Modulation of cytokines and transcription factors (T-Bet and GATA3) in CD4 enriched cervical cells of Chlamydia trachomatis infected fertile and infertile women upon stimulation with chlamydial inclusion membrane proteins B and C

<p>Abstract</p> <p>Background</p> <p>Chlamydial Inclusion membrane proteins (Incs), are involved in biochemical interactions with host cells and infecting Chlamydiae. We have previously reported the role of two Chlamydia trachomatis (CT) Incs, namely IncB and IncC in generating host immunity in CT infected women. Emerging data shows involvement of Inc stimulated CD4 positive T cells in aiding host immunity in infected fertile and infertile women through the secretion of interferon gamma. However the lack of data on the intra-cytokine interplay to these Incs in infected cell milieu prompted us to investigate further.</p> <p>Methods</p> <p>A total of 14 CT-positive fertile, 18 CT-positive infertile women and 25 uninfected controls were enrolled in this study. CD8 depleted, CD4 enriched cervical cells were isolated and upon stimulation with IncB and IncC, modulation of cytokines (Interleukin (IL)-1 Beta, IL-4, IL-5, IL-6, IL-10, Interferon-gamma, IL-12, IL-23, Tumor Necrosis Factor-alpha and Granulocyte macrophage colony-stimulating factor (GM-CSF) and T cell lineage regulating transcription factors T-Bet and GATA3 was determined by real-time reverse-transcriptase (RT)-PCR and ELISA.</p> <p>Results</p> <p>Significant higher expression (P < 0.05) of Interferon-gamma, IL-12, IL-23 and GM-CSF were found in Inc-stimulated CD4 enriched cervical cells of CT-positive fertile women and contrastingly high IL-1 Beta, IL-4, IL-5, IL-6 and IL-10 levels were found in CT-positive infertile women. Positive correlation (P < 0.05) was found between Interferon-gamma and T-Bet levels in CT-positive fertile women and IL-4 mRNA and GATA3 levels in CT-positive infertile patients upon IncB and IncC stimulation.</p> <p>Conclusion</p> <p>Overall our data shows that CT IncB and IncC are able to upregulate expression of cytokines, namely interferon-gamma, IL-12, IL-23 and GM-CSF in CT-positive fertile women while expression of IL-1 Beta, IL-4, IL-5, IL-6 and IL-10 were upregulated in CT-positive infertile women. Our study also suggests that Incs are able to modulate expression of T cell lineage determinants indicating their involvement in regulation of immune cells.</p

Host immune responses to chlamydial inclusion membrane proteins B and C in Chlamydia trachomatis infected women with or without fertility disorders

<p>Abstract</p> <p>Background</p> <p>With an increase in the number of putative inclusion membrane proteins (incs) in chlamydial genomes, there is a need for understanding their contribution in host-pathogen interactions. Thus in this study we determined the host mucosal and peripheral immune responses to incs (IncB and IncC) of Chlamydia trachomatis (CT).</p> <p>Methods</p> <p>Female patients (n = 296) attending the gynaecology out patient department of Safdarjung hospital, New Delhi were enrolled for the study and were clinically characterized into two groups; CT-positive fertile women (n = 38) and CT-positive women with fertility disorders (n = 29). Uninfected healthy fertile women were enrolled as controls (n = 31). Gene specific PCRs were used for detection of incB and incC genes in endocervical samples of CT-positive patients. ELISA and Western blot assay were used for detection of IgA and IgG antibodies to IncB and IncC in cervical washes and sera. Effect of IncB and IncC stimulation of cervical cells and PBMCs on cellular proliferation and cytotoxity was determined using MTT assay and Lactate dehydrogenase (LDH)-cytotoxicity assay respectively. Modulation of cytokines (Interleukin (IL)-1 Beta, IL-4, IL-5, IL-6, IL-10, Interferon-gamma, IL-12, Tumor Necrosis Factor-alpha and Granulocyte macrophage colony-stimulating factor (GM-CSF)) in cervical cells and PBMCs upon stimulation with IncB and IncC was determined by real-time reverse-transcriptase (RT)-PCR and ELISA. Further, CD4 positive T cells were purified from cervical cells and peripheral blood mononuclear cells (PBMCs) and secreted cytokines (Interferon-gamma and IL-4) were evaluated by ELISPOT and real-time RT-PCR.</p> <p>Results</p> <p>Using MTT assay, significantly high proliferative responses (P < 0.05) were observed in inc-stimulated cervical cells and PBMCs from CT-positive fertile women compared to CT-positive women with fertility disorders and controls. Interferon-gamma, IL-12 and GM-CSF were found to be elevated in inc-stimulated cervical cells and PBMCs of CT-positive fertile women compared to CT-positive women with fertility disorders and controls (P < 0.05). In contrast, IL-1 Beta, IL-4, IL-5, IL-6 and IL-10 levels were found to be higher in CT-positive women with fertility disorders compared to CT-positive fertile women and controls (P < 0.05). Interferon-gamma secreting cells and mRNA expression in inc-stimulated cervical and peripheral CD4 positive T cells were significantly higher (P < 0.05) in CT positive fertile women compared to CT-positive women with fertility disorders.</p> <p>Conclusion</p> <p>Our data overall suggests that CT incs, IncB and IncC modulate host immune responses and may have a role in protection/pathogenesis of genital chlamydial infection in women.</p

Higher incidence of persistent chronic infection of Chlamydia pneumoniae among coronary artery disease patients in India is a cause of concern

<p>Abstract</p> <p>Background</p> <p>There is growing evidence that <it>Chlamydia pneumoniae </it>may be involved in the pathogenesis of atherosclerosis, as several studies have demonstrated the presence of the organism in atherosclerotic lesions. <it>C. pneumoniae </it>infections, which are especially persistent infections, have been difficult to diagnose either by serological methods or isolation of the organism from the tissue. Nucleic Acid Amplification tests (NAATs) has emerged as an important method for detecting <it>C. pneumoniae</it>. Inspite of high prevalence of <it>C. pneumoniae </it>specific antibodies in coronary heart disease patients, direct detection of <it>C. pneumoniae </it>in circulating blood of coronary artery disease (CAD) patients by sensitive nucleic acid amplification tests nested PCR (nPCR), multiplex PCR (mPCR) has not been carried out is required. Further correlation of the presence of <it>C. pneumoniae </it>in blood of CAD patients with <it>C. pneumoniae </it>specific IgA and IgG antibodies, which may indicative of the status of infection with the progression of atherosclerosis. This will help in order to prepare strategies for the antibiotic intervention to avoid the progression towards CAD.</p> <p>Methods</p> <p>Venous blood was obtained from 91 CAD patients and 46 healthy controls. Nucleic acid amplification tests <it>viz</it>. nested -, semi-nested – and multiplex PCR were used for detection of <it>C. pneumoniae</it>. ELISA carried out prevalence of <it>C. pneumoniae </it>specific IgG and IgA antibodies.</p> <p>Results</p> <p>29.67% (27/91) patients were positive for <it>C. pneumoniae </it>using nested PCR. The sensitivity and specificity of semi-nested and multiplex PCR were 37.03%, 96.96% and 22.22%, 100% with respect to nested PCR. Positive nPCR patients were compared with presence of <it>C. pneumoniae </it>specific IgA, IgA+IgG and IgG antibodies. Among 27 (29.67%) nPCR <it>C. pneumoniae </it>positive CAD patients, 11(12%) were IgA positive, 13(14.2%) were IgA+IgG positive and only1 (1.1%) was IgG positive. A significant presence of <it>C. pneumoniae </it>was detected in heavy smokers, non-alcoholics and with family histories of diabetes and blood pressure group of CAD patients by nPCR.</p> <p>Conclusion</p> <p>The results indicate synergistic association of <it>C. pneumoniae </it>infection and development of CAD with other risk factors. We also detected increased positivity for <it>C. pneumoniae </it>IgA than IgG in nPCR positive CAD patients. Positive nPCR findings in conjunction with persisting high <it>C. pneumoniae </it>specific antibody strongly suggest an ongoing infection.</p

Original Article Seroprevalence of antibodies against Chlamydia trachomatis inclusion membrane proteins B and C in infected symptomatic women

Background: Proteins in the inclusion membrane of Chlamydia trachomatis (CT) have been anticipated to play pivotal roles in the molecular and cellular interactions between the pathogen and host. However, there is lack of data on host immunity with respect to antibody responses against chlamydial inclusion proteins. Methodology: We used full-length fusion proteins for CT inclusion membrane proteins B and C (IncB and IncC respectively), two earlyinfection phase proteins, to study their role in antibody generation during human infection. Results: Three hundred and fifty-five women (aged 22-36 years) attending the Gynaecology outpatient department, Safdarjang Hospital, New Delhi, India were enrolled in this hospital ethical committee approved study. Out of these, 108 were diagnosed to be cervical CT-positive. Of these 108 patients, 67 (62.03%) showed ELISA positivity for IncB IgG, and 64 (59.25%) for IncC IgG. There was a positive correlation between antibody titres against IncB and IncC and with antibodies against CT major outer membrane protein (MOMP) in CT-positive sera. Our data also showed a positive association between antibody titres against IncB and IncC in patients with cervicitis and pelvic inflammatory disease (PID). Significantly high antibody titres were detected in cervicitis cases compared with PID. There were significantly higher levels of serum cytokines (TNF-, IFN-γ and IL-12) in Inc-positive cervicitis cases than in PID cases. In addition, our study also showed higher IncB and IncC IgG 2 titres in comparison to respective IgG 1, IgG 3 and IgG 4 titres in CT-positive sera. Conclusion: Our data suggested that antibodies against CT IncB and IncC were prevalent in CT-positive women diagnosed with cervicitis o