Kinetics of IFN-Gamma and TNF-Alpha Gene Expression and Their Relationship with Disease Progression after Infection with Mycobacterium Tuberculosis in Guinea Pigs

PURPOSE: Guinea pig is one of the most suitable animal models for Mycobacterium tuberculosis (M. tb) infection since it shows similarities to pulmonary infection in humans. Although guinea pig shows hematogenous spread of M. tb infection into the whole body, immunological studies have mainly focused on granulomatous tissues in lungs and spleens. In order to investigate the time-course of disease pathogenesis and immunological profiles in each infected organ, we performed the following approaches with guinea pigs experimentally infected with M. tb over a 22-week post-infection period. MATERIALS AND METHODS: We examined body weight changes, M. tb growth curve, cytokine gene expression (IFN-gamma and TNF-alpha), and histopathology in liver, spleen, lungs and lymph nodes of infected guinea pigs. RESULTS: The body weights of infected guinea pigs did not increase as much as uninfected ones and the number of M. tb bacilli in their organs increased except bronchotracheal lymph node during the experimental period. The gene expression of IFN-gamma and TNF-alpha was induced between 3 and 6 weeks of infection; however, kinetic profiles of cytokine gene expression showed heterogeneity among organs over the study period. Histophathologically granulomatous lesions were developed in all four organs of infected guinea pigs. CONCLUSION: Although IFN-gamma and TNF-alpha gene expression profiles showed heterogeneity, the granuloma formation was clearly observed in every organ regardless of whether the number of bacilli increased or decreased. However, this protective immunity was accompanied with severe tissue damage in all four organs, which may lead to the death of guinea pigs.

M. tuberculosis Somatic Antigen Specific CD8+T cell Responses in BCG-Vaccinated Subjects / 결핵및호흡기질환

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.

The Utility of Pleural Fluid Cell IFN-gamma Production Assay in the Diagnosis of Tuberculous Pleurisy / 결핵및호흡기질환

BACKGROUND: Diagnosis of tuberculous pleurisy is sometimes difficult using conventional diagnostic methods. We have investigated the utility of pleural fluid cell IFN-gamma production assay in the diagnosis of tuberculous pleurisy. METHODS: We prospectively performed pleural fluid cell IFN-gamma production assay in 39 patients with tuberculous pleural effusions (TPE) and in 26 patients with nontuberculous pleural effusions (NTPE) (13 malignant pleural effusions and 13 parapneumonic effusions). Pleural fluid cells were cultured in DMEM media and stimulated with purified protein derivatives (PPD), and phytohemagglutinin (PHA) for 24 hr. The amount of IFN-gamma released in the culture supernatant was quantitated by IFN-gamma ELISA assay. We have also measured adenosine deaminase (ADA) activities and IFN-gamma concentrations in the pleural fluid. RESULTS: 1) The pleural fluid levels of ADA activity and IFN-gamma concentrations were significantly higher in TPE than NTPE (p<0.01). 2) IFN-gamma production in TPE cells stimulated by PPD (755,266+/-886,636 pg/ml) was significantly higher than NTPE cells (3,509+/-6,980 pg/ml) (p<0.01). By considering the fact that IFN-gamma concentrations over 10,000 pg/ml is a criteria for the diagnosis of TBE, sensitivity and specificity of the test were 97.4 and 92.3%, respectively. 3) The ratios of IFN-gamma production by the stimulation with PPD and PHA (PPD/PHA) were significantly higher in TPE cells (59+/-85) than NTPE cells (5+/-18)(p<0.01). Considering the criteria for the diagnosis of TBE as PPD/PHA ratio over 5, sensitivity and specificity of the test were 76.9 and 92.3%, respectively. CONCLUSION: Pleural fluid cell IFN-gamma production assay may be useful for the diagnosis of tuberculous pleurisy.

The Study of MHC class I Restricted CD8+ T Cell Mediated Immune Responses against Mycobacterium tuberculosis Infection: Evidence of M. tuberculosis Specific CD8+ T Cells in TB Patients and PPD+ Healthy Individuals

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.

Protective Immune Mechanism against Mycobacterial tuberculosis Infection / 나학회지

Because tuberculosis (TB) causes two - three million deaths annually, development of a vaccine to control and eradicate the infection is an important unmet medical need. Analysis of the mechanisms of protective immunity in mouse models has indicated that both MHC class I- and class II-restricted T cells contribute to immunity against TB. MHC class II-restricted CD4+ T cells release lymphokines such as IFN-gamma and tumor necrosis factor-alpha that result in macrophage activation. More importantly, beta(2)-microglobulin (beta(2)m)-deficient mice are unable to develop MHC class I-restricted cytotoxic T lymphocytes (CTL) and rapidly succumb to Mycobacterium tuberculosis (MTB) infection. This pathway of protective immunity appears to involve CD8+ T cells and transporters associated with antigen processing (TAP)-dependent presentation of peptide antigen. Thus, studies of mouse models of tuberculosis (TB) infection have indicated a central role for MHC class I-restricted CD8+ T cells in protective immunity. Furthermore, CD8+ T cells not only are able to lyse MTB infected cells but also can simultaneously kill intracellular bacteria by the release of the antimicrobial peptide granulysin. To define antigens and epitopes of Mycobacterium tuberculosis (MTB) proteins that are presented by infected cells to CD8+ T cells, we screened 40 MTB proteins for HLA class I A(*)0201-binding motifs. Peptides that bound with high affinity to purified HLA molecules were subsequently analyzed for recognition by CD8+ cytotoxic T lymphocytes. We identified three epitopes recognized by CD8+ T cells from patients recovering from TB infection. Those three epitopes were derived from three different antigens: thymidylate synthase (ThyA(30-38)), RNA polymerase beta-subunit (RpoB(127-135)), and a putative phosphate transport system permease protein A-1 (PstA1(75-83)). In addition, CD8+ T cell lines specific for three peptides (ThyA(30-38), PstA1(75-83), and 85B(15-23)) were generated from peripheral blood mononuclear cells of normal HLA-Al(*)0201 donors. These CD8+ T cell lines specifically recognized MTB-infected macrophages, as demonstrated by production of IFN-gamma and lysis of the infected target cells. Finally, CD8+ cytotoxic T lymphocytes reduced the viability of the intracellular MTB, providing evidence that CD8+ T cell recognition of MHC class I-restricted epitopes of these MTB antigens can contribute to effective immunity against tuberculosis. Currently, the frequencies of circulating CD8+ T cells specific for each epitope peptide are being investigated in TB patients, normal PPD+ and PPD- donors in order to evaluate the relationship of epitope-specific CD8+ T cell population to the immune responses to MTB infection.

Identification of Functionally Different Rat IgE in RBL-2H3 Exocytosis

BACKGROUND: IgE is closely related to the development of allergies. However, the poor relationship between the specific IgE level and the severity of allergic diseases suggests the possibility of functionally different IgE isoforms. With this in mind, rat basophilic leukemia (RBL)-2H3 activation was analyzed with each type of rat IgE for two parameters, exocytosis and IL-4 mRNA production. RBL-2H3 has been well documented in the rat mucosal mast cell line. METHODS: RBL-2H3 cells sensitized with each kind of rat IgE was activated by cross-linking FcRI with B5 (monoclonal anti-rat IgE mouse IgG antibodies). The RBL-2H3 exocytosis was measured by analyzing the beta-hexosaminidase level, and the level of IL-4 mRNA synthesis was analyzed using semi- quantitative RT-PCR. Rat IgE, which was produced by a parasite infection (REP), was prepared using either Paragonimus westermani metacercariae (REP-PW) or Anisakis simplex third stage larvae (REP-AS). A rat IgE prototype of IR162 was prepared by a peritoneal injection of immunocytoma. RESULTS: The level of exocytosis showed a linear relationship with the rat IgE concentration when REP-PW or REP-AS was applied. However, it exhibited a biphasic response with IR162. In addition, the time course of heating at 56oC illustrated the similarity between REP-PW and REP-AS, which differed from that of IR162. In contrast, the level of IL-4 mRNA synthesis in the RBL-2H3 cells with IR162 was comparable to that of either REP-PW or REP-AS. CONCLUSION: These results suggest that functionally different rat IgE isoforms exists in RBL-2H3 exocytosis.