Browsing by Department "Division of Clinical Immunology"

Now showing 1 - 8 of 8
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    CD4 and CD8 T-cell responses to acellular pertussis and rotavirus vaccination in breast-fed HIV exposed, uninfected infants
    (2017) Nundalall, Trishana; Jaspan, Heather B; Gray, Clive M
    Introduction: Vaccination is one of the most efficient ways to prevent infectious diseases, however due to the naivety and regulation of immunity found in infants, induction of vaccine-mediated immunity is challenging. Respiratory and diarrheal diseases are major contributors to infant mortality. Additionally, Human Immunodeficiency Virus-1 (HIV) infections increase the risk of mortality. Current advances in Prevention of Mother-to-Child Transmission (PMTCT) have prevented HIV infections in almost 97% of infants being born to HIV-infected mothers. As a result there is an increasing number of HIV exposed, uninfected infants (HEU). HEU infants have a higher rate of infectious disease related mortality and morbidity compared to unexposed infants, the underling causes of these differences are still not understood. In this dissertation, responses to two childhood vaccines, live, attenuated rotaviral vaccine (Rotarix) and acellular Pertussis (aP), were analyzed in HEU infants, with specific focus on T-cell responses to Rotarix and aP, due to the current lack of published data on T-cell responses. Additionally, the influence of feeding mode, that is breast or formula feeding, was also assessed as it is well established that breast fed infants contract fewer infections compared to formula fed infants. Methods: This dissertation included infants from a larger cohort which includes three groups of infants; HIV unexposed breast fed (UBF), HIV exposed breast-fed (EBF) and HIV exposed formula fed (EFF) infants. Infants were recruited at birth and followed up until 36 weeks of age. As no Rotavirus vaccine T-cell assay was previously published, multiple techniques were utilized to attempt to optimize an assay capable of detecting Rotavirus (RV) vaccine-specific T-cell responses. To determine T-cell responses to Bordertella pertussis (BP), blood was collected from infants at each time-point and 200ul was stimulated with BP antigen in a 12-hour whole blood assay. Cells from all assays were fixed and stained for flow cytometric analysis of CD4 and CD8 T-cell responses. The markers used included live/ dead, CD3, CD4 and CD8 for identification of T-cell populations, IFNγ, IL-2 and TNFα cytokines, HLA-DR and Ki67 for activation and proliferation, and CD45RA and CD27 memory differentiation. Data analysis was then completed using Microsoft Excel, Flow.Jo V9, GraphPad prism V6, Pestle 1.7 and Spice V5.33 software packages. Results: Despite multiple attemps it was not possible to optimise an assay capable of consistently detecting Rotavirus vaccine specific responses. This was partly due to interferance from contaminating agents in the protein antigens used, and difficulty in culturing and purification of whole virus. Assessment of aP spcific CD4+ T-cell memory demonstrated an overall increase in terminally differentiated (TD) memory cells accross time. This mirrored the ontogeny of the total T cell pool which showed an overall decrease in naïve T-cell frequencies with a consequent increase in late and terminally differentiated CD4 and CD8 T-cell populations over time through the first months of life. Both total and aP specific CD4+ early differentiated (ED) memory T-cells remained unchanged over time. ED CD8+ memory T-cells peaked at week 15 in EBF infants. A similar observation was found in UBF infants but at a non-significant level. EFF infants had no significant changes in CD8+ naive, ED and late differentiated (LD) memory populations over time. Additionally all infants demonstrated high levels of Ki67 expression at D4-7, which is prior to vaccination and maintained this level of proliferation after vaccination. HEU infants had higher levels of activation compared to HU infants in the first week of life but this normalised to HU infant levels by week 7. Furthermore EFF infants had peak T-cell activation at week 7 as compared to week 15 in EBF infants. In addition HU infants had better cytokine responses than HEU infants at week 7 but similar responses at week 15 and 36. In Addition, EFF infants also had increased vaccine specific CD4+ responses at week 7 and week 36 compared to EBF infants. This was true for overall cytokine expressing CD4 T-cells and single TNFα expressing CD4+ T-cells. Disscussion: Given the important role T-cells play in the clearance of Rotavirus, it is important that an assay capable of detecting RV vaccine specific T-cell responses be developed. Furthermore, T cells play a role in providing help for antibody responses to BP and for killing of intracellular bacteria. Our findings regarding immunity to aP suggest that all infants, regardless of HIV exposure status and feeding mode, are able to mount a T cell response to aP vaccination. However the differing ontogeny of responses seen in all three groups of infants lends some insight on the complex determinants of vaccine T -cell immunogenicity. In this case, age since vaccination, HIV exposure, and feeding mode resulted in apparent changes in vaccine responses as well as T cell differetiation and activation.
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    Characterization of a polypeptide factor that inhibits the growth of a human breast cancer line in vitro
    (1988) Harris, Neil S; Dowdle, Eugene B
    This thesis concerns a melanoma-derived growth regulatory factor that inhibited proliferation of several malignant human cell lines, and, in particular, a line designated UCT-BR-1, which was derived from a human breast cancer metastasis. The work is presented in four chapters. Chapter 1 provides a review of the relevant literature at the time of writing; Chapters 2 and 3 describe the experimental work that was done; and in Chapter 4 I discuss the implications of my results for current and future work in growth factors. Experimental results are presented as Charts (which may be Figures or Tables) and the methods and experimental protocols that I used are described in the Chart legends and not in the main text of the thesis. The Appendix contains details of the tissue culture techniques and descriptions of the cell lines that were used. Sources of the various laboratory materials as well as the methods that were employed for the more routine procedures are also described in the appendix.
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    Diamphotoxin: the arrow poison of the Kung bushmen
    (1983) DE LA HARPE, J; REICH, E; REICH, K; DOWDLE, E
    Purification de la diamphotoxine et étude de ses caractéristiques pharmacologiques et biochimiques.
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    The effects of fibroblast growth factor-2 om human bone marrow cells
    (2003) Hannocks, Melanie-Jane; Wilson, E Lynette
    Bibliography: leaves 163-191.
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    In vivo effects of South African traditional medicines against Mycobacterium tuberculosis in experimental mice
    (2001) Bapela, Nchinya Benedict; Ryffel, Bernhard; Smith, Peter J
    Although it is more than 100 years since Robert Koch discovered the tubercle bacillus, and more than 40 years since effective chemotherapy became available, the incidence of tuberculosis is increasing in much of the developing world and has recently re-emerged as a public health problem in industrialized countries. This problem is compounded by the increase in host susceptibility to tuberculosis caused by co-infection with HIV (Human Immunodeficiency Virus) and the emergence of Mycobacterium tuberculosis strains that are resistant to the front-line drugs. These factors highlight the urgent need for development of new drug classes to counter the threat posed by tuberculosis. The purpose of the present study was to develop a mouse model for Mycobacterium tuberculosis with the aim of determining the antimycobacterial activity of medicinal plants used by traditional doctors to treat tuberculosis in South Africa. Furthermore, the toxic effects of these medicinal plants in uninfected mice were determined. A field trip to the Northern Cape, Western Cape, Eastern Cape and Free State provinces was undertaken and medicinal plants used by traditional doctors to treat tuberculosis or its symptoms were collected, identified and examined for their therapeutic effects against Mycobacterium tuberculosis, determined using the mouse model. In addition, the effects of medicinal plants on the production of cytokines and granuloma formation in infected mice were examined. Six-to-ten week old C57BL/6 mice were infected with 107 viable Mycobacterium tuberculosis H37Rv strain by an aerosol exposure model. Bacterial growth was monitored by sacrificing infected but untreated mice at day 1, week 2 and week 4. Treatment with medicinal plant extracts was started 2 weeks after infection and continued for 2 weeks. An INH-RIF combination was used as positive controls. The bacterial load in infected but untreated mice increased by 1 log unit each week for 2 to 3 weeks. Bacterial loads were not detected in INH-RIF treated mice after 2 weeks of treatment. Treatment of mice with high doses of plant extracts was toxic. None of the tested medicinal plant extracts showed any activity against Mycobacterium tuberculosis. The production of IL-12 at week 4 was suppressed/ decreased when plant extract A was given at different concentrations. The bacterial loads in the lungs of the plant extract A treated mice was higher than that of the untreated mice (p < 0.005). Histological analysis of the lungs also revealed a high number of bacilli and increased size of the formed granuloma. In conclusion, the selected plant extracts obtained by water extraction exhibited no anti-tuberculosis activity in the laboratory mouse model for Mycobacterium tuberculosis infection. Furthermore, it was also shown that some plant extracts suppressed the production of IL-12, which plays an important role in the host's defense against Mycobacterium tuberculosis. However, further work is required to test if treatment for longer periods exhibits antituberculous activity.
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    The penetration protease of the cercariae of Schistosoma mansoni
    (1980) Heussen, Christa; Dowdle, Eugene B
    This thesis is concerned with a study of the proteases released by the cercariae of Schistosoma mansoni while penetrating mammalian skin. The proteases present in secretions collected from the preacetabular glands of cercariae were shown to be active against ¹²⁵I-labelled fibrin but not against undenatured ³H-collagen. A sensitive solid phase radioenzyme assay, with ¹²⁵I-fibrin as the substrate, was used to show that the cercarial protease could be totally inhibited by serine protease inhibitors such as diisopropylfluorophosphate or phenylmethylsulfonyl fluoride, but not by the sulfhydryl reagents iodoacetamide or p-chloromercuribenzoate. Typical trypsin inhibitors such as soy bean trypsin inhibitor, trasylol or benzamidine inhibited the enzyme to a lesser degree. The active-site labels, TLCK, TPCK and AcAAAACK of trypsin, chymotrypsin and elastase respectively had no effect. Calcium and magnesium stimulated protease activity at concentrations below 0,5 mM, but inhibited at higher concentrations, whereas EDTA had no effect. The pH optimum of the protease lay between pH 9,0 and 9,5. From these studies, I have concluded that the major cercarial penetration protease is an alkaline serine protease with trypsin-like specificity, but not acting via the same mechanism. A technique was developed for examining cercarial proteases in polyacrylamide gels containing SDS and copolymerized gelatin substrate. Bands of proteolytic activity could be detected by negative staining. This method was used to show that cercarial secretions contained one major protease with a molecular weight of 35 000 and that crude enzyme preparations are readily contaminated with bacterial proteases. Partial purification of the major cercarial protease was achieved by cation exchange chromatography.
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    The role of the cytolytic mediators, granulysin and perforin, in tuberculosis
    (2008) Semple, Patricia Lynn; Ress, Stanley; Shephard, Enid
    Protective immunity against mycobacterial infection requires an effective cytolytic response, in addition to an intact Type l (Th1) cytokine pathway. Natural killer (NK) cells and cytolytic T-cells (CTL) are essential components of protective immunity against tuberculosis (TB) and mediate granule-dependent killing of infected cells. Granulysin, an antimicrobial protein, and perforin, a pore-forming molecule, have been found to co-localise in the granules of these two cell types. Granulysin has been shown to be directly cytotoxic to extracellular Mycobacterium tuberculosis (M.tb) and, together with perforin, is cytolytic against intracellular mycobacteria. This project evaluated the role of these two cytolytic mediators in TB.
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    Vaginal microbial diversity of the genital tract of South African adolescent females
    (2017) Breetzke, Aerin Olivia; Passmore, Jo-Ann; Jaspan, Heather B; Lennard, Katie
    Young, reproductive-aged women are at highest risk of acquiring human-immunodeficiency virus (HIV). The Women's Initiative in Sexual Health (WISH) study was designed to investigate potential biological reasons for this high risk in HIV negative, South African adolescent females. Little is known about the 'normal' microbiome of this population. As such, the aim of this substudy was to quantify specific bacterial species (L. crispatus, L. jensenii, L. gasseri, L. iners, G. vaginalis and P. bivia) by quantitative real time PCR (qPCR) from adolescent female lateral vaginal wall swabs, and to assess associations between the quantities of these bacteria and bacterial vaginosis (BV) status, inflammation levels, age, hormonal contraceptive usage, and sexually transmitted infections (STIs). Samples were collected from 143 participant adolescent females in total, aged between 16 and 22 years of age, with a median of 18 years of age, from the Masiphumelele Youth Clinic in Cape Town, South Africa. Bacterial DNA was extracted from lateral vaginal wall swabs using the MoBio Powersoil® DNA Isolation Kit after enzymatic digestion. Positive bacterial reference strains were cultured in MRS buffer and Schwedler's broth, after which the DNA was extracted using the Qiagen Blood and Tissue DNA Maxi Extraction Kit. The quality and concentration of the DNA was confirmed using Qubit technology. The positive control DNA was amplified with PCR using species specific primers and the product run on an agarose gel to confirm primer specificity. The positive control DNA was serially diluted from 106 to 10-2 copies/μL to form a standard curve for absolute quantification through qPCR. Multiple steps were taken in order to optimize the qPCR experiments in terms of protocols, initial denaturation and annealing temperatures, cycle length and number, primers, and serial dilutions of the positive control DNA. The optimization for the P. bivia qPCR protocol presented the most issues, with the final quantification results being unreliable and requiring further work. Once the qPCR conditions were optimized for each bacterium; all samples, non-template control and standards were run in triplicate to quantify the number of bacterial copies per ng of DNA for each participant. The average of the three values were used as the final quantities and then used for downstream analyses. The bacterium L. crispatus, L. jensenii and L. gasseri, had median readings of 3.957 copies/ng, 1.568 copies/ng, and 17.58 copies/ng, respectively, with increased L. iners (2807 copies/ng) and G. vaginalis (8540 copies/ng). BV negative participants had increased levels of L. crispatus (p=0.0004, p=0.0002) and L. gasseri (p=0.0016, p<0.0001) in comparison to both BV intermediate and BV positive participants. L. jensenii (p<0.0001) and L. iners (p=0.0461) readings were increased in BV negative participants compared with BV positive and BV intermediate participants, respectively. BV positive participants had increased levels of G. vaginalis in comparison with both BV intermediate (p=0.0059) and BV negative (p<0.0001) adolescents. The 47 immunological factors, assessed via luminex, were categorized into high and low genital inflammation based on the unsupervised analysis by partitioning around medoids (PAM) using an R package 'cluster' with a k-value of 2. The inflammation-low group had increased levels of L. crispatus (p=0.0005), L. gasseri (p=0.033) and L. jensenii (p=0.0046) in comparison to the genital inflammation-high group. In participants with two viral STIs (Herpes Simplex Virus 2 and Human Papilloma Virus), there were increased copies/ng of G. vaginalis in comparison with participants with none (p=0.0098) or one viral STI (p=0.0324). Participants with high-risk HPV subtypes had significantly higher copy numbers of L. crispatus in comparison to the participants with low risk HPV subtypes (p=0.0181). Further, the only association demonstrated between the qPCR-based bacterial levels and the hormonal contraceptive prescribed was indicated by L. jensenii (ANOVA p=0.0222), possibly due to the low copy number readings. In conclusion, BV status, low levels of genital inflammation and the presence of two viral STIs indicate an association with bacterial copy numbers reported in this study, with increased median levels of L. iners and G. vaginalis across all adolescent participants compared to the other reported bacterial copy numbers. This indicates a possible alternate 'normal' microbiota profile of the FGT in adolescents in Masiphumelele.