Chlamydia trachomatis heat shock proteins 60 and 10 induce apoptosis in endocervical epithelial cells.

AIM AND OBJECTIVE: The potential role of chlamydial heat shock proteins (cHSP) 60 and cHSP10 in apoptosis of primary cervical epithelial cells was investigated. METHODS: Primary cervical epithelial cells were stimulated with cHSP60 and cHSP10 for 4 h. Quantitative measurements of apoptosis were made using cytofluorometry, and apoptosis-related genes were analyzed by microarray, real-time PCR and western blotting. Further, levels of proinflammatory cytokines (IL-18 and IL-1ß) were determined by semi-quantitative RT-PCR. RESULTS: After a 4-h incubation in the presence of recombinant cHSP60 or cHSP10, the number of cells exhibiting annexin V binding activity increased 6- and 5-fold, respectively (P < 0.05). A DNA microarray study showed significant (P < 0.05) upregulation of interleukin (IL)-1 ß-convertase, and caspase-3, -8 and -9 genes in cHSP60- and cHSP10-stimulated than in control cells as confirmed by real-time RT-PCR and western blotting. Transcript levels of IL-1ß and IL-18 in cells treated with cHSP60 and cHSP10 were found to be significantly (P < 0.05) higher in stimulated than in control cells. CONCLUSION: cHSP60- and cHSP10-induced caspase expression, proinflammatory cytokine production and apoptosis of primary cervical epithelial cells might play a role in the pathogenesis of infertility in women with persistent chlamydial infection.

Ferritin heavy chain-mediated iron homoeostasis regulates expression of IL-10 in Chlamydia trachomatis-infected HeLa cells.

Chlamydia trachomatis is the leading cause of sexually transmitted infection worldwide, in which disease outcome is determined by the balance between pro- and anti-inflammatory host immune responses. Iron plays important roles in regulation and enhancement of various pro- and anti-inflammatory cytokines. Earlier studies have established essentiality of iron in C. trachomatis infection; however, there is lack of study wherein modulatory effect of iron regulated protein [FHC (ferritin heavy chain)] in regulation of anti-inflammatory cytokine IL (interleukin)-10 has been investigated. In this study, immunoblotting results showed the up-regulation of FHC in C. trachomatis-infected HeLa cells in comparison with mock (in vitro control). Further secretory IL-10 level was significantly increased (P<0.001) or decreased (P<0.001) in response to iron supplementation [FAC (ferric ammonium citrate)] and depletion [DFO (deferoxamine)], respectively. However, in C. trachomatis-infected HeLa cells, levels of IL-10 remain higher, irrespective of availability of iron in comparison with their respective control. These results showed that secretion of IL-10 and expressions of FHC have concordance. Further, to understand interdependence of IL-10 and iron homoeostasis (regulation), the levels of IL-10 were compared with iron-responsive GFP (green fluorescent protein) expression in HeLa-229 cells. The mean fluorescent intensities of GFP were in accordance with levels of IL-10 in C. trachomatis-infected cells. These results showed the association of secreted IL-10, FHC and iron homoeostasis in C. trachomatis-infected HeLa-229 cells. This study provides insight into host-Chlamydia interaction at the crossroad of iron metabolism and immune responses and may help in realizing the potential of iron homoeostasis modulators in treatment of chronic chlamydial infection.

Chlamydia pneumoniae heat shock protein 60 enhances expression of ERK, TLR-4 and IL-8 in atheromatous plaques of coronary artery disease patients.

Chlamydia pneumoniae heat shock protein (cHSP) 60 is produced during chronic chlamydial infection and activate innate immune and inflammatory responses thereby contributing to atherogenesis. However, to date there is no apparent signaling cascade delineated in human atherosclerotic plaques in C. pneumoniae positive coronary artery disease (CAD) patients. Atherosclerotic plaques were obtained from 40 CAD patients (28 men, 12 women) attending Department of Cardio Thoracic and Vascular Surgery Safdarjung Hospital, New Delhi. Atherosclerotic plaques were used for gene expression studies at RNA level by real-time PCR and to study expression of ERK1/2, JNK1/2, NF-kB, IkkB and MCP-1 at protein level by immunoblotting. Significantly higher (p < 0.001) RNA expression was found for IL-8, TLR-2/4, TGF-ß, ICAM1, VCAM1 and MAPKinase genes, whereas significantly lower (p < 0.001) RNA expression for SMAD4, IkkB, BRCA1 and IL-10 was detected in cHSP60-positive atheromatous plaque of CAD patients. Moreover, at proteins level pERK1/2 (p = 0.05), NF-kB (p = 0.017), MCP-1 (p = 0.011) was higher and IkkB expression was lower (p = 0.038) in cHSP60-positive atheromatous plaque of CAD patients. This study by using human atheromatous plaques at RNA and protein levels demonstrated higher expression of TLR-2/4, IL-8, ICAM1, VCAM1, ERK1/2 and NF-kB in cHSP60-positive CAD patients.

Higher expression of ferritin protects Chlamydia trachomatis infected HeLa 229 cells from reactive oxygen species mediated cell death.

Apoptosis plays an important role in modulating the pathogenesis of a variety of infectious diseases. Chlamydial infection protects cells against different forms of apoptosis: extrinsic, intrinsic, and granzyme B mediated. Redox reactions are central to the life and death decision of cells and pathogens and an intimate relationship exists between oxidative stress and iron metabolism. The link between redox status and ferritin was largely unexplored in chlamydia-infected cells. In the present study, we showed that Chlamydia trachomatis (CT) infection induced FHC protein in HeLa cells. FHC induction by CT-infected cells stably expressing FHC blunted ROS production compared with mock infected cells, and the infected cells were relatively resistant to apoptosis induced by H2O2. We also demonstrated that endogenous FHC overexpression correlates well with the stabilization of the mitochondrial membrane potential in CT-infected cells. Increased expression of FHC is independent of iron supplementation (FAC) and depletion (DFO) in CT-infected cells. These data suggest that FHC up-regulation is an acute response of HeLa cells against CT infection and that FHC exerts anti-apoptotic activity against oxidative stress.

Characterization of apoptotic activities during chlamydia trachomatis infection in primary cervical epithelial cells.

Chlamydiae are obligate intracellular bacteria that infect human epithelial cells. It has been reported that Chlamydia trachomatis, induces apoptosis in epithelial cells, however, the molecular mechanisms responsible for host cell death especially in primary epithelial cells remained largely unknown as most of the studies are in cell line like HeLa. In this study we demonstrated that C. trachomatis induces apoptosis signaling pathway and apoptosis in primary cervical epithelial cells in a time and dose dependent manner. Live cervical epithelial cells were isolated from endocervical cells and induction was done with chlamydial EBs. Our results demonstrated that apoptosis in infected epithelial cells was associated with an increased activity of caspase 8; however, caspase 9 was activated to a lesser extent. Analysis of apoptosis pathway revealed that expression level of McL-1, Bcl-2, CASP8, and TRADD genes were found to be significantly upregulated (P < 0.01), where as levels of Caspase 1, Caspase 10 and BRIC2 were found to be significantly downregulated (p < 0.01). Our results showed that Chlamydia induces apoptosis and caspase activation in epithelial cells through caspase 8, with an increased expression of the McL-1, which confers a block at the mitochondrial level.

Decreased susceptibility to azithromycin and doxycycline in clinical isolates of Chlamydia trachomatis obtained from recurrently infected female patients in India.

BACKGROUND: Recurrent genital Chlamydia trachomatis infection often results in serious sequelae and has a major impact on reproductive health. MATERIALS AND METHODS: Recurrent infections were determined in symptomatic female patients. In vitro susceptibility assay was performed for azithromycin and doxycycline using the cell culture technique against 21 clinical isolates obtained from C. trachomatis-positive patients including those who were recurrently infected. RESULTS: Thirteen isolates (61.9%) were found to be susceptible to azithromycin and doxycycline with minimum inhibitory concentration (MIC) values ≤0.125 and ≤0.25 µg/ml, respectively. Eight isolates (38%) were found to be less susceptible to the drugs. Two of them had MICs of 8 µg/ml for both the drugs and could not be completely eradicated as observed by minimum bactericidal concentration assay. CONCLUSIONS: Decreased antibiotic susceptibility to the current first-line drugs (azithromycin and doxycycline) for chlamydial infection treatment was observed in isolates obtained from recurrently infected patients.

Protective or pathogenic immune response to genital chlamydial infection in women--a possible role of cytokine secretion profile of cervical mucosal cells.

Little is known about genital mucosal immune response to chlamydial infection in women with or without sequelae (Chlamydia positive women with or without fertility disorders as infertility and multiple spontaneous abortions). Cervical lymphocytes were stimulated with chlamydial EBs and cytokine secretion was determined by ELISA, RT-PCR and ELISPOT assays. Stimulated cervical cells from women with fertility disorders (FD) secrete significantly (P<0.05) higher levels of IL-1beta, IL-6, IL-8 and IL-10 and cells from fertile women secrete significantly higher levels of IL-12 and IFN-gamma compared to other groups. RT-PCR analysis showed similar results for IFN-gamma and IL-12. For IL-10 and IL-4, mRNA expression levels were significantly higher (P<0.05) in cells obtained from women with FD compared to other groups. Results for ELISPOT assay were similar as those of RT-PCR. The results suggest that cytokine secretion profile of cervical cells may decide whether infection does not hamper fertility or will develop fertility disorder.

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

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Comparison of an in-house PCR assay, direct fluorescence assay and the Roche AMPLICOR Chlamydia trachomatis kit for detection of C. trachomatis.

To improve the control of Chlamydia trachomatis infection in India, a rapid, specific and cost-effective method is much needed. We developed an in-house PCR assay by targeting a unique genomic sequence encoding a protein from the C. trachomatis phospholipase D endonuclease superfamily that produces an amplified fragment of 368 bp. The specificity of the primers was confirmed using genomic DNA from other sexually transmitted disease-causing and related micro-organisms and from humans. The assay was highly sensitive and could detect as low as 10 fg C. trachomatis DNA. Clinical evaluation of the in-house-developed PCR was carried out using 450 endocervical specimens that were divided in two groups. In group I (n=274), in-house PCR was evaluated against the direct fluorescence assay. The resolved sensitivity of the in-house PCR method was 97.22 % compared with 88 % for the direct fluorescent antibody assay. In group II (n=176), the in-house PCR was compared with the commercial Roche AMPLICOR MWP CT detection kit. The resolved sensitivity of the in-house PCR assay reported here was 93.1 % and the specificity was 97.46 %, making it a cost-effective alternative for routine diagnosis of genital infection by C. trachomatis. The method should facilitate early detection leading to better prevention and treatment of genital infection in India.

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.

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-gamma 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-gamma 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-gamma 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-lambda were lowered. Suggesting the important role of these cytokines in progression of CAD.

Persistently elevated level of IL-8 in Chlamydia trachomatis infected HeLa 229 cells is dependent on intracellular available iron.

Chlamydia trachomatis is a leading cause of sexually transmitted infection worldwide and responsible for myriad of immunopathological changes associated with reproductive health. Delayed secretion of proinflammatory chemokine interleukin (IL)-8 is a hallmark of chlamydial infection and is dependent on chlamydial growth. We examined the effect of iron chelators on IL-8 production in HeLa 229 (cervix epitheloid cell, CCL2) cells infected with C. trachomatis. IL-8 production was induced by Iron chelator DFO and Mimosine, however, synergy with chlamydial infection was obtained with DFO only. Temporal expression of proinflammatory secreted cytokines IL-1beta, TNF-alpha, and IL-8 did not show synchrony in Chlamydia trachomatis infected cells. Secretion of IL-8 from Hela cells infected with C. trachomatis was not dependent on IL-1 beta and TNF- alpha induction. These results indicate towards involvement of iron in chlamydia induced IL-8 production.

Cervical epithelial cells from Chlamydia trachomatis-infected sites coexpress higher levels of chlamydial heat shock proteins 60 and 10 in infertile women than in fertile women.

BACKGROUND/AIMS: The objective of the present study was to examine the possible relationship between the chlamydial heat shock proteins (cHSP) 60 and 10 expression and the damaging sequelae of a Chlamydia trachomatis infection, such as infertility. METHODS: Seven fertile and 7 infertile female patients infected with C. trachomatis attending the gynecology outpatient department of Safdarjung hospital (New Delhi, India) were enrolled. The relative transcript levels and intracellular expression of cHSP60 and cHSP10 in cervical cells were assessed using real-time RT-PCR and flow cytometry, respectively. RESULTS: Quantitative real-time RT-PCR analysis showed that transcript levels of both cHSP60 (p = 0.007) and cHSP10 (p = 0.0006) were higher in infertile women than in fertile women. Flow cytometric analysis showed significantly higher intracellular levels of cHSP60 (p = 0.0006) and cHSP10 (p = 0.0041) in fertile women infected with Chlamydia than in infertile women. However, the percentage of double-positive cells (both cHSP60- and cHSP10-expressing cells) were higher (p = 0.0006) in infertile women than in fertile women. CONCLUSION: Our results suggest that cHSP60 and cHSP10 have a different pattern of expression in infertile women compared to fertile women reflecting a probable difference in the metabolic state of Chlamydia with the presence of an abnormal cryptic form of C. trachomatis in infertile women.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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. beta-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.

Serovar-specific immune responses to peptides of variable regions of Chlamydia trachomatis major outer membrane protein in serovar D-infected women.

The role of major outer membrane protein (MOMP) variable regions in the interaction of chlamydiae and host cells has been evaluated and their role in neutralization of antibodies has been clearly demonstrated. There are also studies that delineate the contribution of these regions to the cell-mediated immune response of the host and suggest that serovar E elicits serovar-specific immune responses in infected humans. However, further studies with other serovars are required to confirm these findings and to elucidate the role and importance of serovar-specific responses of variable regions of MOMP in other serovars. We, therefore, performed a detailed analysis of the humoral and cellular immune responses against the serovar D-specific variable segments (VS) of MOMP in women infected with Chlamydia trachomatis. We found that VS4 elicits significantly higher responses (both humoral and cellular) than other VS peptides (VS1, VS2 and VS3). VS4 elicited significantly higher (P < 0.0001) proliferative responses, interferon-gamma levels (P < 0.0001) as well as higher prevalence (P < 0.0001) of IgG antibodies against VS4 in serovar D-infected patients as compared to patients infected with other serovars, suggesting its role in serovar-specific immune responses.

High immunoglobulin A seropositivity for combined Chlamydia pneumoniae, Helicobacter pylori infection, and high-sensitivity C-reactive protein in coronary artery disease patients in India can serve as atherosclerotic marker.

Atherosclerosis is increasingly recognized as a chronic inflammatory disease. A variety of infectious agents (Chlamydia pneumoniae, Helicobacter pylori, and cytomegalovirus [CMV]) and inflammatory marker such as high-sensitivity C-reactive protein (hs-CRP) have been found to be associated with atherosclerosis and its consequences. There is a need to know about the type and burden of infection in coronary artery disease (CAD) patients and the level of hs-CRP in India as there is growing evidence that a variety of pathogens are participating in the development and/or acceleration of at least pre-existing atherosclerosis. In addition, there is a need to find the association between these pathogens and conventional risk factors among CAD patients in India, to possibly identify a prognostic marker. In this study 192 patients with incident or prevalent CAD attending the Cardiology Outpatient Department of Safdarjung Hospital, New Delhi, India, were enrolled. In addition, 192 age-and sex-matched controls were also included. Cases and controls differ significantly in seropositivity to C. pneumoniae immunoglobulin IgA (154 vs 76) and IgG (71 vs 48) (P < 0.001, P < 0.015), H. pylori IgA (98 vs 57) and IgG (77 vs 43) (P < 0.001, P < 0.001), CMV IgG (62 vs 38) (P = 0.01) and with hs-CRP (114 vs 60) (P < 0.001), respectively. The level of hs-CRP was higher in CAD patients with IgA seropositivity of C. pneumoniae and H. pylori (5.18 and.65 mg/l) than the IgG of these bacteria (3.73 and 3.36 mg/l), respectively. These findings support an association between specific infectious agents, namely, C. pneumoniae, H. pylori, CMV, and hs-CRP in CAD patients. Association of hs-CRP with IgA specific for C. pneumoniae and H. pylori suggests the role of chronic infection in the development of CAD and may be used as a marker to target the population.

Cervical cytokine responses in women with primary or recurrent chlamydial infection.

Little is known about concurrent expression of cervical cytokines and their regulation by sex hormones during primary or recurrent chlamydial infections in humans. Cytokine (interleukin-1beta [IL-1beta], IL-6, IL-10, interferon-gamma [IFN-gamma], and tumor necrosis factor-alpha [TNF-alpha]) concentrations in cervical washes and serum samples, along with levels of beta-estradiol and progesterone in women with primary or recurrent chlamydial infections and healthy controls, were measured by ELISA. Women with recurrent infections had significantly higher levels of IFN-gamma in cervical washes than did women with primary infections. Significant negative correlation was found between IL-1beta and progesterone levels during recurrent infections. Beta-estradiol levels in women with primary infections showed significant negative correlations with cervical concentrations of IL-10, IL-1beta, and IL-6. Our study suggests that Chlamydia trachomatis infection in the female genital tract may be regulated by both the synergistic actions of the cytokines and the sex hormones beta-estradiol and progesterone.

Primary and secondary immune responses of mucosal and peripheral lymphocytes during Chlamydia trachomatis infection.

Chlamydia trachomatis infection is followed by the development of antigen-specific cell-mediated immunity, which is detectable as a positive lymphocyte proliferation response to the chlamydial major outer membrane protein (MOMP) antigen. To date, however, there have been no studies on the mucosal immune responses to chlamydial antigens. This study aimed to study the primary and secondary immune responses of cervical lymphocytes in response to the chlamydial antigen. Median proliferative responses were found to be significantly (P<0.05) higher in patients with chlamydial infections than in controls. The chlamydial MOMP induced significantly higher IL-6 and IL-10 and lower interferon-gamma (IFN-gamma) secretion in cervical lymphocytes of Chlamydia-positive women, resulting in a T helper 2 response. On stimulation of peripheral blood mononuclear cells (PBMC) obtained from Chlamydia-positive women with the chlamydial antigen, the median levels of IL-10, IL-12 and IFN-gamma were higher than in controls, but the differences were not significant. Our study suggests that the mucosal immune responses towards Chlamydia trachomatis are different from those of PBMCs and are more helpful in understanding the cytokine responses in the female genital tract during chlamydial infection.