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 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.

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.

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*

BACKGROUND: The protective immunity against tuberculosis (TB) involves both CD4+ T cells and CD8+ T cells. In our previous study, we defined four Mycobacterium tuberculosis derived peptide epitopes specific for HLA-A*0201 restricted CD8+ T cells (ThyA30-38, RpoB127-135, 85B15-23, PstA175-83). In this study, we investigated the immune responses induced by these peptide specific CD8+ T cells in latently and chronically infected people with TB. METHODS: We characterized these peptide specific CD8+ T cell population present in PBMC of both TB patients and PPD healthy people using IFN-gammaelispot assay, intracellular staining and HLA-A2 dimer staining. RESULTS: The frequency of peptide specific CD8+ T cell was in the range of 1 to 25 in 1.7x10(5) PBMC based on ex vivo IFN-gamma elispot assay, demonstrating that these peptide specific CD8+ T cell responses are induced in both TB patients and PPD people. Short term cell lines (STCL) specific for these peptides proliferated in vitro and secreted IFN-gamma upon antigenic stimulation in PPD+ donors. Lastly, HLA-A*0201 dimer assays indicated that PstA175-83 specific CD8+ T cell population in PPD+ healthy donors is heterogeneous since approximately 25~33% of PstA175-83 specific CD8+ T cell population in PPD+ healthy donors produced IFN-gamma upon peptide stimulation. CONCLUSION: Our results suggest that MHC class I restricted CD8+ T cell mediated immune responses to M. tuberculosis infection are induced in both TB patients and PPD+ people; however, the CD8+ T cell population is functionally heterogeneous.
Cell Line ; Enzyme-Linked Immunospot Assay ; Epitopes ; HLA-A2 Antigen ; Humans ; Mycobacterium tuberculosis* ; Mycobacterium* ; Peptides ; T-Lymphocytes* ; Tissue Donors ; Tuberculosis*

BACKGROUND/AIMS: We evaluated the proposed clinical application of the combined interpretation of host factors and viral factors in two different cytomegalovirus (CMV) co-infection models. METHODS: We prospectively enrolled all human immunodeficiency virus non-infected patients with confirmed Pneumocystitis jirovecii pneumonia (PCP) and those with suspected gastrointestinal CMV disease in a tertiary hospital. All patients underwent CMV interferon-γ releasing assay (IGRA) for CMV (T-track CMV, Lophius Biosciences). We created the 2-axis model with the CMV IGRA results as the x-axis and the results for CMV virus replication as the y-axis, and hypothesized that cases falling in the left upper quadrant (high viral load and low CMV-specific immunity) of the model would be true CMV infections. The CMV IGRA results were concealed from the attending physicians. RESULTS: Of 39 patients with PCP, four (10%) were classified as combined CMV pneumonia, 13 (33%) as bystander activation, and the remaining 22 (56%) as no CMV infection. The data for all four patients with PCP and CMV pneumonia fell in the left upper quadrant of the 2-axis model. Of 24 patients with suspected gastrointestinal CMV disease, 12 (50%) were classified as gastrointestinal CMV disease and the remaining 12 (50%) as bystander activation with no gastrointestinal CMV disease. The data for 11 of the 12 patients (92%) with gastrointestinal CMV disease were located in the left upper quadrant of the 2-axis model. CONCLUSIONS: Cases yielding low CMV IGRA results and high CMV viral replication appear to be true CMV infections. Further studies with large number of cases in different types of CMV disease should be proposed.
Accidental Falls ; Coinfection ; Cytomegalovirus* ; Enzyme-Linked Immunospot Assay ; HIV ; Humans ; Pneumonia ; Prospective Studies ; Tertiary Care Centers ; Viral Load ; Virus Replication

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

BACKGROUND: Interferon-gamma assays based on tuberculosis (TB)-specific antigens have been utilized for diagnosing and ruling out latent TB and active TB, but their utility is still limited for TB incidence countries. The aim of this study is to understand the clinical utility of enzyme-linked immunospot (ELISpot) assays among patients with clinically suspected TB and healthy adults in clinical practices and community-based settings. METHODS: The ELISpot assays (T SPOT.TB, Oxford Immunotec, UK) were prospectively performed in 202 patients. After excluding those with indeterminate results, 196 were included for analysis: 41 were TB patients, 93 were non-TB patients, and 62 were healthy adults. RESULTS: The sensitivity and negative predictive values of the T SPOT.TB assays for the diagnosis of TB were 87.8% and 89.1%, respectively, among patients with suspected TB. The agreement between the tuberculin skin test (10-mm cutoff) and the T SPOT.TB assay was 66.1% (kappa=0.335) in all participants and 80.0% (kappa=0.412) in TB patients. Among those without TB (n=155), a past history of TB and fibrotic TB scar on chest X-rays were significant factors that yielded positive T SPOT.TB results. There was a significant difference in the magnitude of T SPOT.TB spot counts between TB patients and non-TB patients or healthy adults. CONCLUSION: The T SPOT.TB assay appeared to be a useful test for the diagnostic exclusion of TB. A positive result, however, should be cautiously interpreted for potential positives among those without active TB in intermediate TB incidence areas.
Adult* ; Cicatrix ; Diagnosis ; Enzyme-Linked Immunospot Assay* ; Humans ; Incidence ; Interferon-gamma* ; Prospective Studies ; Skin Tests ; Thorax ; Tuberculin ; Tuberculosis*