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

In infertile women, cells from Chlamydia trachomatis infected site release higher levels of interferon-gamma, interleukin-10 and tumor necrosis factor-alpha upon heat shock protein stimulation than fertile women

<p>Abstract</p> <p>Background</p> <p>The magnitude of reproductive morbidity associated with sexually transmitted Chlamydia trachomatis infection is enormous. Association of antibodies to chlamydial heat shock proteins (cHSP) 60 and 10 with various disease sequelae such as infertility or ectopic pregnancy has been reported. Cell-mediated immunity is essential in resolution and in protection to Chlamydia as well as is involved in the immunopathogenesis of chlamydial diseases. To date only peripheral cell mediated immune responses have been evaluated for cHSP60. These studies suggest cHSPs as important factors involved in immunopathological condition associated with infection. Hence study of specific cytokine responses of mononuclear cells from the infectious site to cHSP60 and cHSP10 may elucidate their actual role in the cause of immunopathogenesis and the disease outcome.</p> <p>Methods</p> <p>Female patients (n = 368) attending the gynecology out patient department of Safdarjung hospital, New Delhi were enrolled for the study and were clinically characterized into two groups; chlamydia positive fertile women (n = 63) and chlamydia positive infertile women (n = 70). Uninfected healthy women with no infertility problem were enrolled as controls (n = 39). cHSP60 and cHSP10 specific cytokine responses (Interferon (IFN)-gamma, Interleukin (IL)-10, Tumor Necrosis Factor (TNF)-alpha, IL-13 and IL-4) were assessed by ELISA in stimulated cervical mononuclear cell supernatants.</p> <p>Results</p> <p>cHSP60 and cHSP10 stimulation results in significant increase in IFN-gamma (P = 0.006 and P = 0.04 respectively) and IL-10 levels (P = 0.04) in infertile group as compared to fertile group. A significant cHSP60 specific increase in TNF-alpha levels (P = 0.0008) was observed in infertile group as compared to fertile group. cHSP60 and cHSP10 specific IFN-gamma and IL-10 levels were significantly correlated (P < 0.0001, r = 0.54 and P = 0.004, r = 0.33 respectively) in infertile group.</p> <p>Conclusion</p> <p>Our results suggest that exposure to chlamydial heat shock proteins (cHSP60 and cHSP10) could significantly affect mucosal immune function by increasing the release of IFN-gamma, IL-10 and TNF-alpha by cervical mononuclear cells.</p

Chlamydia trachomatis Alters Iron-Regulatory Protein-1 Binding Capacity and Modulates Cellular Iron Homeostasis in HeLa-229 Cells

Chlamydia trachomatis (CT) is the leading cause of diseases related to reproductive health and iron plays important role in chlamydial pathogenesis. Iron homeostasis in chlamydia-infected cells is not clear thus far. This study shows that expression of the transferrin receptor (TfR) is downregulated, whereas expression of the ferritin heavy chain is upregulated in CT-infected HeLa-229 cells. Expression of iron-regulatory protein (IRP)-1 predominates over IRP-2 in infected cells. In infected cells, attenuated binding activity of IRP-iron responsive elements (IREs) is observed using the electrophoretic mobility-shift assay. These results suggest that iron homeostasis is modulated in CT-infected HeLa cells at the interface of acquisition and commensal use of iron

Role of cervical dendritic cell subsets, co-stimulatory molecules, cytokine secretion profile and beta-estradiol in development of sequalae to Chlamydia trachomatis infection

BACKGROUND: Chlamydia trachomatis infection of the female genital tract can lead to serious sequelae resulting in fertility related disorders. Little is known about the mechanism leading to Chlamydia induced pathology and factors responsible for it. As only some of the women develops reproductive disorders while majority of the women clears infection without any severe sequalae, mucosal immune response in women with or without fertility disorders was studied to identify factors which may lead to final clinical outcome of chlamydial infection. METHODS: Myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) populations in cervical mucosa and peripheral blood were analyzed in controls and Chlamydia positive women with or without fertility disorders with multicoloured flow cytometric analysis. Cervical cytokines (IL-6, IL-8, IL-10, IL-12, TNF-alpha and IFN-gamma), C-reactive protein levels and sex hormone levels in serum were quantified by ELISA. RESULTS: In cervix of Chlamydia positive women with fertility disorders, significantly high (P < 0.05) numbers of pDCs were present with increased CD80 expression. pDCs correlated significantly with C-reactive protein levels, IL-6 and IFN-gamma levels in women with fertility disorders. In contrast, mDCs showed significant upregulation of CD1a during chlamydial infection and correlated significantly with IL-12 levels in Chlamydia positive fertile women. β-estradiol levels were significantly higher in women having fertility disorders as compared to fertile women and have significant correlations (r = 0.65; P < 0.05) with pDCs numbers, CD80 expression, IL-6 levels and IFN-gamma levels in these women. CONCLUSION: These results suggest that development of sequalae in some women can be a result of interplay of many factors including type of dendritic cell, co stimulatory molecule expression, cytokine secretion pattern and hormone levels

Association of Plasma Circulatory Markers, Chlamydia pneumoniae, and High Sensitive C-Reactive Protein in Coronary Artery Disease Patients of India

Plasma inflammatory markers have been shown to be predictors for cardiovascular risk, however, there is no study where the levels of plasma circulatory markers have been evaluated in coronary artery disease patients (CAD pts) positive for C. pneumoniae IgA and high sensitive C-reactive protein (hsCRP) which may help in better understanding of disease pathogenesis. A total of 192 patients and 192 controls attending the Cardiology Outpatient Department of Safdarjung Hospital were enrolled. The levels of plasma circulatory inflammatory markers were evaluated by ELISA. The levels of circulatory plasma markers (IL-4, IL-8, IL-13, ICAM-1, and VCAM-1) were significantly higher, whereas, levels of IL-10 and IFN-γ were significantly lower in CAD pts compared to healthy controls. The levels of IL-4, IL-8, and ICAM-1 (P = .007, .015, and .048) were significantly higher, however, IL-10 and IFN-γ were significantly lower (P < .001, < .001) in C. pneumoniae IgA positive CAD pts. The levels of IL-4, IL-8, IL-13, ICAM-1, and VCAM-1 were higher but not significant and levels of IL-10 and IFN-γ were significantly (P < .001, < .001) lower in hsCRP positive CAD pts. Our study suggested that circulatory cytokines, namely, IL-4, IL-8, and adhesive molecules like ICAM-1 were enhanced after infection with C. pneumoniae whereas in contrast to this IL-10 and IFN-λ were lowered. Suggesting the important role of these cytokines in progression of CAD

Chlamydia pneumoniae heat shock protein 60 is associated with apoptotic signaling pathway in human atheromatous plaques of coronary artery disease patients

SummaryBackgroundChlamydia pneumoniae heat shock protein (HSP) 60 is known to contribute to the activation of inflammation. In addition, there are contradictory reports on C. pneumoniae and their role in activation of pathways (apoptotic/antiapoptotic/necrosis) in coronary artery disease (CAD). Hence, more studies are required to know the actual role of C. pneumoniae in activation of apoptotic/antiapoptotic/necrosis pathways.Methods and resultsIn this study, two sets of patient groups (cHSP60 positive and cHSP60 negative) were included and gene expression was studied by cDNA micro array and real time polymerase chain reaction arrays. Expression of Caspase-3, 8, 9, c-FLIP, PPAR-γ, PGC-1α, and Gsk-3b were also evaluated at protein level by immunoblotting. In cHSP60 positive CAD patients significantly higher (p<0.001) mRNA expression was found for CCL4, CXCL4, CXCL9, IL-8, CD40LG, CD8, TGFβ1, TGFβ2, APOE, EGR1, CTGF, APOB, LDLR, LPA, and LPL, whereas significantly lower (p<0.001) mRNA expression was detected for CD4, IL1F10, IFNA2, and IL-10 as compared to cHSP60 negative CAD patients. Additionally, at protein level expression of Caspase-3 (p=0.027), 8 (p=0.028), and 9 (p=0.037) were higher and c-FLIP (p=0.028) and PPAR-γ (p=0.95) expression were comparable in cHSP60 positive CAD patients compared to cHSP60 negative CAD patients.ConclusionGenes/proteins of pre-apoptotic caspase dependent/independent pathways, chemokines, and inflammatory cytokines receptors were significantly up-regulated in human atheromatous plaques of cHSP60 positive CAD patients suggesting an association of cHSP60 with CAD

Oxidation resistance of graphene-coated Cu and Cu/Ni alloy

The ability to protect refined metals from reactive environments is vital to many industrial and academic applications. Current solutions, however, typically introduce several negative effects, including increased thickness and changes in the metal physical properties. In this paper, we demonstrate for the first time the ability of graphene films grown by chemical vapor deposition to protect the surface of the metallic growth substrates of Cu and Cu/Ni alloy from air oxidation. SEM, Raman spectroscopy, and XPS studies show that the metal surface is well protected from oxidation even after heating at 200 \degree C in air for up to 4 hours. Our work further shows that graphene provides effective resistance against hydrogen peroxide. This protection method offers significant advantages and can be used on any metal that catalyzes graphene growth