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No description available.

No description available.

<p>Medulloblastoma (MB) remains incurable in one third of patients despite aggressive multi-modality standard therapies. The heterogeneity of MB molecular subtypes as well as the failure of standard therapies to treat metastatic or recurrent disease necessitates more potent targeted approaches that minimize collateral toxicity. Immunotherapy presents a promising strategy by specifically targeting cancer cells and to date, there have been few successful immunologic applications targeting MB. Emerging evidence from integrated genomic studies has suggested MB variants arise from deregulation of pathways affecting the proliferation and differentiation of progenitor cell populations within the developing cerebellum. To test the developing cerebellum as a source of tumor rejection antigens, we adapted two animal models of MB recapitulating human Sonic Hedgehog (SHH) and Group 3 tumors for immunotherapeutic evaluation. Immunologic characterization of these murine models revealed subtype-specific differences in the tumor microenvironment and a differential response to immune checkpoint blockade. We used total embryonic RNA from the developing mouse cerebellum (P5) to generate antigen-specific T cells and confirmed the immunogenicity of targeting developmentally regulated antigens in vitro. Developmental antigen-specific T cells produced high levels of Th1-type cytokines in response to two immunologically distinct subtypes of MB. Interestingly, developmental antigen specific T cells did not show any cross reactivity with the normal brain or subsequent stages of the developing brain after P5. Targeting developmental antigens conferred a significant survival benefit and long term cures in intracranial treatment models of SHH and Group 3 tumor bearing animals. We additionally tested whether the enrichment of select developmental antigens through the exclusion of normal brain transcripts would potentiate antitumor responses in both animal models. Finally, we evaluated the relevance of targeting fetal antigens across human MB subtypes. Our studies demonstrate that developmental antigens can safely target multiple MB subtypes and can be further refined to preferentially target individual subgroups. Further studies targeting immunogenic developmental antigens and leveraging this strategy with specific immune modulatory interventions represent a novel approach at utilizing patient molecular classification information to mediate safe and effective immunotherapy.</p> / Dissertation

Plasmodium falciparum is the etiological agent of severe human malaria. The virulence of the parasite is dependent on a complex life cycle supported by a diverse repertoire of stage specific surface antigens. Notably, members of the 6-Cys s48/45 protein family are differentially presented on the parasite surface of each life cycle stage and known to play important biological roles, though the underlying molecular mechanisms are not well understood. Of the 6-Cys antigens, Pf41 is localized to the surface of the blood-stage merozoite through its interaction with Pf12 and is a target of the host immune system; accordingly, Pf41 is one of the five top-ranked potential malaria vaccine candidates. Pfs47 is localized to the surface of the sexual-stage gametocyte through its glycophosphatidylinositol-anchor and is currently being investigated as a transmission blocking vaccine. Intriguingly, both Pf41 and Pfs47 are predicted to adopt a three domain architecture. Prior to the studies presented here, only a single two domain 6-Cys protein had been structurally characterized. During my graduate studies, the structure of Pf41 was also determined by Dr. Michelle Parker in the Boulanger lab and I was able to perform the structural interpretation. Structural analysis revealed an unexpected topology where domains 1 and 2 are juxtaposed and the predicted central domain, which was largely proteolyzed during the crystallization process, is inserted as an extended loop in domain 1. Data from my ITC binding studies and protease protection assays suggest this inserted domain-like region (ID) plays an essential role in promoting assembly with Pf12. Despite several attempts, I was unable to crystallize Pfs47. Thus, to obtain architectural information describing Pfs47, a chemical cross linking experiment coupled with mass spectrometry was performed. The resulting data led me to predict that Pfs47 also incorporates an ID (Ser155 to Gln267) within D1. An engineered Pfs47 construct lacking the predicted ID was purified as a monomer, indicating that the predicted ID is expendable for stability of the overall structure. Collectively, these data provide important insight into the overall architecture of the biologically important Plasmodium 6-Cys proteins, which enables us to support ongoing collaborative vaccine design efforts. / Graduate

This investigation was concerned with the relationship of the slime layer material of Pseudomonas aeruginosa, Verder and Evans strain 1369, to the presumably somatic "O" type of antigen used by these authors as the base for their serological schema.

Modified vaccinia virus Ankara (MVA) has been used extensively as a recombinant vector for delivery of antigens from diverse pathogens. Its ability to generate strong antigen specific CD8+ T cell responses in humans has been shown in clinical trials of novel vaccines against malaria, tuberculosis, HIV I AIDS, influenza and cancer. The work in this thesis describes the use of BAC recombineering technology to harness the endogenous regulatory signal (promoter) that drives the expression of non-essential open reading frames (ORFs) in MVA for immunogenic expression of a recombinant antigen. Replacement of the ORFs of four non-essential genes in MVA; C11R, F11l, A44L and B8R with an epitope tagged luciferase positioned to use the same endogenous promoter showed early transgene expression equal to or slightly higher than traditional p7.5 and short synthetic promoter (SSP) constructs. The frequency of antigen-specific CD8+ T cell induced in mice by single dose MVA or adenovirus-prime, rMVA-boost vaccination showed equivalent or slightly higher responses by the endogenous promoters compared to the traditional p7.5 and SSP constructs. Assessment of the growth rate of these viruses showed they were unimpaired and the insertions were genetically stable. Furthermore, the endogenous promoter driven insertion loci of B8R and C11R were used for the construction of a bivalent MVA expressing an epitope tagged luciferase (rLucPb9) and a Photinus pyralis (pLuc) luciferase. The frequency of antigen-specific CD8+ T cells induced in mice by bivalent MV A was equivalent to single-pLuc and single-Pb9 recombinants co-administered as a mixture, at separate sites or administered alone following single dose MV A vaccination but slightly lower for Pb9-specific CD8+ T cell following adenovirus-prime, rMVA-boost.

Being a dissertation presented in fulfilment of the requirements governing the degree of Masters of Science in the Faoulty of Medicine, University of the Witwatersrand / Recent investigations have shown enhanced or aberrant expression of major histocompatibility system (MHC) antigens on cells lines derived from human hepatocellular carcinoma (HCC) in vitro and HCC in vivo. ( Abbreviation abstract ) / AC2017

Introduction: This study is a secondary data analysis of HIV/AIDS patients on Anti-retroviral Therapy (ART), at Themba Lethu HIV/AIDS clinic, who have had the first break in the continuity of taking their Antiretrovirals (ARVs) of more than 10 days, measured by patient missing the refill appointment for more than 10 days. The clinic started in 2004. HIV/AIDS is high in South Africa with about 400,000 AIDS patients on ARVs. For ARVs to be most effective they must be taken continuously without breaks, and for life. Without this, there is risk of ARVS drug resistance development and consequent failure of the ART program. Some patients may break this continuity and this seems to be a problem in South Africa. Where the patients develops side-effects or is not responding well to treatment, clinicians may also cause a break in the therapy. This study described the first break as when it occurred and for how long it lasted, investigated the factors associated with this break and the association of the first break and the last CD4 count. Materials and methods: 7,930 adults (≥18 years, either gender) on ART and baseline CD4 <250 cells/μl were included in the study. The study group were patients who had first break in continuity of therapy of more than 10 days. The first break was described as when it occurred after months of ART initiation and how long(days) the first break lasted. Patients on Post- Exposure Prophylaxis, single-dose Nevirapine, Prevention-of mother-To-Child- transmission therapy, and those with breaks in therapy of more than 364 days were excluded. Outcome variables was the last CD4 count. Analyses were in STATA 10, at 95% confidence interval. Median and quartile ranges were used to describe participants in the study. T-test, Fishers exact test and chi-square were used to compare groups. Regression was used to determine demographic and clinical factors associated with first break in therapy and also to determine the association of first break in therapy and the last CD4 count. Results: The median duration on ART for the patients was 764 days. 63% of patients had a break in ART. 47.5% of patients had their first break in therapy within the first 2 years of being on the ART program, with the largest proportion within the first 6 months of therapy. Most patient came with advanced disease(CD4 <100cells/μl, WHO clinical staging IV). Women were twice more than men. They tended to come earlier for therapy, took longer to improve and delayed in having the first break compared to men (254 vs. 205 days). Baseline hemoglobin and unemployment were factors associated with when the first break occurred. The median length of first break was 21 (Q1-Q3 7-43) Unemployment and baseline hemoglobin were associated with length of first break. The first break in therapy was associated with the last CD4 count. The longer the patient stayed on ART without the first break, the higher the last CD4 would be. Peripheral neuropathy had a statistically significant positive association with the last CD4 count. However, baseline CD4, Age, baseline BMI, WHO stage IV, baseline hemoglobin and unemployment had a statistically significant but negative association with the last CD4 count. The weakness of using the missing appointment system is that it does not inform clinician whether patients is really taking or not taking ARVs at home. Its strength over the self reported adherence system is that it is free of recall bias. Conclusion: Though Themba Lethu clinic has a follow-up system in place for patients missing refill appointment, up to 63% patient missed their appointment to collect medicine on time and this had a negative effect on the last CD4. There is need to strengthen existing follow-up method besides decentralising the ART services in Johannesburg.

A dissertation submitted to the Faculty of Health Sciences University of the Witwatersrand, Johannesburg for the degree of Master of Science. / The Special Pathogens Unit (SPU) of the National Institute for Virology has diagnosed a total of 158 cases of Crimean-Congo haemorrhagic fever (CCHF) from the time that the disease was first recognized in South Africa in 1981 up until the end of 2000. The virus has a propensity to cause nosocomial infections, and consequently rapid diagnosis is important for the isolation of the patient and the institution of barrier-nursing to protect medical staff and the community at large. Thus it is essential that the SPU should have the latest diagnostic and research tools available. Diagnosis of CCHF is generally confirmed by isolation of the virus, detection of viral RNA amplified by reverse transcriptase polymerase chain reaction (RT-PCR), demonstration of seroconversion or a >4-fold increase in IgG antibody titre, or detection of specific IgM antibody activity. Virus can be isolated in 1-6 days in cell culture, but the method is less sensitive for the isolation of low concentrations of virus than the use of suckling mice which, however, takes 7-9 days.