identification of patients for their risk of organ rejection and their suitability for drug weaning. The clinical assessment of tolerance has been attempted with immunological tools, including the analysis of immune cell subsets, regulatory T cells, ELISPOT, and trans-vivo delayed-type hypersensitivity assays. These methods are promising tools for diagnosis; however, their ability to determine the presence and persistence of responsiveness to their transplant is not available. As the "omics" technologies advance, comprehensive and high-throughput biomarker studies have become more accessible, more affordable, and more customizable. Validating the use of microarray analysis has important implications for monitoring patients for the development of tolerance following transplantation as well as for determining the mechanisms by which tolerance is induced and maintained. Finally, collaborative efforts are needed to design and perform prospective multicenter trials to validate the identified biomarkers across different laboratories.
Biomarkers ; Enzyme-Linked Immunospot Assay ; Humans ; Hypersensitivity ; Immune Tolerance ; Microarray Analysis ; Organ Transplantation ; Rejection (Psychology) ; T-Lymphocytes, Regulatory ; Transplants ; Weaning