Background. Rheumatoid Arthritis (RA) is an autoimmune inflammatory disease that, without powerful treatment, may lead to irreversible joint damage. During the past decade, anti-cytokine therapy has become available, e.g., infliximab, a chimeric antibody targeting the pro-inflammatory cytokine TNF that has a central role in the inflammatory process in RA patients. Systemic Lupus Erythematosus (SLE) is a systemic autoimmune disease that may affect all organs and is characterized by a massive antibody production. Chemokines, chemokine receptors and lipoprotein receptor-related protein 1(CD91) are regulators of inflammation in autoimmune diseases and T-cell migration. Objectives. The aim of this study was to get a deeper understanding how TNF blocking treatment influences inflammatory mechanisms and autoantibody formation in RA with special reference to similarities and differences with SLE. Methods. In patients with RA treated with anti-TNF, and in SLE patients (ACR criteria) clinical evaluation was performed and blood samples analyzed. Autoantibodies were analyzed using indirect immunofluorescence, ELISA and multiplex flow cytometry in samples from anti-TNF treated RA patients (n=59) followed longitudinally for 54 weeks, in pre-diseased samples from SLE patients (n=38) and matched population-based controls (n=152). T-cell expression of chemokine receptors and CD91 was analyzed by flow cytometry, whilst serum levels of chemokines were determined using ELISA in anti-TNF treated RA-patients (n=24) followed longitudinally (30 weeks), and cross-sectionally in SLE-patients (n=23). Expression of mRNA for chemokines was analyzed in T-cells from SLE-patients (n=10) using PCR. Results. After treatment with infliximab, RA patients produced ANA, anti-dsDNA and anti-nucleosome antibodies, but not anti-ENA antibodies. Although these antibodies are considered typical for SLE only one patient developed a transient lupus-syndrome. Antibodies against cell nuclear antigens, including ENA, were detected several years before the first clinical symptom of SLE; anti-SSA was the earliest detectable antibody. In RA-patients before infliximab treatment, the T-cell expression of several chemokine receptors was elevated compared with healthy controls. In contrast, only one soluble chemokine, IP-10 was elevated. After treatment the levels of soluble MIP-1β, MCP-1 and IP-10, and the T-cell expression of CCR2 were decreased. In SLE-patients MIP-1β, MCP-1, SDF-1, IP-10 and RANTES in blood were elevated, whilst expression of CXCR5 and CCR6 on T-cells was lower than in healthy controls. T-cell expression of CXCR2 and CCR1 was elevated in active disease (measured as SLEDAI index), whereas the CXCR5 and CCR2 expression was lower in inactive SLE. In SLE patients with nephritis IP-10 was lower and T-cell expression of CXCR3 and CCR3 elevated compared with patients without nephritis. The expression of CD91 was higher on T-cells from patients not responsive to infliximab treatment compared with responders. Conclusion. These findings indicate that anti-TNF (infliximab) treatment in RA-patients has a major impact on the production of autoantibodies and chemokines. The autoantibody profile in infliximab-treated patients was similar to that predating disease onset in SLE patients with the exception of anti-ENA being detectable in SLE, but the development of lupus-syndromes was rare. The expression of CD91 on T-cells may predict responsiveness to infliximab. The expression of chemokine receptors in SLE- patients seemed to be related to disease activity. Anti-nuclear antibodies were detectable years before clinical disease onset in patients who developed SLE suggesting a gradual pathogenic process.

Certain HLA-B-restricted CD8+ T cell responses are associated with control of viremia whereas HLA-Cw* restricted responses, including Gag epitopes are associated with high viremia. To better understand the role of HLA-Cw* restricted epitopes in viral control, HLA-Cw* restricted epitopes were optimally defined. Seventy eight study subjects from a cohort of 451 chronically infected participants had HLA-Cw* restricted CD8+ T cells responses as quantified by intracellular cytokine staining assessing IFN-γ secretion. Fine mapping and HLA restriction of the optimally defined HLA-Cw* restricted epitopes were performed using ELISPOT assay. Functional avidity of responses was assessed by peptide dilution in an ELISPOT assay. Two novel HLA-Cw* restricted epitopes Cw*04- TF10 (in reverse transcriptase) and Cw*08-RM9 (in gp120) were optimally defined. A previously described epitope, Cw*07- KY11 (Nef) was the most frequently targeted epitope in this cohort (30/78) and has high functional avidity compared to other HLA-Cw restricted CD8+ T cell responses. The polyfunctionality of HLA-B*57/5801-restricted Gag-specific HIV-1 CD8+ T cell responses and HLA-Cw*07-KY11 restricted CD8+ T cell responses within the same study subject was determined. Polyfunctionality of CD8+ T cell responses to HLAB* 57/5801 and HLA-Cw*07 restricted epitopes were determined in nine study subjects assessing IFN-γ, TNF-α, IL-2, MIP-1β, and CD107a by multicolour flow cytometry. Additionally gag and nef genes were sequenced from plasma. HLA-B*57/5801-restricted IFN-γ-producing CD8+ T cell responses were of lower magnitude than HLA-Cw*07 responses (p=0.0012) for the nine subjects. The majority of responses were monofunctional (75%), irrespective of HLA restriction. HLA-B*57/5801 and HLACw* 07 restricted CD8+ T cells did not differ significantly in polyfunctionality (p=0.84). Possession of ≥3 functions correlated positively with CD4+ T cell counts (r=0.85; p=0.006). The percentages of monofunctional CD8+ T cells inversely correlated with CD4+ T cell counts (r=-0.79; p=0.05). There was no correlation between polyfunctionality and viral load and sequence variation within targeted epitopes did not impact polyfunctionality. These results suggest that polyfunctionality of HIV-1-specific CD8+ T cells is associated with disease progression independent of restricting HLA alleles, and that loss of these polyfunctional cells correlates with increased in the frequency of monofunctional virus-specific CD8+ T cells. In addition, sequence variation does not appear to significantly impact CD8+ T cell polyfunctionality in chronic HIV infection. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2010.

1,25-dihydroxyvitamin D3 (Vitamin D) is a potent immunomodulator capable of generating regulatory T cells (Tregs) and contributing to immune tolerance. Additionally, vitamin D has been shown to promote mucosal immunity when used as a vaccine adjuvant. We show here that pretreatment of an area of skin with the synthetic vitamin D analog calcipotriol combined with transcutaneous immunization results in the induction of CD4⁺CD25⁺ Tregs capable of inhibiting the elicitation of a contact hypersensitivity response. We also demonstrate that topical calcipotriol has significant effects on the immune response to subcutaneously injected vaccines, and compare it with another common topical immunosuppressant, the corticosteroid betamethasone-17-valerate (BMV). Functionally, calcipotriol and BMV treatment both result in the suppression of CD8⁺ T cell priming in response to subcutaneous vaccination, despite the topical co-administration of the potent Th1 inducing TLR9 agonist unmethylated CpG DNA. The effects of calcipotriol on the humoral response are subtler as we observe marginally increased production of antigen-specific IgG1 immunoglobulins along with a strong suppression of the IgG2a isotype. This is in contrast to pretreatment with BMV, which instead suppresses the production of IgG1 and IgA antibodies. In the draining lymph nodes of calcipotriol treated animals, we see no change in the percentage of Foxp3⁺ CD4⁺ T cells post-immunization, but show that tolerance is transferable with the adoptive transfer of CD4⁺CD25⁺ cells. Despite a decrease in the percentage of antigen-bearing APCs in the DLN of calcipotriol treated animals, the DCs maintain high expression of co-stimulatory markers and can induce CD4⁺ T cell proliferation ex vivo. Our data indicate that calcipotriol has distinct effects on immune responses to subcutaneous vaccines consistent with its role as an immunomodulator, although the mechanism(s) through which it is acting remain unclear. We believe that further research is warranted into its potential use as part of a treatment modality for allergy and autoimmune disorders.

No description available.

Human Immunodeficiency Virus (HIV) infection is characterized by immune dysfunction that predisposes infected individuals to opportunistic infections such as Mycobacterium Tuberculosis (MTB). The result of this is an exacerbation of HIV-TB related deaths annually. Therefore there is an imperative need for HIV-TB focused research that aims to identify immunological factors that are involved in the control of MTB and HIV in both mono- and co-infected individuals. The CD161+ CD4+ T cell subset is linked to a distinct phenotypic and functional profile. Importantly, these CD161+ T cells may act as an important component of immunological defense and provide protection in infected tissues. CD161+ CD4+ T cells have also been identified as the precursor population of Th17 cells and it has been previously reported that reduction of CD161+ CD4+ T cells during HIV infection may limit Th17 reconstitution (Prendergast et al., 2010). This may ultimately contribute to impairment of mucosal immunity leading to the acquisition of opportunistic infections such as MTB and disease progression in HIV infected individuals. Our study aimed to comprehensively characterise the impact of HIV and MTB infection on the CD161+ CD4+ T cell subset and to assess the frequency, phenotype and function of these cells. The study also aimed to correlate the longitudinal variation in frequency, phenotype and function with markers of HIV disease progression. Methods The frequency, phenotype and function of the CD161+ CD4+ T cell subset was measured by flow cytometry. For the frequency and phenotypic assessment, whole blood was collected from HIV negative and HIV/MTB mono and co-infected subjects (n = 17 per patient group). Whole blood was surface stained with antibodies specific to CD3, CD4, CD8, CD161 and chemokine receptors CD103, CCR6, CXCR4, CCR5 and CXCR6. The percentage positive expression of CD161 on CD4+ T cells and chemokine receptor expression was measured. The functional assessment of CD161+ CD4+ T cells involved PBMC stimulation with antigenic stimulant, phorbol 12-myristate 13-acetate (PMA) and ionomycin or ESAT-6/CFP-10, GAG, TB10.4 and Ag85a followed by intracellular cytokine staining for IFN-γ, IL-17A, IL-22 and TNF-α. A subgroup of HIV negative (frequency and phenotype, n = 10, function n = 7) and HIV mono-infected subjects (frequency and phenotype, n = 10, function n = 7) were longitudinally followed to assess variations in the frequency, phenotype and function of CD161+ CD4+ T cells over time. Results The CD161+ CD4+ T cell subset demonstrated high-level expression of chemokine receptors CCR5, CCR6, CXCR4 and low-level expression of CD103 and CXCR6. The subset also demonstrated the ability to produce cytokines IFN-γ, IL17A, IL-22 and TNF-α in healthy subjects. Analysis of HIV infected samples revealed a significant reduction in the frequency of the CD161+ CD4+ subset (median = 06.86%, p < 0.0001) compared to that of healthy individuals (median = 14.75%). Correlation of the subset frequency to markers of disease progression revealed a positive trend to CD4 count (r = 0.2590, p = 0.0787) and a significant negative correlation to viral load (r = -0.3522, p = 0.0152). Unlike with HIV infection, no significant changes in CD161+ CD4+ T cell frequency was observed in individuals with LTBI (mono- or HIV co-infected) or active TB disease compared to that of the healthy patient group. However, the exception to this was HIV infected individuals with active TB disease (co-infected) (median = 03.80%, p < 0.0001). Decreased CCR6 expression on CD161+ CD4+ T cells was observed in HIV monoinfected (p = 0.0065) and HIV infected individuals with active TB disease (p = 0.007). No functional changes were observed in both the HIV and MTB mono- and co-infected cohorts following non-specific stimulation. An interesting positive trend in correlation between IFN-γ production and CD4 count (r = 0.2727, p = 0.0733) was demonstrated with a significant negative correlation between IFN-γ production and viral load observed following non-specific antigenic stimulation (r = -0.3705, p = 0.0133). CD161+ CD4+ T cells demonstrated antigen-specific T cell responses to peptides ESAT-6/CFP-10, TB10.4, Ag85a and GAG in a small proportion of 69 study participants with variable ranges in magnitude of the responses observed. The longitudinal assessment of CD161+ CD4+ T cell frequency and phenotype demonstrated low-level proportion of CD4+ T cells expressing CD161 and CCR6 expression longitudinally maintained in HIV mono-infected compared to that of healthy individuals. Conclusion The phenotypic and functional profile of the CD161+ CD4+ T cell population indicates that it may be an important component of immunological defense that may provide mucosal defense and protection at epithelial sites and tissues e.g. expression of tissue homing markers like CCR6 and the production of cytokines such as IL-17A and IL-22 (important in mucosal immunity). HIV infection is associated with a reduced frequency of CD161+ CD4+ T cells. The correlation between CD161+ CD4+ T cell frequency and markers of disease progression suggests that the observed low-level frequency in HIV infected individuals may in part be a result of non-specific HIV-mediated depletion of CD4+ T cells. However, lower levels of CD161+ CD4+ T cells in HIV infected individuals could also be a result of naturally lower levels being present in individuals prior to infection, thereby making these individuals more susceptible to HIV infection. The significantly reduced levels of CCR6 expression on CD161+ CD4+ T cells in HIV monoinfected individuals may also be an indication of cell subset migration to gut associated lymphoid tissue (GALT, target site of HIV replication) during HIV infection. Given their potential role in mediating signals that are essential for immune responses to microbes and microbial products, migration of CCR6+ CD161+ CD4+ T cells to target sites of HIV infection could serve as a protective measure in the fight against HIV infection. Although there were no observable changes in the functional capacity of the CD161+ CD4+ T subset in HIV infection, we believe that the reduction in frequency may contribute to HIV disease progression and susceptibility to opportunistic infections such as MTB or active TB disease. Unlike with HIV infection, infection with MTB appeared to have no significant impact on CD161+ CD4+ T cells as there were no observable differences in frequency or the functional capacity of the cell subset following PMA stimulation. However, MTB and HIV antigen-specific responses were observed in a small proportion of the total 69 subjects tested. This therefore indicates that a subset of CD161+ CD4+ T cells may act in an HIV and MTB-specific manner. Additional MTB and HIV-specific responses may be present in this CD161+ CD4+ population and may only be identified through stimulation with additional antigenic targets. Further investigation of CD161+ CD4+ T cells should be performed at the actual sites of infection to investigate if CD161+ CD4+ T cells are concentrated at sites of disease. Also it may be important to investigate the polyfunctionality of CD161+ CD4+ T cells to understand the multifunctional capacity of the cell subset in providing immunological defense to pathogens such as HIV and MTB. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2012.

Chronic myelogenous leukemia (CML) is a clonal hematopoetic stem cell disorder characterized by proliferation of cells expressing BCR-ABL fusion protein. In the BCR-ABL+ leukemia murine model, 12B1, we explored the therapeutic applicability of chaperone-rich cell lysate (CRCL) in the context of syngeneic hematopoietic cell transplantation (HCT) to treat pre-existing leukemia. Our results demonstrate that tumor growth is significantly delayed in mice receiving syngeneic HCT from 12B1 tumor CRCL immunized donors compared to animals receiving HCT from non-immunized donors. CRCL immunization post-immune HCT further hindered tumor growth when compared to immune HCT without post-transplant vaccination. The magnitude of the immune response was consistent with the anti-tumor effects observed in vivo. We also demonstrated that cured mice had developed long-term tumor specific immunity against 12B1 tumor cells. In addition, we documented that both T cells and NK cells contributed to the anti-tumor effect of CRCL vaccination as depletion of either subset hampered tumor growth delay. Thus, our results suggest that CRCL represents a promising vaccine capable of generating specific immune responses. This anti-tumor immunity can be effectively transferred to a host via HCT and further enhanced post-HCT with additional tumor CRCL immunizations.CD4+CD25+ regulatory T cells (Treg) play a central role in the prevention of autoimmunity and in the control of immune responses by down-regulating the function of effector CD4+ or CD8+ T cells. The role of Treg in Mycobacterium tuberculosis infection and persistence is inadequately documented. Therefore, the current study was designed to determine whether CD4+CD25+ FoxP3+ regulatory T cells may modulate immunity against human tuberculosis (TB). Ourresults indicate that the number of CD4+CD25+FoxP3+ Treg increases in the blood or at the site of infection in active TB patients. The frequency of CD4+CD25+FoxP3+ Treg in pleural fluid inversely correlates with local MTB-specific immunity(p<0.002). These CD4+CD25+FoxP3+ T lymphocytes isolated from the blood and pleural fluid are capable of suppressing MTB-specific IFN-γ and IL-10 production in TB patients. Therefore, CD4+CD25+FoxP3+ Treg expanded in TB patients suppress Mycobacterium tuberculosis immunity and may therefore contribute to the pathogenesis of human TB.

Studies have shown that the traditional healers have used Tulbaghia violacea (TV) (also known as ‘wild garlic’) for the treatment of a number of ailments including fever, tuberculosis, stomach problems, and oesophageal cancer. However, little is known with regards to the anticancer and antiproliferative properties of this plant. Therefore, this study investigated the effects of TV and domesticated garlic extracts on Jurkat cells, in order to determine whether or not these extracts possess anti-proliferative properties. Cultured Jurkat cells were treated with IC50 concentrations of garlic (14μg/ml), TV leaf (256μg/ml), TV bulb (225μg/ml) and TV stalk (216μg/ml) extracts as determined by the methylthiazol tetrazolium assay. Free radical production was measured using the thiobarbituric acid reactive substance (TBARS) and nitric oxide (NO) assays, while glutathione (GSH) concentration was measured using the GSH-Glo™ assay. The apoptosis inducing properties of each extract were measured using flow cytometry (Annexin V- Fluos and JC-1 assays) and luminometry (caspases 3/7, 8, 9 and ATP). Western blots were run to determine protein expression, while comet and DNA fragmentation assays were used to determine the level of DNA damage induced. Wild and domesticated garlic extracts induced a significant increase in malondialdehyde concentration ([MDA]), with TV bulb extract inducing the highest concentration (p<0.0001). A significant increase in NO concentration was observed in the bulb (p<0.0001) and stalk (p<0.001) extracts, and leaf (p<0.05) and stalk (p<0.05) TV extracts significantly increasing GSH concentration. The longest comet tails were observed in TV bulb extracts (p<0.0001) and comprised mainly of single strand breaks, while the comets induced following garlic exposure contained double strand breaks. All extracts, except TV leaf, increased the percentage of cells undergoing apoptosis. Tulbaghia violacea leaf induced a significant (p<0.0001) increase in percentage of cells undergoing necrosis, whereas TV bulb resulted in a significant (p<0.0001) decrease. All TV extracts induced caspase 3/7 and 9 activity, with the most significant increase in caspase 9 activity observed for TV leaf and bulb. No significant change in caspase 3/7 activity was evident for domesticated garlic. Cleavage of PARP and expression of NF B and HSP 70 occured for all extracts. However, HSP 70 was not differentially expressed. Exposure to wild and domesticated garlic extracts induced peroxidative lipid and DNA damage within the cells, indicating oxidative stress. This damage occurred in conjunction with increased percentage of cells undergoing apoptosis and expression of caspase 3/7. Therefore, these findings suggest that TV is inducing cell death through apoptosis in Jurkat cells using a number of mechanisms, including the induction of oxidative stress. This is of clinical significance, as cell death through apoptosis is the preferred method of action for anti-cancer drugs. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2012.

Ebola virus (EBOV) is a zoonotic pathogen which causes a fulminant hemorrhagic fever and results in up to 90% fatality. Despite efforts over the past 38 years, a licensed prophylactic or post-exposure option remains unavailable. Several experimental vaccines have already demonstrated protection from lethal EBOV disease in nonhuman primates (NHPs). However, attempts to translate this research from bench to bedside have been hampered, since the immune responses that correlate with protection against EBOV are not well-defined. Without this information, it is not possible to reliably predict the efficacy of vaccines or treatments in humans without lethal challenge. The goal of this thesis is to determine the immune parameters that are predictive of protection against EBOV. Due to the rapid speed of EBOV pathogenesis, it was originally hypothesized that a rapid and robust CD8+ T-cell response must be crucial for survival. Using a previously-characterized adenovirus-vectored Ebola vaccine (Ad5-optGP), transgenic/knockout C57BL/6J mice with ablation for selected immune responses were vaccinated and challenged 28 days later. Surprisingly, while CD8+ T-cell knockout mice survived infection, B-cell knockout mice did not, indicating that the antibody response played a critical role in protection. Humoral and cell-mediated responses were compared between survivor and moribund guinea pigs and NHPs from previous vaccination and post-exposure therapy experiments, either with Ad5-optGP or a vesicular stomatitis virus (VSV)-vectored vaccine (VSVΔG/EBOVGP). Circulating EBOV glycoprotein (GP)-specific IgG antibody levels were the best correlation for protection independent of vaccine platform or timing of exposure (p<0.0001), strongly supporting the role of antibody responses in the control of EBOV infection. To demonstrate that antibodies are also responsible for protection, three previously-characterized monoclonal antibodies recognizing different GP epitopes were combined into a cocktail (ZMAb). Initiation of ZMAb treatment 1 and 2 days post infection (dpi) with EBOV protected 100% and 50% of NHPs, respectively. These results provide a reliable measure for predicting protection from EBOV in three commonly used animal models, and present a strong case for the use of antibodies as an effective post-exposure treatment. This knowledge will ultimately help in the development and validation of a clinical product against EBOV infection.

Two areas of the HTLV-I genome were targeted for an in-house molecular diagnostic test, namely the pol and env regions. The pol primers proved the most sensitive (100%)and specific (100%). Amplification using the env primer pair was not reproducible, and was not pursued further. The AmpliSensor assay (Acugen Systems, Lowell, MA) was also tested. The assay was very specific, but not as sensitive as our in-house PCR. To investigate the predominant HTLV-I subtype in the region, a 1535 by env gene was isolated from peripheral blood obtained from five local HTLV-I seropositive patients. Four of the patients presented with HAM/TSP, and the fifth presented with a skin disease. Nucleotide sequencing of the amplified products revealed the local strains to be very conserved, differing by 0.1% to 0.9% among themselves. No apparent difference was noted for the two clinical manifestations. Phylogenetic analysis was performed using repesentative strains from around the world. The local strains clearly fell within the cosmopolitan subtype. The local strains were most closely related to the North American strains suggesting an unexpected link between the two countries. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1998.

T cells are important effectors of the immune system that act to regulate the immune response and participate in the destruction of foreign cells. Of the two major populations of T lymphocytes, the αβ population is well studied whereas γδ cells display a number of idiosyncratic properties. γδ T lymphocytes are the first to appear in ontogeny [1], have the ability to present antigen [2] and are able to recognize pathogen associated molecular patterns [3], stress molecules [4] and phosphoantigens [5]. γδ T cells preferentially localize to epithelial sites and are expressed in high percentages in the peripheral blood of certain species such as chickens and cattle [6, 7]. To investigate if high expression of γδ T cells in cattle is due to an alteration in the αβ or γδ TCR gene structure, 0.7 megabases of bovine genomic DNA sequence was acquired that encompasses the J and C regions of the TCR α and β loci as well as the entire γ locus. Annotation and analysis of these loci revealed that both the TCR α and β loci contain well conserved joining (J) gene segments, recombination signal sequences (RSS) and constant (C) regions. This study identified a novel constant region (C3) in the TCR β locus. It was also verified that the bovine TCR γ locus exists in two separate sections on the chromosome. The TCR γ gene locus has weak conservation of canonical J gene segments. Expression analysis of the bovine TCR loci indicates that although α, β and γ all display preferential gene segment usage, the TCR γ locus utilized preferential expression considerably more than either TCR α or β. A particular TCRGC region that is highly utilized (C6) contains four copies of a TTEPP ruminant specific repeat and it is postulated that the function of this repeat, which is not presently known, may relate to high peripheral blood γδ T cell expression. A second study was performed to correlate high peripheral blood γδ T cell expression with digestive system type. A flow cytometry study performed with 42 different species of animals identified anti-TCR αβ, γδ, CD3 and WC1 antibodies that were cross-reactive with 30 different species including mammals and reptiles. Using cross-reactive antibodies from this experiment, 15 species were immunophenotyped to calculate peripheral blood γδ T cell populations. The results of the immunophenotyping experiment revealed that high expression of peripheral blood γδ T cells cannot be correlated with digestive system type, as evidenced by the information that ruminant and non-ruminant primates contain the same percentages of γδ T lymphocytes. As there is no correlation with digestive system type, it is hypothesized that high 76 T cell percentages may be connected with placenta use. Animals with epitheliochorial placentae are unable to transfer antibodies from mother to fetus; therefore, the neonate is born with no passive immunity [8]. It is postulated that since γδ T cells are expressed highly in young animals that these cells may act as the early immune system in the neonate. It is recommended that future studies investigating placentation and γδ T cells begin with an analysis of γδ T cell percentages in epitheliochorial primates of the infraorder Strepsirhini (lemurs and lorises) as these species are born without passive immunity.