RAG1-/- mutant zebrafish lack T and B lymphocytes. However, when re-exposed to homologous bacteria, these fish mount a response that provides specific protection. To further define this response, we utilized microarray analyses to determine the mechanisms underlying innate immune system memory in zebrafish. We also analyzed interferon (IFN) gamma by qRT-PCR. It is produced by activated NK cells and could indicate if this cell mediates the protective response seen in lymphocyte deficient zebrafish. Pathological studies and in situ hybridizations were performed to observe tissue changes and location of the cells that produced IFN gamma. Following bacterial re-exposure, zebrafish transcripts in cell receptor activation, cell proliferation and cytotoxic function categories were differentially expressed. We found high expression of IFN gamma in the lymphocyte like cell population after bacterial exposure and this was induced to a higher level in fish that had been vaccinated. The phagocytic cell population showed no induction of INF gamma. Over-all, the pathological response was much less severe in the vaccinated (48 hps) fish. Our microarray and pathological findings indicate that the primary immune response of mutant zebrafish is not impaired, and they demonstrate an enhanced innate immune response following secondary bacteria exposure. Following homologous secondary exposure, mutant zebrafish have a cell population that is undergoing upregulated cell receptor activation, cell cytotoxic functions and cell proliferation. This cell population expresses INF gamma. Activated T cells, NK-T cells and NK cells express INF gamma. Since RAG1 deficient zebrafish do not have T or NK-T cells, this cell population is most likely NK cells.

No description available.

The Human immunodeficiency virus (HIV) pandemic has resulted in a heavy global disease burden, and clinically causes Acquired Immuno-Deficiency Syndrome (AIDS). The development of highly active antiretroviral therapy (HAART) has achieved remarkable control of the rapidly evolving HIV. However, HIV remains neither curable nor preventable by vaccine, and in the developing regions worst affected by HIV, HAART remains inaccessible to most patients. Furthermore, the change in both immunology and viral evolution during chronic HIV infection and its relation to AIDS pathogenesis remains unknown. Following the failure of recent HIV vaccines, it is believed that a better understanding of host-pathogen interaction is vital to advance therapeutic (vaccine and drug) design. In this thesis, I have performed an investigation of viral adaptation in response to different selection forces during advanced HIV infection and AIDS. The thesis first examined a case study that reveals the potential role of B cell-mediated neutralising antibody (NAb) in chronic HIV infection through the unexpected effect of B cell depletion agent, anti-CD20 (Rituximab). Here, longitudinal results have shown that viral load (VL), env gene diversity, and NAb sensitive strains increased during B cell and NAb depletion as a result of Rituximab administration, and reversed as B cells recovered. The study provides preliminary evidence to support the idea that NAb may be effective at suppressing HIV. The rest of the thesis focused on the cross-sectional cohort at Bloemfontein, South Africa (n=1491), a resource-limited region affected by the pandemic. Here, we used methods that include molecular and pretherapy drug resistance epidemiology, mathematical modelling, phylogenetically adjusted bioinformatics analysis and in vitro viral replication capacity (VRC) assay to study materials including cohort demography, plasma samples, CD4 cell count, VL, viral genetic sequences and host human leukocyte antigen (HLA) tissue types. Our analysis was further augmented by the additional data kindly contributed by our neighbouring Durban cohort collaborators (n=775), which also includes an IFN! ELISPOT assay that measures cytotoxic T lymphocyte (CTL) responses. Using the HIV pol sequencing data and phylogenetic analysis we confirmed that the local molecular epidemiology is similar to the circulating strains documented in the regional database. However, the pretherapy drug resistance mutation screening results have revealed an unexpected high incidence of drug-induced viral mutants in the AIDS patients with CD4 counts <100 cells/μl. According to mathematical modelling, this finding is attributable to additional sources of antiretroviral therapy exposure, which warrants public health caution. The investigation then focused on studying the changes in HLA class I mediated CTL selection and viral evolution as CD4 counts are reduced in AIDS. Interestingly we have noted evidence that suggest weakening CTL immune selection against gag during AIDS is associated with increased viral fitness (measured by VRC) and reversion of previous immune-escape mutations which conferred high fitness costs. In conclusion, this thesis compared different sources of host and drug mediated HIV selection and its implication for viral evolution. The identification of more bottleneck sites conferring high fitness costs to the selection of escape mutants is expected to be helpful in the design of future therapeutics (via vaccine, drug, immune therapy, or public health strategy). As we have learnt from the principle of combinational ARV, it would be desirable for a vaccine to select HIV at multiple sites of high escape-mutation fitness cost, hence offering protective effect.

Thesis (MScMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Background: In South Africa alone, 30% of women of child-bearing age are infected with HIV. With the increasing focus and success of prevention of mother-to-child transmission (PMTCT) programmes, an estimated 300 000 infants are born exposed to HIV every year. The underlying impact of in utero HIV exposure on infant immune health has not been extensively characterised. Clinical follow-up of these HIV-exposed uninfected (HEU) infants reveals increased infectious morbidity and mortality compared to their unexposed (UE) counterparts. Objectives: (i) To evaluate and characterise adaptive immune properties by measuring vaccine-specific antibody levels in children from 2 weeks to 2 years of age in the presence and absence of maternal HIV infection. (ii) To investigate specific cellular markers of immune activation, immune regulation, apoptosis and B cell memory on T and B cell populations in HEU and UE children measured at 18 and 24 months of age. Methods: This sub-investigation formed part of a collaborative pilot study between the universities of British Columbia (Vancouver, Canada) and Stellenbosch. A total of 95 HIV-positive and HIV-negative mothers were recruited after delivery at Tygerberg Hospital, and signed informed consent for their infants to be included in the study. Of these infants, only 27 HEU and 30 UE infants were eventually enrolled and followed up at various time points, starting at two weeks of age. Four of these infants were confirmed to be HIV-positive at 2 weeks and clinically followed up according to the protocol, but were excluded from statistical data analyses. Blood was collected at 2, 6 and 12 weeks and again at 6, 12, 18 and 24 months of age. Quantitative IgG-specific antibodies to Haemophilus influenzae B (Hib), Bordetella pertussis, tetanus and pneumococcus were measured at each time point, using commercially available ELISA (Enzyme-Linked ImmunoSorbent) kits. Cellular markers of immune activation, immune regulation, apoptosis and memory were measured in various populations of T and B cells at 18 and 24 months only, by using four-colour flow cytometry and validated whole-blood staining methods. In addition, a functional assay was developed to evaluate cell susceptibility to apoptosis (spontaneously) by measuring the expression of Annexin V on both CD4+ T and CD20+ B cells after 16 and 24-hour incubation periods. The statistical analysis of the antibody data was conducted by repeated-measures ANOVA (i.e. analysis of variance), using a mixed-model approach. Differences in the expression of the two groups’ cellular markers were compared by employing one-way ANOVA. An F test p value (which assumes normality) was reported, while the non-parametric Mann-Whitney U test served as confirmatory tool. Repeated-measures ANOVA was used for the evaluation of the functional spontaneous apoptosis assay at three time points (ex vivo, 16 and 24 hours) on the 18-month samples, while one-way ANOVA was used for the 24-month samples. Results: The HEU group (n = 23) displayed significantly lower levels of antibodies to pertussis (20.80 vs 28.01 Food and Drug Administration [FDA] U/ml; p = 0.0237), tetanus (0.08 vs 0.53 IU/ml; p < 0.001) and pneumococcus (31.67 vs 80.77 mg/l; p = 0.003) than the UE group (n = 23) at 2 weeks of age. No statistical differences were noted for Hib antibody levels between the two groups at this time point. At 6 weeks of age, HEU infants displayed lower mean levels of all antibodies measured; however, these differences did not reach statistical significance. Following vaccination, compared to UE controls, the HEU group presented with statistically significantly higher antibody levels to pertussis at 6 months (155.49 vs 63.729 FDA U/ml; p = 0.0013), 12 months (26.54 vs 8.50 FDA U/ml; p < 0.001) and 18 months of age (1658.94 vs 793.03 FDA U/ml; p = 0.0362). A significant difference in tetanus antibody levels between the two groups was only evident at 24 months, with the HEU group displaying higher levels (3.28 vs 1.70 IU/ml; p = 0.018) than the UE group. No differences were observed between the two groups following vaccination for Hib. At 18 and 24 months, the HEU group showed increased expression of cellular markers of immune activation (CD69 and CD40L) on CD4+ T cells compared to UE controls. The two groups showed similar expression of the cellular marker of activation CD38 on CD8+ T cells. The HEU group displayed significantly higher levels of CD127, the interleukin (IL) 7 receptor, on CD4+ T cells compared to UE controls at 18 months of age. The HEU group also showed increased expression of cellular markers of apoptosis on both CD4+ T and CD8+ T cells. No statistical significance was noted for the expression of Fas on CD4+ T cells at 18 and 24 months of age. However, at 24 months, the HEU group showed significantly increased expression of FasL on both CD4+ T and CD8+ T cells. During cell culture experiments, the HEU group displayed increased susceptibility to spontaneous apoptosis shown by increased Annexin V expression on CD4+ T cells after a 16-hour incubation period at both 18 and 24 months. At 18 and 24 months, no difference was noted in the two groups’ immune regulation as measured by the expression of CTLA-4. The HEU group displayed increased levels of the cellular markers of immune activation CD80 on CD20+ B cells at 18 and 24 months of age. The HEU group also showed significantly increased levels of CD69 on CD19+ B cells at 24 months. No statistical significance was reached for the expression of CD62L and CD10 at either 18 or 24 months. Although the HEU group displayed increased levels of apoptosis (Fas) on CD20+ B cells, no statistical significance was reached at 18 or 24 months of age. In addition, the HEU group showed no difference in the expression of programmed death 1 (PD-1) at 18 and 24 months. HEU and UE groups showed similar expression of Annexin V after 16 hours of incubation in the 18 and 24-month samples. The expression of the biomarker of B cell memory CD27 on CD20+ B and CD19+ B cells was comparable between the two groups at both time points. Conclusion: At 2 and 6 weeks, lower mean antibody responses in HEU infants suggest poor placental transfer due to maternal HIV infection, while increased responses to specific antibodies may reflect an exaggerated immune response to immunisation. These robust responses may be due to the lack of competition with maternal antibodies, or may be ascribed to indirect stimulation of B cells via the activation of T cells. A hyper-inflammatory state is an imminent danger, with increased expression of cellular markers of immune activation and apoptosis that may be consistent with early HIV exposure that persists following infancy. These observations may serve as contributing factors to the extensively documented increased susceptibility to infections in the HEU population. Although these findings are consistent with a primed immune system, larger studies are required to confirm these observations in relation to clinical outcomes and to assess further whether these differences persist in later years. / AFRIKAANSE OPSMOMMING: Agtergrond: In Suid-Afrika alleen het 30% van vroue van ʼn vrugbare leeftyd MIV. Met die toenemende fokus en sukses van programme vir die voorkoming van moeder-na-kind-oordrag (sogenaamde PMTCT-programme) word ongeveer 300 000 babas jaarliks aan MIV blootgestel. Die onderliggende impak van intra-uteriene MIV-blootstelling op ʼn baba se immuunstelsel is nog nie omvattend beskryf nie. Kliniese opvolgondersoeke van hierdie MIV-blootgestelde dog onbesmette babas (sogenaamde HEU’s) dui op ʼn hoër siekte- en sterftesyfer weens infeksies as hul nieblootgestelde eweknieë (sogenaamde UE’s). Doelstellings: (i) Om kinders met MIV-positiewe en MIV-negatiewe moeders se aangepaste (verworwe) immuuneienskappe te beoordeel en te beskryf deur hulle vaksienspesifieke teenliggaamvlakke vanaf die ouderdom van twee weke tot twee jaar te meet. (ii) Om ondersoek in te stel na bepaalde sellulêre merkers van immuunaktivering, immuunregulering, apoptose en B-selgeheue by die T- en B-selgroepe van sowel HEU’s as UE’s op die ouderdom van 18 en 24 maande. Metodes: Hierdie subondersoek het deel uitgemaak van ʼn samewerkende loodsondersoek tussen die universiteite van Brits-Columbië (Vancouver, Kanada) en Stellenbosch. Altesaam 95 MIV-positiewe en MIV-negatiewe moeders is gewerf nadat hulle by Tygerberghospitaal geboorte geskenk het, en het ingeligte toestemming verleen dat hul babas by die studie ingesluit kon word. Van dié babas is slegs 27 HEU’s en 30 UE’s uiteindelik in die studie opgeneem en in verskillende stadia vanaf die ouderdom van twee weke opgevolg. Vier van die babas is op twee weke as MIV-positief bevestig en volgens die protokol klinies opgevolg, maar is van die statistiese dataontleding uitgesluit. Bloedmonsters is op twee, ses en 12 weke en weer op ses, 12, 18 en 24 maande geneem. Kwantitatiewe IgG-spesifieke teenliggame teen Haemophilus influenzae B (Hib), Bordetella pertussis, tetanus en pneumokokkus is telkens met behulp van kommersieel verkrygbare ELISA- (“Enzyme-Linked ImmunoSorbent”-)stelle bepaal. Sellulêre merkers van immuunaktivering, immuunregulering, apoptose en geheue is op slegs 18 en 24 maande by verskillende populasies T- en B-selle deur middel van ʼn vierkleurvloeisitometrie en geldig verklaarde volbloedkleuringsmetodes bepaal. Voorts is ʼn funksionele toets ontwikkel om selvatbaarheid vir apoptose te bepaal deur die ekspressie van Annexin V op sowel CD4+ T- as CD20+ B-selle ná 16 en 24 uur van inkubasie te meet. Die statistiese ontleding van die teenliggaamdata is met behulp van herhaaldemetings-ANOVA (d.w.s. afwykingsontleding) volgens ʼn gemengdemodel-benadering gedoen. Verskille in die twee groepe se sellulêre merkervlakke is deur middel van eenrigting-ANOVA vergelyk. ʼn F-toets-p-waarde (wat normaliteit veronderstel) is bereken, terwyl die nieparametriese Mann-Whitney-U-toets as bevestigende instrument gedien het. Vir die 18 maande-monsters is herhaaldemetings-ANOVA gebruik om die funksionele toets vir spontane apoptose in drie stadia (ex vivo, op 16 uur en op 24 uur) te beoordeel. Vir die 24 maande-monsters is eenrigting-ANOVA gebruik. Resultate: Op die ouderdom van twee weke het die groep HEU’s (n = 23) aansienlik laer teenliggaamvlakke teen kinkhoes (20.80 vs 28.01 Food and Drug Administration [FDA] U/ml; p = 0.0237), tetanus (0.08 vs 0.53 U/ml; p < 0.001) en pneumokokkus (31.67 vs 80.77 mg/l, p = 0.003) as die UE-groep (n = 23) getoon. In dié stadium is geen statistiese verskille in Hib-teenliggaamvlakke tussen die twee groepe opgemerk nie. Op ses weke het die groep HEU’s laer gemiddelde vlakke van ál die betrokke teenliggame getoon, hoewel hierdie verskille nie statisties beduidend was nie. In vergelyking met die UE-kontrolegroep het die groep HEU’s ná inenting statisties beduidend hoër teenliggaamvlakke teen kinkhoes getoon op ses maande (155.49 vs 63.729 FDA U/ml; p = 0.0013), 12 maande (26.54 vs 8.50 FDA U/ml; p < 0.001) én 18 maande (1658.94 vs 793.03 FDA U/ml; p = 0.0362). ʼn Beduidende verskil in die twee groepe se tetanus-teenliggaamvlakke het eers op 24 maande geblyk, met die groep HEU’s s’n hoër (3.28 vs 1.70 IE/ml; p = 0.018) as die UE’s s’n. Ná inenting teen Hib is geen verskille tussen die twee groepe waargeneem nie. Op 18 en 24 maande het die HEU’s verhoogde ekspressie van sellulêre merkers van immuunaktivering (CD69 en CD40L) op CD4+ T-selle getoon in vergelyking met die UE-kontrolegroep. Soortgelyke vlakke van die sellulêre merker van aktivering CD38 is ook op die CD8+ T-selle van die twee groepe opgemerk. Op 18 maande het die HEU-groep ʼn beduidend verhoogde ekspressie van CD127, die IL-7-reseptor, op CD4+ T-selle getoon in vergelyking met die UE-kontrolegroep. Die HEU groep het ook verhoogde ekspressie van sellulêre merkers van apoptose op sowel CD4+ T- as CD8+ T-selle getoon. FAS-ekspressie op CD4+ T-selle op 18 en 24 maande was nie statisties beduidend nie, hoewel die HEU-groep op 24 maande beduidend verhoogde ekspressie van FasL op CD4+ T- sowel as CD8+ T-selle getoon het. In selkwekingseksperimente het die HEU-groep ʼn verhoogde vatbaarheid vir apoptose getoon na aanleiding van die ekspressie van Annexin V op CD4+ T-selle ná 16 uur van inkubasie op sowel 18 as 24 maande. Op 18 en 24 maande was immuunregulering, aan die hand van die ekspressie van CTLA-4, bykans dieselfde by albei groepe. Op sowel 18 as 24 maande toon die HEU’s verhoogde ekspressie van die sellulêre merker van immuunaktivering CD80 op CD20+ B-selle. Op 24 maande het die HEU’s ook aansienlik hoër vlakke van CD69 by CD19+ B selle getoon. Op nóg 18 nóg 24 maande was die ekspressie van CD62L en CD10 statisties beduidend. Hoewel verhoogde vlakke van apoptose (Fas) by CD20+ B-selle by die HEU-groep opgemerk is, was dit nie statisties beduidend op 18 óf 24 maande nie. Daarbenewens was daar ook geen verskil in die ekspressie van geprogrammeerde seldood 1 (PD-1) op 18 en 24 maande nie. Op 18 en 24 maande het die HEU’s en UE’s ʼn soortgelyke ekspressie van Annexin V ná 16 uur van inkubasie getoon. Op sowel 18 as 24 maande was die twee groepe se ekspressie van die biomerker van B-selgeheue CD27 op CD20+ B- en CD19+ B-selle vergelykbaar. Gevolgtrekking: Op twee en ses weke dui laer gemiddelde teenliggaamreaksies by HEU’s op swak plasentale oordrag weens die moeder se MIV-infeksie, terwyl verhoogde reaksies op bepaalde teenliggame weer op oordrewe immuunreaksie op inenting dui. Hierdie robuuste reaksie kan toegeskryf word aan die gebrek aan mededinging met die moeder se teenliggame, of kan deur indirekte stimulasie van die B-selle via die aktivering van die T-selle veroorsaak word. ʼn Hiperinflammatoriese toestand is ʼn dreigende gevaar, met verhoogde ekspressie van sellulêre merkers van immuunaktivering en apoptose wat met vroeë MIV-blootstelling met ʼn latere nawerking verbind kan word. Hierdie waarnemings kan bydraende faktore wees tot HEU’s se goed gedokumenteerde verhoogde vatbaarheid vir infeksies. Hoewel hierdie bevindings met ʼn geaktiveerde immuunstelsel strook, moet groter studies dit aan die hand van kliniese uitkomste bevestig en ook bepaal of hierdie verskille in later jare voortduur. / The Harry Crossley Foundation, Poliomyelitis Research Foundation (PRF) / NHLS Research Grant Trust

Keloid disease (KD) is a benign fibroproliferative dermal disease of unknown aetiopathogenesis that occurs in genetically susceptible individuals. KD shows high heterogeneity within the lesion, harbouring different immune cell profiles, which are poorly characterised in KD at different lesional sites. Although, it has long been appreciated that chronic inflammation and dermal fibrosis is associated with other fibrotic diseases (e.g. scleroderma), this link has not, yet, been established in KD through direct evidence. Additionally, the limited availability of a simple KD animal model has hindered our understanding of the underlying pathogenesis of KD. Therefore, the main objectives were a) to identify and profile different immune cells at defined KD lesional and histological sites, b) to further characterize the potential contribution of viral particles in KD by investigating the gene and protein expression profile of toll like receptors that recognise viral particles in KD, and c) to develop an optimized long-term serum-free organ culture (OC) model for KD research as a tool for probing novel hypotheses in KD pathobiology deduced from a) and b) and to also validate the reliability and instructiveness of this novel ex vivo KD model with conventional (e.g. dexamethasone) and potential future anti-KD compounds [(-)-epigallocatechin-3-gallate (EGCG) and plasminogen activator inhibitor-1 (PAI-1) knock-down by siRNA]. To achieve above objectives, different cellular and molecular techniques were applied. Immune profiling of KD (chapter 2) at defined lesional and histological sites generated the first comprehensive analysis of KD-associated inflammatory cell infiltrates. This work demonstrated for the first time the presence of specific type of chronic inflammation in KD that resembles the formation of tertiary lymphoid tissues (TLTs) (in 14.7%, out of 68 KD cases). Although, these TLTs are not strictly linked to defined lesional sites within the KD, they are similar in structure to mucosa-associated lymphoid tissue (MALT). Therefore, we named this phenomenon as keloid-associated lymphoid tissue (KALT). Immunophenotyping of KD lesional sites also showed a predominance of T-cells, B-cells, M2 macrophages and OX40L+ degranulated mast cells in intralesional and perilesional sites of KD compared to normal skin and normal scar tissue. In the epidermis, Langerhans cells showed no changes, whereas the intra-epidermal T-cells were significantly increased in both the intralesional and perilesional sites of KD with an increased CD4:CD8 ratio. Intra-epidermal B-cells were only rarely found in KD. Interestingly, there was no significant statistical difference between intralesional and perilesional sites of KD immunophenotyping. These abnormal immune profiles suggest the persistence of non-resolving inflammation presence towards unknown stimuli, which require further investigation. The chronic inflammation could be followed by a reparative phase in a repetitive manner leading to KD formation. Evaluation of toll-like receptor (TLR) gene and protein expression in KD showed a significant increase in the expression of intra-epidermal TLR-6, -7 and dermal TLR-8. Since these TLRs are typically up regulated during anti-viral responses, these results further support the hypothesis that certain viruses or yet unidentified ligand may play a role in KD pathogenesis (chapter 3). A successful long-term, serum-free keloid OC model was established using a 4 mm sized punch biopsy embedded in collage matrix as air liquid interface in supplemented William’s E medium for up to 6 weeks (Chapter 4).