The protozoan parasites Trypanosoma congolense and T. vivax cause trypanosomosis in cattle. The major lysosomal cysteine proteinase of T. congolense, congopain, may contribute to pathogenesis of the disease, and antibody-mediated inhibition of this enzyme may contribute to mechanisms of trypanotolerance. Oligopeptidase B, a trypanosomal serine peptidase, is also a potential virulence factor in African trypanosomes because it is released into the host circulation by dead or dying parasites, where it retains catalytic activity due to the enzyme's insensitivity to serum protease inhibitors. The vaccine potential of the catalytic domain of congopain, C2, and oligopeptidase B complexed with 0'2-macroglobulin (0'2M) was evaluated by producing antibodies in rabbits. Inhibition of congopain and oligopeptidase B activity by these antibodies was assessed. The oligopeptidase B open reading frame from T. congolense and T. vivax was cloned and expressed in Escherichia coli, from which active recombinant enzymes were purified. These recombinant enzymes exhibited trypsin-like specificity for peptide substrates, cleaving on the carboxy side of basic amino acid residues such as arginine and lysine. Enzymes were found to be optimally active between pH 8 and 10, optimally stable at pH 6, and showed activation by reducing agents and sensitivity to ionic strength. The enzymes showed typical oligopeptidase B-like inhibitor profiles, except that they were not inhibited by thiol sensitive inhibitors such as iodoacetamide and Nethylmaleimide. High yields of bovine and rabbit 0'2M were isolated by a three-step procedure of fractionation by PEG 6000, and zinc chelate and Sephacryl S-300 HR chromatography. Congopain, its catalytic domain C2, papain and cathepsin L all cleaved the bait region of bovine 0'2M and became trapped inside the 0'2M molecule, where their activity against large molecular weight substrates was inhibited. C2 could thus be complexed with 0'2M directly or used to form C2-0'2M-oligopeptidaseB complexes for immunisation purposes. iv The catalytic domain of congopain, C2, was used to immunise rabbits either without adjuvant, as a water-in-oil emulsion with Freund's adjuvant, or in a complex with either bovine or rabbit U2M. Freund's adjuvant elicited the highest anti-C2 antibody response. However, the greatest inhibition, 65%, of C2 activity against Z-Phe-Arg-AMC was obtained with antibodies produced by rabbits receiving C2-U2Mcomplexes. In a second study, C2 and oligopeptidase B were used to immunise rabbits , either in alum, or complexed to bovine U2M. Anti-C2 antibody levels were highest in rabbits immunised with the free proteins in alum, whereas anti-oligopeptidase B antibody levels were comparable for each adjuvant system. Anti-oligopeptidase antibodies produced with alum gave 100% inhibition of oligopeptidase B activity. In contrast, antibodies produced against C2-u2M-oligopeptidase B complexes had little effect on oligopeptidase B activity. However, these antibodies inhibited 55% of C2 activity. Alum was a slightly less efficient adjuvant for C2 and 50% inhibition of C2 activity was observed. It appeared that immunisation of rabbits with C2 complexed to U2M resulted in the production of antibodies that were better able to neutralise the proteolytic activity of C2 and congopain in vitro than that with conventional adjuvants . The immunisation of C2 complexed to bovine u2-macroglobulin therefore has the potential to neutralise parasite congopain in vivo, and may contribute to an anti-disease vaccine against African trypanosomosis. Complexation of oligopeptidase B to u2M offers no benefit, since antibodies produced against this complex are not able to inhibit the activity of oligopeptidase B. Immunisation with oligopeptidase B in alum is sufficient to produce efficient enzyme-inhibiting antibodies in the context of an anti-disease vaccine against African trypanosomosis. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.

Effective case management of malaria is hampered by the spread of parasite resistance to nonartemisinin antimalarials. To counteract the impact of drug resistance, the World Health Organization (WHO) has endorsed artemisinin-based combination therapy (ACT) as the first-line treatment for uncomplicated Plasmodium falciparum malaria. Currently recommended ACTs are artemether-lumefantrine, artesunate plus amodiaquine, artesunate plus mefloquine, artesunate plus sulfadoxine-pyrimethamine and dihydroartemisinin-piperaquine. This study sought to review evidence of the efficacy and safety of different non-artemisinin antimalarials in combination with artesunate, artemether or dihydroartemisinin for the treatment of uncomplicated P. falciparum malaria in non-pregnant adults and children. The search for randomized controlled trials (RCTs) was conducted in the Cochrane Central Register for Controlled Trials (CENTRAL), MEDLINE, EMBASE and in ClinicalTrials.gov in January 2009. The eligibility and the methodological quality of trials were assessed and data were extracted, using standard forms. Data were captured and analyzed in Review Manager Software, versions 4.2 and 5.0. The outcomes assessed were: treatment failure, fever and parasite clearance time, calculating the relative risk (RR) and a weighted mean difference (WMD) with a 95% confidence interval and p-values, indicating statistical significance at 0.05. Thirty-seven trials with 6862 participants were included. Artesunate combined with amodiaquine had a statistically significant lower risk of treatment failure compared to the combination of artesunate with sulfadoxine-pyrimethamine (RR=0.57, 95% CI [0.33, 0.97], p=0.04, seven trials, N=1341). In addition, treatment with artesunate plus mefloquine was significantly associated with a lower risk of treatment failure compared to artesunate plus azithromycin (RR=0.04, 95% CI [0.00, 0.64], p=0.02, one trial, N=54). There was no significant difference when either mefloquine or atovaquone-proguanil were combination partners with artesunate (RR=2.6, 95% CI [0.93; 7.24], p=0.07, one trial, N=1066). When artesunate was combined with chloroquine, primaquine or azithromycin and compared with artesunate monotherapy, there was no statistically significant difference in the risk of unadjusted treatment failure. Each of these comparisons had one trial each. Artesunate plus chloroquine was quicker at clearing fever compared to artesunate plus sulfadoxinepyrimethamine (WMD= -7.20, 95% CI [-12.53, -1.87], one trial, N=132). Few trials adequately reported adverse events. There was no significant difference observed in the risk of adverse events between artesunate plus amodiaquine compared with artesunate monotherapy, however, adverse events were significantly less in artesunate plus amodiaquine compared to artesunate plus methylene-blue. Artesunate plus amodiaquine on the other hand had significantly more adverse events reported compared to artesunate plus sulfadoxine-pyrimethamine. The findings of this study support the implementation of artemisinin-based combination therapy for the treatment of uncomplicated malaria. Most crucially, this review found a greater advantage of combining amodiaquine with artesunate compared to sulfadoxine-pyrimethamine. The efficacy of artesunate plus mefloquine was superior to that of artesunate plus azithromycin. Furthermore, the combination of artemisinins with chloroquine, primaquine and azithromycin has shown very low efficacy and these combination therapies should not be recommended. The reporting of efficacy was not standardized as many trials did not differentiate between re-infections and recrudescences. Adverse events were also not adequately reported. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

The molecular biology of human genetic disorders is under intensive investigation at present. In those cases where the disorder is clearly defined in terms of altered gene structure, possibilities may exist for the correction of the disorder by insertion of normal genes through the process of DNA transfection. A possible method for the transfer of genetic material is by attempting to attach DNA to a protein which has specific receptors on cells and which undergoes receptor-mediated endocytosis. By this means one might be able to get DNA into cells. This thesis deals with experimental work on the chemical modification of human serum transferrin by means of water-soluble carbodiimides. The resulting N-acylurea transferrins bind DNA in a reversible manner. Characteristics and properties of the binding interactions are dealt with in detail. N-acylurea derivatives of transferrin were prepared with the water-soluble carbodiimides, N-ethyl-N' -(3-dimethylaminopropyl) carbodiimide and N-ethyl-N' -(3-trimethylpropylammonium) carbodiimide iodide. Reactions were carried out under mild conditions at room temperature for 48-72 hours. [³ H] N-ethyl-N' -(3-trimethylpropylammonium)carbodiimide iodide was used for the determination of covalently attached N-acylurea groups in the protein. Changes in charge properties were determined by agarose gel electrophoresis. Carbodiimide modification of proteins is thought to occur at side chain carboxyl groups of glutamic and aspartic acid residues. This was confirmed by the use of Staphylococcus aureus V8 protease, which cleaves peptide bonds at the carboxyl side of glutamic and aspartic acid residues, but not in the case of substituted side chain carboxyl groups. Through the use of puromycin as a nucleophile it has been shown that other functional groups were not activated upon reaction of transferrin with carbodiimide. The carbodiimide-modified proteins bind various types of DNA and RNA in a reversible manner. Low concentrations of N-acylurea transferrin retarded the migration of pBR322 DNA, M 13 mp 8 single-stranded DNA and Pst 1 restricted lambda DNA on agarose gel electrophoresis, while at higher concentrations the DNA was unable to enter the gel. Nitrocellulose filter binding assays showed that binding of DNA to Nacylurea transferrins was rapid, dependent on concentration of the modified transferrin and sensitive to ionic conditions. Binding was found to occur mainly through electrostatic interactions between phosphate groups of DNA and N-acylurea groups. These conclusions were based on experiments which showed that protein-DNA complexes were dissociated by increasing salt concentrations and by heparin. Non-electrostatic interactions such as hydrophobic interactions and hydrogen bonding are also involved in binding, since half dissociation of complexes, induced by chaotropic salts, KSCN and NaC10₄occurs at lower concentrations of salt than in the case of NaCl. Also RNA polynucleotides inhibit binding of DNA to Nacylurea transferrins to varying extents. The N-acyl urea transferrins have been shown to bind certain specific restriction endonuclease cleavage sites on pBR322 DNA. The N-acylurea transferrin-DNA complexes would thus be suitable for experiments in cell transfections using cells which have transferrin receptors. / Thesis (M.Sc.)-University of Durban-Westville, 1986.

In order to develop a cheaper alternative to the conventional enzyme-linked immunosorbent assay system, application of dye molecules as labels in immunoassay was investigated in this study. This chromogenic dye-antibody conjugate could be used in colourimetric immunodetection diagnostic assays that could be used in a rural African setting. The chemistry of the interaction between twenty-six dyes of anionic, cationic and ligand dye classes with IgY and other proteins were studied for protein detection and conjugation to antibodies. Out of the twenty-six dyes studied, Direct Red 81 proved to be a good protein stain on nitrocellulose and polyacrylamide gels with comparable sensitivity to Coomassie Blue R 250. Direct Red stained proteins faster (< 5 min) than Coomassie Blue R 250 in polyacrylamide gels. Aurintricarboxylic Acid, Ethyl Red and Gallocyanine with carboxylic acid and/or hydroxyl functional groups were selected, activated with carbonyldiimidazole (CDI) to form amine reactive-imidazole intermediates and conjugated to anti-rabbit albumin IgY. Gallocyanine gave the best molar coupling ratio with IgY (76:1 dye:IgY). The dye-antibody conjugates were used to detect rabbit albumin on nitrocellulose. Aurintricarboxylic Acid-IgY and Gallocyanine-IgY detected 50 ng of rabbit albumin on nitrocellulose, which was 10 fold less sensitive than HRPO-IgY conjugate. Cross-linking of the antibodies by the dyes compromised the immunoreactivity of the Aurintricarboxylic Acid-IgY and Gallocyanine-IgY conjugates. The immunoreactivity of Ethyl Red-IgY was not compromised. Anti-rabbit albumin IgY was conjugated to derivatized dextran as an alternative immunoassay reagent and used to detect rabbit albumin on nitrocellulose by staining the polysaccharide (dextran) in the immune complex with PAS reagent. IgY-dextran complex was able to detect 25 ng of rabbit albumin on nitrocellulose, but PAS staining resulted in high background staining of the nitrocellulose membrane. Dextran-antibody conjugates may have better potential as immunodetecting reagent than dye-IgY conjugates, if a more sensitive and specific method of detecting the dextran in the Ag:Ab-dextran immune complex is developed. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

Mannoproteins form the outermost layer of the cell wall in Saccharomyces cerevisiae. These glycoproteins are first synthesized in the endoplasmic reticulum and undergo posttranslational modification before they are transported through the secretory pathway. Some of the glycosylphosphatidyl inositol (GPI) anchored proteins are incorporated into the cell wall where their GPI-anchor is first trimmed off before they are anchored into the β-glucan network in the cell wall. The yeast cells are constantly faced with different environmental conditions and the cell surface mannoproteins are responsible for different morphological transitions that allow the cell to survive harsh conditions. The Flo proteins or adhesins encoded for by the family of FLO genes are known to confer adhesion to biotic and abiotic surfaces, hydrophobicity, biofilm and pseudohyphal filamentation. These phenotypes are suggested to be passive mechanisms employed by the cells to escape from stress or to prevent being washed away. The adhesion properties conferred by the adhesins are important in biotechnological processes. Identification of genes that have the potential to release more adhesins into the culture media will facilitate studies on the fine structural details and functional domains in these glycoproteins. The knowledge will also help in the formulation of fungal drugs since the adhesion of fungal pathogens to host such as humans is known to be the first step of infection. In this study, yeast strains with a deletion in KNR4 or GPI7, which are genes related to the biogenesis of the cell wall were employed to over-express FLO genes. Flocculation intensity and hydrophobicity of cells in the stationary phase were used as a measure of phenotypic changes of the cell surface. The effects of these deletions on the cell surface phenotypes in transgenic strains over-expressing FLO genes were assessed. We found that the KNR4 deletion resulted in a 50% decrease in cell-cell adhesion compared to the wild type. The deletion in GPI7 was found to have no effect in flocculation or the cell initiated a response that resulted in the expression of other genes to compensate for the loss of GPI7. The ability of the yeast cells to invade agar surfaces was not affected by the deletion of GPI7 or KNR4. The observed flocculation intensity was found to correlate with cell surface hydrophobicity. A decrease in the level of flocculation was also accompanied by a decrease in cell surface hydrophobicity. The results of this study indicate that deletion of the KNR4 gene affects the adhesins more than the deletion of the GPI7 gene. A screen of other genes related to cell wall biosynthesis will allow for a selection of genes with the potential to release adhesins to the cell culture medium. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.

Microorganisms may evade killing by neutrophils (PMNs) by altering signal transduction and hence phagosome maturation. Secreted, active matrix metalloproteinases (MMPs) appear to be required for PMN killing of pseudomonas microorganisms, via an MMP and complement-dependent, but otherwise unknown mechanism. This also depends on the absence of the inhibitor of MMPs, tissue inhibitor of metalloproteinases-1 (TIMP-1). By altering their particular complement opsonin and hence the PMN complement receptor bound, microorganism may evade killing, as not all PMN complement receptors trigger phagosome maturation and hence killing of microorganisms. C1 inhibitor of the classical complement cascade, required for the exposure of C1q and further assembly of complement factors on the bacterial surface and hence binding to specific PMN receptors, is MMP sensitive. MMP secretion may, therefore, not only facilitate the killing of microorganisms, but inappropriate secretion, induced by pathogens, may prevent complement assembly and killing via complement-mediated pathways. It was, therefore, decided to assess MMP-9 and TIMP-1 secretion in the presence of C1q-opsonized polystyrene beads and subsequently upon stimulation with pseudomonas organisms, and explore the relationship between secretion of PMN MMPs (specifically MMP-9) and TIMP-1 and phagocytic uptake and maturation of the PMN phagosome into a killing body. MMP-9 and TIMP-1 secretion was seen to occur at low levels under most conditions. However, in the presence of serum, and hence complement, MMP-9 secretion was found to be upregulated during uptake of C1q-coated beads. MMP-9 possibly inactivates C1 inhibitor at this stage, causing local tissue swelling (normally associated with the inactivation of C1-inhibitor), entry of various white blood cells and further complement into the area of infection, assisting in the extracellular killing of microorganisms. MMP secretion may simultaneously down-regulate the activation of further PMNs via inactivation of C1q assembly and hence phagocytic uptake and activation of PMNs. Unlike MMP-9, secretion of TIMP-1 was not upregulated by C1q receptor binding, implying that any secreted MMP-9 may, therefore, be in excess and hence uninhibited by TIMP-1. A distinct regulatory mechanism seems to be responsible for the release of TIMP-1, though TIMP-1 secretion was upregulated by extracellular calcium levels, partially contradicting previous findings which suggested that TIMP-1 was not calcium regulated. It seems unlikely that extracellular calcium levels would be the only mechanism by which TIMP-1 is regulated, however, and further surface receptor mediated agonists should be explored. Levels of MMP-9 and TIMP-1 secretion in the presence of pseudomonas microorganisms now need to be assessed to see whether these secretion patterns are altered to favour the evasion of opsonization by C1q. Uptake of C1q-opsonized beads was also increased by the presence of serum, possibly due to presence of complement. MMP-9 and TIMP-1 secretion patterns still need to be correlated with phagosomal uptake and killing of microorganisms, before their role in killing of microorganisms becomes fully evident. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

The correction of human genetic disorders by transfer of genetic material to cells is under intensive investigation in a number of 1aboratories. One possible way of trying to achieve the transfer of nucleic acid is by attaching DNA to a protein which has specific receptors on cells and which undergoes receptor-mediated endocytosis. In order to make use of the ligand protein-receptor approach for DNA transfer, iron-loaded human serum transferrin has been modified with the water soluble carbodiimides N-ethy1-N I -(3-dilllethy1aminopropyl) carbodiimide (CDI) and its quaterary analogue (ECDI) to give modified N-acy1urea transferrins. N-Acy1urea CDI (Fe 3+) transferrin and N-acy1urea CDI (Fe ) transferrin have been found to interact with and bind DNA in a reversible manner which i! dependent on ionic strength. [1251] N-Acy1urea CDI+(Fe3+) transferrin binds to transferrin receptors on Hea cells in culture and undergoes internalization through receptor-mediated endocytosis. Binding of the modified transferrin in the presence of calf thymus DNA to transferrin receptors also takes place. However, although internalization in the presence of DNA doe! appear to take place, the results of the internalization are not fully understood. Transfection studies with N-acy1urea CDI (Fe ) transferrin and plasmid pBR322 DNA as well as plasmid ptkNEO DNA complexes in the HeLa cell system are reported. The results of a number of transfection experiments suggests that N-acy1urea transferrins are capable of transfecting DNA (ptkNEO DNA), carrying genes for resistance to the antibiotic Geneticin (G41S) in the HeLa cell system. However, further development of the transfection system is necessary in order that consistantly reproducible results may be achievd. / Thesis (M.Sc.)-University of Durban-Westville, 1986.

African animal trypanosomiasis is a major threat to African agriculture causing a loss estimated to 4.5 billion US$ per annum. Trypanosoma congolense is the major causative agent in African animal trypanosomiasis and is transmitted by tsetse flies of the Glossina spp. Congopain, a major cathepsin L-like cysteine peptidase in T. congolense is associated with trypanotolerance in N‘Dama cattle and is a target for an anti-disease vaccine. It is suggested that trypanotolerant cattle control the disease by antibody mediated neutralisation of congopain, and that immunisation of cattle against congopain can mimic trypanotolerance resulting in minimised disease pathology. Susceptible cattle immunised with recombinant catalytic domain of congopain, C2, produced high levels of anti-congopain IgG specific antibodies against congopain, maintained weight and exhibited less severe anaemia. However, there was no effect on the establishment of T. congolense infection and acute anaemia development in trypanosusceptible cattle. It has been suggested that failure of congopain to give full protection of the host may be due to poor presentation to the immune system by conventional adjuvants used in previous studies. The aim of the present study was to improve the presentation of the catalytic domain of congopain (C2) to the immune system, by linking it to the proposed molecular adjuvant, BiP, an ER localised HSP70. A further aim was to localise the domain(s) of BiP where the adjuvant properties reside. BiP consists of an ATPase domain (ATPD), a peptide binding domain (PBD) and a C-terminal domain (C-term). Consequently, BiP69, BiP69 lacking the C-terminal domain (BiP60), BiP coding fragments (ATPD, PBD and C-term) and the C2 coding sequence were amplified by PCR from either genomic T. congolense DNA or plasmid DNA. The PCR products were each sub-cloned into a pTZ57RT vector, and C2 cloned into a pET-28a expression vector. The BiP coding fragments were inserted into the recombinant pET-28a-C2 vector, resulting in pET-28a-BiP69-C2, pET-28a-BiP60-C2, pET-28a-ATPD-C2, pET-28a-PBD-C2 and pET-28a-C-term-C2 coding chimeras. The fusion proteins were expressed in an E. coli system as insoluble inclusion bodies at the expected sizes of 96 kDa (BiP69-C2), 88 kDa (BiP60-C2), 47 kDa (PBD-C2), 34 kDa (C-term-C2) and 27 kDa (C2). However, the ATPD-C2 fusion protein was expressed at a larger and smaller size in different attempts. Protein expression was confirmed by western blots using anti-BiP antibodies and anti-congopain N-terminal peptide antibodies. Recombinantly expressed peptide binding domain (PBD)-C2, C-terminus-C2, BiP69-C2, BiP60-C2 chimeras and a BiP69 fusion protein were purified and refolded by a Ni-NTA based one-step on-column refolding method. Bacterial proteins co-purifying with BiP69-C2 and BiP60-C2 chimeras were removed by incubation with 5 mM ATP in the dissociation buffer, but poor yields resulted in using these chimeras as non-pure proteins. Immunisation of Balb/c mice with the BiP69-C2 fusion protein chimera induced a higher antibody response to C2 compared to immunisation with the BiP69/C2 mixture or with C2 in Adjuphos/Quil A. BiP69-C2 and PBD-C2 chimeras and BiP69/C2 mixture induced a robust antibody response to BiP69, but no correlation could be made with the contribution to control of parasitemia and disease induced pathology. Mice immunised with BiP69-C2 and PBD-C2 chimeras showed a better booster effect of T. congolense infection with higher anti-C2 antibody stimulation compared to control groups. Immunisation did not change the establishment of T. congolense infection and anaemia development in most immunised groups. However, mice immunised with the BiP69/C2 mixture and with the PBD-C2 chimera produced anti-C2 antibodies possible contributing to clearing parasites 10 days and 16 days earlier respectively, than mice immunised with BiP69-C2, C-term-C2 and BiP60-C2 chimeras and PBS, C2 and C2 in Adjuphos/Quil A control groups and showed no clinical symptoms of the disease. There was no significant difference in percentage mice survival between BiP-C2 chimera immunised mice and control groups immunised with C2 alone or with a mixture of Adjuphos/Quil A or immunised with PBS. In the present study, it was shown that BiP69 has adjuvant effects when linked to C2 and that its peptide binding domain acts as an adjuvant. It is possible that the removal of the C-terminal domain reduced the adjuvant potency of the peptide binding domain suggesting a prominent role in the adjuvant effect of the BiP molecule. Finding the exact role of the C-terminal domain in the adjuvant effect of BiP would be of utmost interest, and would involve comparing anti-C2 antibody response produced by immunisation with C2 linked to the peptide binding domain with or without the C-terminal domain. Future work includes repeating this study in trypanosusceptible cattle to confirm these findings. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Infectious bursal disease virus (IBDV) is a chicken pathogen that infects the bursa of Fabricius, an organ involved in the development of the immune system in chickens. Infection by the virus leads to destruction of the bursa and immunosuppression. Infection by virulent strains may result in mortality. Current methods to combat the virus involve the use of vaccines. These are usually a mixture of live attenuated and oil inactivated virus. Variant strains of the virus are able to escape the vaccine-generated antibodies. In addition, the vaccines result in damage to the bursa. Identification of a receptor for IBDV could result in the development of either treatment for the virus or superior vaccines by interfering with the attachment of the virus to host cells. Several methods for identifying IBDV binding proteins from the membranes of cells from the bursa of Fabricius were examined. Affinity chromatography of IBDV binding proteins with a matrix consisting of IBDV cross-linked to Sepharose 4B allowed separation of a number of virus binding proteins. In contrast, virus overlay protein blot assay (VOPBA) and reversible cross-linking with 2-iminothiolane proved less; conclusive. Predominant proteins in the affinity-separated fraction were of 40 and 32 kDa. These were further examined by N-terminal amino acid sequencing of the whole protein and N-terminal sequencing of peptides produced by endoproteinase Lys-C digestion of the protein respectively. The 40 kDa protein showed homology with human synovial stimulatory protein involved in the formation of autoantibodies in rheumatoid arthritis. Virus was also shown to bind to a 440 kDa protein complex. This 440 kDa protein complex appeared to consist primarily of a 40 kDa protein when examined by reducing Tris-Tricine SDS-PAGE. Analysis of bursal membrane proteins by Western blots using sera from rheumatoid arthritis patients revealed interactions between several IBDV proteins and the antibodies from rheumatoid arthritis patients. Using serum from one of the five patients showed a strong interaction at approximately 80 kDa and a weaker interaction at approximately 40 kDa. This may indicate an immune reaction between a chicken homolog of the synovial stimulatory protein and antibodies in rheumatoid arthritis sera. The 32 kDa protein showed homology to a Pseudomonas fluorescens protein. A section of this sequence was amplified by PCR from chicken DNA and RT-PCR from chicken RNA using degenerate primers constructed from the established N-terminal amino acid sequences and chicken codon usage tables. The fragment produced upon amplification from chicken DNA and RNA did not correspond to the predicted size of 177 bp. In contrast, when the RT-PCR product was heated and snap cooled before examination by agarose gel electrophoresis, the product consisted of two fragments, one of approximately 400 bp in size and one of approximately 200 bp in size. The establishment ofthe 40 and 32 kDa chicken bursal membrane proteins as possible receptors for the virus could allow for the development of vaccines and/or treatment strategies for the virus. Treatment strategies or vaccines would be based on blocking of the interaction between IBDV and chicken host cells. Peptide mimics of the epitopes involved in such interactions could possibly achieve this. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.

Cathepsin, matrix metalloproteinase (MMP) enzyme and tissue inhibitor of MMP (TIMP) distribution in J774 mouse macrophages has not been comprehensively studied. The distribution and vesicle regulation, trafficking and release of these is important, possibly suggesting drug targets for the therapeutic regulation of inflammatory disease and phagosomal killing of pathogenic organisms in J774 and other macrophages. Percentage immunofluorescence and ultrastructural enzyme and inhibitor distribution, together with LysoTracker (acidity) and lysosome-associated membrane proteins (LAMPs) colocalisation (both indicating late endosome or “lysosomal” association), western blot estimates of percentage processed- and unprocessed intracellular and secreted enzyme and inhibitor, and vesicle size was used to assign enzyme and inhibitor to “classical” vesicle types. Antibodies against TIMP-1 and TIMP-2 were raised and all antibodies characterised for this purpose. Together these were used to assign cathepsins H, S, D, B and L to possible secretory vesicles (±20 nm, non-acidic, LAMPs-negative, containing precursor enzymes) and identify at least 6 other endosome-“lysosome-like” vesicles. Cathepsin H appears to be present in classical early endosomes (±100 nm, non-acidic, LAMPs-negative) and cathepsin S in late endosomes(±50 nm, acidic, LAMPs-positive) and possibly “lysosomal” (“hybrid” or digestive organelles) (±150-200 nm, acidic, LAMPs-positive). Both cathepsins H and S, however, seem only reliable markers if used together with additional markers. Cathepsin D appearsmainly associated with “lysosomal” (“hybrid” or digestive organelles) (±150-200 nm, acidic, LAMPs-positive), possibly consisting of further subpopulations which requires furtherinvestigation e.g. labelling for LAMP-1 and LAMP-2 and cathepsin D. Cathepsins B and Lmay occur in late endosomes and/or hybrid organelles and “secretory lysosomes” containing cathepsins B, D and L may also exist (±30-50 nm, acidic, LAMPs-positive). The distribution of MMP-9, TIMP-1 and -2 in vesicles (non-acidic, LAMP-2-negative) thatappear novel and distinct from late endosome-“lysosome” vesicles were also demonstrated. In LPS-stimulated cells, the identity of the large (±450 nm), possible recycling endosomes (Rab11-positive, LAMPs-negative), containing colocalised MMP-9 and TIMP-1, needs investigation i.e., requires further verification with triple labelling and EM. Possible cell membrane and recycling endosome localisation of TIMP-2 needs confirmation with labelling of non-permeabilised cells and labelling for MT1-MMP and proMMP-2, respectively. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.