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Prototype Proposal I

Prototype Proposal I






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    Prototype Proposal I Prototype Proposal I Document Transcript

    • Outline of the Proposed Topic of Research Name of Candidate : *********** ID No. : *********** Place of Research Work and Organisation : Institute of ********, New Delhi Proposed Supervisor’s Details Name : *************** Qualification : M.Sc., Ph.D Designation : *************** Organization : Institute of *********, New Delhi Proposed Topic of Research Role of Antigen Presenting Cells (APCs) and Toll like Receptors in Providing a Protective Immune Response during Chlamydia trachomatis Infection Objective of the Proposed Research 1. In vitro study of processing and presentation of chlamydial antigens by Dendritic cells (DC’s) and monocytes/macrophages to CD8+ and CD4+ T lymphocytes. 2. Differential regulation of cytokine production by CD8+ and CD4+ T lymphocytes. 3. Role of Toll like receptors 2 and 4 in recognition of Chlamydial antigens and regulation of cytokine production. 4. Regulation of nitric oxide production by chlamydial antigens. Background of the Proposed Research Introduction Worldwide, an estimated 90 million sexually transmitted Chlamydia trachomatis infections occur each year. Sexually transmitted C. trachomatis infection is an important public health concern because of its adverse effects on reproduction [1]. In India alone a high chlamydial prevalence rate (28%) was found in symptomatic patients [2]. In women, infection with C. trachomatis causes pelvic inflammatory disease (PID) and has long term consequences – such as 1
    • infertility, ectopic pregnancy and chronic pelvic pain- that are secondary to scarring of the fallopian tubes (caused by salpingitis) and ovaries. In addition, infection with C. trachomatis felicitates the transmission of HIV [3] and might be a co factor in human papilloma virus (HPV)- induced cervical neoplasia [4-5]. The pathological mechanism by which C. trachomatis induces scarring is not well understood. In all cases the pathology seems to be related to a chronic inflammation caused by a persistent chlamydial infection or by repeated infections with the bacterium. After initial infection of the host with C. trachomatis, Dendritic cells (DC) are the first professional antigen presenting cells (APC’s) encountering the bacteria. DC’s are present in the epithelium of cervix and vagina [6] and they prime the T cells to modulate the type of T-cell responses (Th1/Th2), contributing to the inflammatory response which largely depends on the up-regulation of co-stimulatory and adhesion molecules and on secretion of inflammatory cytokines [7-9]. Protection against chlamydial infection has been shown to be primarily mediated by IFN-γ producing T-cells [10,11] and it has been shown that DC can process and present chlamydial antigens to T cells [12-14]. Little is known, however, about the interaction between human DC and Chlamydia species. Review Chlamydia trachomatis is an obligate intracellular gram –ve bacterium which causes a wide spectrum of human diseases. C. trachomatis infection of genital tract is now recognized as one of the most common major cause of sexually transmitted diseases in developed countries [1]. Without treatment the chlamydial genital infections can cause pelvic inflammatory disease (PID) and its sequelae of ectopic pregnancy and infertility. The chlamydial developmental cycle involves a metabolically inactive non replicating infectious form called elementary body (EB), that, after entry into the host cells differentiate into metabolically active Reticulate Body (RB). The organism infects the epithelial cells often inducing an acute inflammatory response, caused by persistent chlamydial infections, or by repeated infections with the bacterium, however, protective immunity is limited. C. trachomatis infection remains sub clinical in a high proportion of infected individuals (70-90% of women and 30-50% of men). Clinical symptoms if present include dysuria, abnormal vaginal discharge and lower abdominal pain. Infection ascends the endometrial epithelium to the fallopian tubes, where C. trachomatis can establish persistent infection and can cause PID. 2
    • Overall, 11% of women with PID develop tubal factor infertility and 9% develop ectopic pregnancies. Both humoral and cellular responses can be readily detected in patients suffering from C. trachomatis infection. Since infection is intracellular, neutralizing antibodies have little relevance in resolving infection [10]. An early epidemiological observation suggested an inverse correlation between the amount of IgA in cervical secretions and the amount of C. trachomatis recovered from the cervix of infected women [15]. In vitro, antibodies specific for C. trachomatis can neutralize infection in tissue culture [16], however, in humans high titers of C. trachomatis specific antibodies do not correlate with resolution of infection and , in fact, more strongly correlated with increased severity of sequelae of infection, such as tubal infertility [17]. In contrast, there is good evidence that T-cell mediated immune (CMI) responses play a major role in clearance and resolution of chlamydial infections and T-cell responses are critical in host resistance to C. trachomatis. Transfer of T lymphocytes into naïve mice have been shown to protect the mice against C. trachomatis infection [18]. After infection of host with C. trachomatis, dendritic cells (DC) and macrophages/monocytes are the main antigen presenting cells (APC’s) encountering the bacteria. DC’s are key players in immunity that dictate the type of immune response generated to a particular antigen. DC’s are professional antigen presenting cells that have extraordinary capacity to stimulate naive T-cell and initiate primary immune response. Immature DC present in the epithelium of cervix and vagina capture microbial antigens, process them and present them to T lymphocytes. Mature DC’s are highly effective at presenting antigen and priming protective adaptive immune responses. TLR’s comprise a family of cell surface receptors that recognize pathogen associated molecular patterns (PAMP’s), including lipopolysaccharide (LPS) and hypomethylated CpG- rich DNA as well as double stranded and single stranded RNA. Toll like receptors detect microbial infection and have an essential role in the induction innate and adaptive immune responses [19]. Of the 10 cloned mammalian TLRs, TLR2 and TLR4 are the best characterized with respect to innate responses to bacteria. TLR4, in association with accessory molecules MD-2 and CD 14, is the signal transduction receptor for gram- negative bacterial lipopolysaccharide and heat shock proteins. A broader range of microbial products activate immune responses through engagement of TLR2, including peptidoglycan from gram-positive bacteria, bacterial lipopeptides, and 3
    • zymosan. A recent hypothesis states that differential expression and engagement of TLR family members at the surface of dendritic cells and macrophages influences the type of immune response that is induced by a microbial pathogen. Infection with Chlamydia muridarum has been shown to stimulate DC’s to produce IL-12 (TH1 type response) [20]. It is not confirmed which particular TLR’s expressed by dendritic cells are engaged by Chlamydia spp., TLR2 might have an important role in the activation of DC’s by C. pneumoniae [21]. Furthermore, signaling through TLR2 , but not TLR4, is associated with increase fallopian tube pathology in C. muridarum infected mice [22], indicating that engagement of TLR2 is a potential common pathway in both the immunity and immunopathology induced by Chlamydia spp. Given the high level of expression of TLR’s by DC’s and there ability to polarize immune responses, the identification of the role of DC’s in Chlamydia specific immune responses is crucial for understanding the type of immune response that is elicited and therefore also for designing a vaccine against infection with Chlamydia trachomatis [23]. Studies in the animal models have clearly established that T cells have a crucial role in the resolution of infection with Chlamydia spp [24]. Nude mice cannot control infection and adoptive transfer of Chlamydia specific CD4+ or CD8+ cells allow these mice to successfully control infections. Specifically protection seems to be mediated by CD4+ T cells that produce IFN-γ [25]. The role and effector mechanisms of Chlamydia positive CD8+ T cells are less clear. MHC class I peptide presentation to CD8+ T cells is not essential for clearance of infection with Chlamydia spp. In some situation CD8+ T cells might be important in elimination of cells infected with Chlamydia spp. [26]. Also adoptive transfer of CD8+ T cell lines specific for serovar L2 of C. trachomatis protected mice against infection with C. trachomatis through a mechanism involving the production of IFN-γ [27]. Thus to establish the real role played by CD8+ T cells in C. trachomatis infection further studies are required. Stimulated T cells produce a variety of cytokines for clearance of Chlamydial infection including IFN-γ and IL-12. Most of the cytokines secreted by T cells and macrophages are T helper1 (TH1) cytokines, which have a role in polarizing the immune response to Chlamydia spp towards a protective TH1 type response. By contrast, cytokines such as TNF, IL-1α and IL-10 might be involved in the pathology associated with infection with Chlamydia spp. IFN-γ, the main TH1 type cytokine is essential for the clearance of Chlamydial infections from genital tract. It controls the in vitro growth of C. trachomatis through inducing production of enzyme indoleamine-2,3- 4
    • dioxygenase (IDO) [28]. Activation of IDO by IFN-γ leads to degradation of tryptophan and lack of this essential amino acid causes the death of C. trachomatis through tryptophan starvation [28]. Additional immune effector mechanisms induced by IFN-γ include induction of nitric oxide production, which inhibits growth of C. muridarum [29] and the promotion of TH1 type protective immune response, which downregulate non protective TH2 type responses, thereby, promoting persistent infection [30]. Persistent infection might induce the secretion of proinflammatory cytokines, leading to chronic inflammatory cellular response and tissue damage [31, 32]. Overall, these data show that Chlamydia specific CD4+ TH1 cells and to a more limited extent CD8+ T cells are required to control C. muridarum infection in genital tract of mice [33]. Observations from humans infected with C. trachomatis indicate that similar immune effector mechanisms occur in humans [33]. Gap in Existing research More than two-third of the Chlamydial infections cases occur in the developing countries, where diagnostic and treatment services are almost absent. An estimated 15 million new cases are occurring in Africa and 45 million new cases in Southern Asia every year [34]. Because of its effect on reproduction, programmes to control C. trachomatis have been implemented in many developed countries but many regions are now showing an increase in the number of infected individuals [35]. Since current programmes for the control of C. trachomatis are not affordable for much of the developing countries, vaccine development have been identified as an essential to controlling infection with C. trachomatis. Mouse models of chlamydial infections with C. muridarum have provided information on the immune mechanisms of clearance of infection and resistance to reinfection, but there are several important differences between C. muridarum and C. trachomatis that might effect the immunobiology of infection. Firstly, C. trachomatis infection in humans is much more prolonged than C. muridarum infection in mice [36]. Secondly, immune-evasion mechanisms also differ. These differences limit the direct extrapolation of findings from C. muridarum infection to C. trachomatis infection. Thus, a better definition of human immune response correlates with C. trachomatis protective immunity and disease pathogenesis needs to remain an important research priority if we are to develop a vaccine against C. trachomatis infection that has protective and not deleterious effects. 5
    • Methodology For carrying out the proposed research work facilities such as tissue culture room equipped with bio safety hood and incubator, thermocyclers for Polymerase Chain reaction, cell sorter, flow cytometer, confocal microscope and computers for analysis and storage of data are required which are available at the Institute of Pathology. Phase 1: This phase will comprise of Literature survey. Phase 2: (A) Enrollment of patients: Symptomatic female patients attending the Gynecology Out Patient Department of Safdarjung Hospital, New Delhi, and having complaints of cervical/ vaginal discharge, abdominal pain, dysuria or infertility will be enrolled. Cervical lavage samples and peripheral blood will be collected from these women. Diagnosis for Chlamydia trachomatis and other STD pathogen will be done using standard methodology. (B) Flow Cytometry: Quantification of different T-cell subsets, monocytes, dendritic cells and expression of TLR’s on the surface of dendritic cells and monocytes will be performed by standard flow cytometry methodology. Phase 3: Culture of Monocyte derived Dendritic Cells (MDDC) Peripheral blood mononuclear cells (PBMC’s) will be purified on Ficoll gradients and will be washed three times with RPMI 1640. They will be then stained with CD14 microbeads and will be separated using a cell sorter. These CD14 positive cells will be inoculated (1-2 x 10 6/ml) into each well of a six well plate and will be cultured in RPMI 1640 supplemented with 10% heat inactivated Fetal Calf Serum, 2mM L-glutamine, 25mM HEPES, 0.02M 2-mercaptoethanol, 10µg/ml Gentamycin and 2µg/ml Amphotericin B at 370c in a 5% CO2 incubator, in the presence of 50 ng/ml Granulocyte Macrophage Colony Stimulating factor and 20 ng/ml IL-4 for 6-7 days. After 6-7 days immature MDDC’s will be washed and analyzed for CD14 and CD1a expression (marker for immature DC’s) Phase 4: Coculture of Dendritic cells with CD8 and CD4 T lymphocytes Immature Dendritic cells and Monocytes will be infected with live C. trachomatis EB’s and cultured in RPMI 1640 for 4 days at 370C. Autologous CD4+ and CD8+ T cells will be separated 6
    • using a cell sorter and will be cocultured with the EB pulsed dendritic cells and monocytes for further 4 days in the presence of recombinant IL-2 for generation of T-cell clones. The culture supernatants will be then analyzed for production of various cytokines. Phase 5: (A) Blocking of Toll like receptors on dendritic cells and monocytes Toll like receptors 2 and 4 present on the surface of Dendritic cells and monocytes will be blocked with the help of blocking peptides before pulsing them with chlamydial EB’s and will then be cocultured with CD8+ and CD4+ T cells. The culture supernatants will be again analyzed for production of various cytokines. Changes in the gene expression patterns of Toll like receptors, upon infection with Chlamydial EB’s will be done with RT-PCR (reverse transcriptase Polymerase chain reaction). (B) Regulation of nitric oxide production by chlamydial antigens Nitric oxide production by antigen presenting cells and T cells will be analyzed in the culture supernatants and its regulation by IFN-γ and Toll like receptors will be studied. Phase 6: Conclusion and Thesis writing Results will be concluded with the help of the data, which we obtained throughout our research work and will be compiled in thesis. Work Plan : PHASE 6 A PHASE 5 C T I PHASE 4 V I PHASE 3 T Y PHASE 2 PHASE 1 0 6 12 18 24 30 36 42 48 Duration in Months 7 PHASE 1 PHASE 4
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