The relationships among parasite growth, responses to infection and host genetic factors were examined in rodents infected with Echinococcus multilocularis. Mongolian gerbils, cotton rats and C57L/J mice were relatively susceptible to the infection, whereas five other inbred strains of mice, and hybrids and backcrosses between C57L/J and C57BL/6J mice were more resistant. In mice, susceptibility to E. multilocularis was controlled by multiple, non-H-2-linked genes, as were pathological, inflammatory and specific (antibody) responses to the infection. These responses were also affected by the degree of parasite growth in individual hosts. Antibodies, natural killer cells and hematological responsiveness were ruled out as contributing to resistance to E. multilocularis. Studies on peritoneal leukocytes from infected animals suggested that infections with E. multilocularis were controlled by cells in two phases: an acute phase involving neutrophils and mononuclear cells and a chronic phase involving eosinophils and mononuclear cells.

No description available.

This thesis describes the development of serum and whole blood immunodiagnostic assays for Maedi-Visna virus (MVV). All previously described recombinant MVV ELISA assays utilised either the core p25 or transmembrane (TM) proteins alone, or combined, but as individual proteins. The p25 and TM genes of MVV were cloned individually into the pGEX-2T expression vector. Both proteins were expressed as a combined fusion protein in frame with glutathione S-transferase (GST). The purified recombinant antigens (GST-TM and GST-TM-p25) were used to develop a MVV ELISA. Sera from 46 positive and 46 negative sheep were tested using the GST-TM and GST-TM-p25 ELISAs and a commercial p25 EIA kit. A two-graph receiver operating characteristic (TG-ROC) analysis program was used to interpret the data. The GST-TM-p25 ELISA was more sensitive than the commercial assay which is based on the p25 antigen alone and more specific than the GST-TM ELISA. The GST-TM-p25 ELISA showed a sensitivity and specificity of 100%. The human AIDS lentivirus transmembrane (TM) glycoprotein portion of the envelope viral protein has been identified as the antigen most consistently recognised by antibodies. There is suggestive evidence that the same applies to MVV as the GST-TM fusion protein, expressed in E. coli, has comparable sensitivity to the GST-TM-p25 fusion protein, but lacks specificity. However, due to the hydrophobic nature of the MVV TM protein, purification of the expressed fusion protein required lengthy purification protocols. This was despite the fact that only a truncated version of the TM protein was expressed. This prompted investigating an alternative expression system that could possibly circumvent the above mentioned problems. The yeast Pichia pastoris is known to be suitable for the high-level expression of heterologous proteins which are secreted into the culture supernatant. These features made P. pastoris an attractive host for the expression of the hydrophobic TM protein of MVV. However, limited success was achieved as only low expression levels were obtained and detection and quantification was only accomplished by means of ELISA. Evaluation of the diagnostic performance of the P. pastoris expressed MVV TM-polypeptide was performed using a panel of 36 confirmed negative and positive sera, and evaluated using a TG-ROC analysis programme, which yielded an equal Se and Sp of 83%. The use of a novel rapid immunoassay system, which allows the detection of circulating antibodies in whole blood, has been investigated for use as a MVV diagnostic assay. The central feature of this immunoassay lies in a monoclonal antibody against a glycophorin epitope present on all sheep erythrocytes. A Fab'-peptide conjugate was constructed by coupling a synthetic peptide, corresponding to a sequence from MVV TM protein, to the hinge region of the Fab' fragment of the antisheep erythrocyte antibody. Within the limited number of 10 seronegative and 10 seropositive samples the autologous red blood cell agglutination assay had a sensitivity of 90% and a specificity of 80%. Despite the limitations and difficulties encountered, the use of such rapid whole blood immunodiagnostic assays for MVV holds promise. / Thesis (Ph.D.)-University of Natal, Durban, 1997.

Trypanosomosis is a parasitic disease in man, domestic and wild animals and is of major economic importance in many parts of the world, particularly in Sub-Saharan Africa. Trypanosoma congolense, T vivax and T brucei brucei are the major pathogenic trypanosomes infecting cattle in sub-Saharan Africa. The parasite itself is not directly responsible for the disease, but rather causes illness through the release of pathogenic factors. One of the major pathogenic factors released by trypanosomes is proteinases. Trypanotolerant cattle produce antibodies against a trypanosomal proteinase, congopain, that inhibit congopain activity. Congopain thus has vaccine potential. This study describes the mapping of immunogenic epitopes of congopain to identify peptide regions of the protein that induce enzyme inhibitory antibodies for inclusion in a trypanosome vaccine. This vaccine approach targets the disease, rather than the parasite by focusing on a pathogenic factor. These peptides also have potential for use in diagnostic assays. Peptides from the catalytic domain of a trypanosomal cysteine proteinase, congopain, were selected using an epitope prediction program. Peptides selected were from the two forms of congopain called CP1 and CP2. Antibodies against peptide-carrier conjugates were produced in chickens. The antibodies recognised native congopain, recombinant CP2 and the recombinant catalytic domain (C2). This suggests that the peptides selected have promise for use in vaccines. The peptides were also used to determine whether they are natural immunogenic epitopes of CP2 and thus have potential for use in diagnostic assays. Antibodies in the sera from T. congolense infected cattle recognised all the peptides in an ELISA. Antibodies in the sera from C2-immunised, non-infected cattle recognised most of the peptides in an ELISA. In order to distinguish between T. congolense and T vivax infection, two different peptides from the C-terminal extensions of CP2 and vivapain were used in ELISA tests with sera from infected cattle. Although anti-peptide antibodies produced against the two C-terminal extension peptides were specific for their respective peptides, thereby indicating the discriminatory power of the peptides selected, there was cross-reactivity by the sera from T. congolense and T. vivax infected cattle. Optimal antibody binding peptide sequences of these two peptides need to be identified by testing modified sequences of these two peptides to improve the sensitivity of this assay. In addition to attempting to define the epitopes of congopain, preliminary studies to increase the immunogenicity of congopain were also undertaken. Alpha 2-macroglobulin is a natural host inhibitor of proteinases. Inhibition occurs by entrapment of an active proteinase within the alpha 2-macroglobulin cage. In addition, it has been demonstrated that antigen complexed with alpha 2-macroglobulin becomes more immunogenic, resulting in enhanced antigenic presentation of an entrapped antigen. This study reports the interaction between congopain and alpha 2-macroglobulin. The preliminary results of this study showing congopain-alpha 2-macroglobulin interaction could be used to explore the possibility of increasing the immunogenicity of congopain and congopain epitopes by complexing these to alpha 2-macroglobulin. Congopain epitopes complexed with alpha 2-macroglobulin could be used to form a peptide-based vaccine. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

Malaria is a devastating disease caused by one of the world's most pathogenic parasites, Plasmodium. Five species of Plasmodium infect humans: P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi. P. falciparum is the most pathogenic and causes the greatest numbers of deaths. To date, no licensed vaccine against malaria is available, although there are numerous vaccine candidates in various stages of development. Pca 96 is a 96 kDa Plasmodium chabaudi adami protein shown to have a protective property in mice challenged with P. chabaudi adami. Thus, a P. falciparum orthologue of Pca 96 may be useful in vaccine development. BLAST searches with the Pca 96 amino acid and nucleotide sequences revealed proteins with high sequence identity to Pca 96 including the hypothetical P. falciparum PFC0760c and P. yoelii yoelii PY05757 proteins. A peptide sequence FKLGSCYLYIINRNLKEI was found to be conserved in all homologues of Pca 96, including PFC0760c, PY05757 and in the sequences of proteins from 5 other Plasmodium species. Bioinformatic approaches were explored to attempt to find a possible role of the protein and the possible importance of the conserved sequence. The conserved sequence was predicted to be an alpha helix and to contain possible HLA-DRB1*1101 and HLA-DRB1*0401(Dr4Dw4) T-cell epitopes (GSCYLYIINRNLKEI) in addition to a possible H2-Kd T-cell epitope (CYLYIINRNL). Protein-protein interaction predictions revealed that PFC0760c was likely to interact with proteins involved with nucleic acid binding. PFC0760c was predicted to have a domain found in proteins involved in the structural maintenance of chromosomes, which may suggest the protein is involved in chromatid cohesion during mitotic chromosome condensation. PFC0760c was also predicted to be located in the nucleus by the sub cellular prediction program, SubLoc. Anti-peptide antibodies were raised against the conserved amino acid sequence and against a peptide specific for PY05757 (SDDDNRQIQDFE). Both antibodies detected native antigens with immunofluorescence microscopy. The fluorescent signal appeared throughout the parasite cytoplasm and as an intense signal in the parasite nucleus. These immunofluorescence data supports the predicted nuclear location of the protein. A 822 bp portion of PFC0760c gene was expressed as a maltose-binding protein fusion protein (Pf33-MBP). Pf33-MBP was expressed and purified. Reducing SDS-PAGE and western blotting analysis revealed the fusion protein to be expressed at low levels as four bands (79, 60, 45 and 37 kDa). The purified fusion protein was cleaved with Factor Xa. MBP and Pf33 were of similar molecular mass after cleavage. To attempt to obtain better expression and purification, the 822 bp insert from pTS822 was sub-cloned into pGEX4T1. A glutathione-S-transferase (GST)-fusion protein (Pf33-GST) was expressed. The level of expression was poor and therefore not pursued. To take the study further, potential proteins that interact with PFC0760c and Pf33 need to be identified. In addition, immunisation of mice with the protein and subsequent Plasmodium challenge needs to be performed to ascertain the protective potential of the protein. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

No description available.

Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / Department of Physiology and Health Science

A suppressed immune system is a complicating health factor in cancer patients that keeps them from achieving the highest quality of life possible. Moderate exercise is thought to boost the immune system in cancer patients. The aim of this project was to determine the effects of an eight week aerobic exercise program on the mucosal immune system of cancer survivors compared to non-cancer patients. Our hypothesis was that the immune system of the cancer patients would positively respond to a moderate exercise program, specifically increasing antibody production. To examine our hypothesis, five cancer and six non-cancer patients undertook a supervised moderate aerobic exercise program at the University of Northern Colorado. The subjects performed an incremental peak treadmill test to exhaustion at the start of the program and after 8 weeks of training. Saliva samples were taken at specific times for each peak exercise test: prior to testing, immediately after testing, and 30 minutes post-test. Enzyme-Linked ImmunoSorbent Assays (ELISA) were performed at Ball State University to analyze the levels of immunoglobulins (IgA, IgG, IgM) in saliva samples of cancer and non-cancer patients. Our findings demonstrated there was a significant increase in IgG after 8 weeks of moderate exercise in non-cancer patients 30 minutes after the treadmill test. A significant increase was also seen in salivary IgA levels after 8 weeks of moderate exercise in cancer patients 30 minutes after the treadmill test was administered, supporting our hypothesis that exercise enhances immune function. Eight weeks of moderate exercise has been shown to enhance immune function demonstrated by the increase of IgA and IgG levels in saliva. / Department of Biology

Group A streptococcal disorders can result from autoantibodies generated against M proteins. These autoantibodies cross react with the basal ganglia resulting in movement disorders. Previously, we demonstrated binding of streptococcal mAb10F5, with CPu and phospholipids. To determine if mAb10F5 binding to basal ganglia and phospholipids is due to virulence of the antibody or antibody subtype, rats were injected with control IgG2 antibodies and euthanized after 24, 48, or 72 hours. Brains were harvested and immunofluorescence was used to analyze brain slices. Control IgG2 rats showed significantly less fluorescence in the CPu than mAb10F5 injected rats at every time point. These findings reaffirm 10F5 is an anti-basal ganglia antibody. To evaluate mechanism of antibody entry, mAb10F5 was examined for anti-phospholipid activity. MAb10F5 displayed greater affinity to phospholipids when compared to IgG2 controls. Our findings support mAb10F5 is an anti-basal ganglia and anti-phospholipid antibody due to its own virulence. / Access to thesis permanently restricted to Ball State community only / Department of Physiology and Health Science

Autoantibodies generated against M proteins can cause post-streptococcal disorders such as Rheumatic Fever. A severe complication of rheumatic fever is rheumatic heart disease which may involve both cardiomyopathy and valvulitis. Rheumatic fever has been associated with the class I M protein epitope of Group A streptococcus (GAS). This epitope can be recognized by monoclonal antibodies (mAbs) 10B6 and 10F5. Previously, we demonstrated binding of streptococcal mAb10F5 in the heart tissue (apex, atria, and valves) of Lewis rats as compared to anti-myosin binding. To determine if mAb10F5 binding in the heart is due to virulence of the antibody or antibody subtype, rats were injected with control IgG2 antibodies and euthanized after 24, 48, or 72 hrs. Hearts were harvested and immunofluorescence was used to analyze the hearts. The immunofluorescence intensities for IgG2b were compared to mAb10F5 using previously acquired data. Control IgG2b rats showed significantly less immunofluorescence intensities in the heart regions than mAb10F5 injected rats at the 48 and 72 hr time points. These findings reaffirm mAb10F5 as an anti-cardiac antibody thatbinds heart tissue due its own virulence. To differentiate between the two IgG subtypes, binding intensities of IgG2a were compared to the binding intensities of IgG2b. The binding intensities of IgG2a increased with time. This finding was supported by previous work in our laboratory suggesting IgG2a remained in the bloodstream longer than the IgG2b. / Access to thesis permanently restricted to Ball State community only. / Department of Physiology and Health Science