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Φιλομήλ Ηλιόπουλος
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1 Table of Content CHAPTER INTRODUCTION... 1 CHAPTER REVIEW OF LITERATURE History of Filariasis Discovery of Symptoms ( ) Discovery of Microfilariae (1863 and 1866) Discovery of Adult Worm (1876) Discovery of Filarial Life Cycle (1877) Discovery of Transmission (1900) Causative agent... 7 I. Wuchereria bancrofti... 7 II. Brugia malayi... 7 III. Brugia timori Life cycle of B. malayi Development in intermediate host (mosquito) Development in definitive host (humans or rodents) Periodicity Epidemiology Global Distribution Current situation in India Economic burden of disease Disability adjusted Life years (DALY) Hold in endemic countries Symptoms and pathology Development of the disease The pre-patent period (biological incubation period) The patent (symptom-less) period The acute or allergic stage The chronic manifestations Categories of filarial subjects in filaria endemic area Mf carriers Symptomatic Endemic normal Other forms of filariasis Pathogenesis Disease management Diagnosis... 22

2 New advances in diagnosis Treatment and control Global Programme to Eliminate Lymphatic Filariasis (GPELF) Vector control Drug targets Newer targets from Brugia genome Vaccine development Vaccine targets Immune response against filarial infection Host-parasite interactions Humoral immune responses Cellular immune responses Molecular basis of immune responses Cells involved in inflammatory response Immune modulation: in vivo mechanisms Role of DIM-1 protein CHAPTER MATERIALS AND METHODS Animals Parasite Animal models Maintenance of B. malayi infection Rearing and breeding of A. aegypti colony Feeding of mosquitoes on mf positive M. coucha Isolation of L3 from mosquitoes Inoculation of L3 to M. coucha/jird and assessment of the infection Cloning Gene selection Design of primer for the gene mrna isolation Isolation of adult parasites mrna isolation by Trizol method cdna synthesis PCR amplification of gene Elution of amplified DNA (gene product) from the gel Cloning of DNA in TA vector Ligation of PCR product and TA vector Transformation in DH5α competent strain of E.coli Confirmation of positive colony by colony PCR Sequencing of gene... 70

3 3.5.7 Sub cloning of DIM-1 TA clone in expression vector (ptriex-4) Isolation of plasmid DNA from transformed E. coli Expression vector (ptriex-4) Restriction digestion of plasmid of TA clone and plasmid of expression vector (ptriex-4) Elution of digested plasmid from the gel Ligation of digested expression vector with amplified product Transformation Confirmation of clone Isolation of plasmid from transformed DH5α cells Over expression of DIM-1 in expression cell (BL21-DE3 cells) Transformation of isolated plasmid in BL21-DE3 cells Confirmation of clones by colony PCR Expression of proteins from transformed E. coli BL21-DE3 cells Checking of protein solubility Purification of recombinant DIM-1 of B. malayi (rdim-1bm) by Ni NTA-column (affinity chromatography) Protein estimation by Bradford method Western blotting for transfer of resolved proteins on nitro cellulose paper (NCP) Confirmation of r-protein with anti-histidine antibody in western blot Bacterial endotoxins test for recombinant DIM Sequence and phylogenetic analysis In silico modeling studies Immuonolocalization of DIM-1 in B. malayi adult parasite by confocal microscopy Reactivity of antibodies to rdim-1bm with resolved fractions of mf, L3, L4 and adult worm stages by immunoblotting Isolation of parasites (adult and L4) and preparation of parasite extract Isolation of L3 and preparation of parasite extract Isolation of mf and preparation of mf extract Fractionation of different stages of B. malayi by SDS-PAGE Immunoblotting of resolved component of mf, L3, L4 and BmA with sera of rdim- 1bm immunized M. coucha Immune responses of rdim-1bm immunized animals Grouping and immunization of animals Immunization and experimental schedule Assessment of infection status Weekly body weight Rectal temperature Hematology (at autopsy)... 94

4 Collection of Blood Total leukocyte count (TLC) Differential leukocyte count (DLC) Packed cell volume (PCV) Erythrocyte sedimentation rate (ESR) Haemoglobin concentration (Hb) Humoral mediated immune response Determination of IgG antibody and subtypes Determination of specific IgE Determination of specific IgM Determination of specific IgA Cell mediated immune response Nitric oxide production by macrophages Cellular proliferation (lymphocyte transformation test; LTT) Cytokine determination by ELISA Macrophage function test Histopathology of lymph nodes Statistical analysis CHAPTER RESULTS Cloning of DIM Amplification of DIM-1 gene Sub cloning of amplified product in ptz57r/t vector Sub cloning of gene in the expression vector (ptriex4) Over expression and purifiaction of rdim-1bm In silico DIM-1bm sequence analysis Molecular modeling studies Confocal microscopy to localize rdim-1bm in adult B. malayi Reactivity of rdim-1bm with resolved fractions of various stages of B. malayi NO release, cellular proliferation, proinflammatory (TNF -α, IFN- and IL-6) and antiinflammatory (IL-10) cytokine production in splenocytes of normal M. coucha stimulated with rdim-1bm Nitric oxide (NO) release Cellular proliferation response Cytokines release Effect of repeated exposures of M. coucha to rdim-1bm Dose immunization schedule (standard immunization protocol for vaccination) In-life observations Behavior Body weight

5 Rectal temperature Organ weight Hematological parameters Parasitological responses Effect of rdim-1bm on L3 induced infection Immunological responses Effect on humoral responses: IgG and its subclass, IgE, IgM and IgA Effect on cell mediated immune responses Lymphocyte transformation test (LTT) Macrophage phagocytic activity test (MAT) NO release Cytokine release Week immunization schedule (non-standard procedure) In-life observations Behavior Body weight Rectal temperature Organ weight Hematological parameters Parasitological responses Effect of rdim-1bm on L3 induced infection Immunological responses Effect on humoral responses: IgG and its subclass, IgE, IgM and IgA Effect on cell mediated immune responses Lymphocyte transformation test (LTT) Macrophage phagocytic activity test (MAT) NO release Cytokine release Week immunization schedule (non-standard procedure) In-life observations Behavior Body weight Rectal temperature Organ weight Hematological parameters Parasitological responses Effect of rdim-1bm on L3 induced infection Immunological responses

6 Effect on humoral responses: IgG and its subclass, IgE, IgM and IgA Effect on cell mediated responses Lymphocyte transformation test (LTT) Macrophage phagocytic activity test (MAT) NO release Cytokine release Histopathological studies CHAPTER DISCUSSION CHAPTER SUMMARY CHAPTER BIBLIOGRAPHY LIST OF PUBLICATIONS...195

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