Theses (PHD)

Candida spp. Are the most common fungal pathogens of systemic candidiasis. As there are pathognomonic signs or symptoms of systemic candidiasis, the diagnosis of invasive candidiasis remains a laboratory and clinical challenge. Thus, diagnosis assays to detect systemic candidiasis and to identify Candida virulence factors and associated pathogenesis through immunohistochemistry using specific monoclonals and polyclonals will be useful. Balb/c mice were immunized with C. albicans and C. parapsilosis and blood were checked for the presence of reactive antibodies using ELISA. Fusions were performed using the harvested spleen cells and NSI mouse myeloma cells and the clones were screened for the presence of antibodies producing hybrid cells by dot-blot. A total of forty and fifty clones producing monoclonal antibodies against C. albicans and C. parapsilosis respectively were obtained. Western blot analyses showed that the monoclonal antibodies against non heat-shocked (IgM) and heat-shocked (IgG3) C. albicans were reactive against the antigens with MW at 30 and 59 kDa. Experimental systemic candidiasis in mice and rats were induced through intravenous injection of C.albicans and C. parapsilosis. The liver, spleen, kidneys, brain, heart and lungs were collected for histopathological examination. Cystic lesions with large clumps of fungus consisting mainly of hyphae and some yeast were observed in the kidneys. Clusters of yeast cells were detected in multiple organs. The female mice and rats were more resistant to the C. albicans infection than the male counterparts. The rats were found to be more susceptible to C. albicans infection than Balb/c mice. C. parapsilosis was found to be less virulent than C. albicans. The animals infected with C. parapsilosis had mild infection in spleen and lungs only. However, all the majority of the organs in animals that succumbed to the C. albicans or C. parapsilosis infection had fungal invasion. Histopathhological examination revealed that all dead but no those surviving or control animals had mild to serve fungal colonization in brains. Fungal colonization in the brain may be a determinant factor for mortality in experimental systemic. Candidiasis. Two Isolates of fungal were obtained from blood culture on Sabouraud dextrose agar and were found to be of the same species with those used for the infection, based on restrictive length polymorphism and DNA alignment analysis. The monoclonal antibodies were purified, characterized and used in the immunoperoxidase and immunofluorescence techniques. The monoclonal antibodies reacted to surface epitopes on the yeast cells, germ tubes, hyphae and to immune complexes. The monoclonal antibodies obtained were used for the detection of antigens, together with the polyclonal antibody, in the experimental model and were found able to detect antigens present in the sera at 0.2µg/µL. Antibody levels were also determined using the ELISA method. The antibody levels against C. albicans or C. parapsilosis in infected mice or rats were increased compared with uninfected animals. The Circulating antigen level in the infected animals increased initially but declined subsequently. The cytokine levels (IL-4, IL-6, IL-10, IFNƴ and TNF) induced in the models were measured using flow cytometry. C. albicans and C.parapsilosis elicited different cytokine expression in mice and rats. The groups infected with C. albicans had higher IL-6 and TNF, but unchanged IL-4, IL-10 and IFNƴ levels when compared with uninfected mice. Elevated levels of TNF were seen in both mice and rats infected with C. albicans but not in those infected with C. parapsilosis. The level of IL-10 was lower for all the C. albicans and C. parapsilosis infected rodents compared with unifected rodents except for rats infected with C. albicans at the later stage of infection. In conclusion, monoclonal antibodies were produced and user for the detection of antibody, circulating antigen and immune complexes in the experimental systemic candidiasis model. These monoclonal antibodies may serve as potential primary capture antibodies for the development of rapid diagnostic test for human systemic Candida infection Fungal colonization in different organs and differential host immune responses against C. albicans and C. parapsilosis were shown in the present study.

Mites are eight-legged acari that are classified under the class of Arachnida. Mites are diverse groups of invertebrate that can survive in different habitats. Domestic mites include the house dust mites (HDM) and storage mites that are associated with the home environment. These are the mites of medical and economic importance. Inhalation of dead or live mites, their faecal matter and other byproducts from our living environment can trigger asthma, rhinitis and contact dermatitis. The two species of mites under this study, Dermatophagoides pteronyssinus and Tyrophagus putrescentiae, belong to the group of HDM and storage mites respectively. HDM infestation occurs at homes, including in carpets, mattresses and bedding, clothing, stored food products, and house hold pets. HDM is a major causative agent for asthma and rhinitis in Malaysia. Storage mites represent one of the occupational hazards for farmers, field hands, mill workers, warehouse operators and other food industrial workers. Group 10 allergen of mite is a muscle protein, tropomyosin, which has received attention due to its cross-reactivity with some allergens from invertebrates, particularly in seafood, responsible for severe systemic anaphylaxis. The objectives of this study were to produce recombinant Group 10 allergens of D. pteronyssinus and T. putrescentiae and to assess their IgE binding reactivity with human sera; to produce monoclonal antibodies against these recombinant allergens and to apply them in the localisation of inhaled mite Group 10 allergens in respiratory system of animal models. Two species of mites, D. pteronyssinus and T. putrescentiae were cultured and harvested. The mites were homogenised and the crude mite proteins were used to immunise the mice for the production of anti-mite polyclonal antibodies. Total RNA of mites was isolated and cDNA was constructed. Specific primers were designed for the amplification of group 10 allergen genes of both species of mite (Der p 10 and Tyr p 10) through polymerase chain reaction (PCR). The PCR produced a 855 bp band, which is in accordance with the reported molecular weight of mite tropomyosin in previous studies. DNA sequencing of the PCR products further confirmed that they are the group 10 allergen genes. The PCR products were purified and digested with restriction enzymes before they were cloned into the expression vector pRSET C. The recombinant plasmids (pRSET C/Der p 10 and pRSET C/Tyr p 10) were purified and transformed into the expression host E. coli, BL21. Colony PCR was carried out to screen and to select the colonies of BL21 that carried the recombinant plasmids. The selected positive BL21 clones were subjected to recombinant allergen protein expression under the induction of IPTG. The recombinant allergens were screened in western blot by using anti-His antibody and sera of immunised mice. A protein band of about 38 kDa which represent the group 10 mite allergen was detected. The recombinant allergens were purified with nickel-nitrilotriacetic acid (Ni-NTA) resin affinity column. Most of the unwanted bacterial proteins were removed throughout the purification process leaving mainly the recombinant allergen protein (38 kDa) in the elution fraction. A panel of 44 human sera from allergic and normal human subjects was used to assess the IgE binding reactivity of the recombinant mite tropomyosin by dot blot immunoassay. The results showed 4.5% (2 / 44) and 2.3% (1 / 44) positive reaction to recombinant Der p 10 and recombinant Tyr p 10 respectively. The purified recombinant mite allergens were used to immunise the mice for the production of monoclonal antibodies against group 10 allergen. The spleens of the mice were removed and splenocytes were fused with NS1 myeloma cells. The hybridoma cells were screened for the presence of anti-mite tropomyosin specific antibody using dot blot immunoassay and western blot. One positive hybridoma clone for each species of mites was selected for the study. The selected positive hybridoma clones were DP10-F11 (D. pteronyssinus) and TP10-C5 (T. putrescentiae). The result of antibody isotyping showed that both of them were IgG1 with κ light chains. DP10-F11 antibody was found to be cross-reactive against Tyrophagus putrescentiae (68%), Glycycometus malaysiensis (50%), Aleuroglyphus ovatus (51%), Blomia tropicalis (62%), D. farinae (102%) and shrimp (56%). TP10-C5 antibody demonstrated cross-reactivity with Dermatophagoides pteronyssinus (63%), Blomia tropicalis (59%), Dermatophagoides farinae (82%) and shrimp (58%). However, both of these antibodies did not cross-react with E. coli (< 50%). The hybridoma cells of DP10-F11 and TP10-C5 were intraperitoneally injected into the pristine-primed BALB/c mice and allowed to propagate inside them for the production of ascites fluid. The ascites fluids collected from mice were purified using the HiTrap Protein G HP column and fractioned protein liquid chromatography (FPLC). Both the purified monoclonal antibodies from ascites fluids showed positive reactivity to the recombinant mite tropomyosin in dot blot immunoassay and indirect ELISA. Female inbred Balb/c mice (n=6) were intranasally challenged with purified recombinant Der p 10 and recombinant Tyr p 10 for 10 consecutive days. Positive and negative control groups (n=6 each) were challenged with ovalbumin and PBS respectively. The mice from each group were re-challenged on day 17 for 3 consecutive days. The mice were sacrificed via cardiac puncture on the next day after re-challenge. Anti-mite tropomyosin polyclonal antibodies were detected in the sera of mice intranasally challenged with recombinant allergens. Histopathological changes such as infiltration of inflammatory cells and hypertrophy of airway epithelial cells were observed in the lung and trachea of mice challenged with recombinant allergens. The results of immunohistochemistry staining showed that both DP10-F11 and TP10-C5 antibodies demonstrated positive immunoreactivity at the alveolar epithelium in the lung tissues and fibrocartilaginous areas of the trachea. The recombinant group 10 allergens (tropomyosin) of D. pteronyssinus and T. putrescentiae were successfully produced in this study. Both of these recombinant allergens demonstrated their allergenicity through IgE binding reactivity with human sera. Monoclonal antibodies against recombinant group 10 allergens of D. pteronyssinus and T. putrescentiae were produced and were able to localize the inhaled allergens of D. pteronyssinus and T. putrescentiae in the respiratory system of mice. Both the research hypotheses of this study were accepted and all the objectives were achieved.