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Background: South Africa has one of the highest HIV prevalence rates in the world and high contraceptive use among women aged 15–49 (65.3%). Family planning (FP) services remain a missed opportunity to integrate services for HIV. Recent reviews highlighted the lack of rigorously conducted studies of the effectiveness of integrated services. A cluster randomised trial (CRT) was conducted to evaluate the effectiveness of a model of integrating HIV into FP services compared with standard practice. The study sought to measure the effect of integrated services (Balanced Counselling Strategy Plus) on HIV testing in the previous year; use of dual protection and quality of HIV and FP care. Methods: A CRT was conducted in 12 clinics in North West province, South Africa. Structured clientprovider observations (CPOs) and client exit interviews (EIs) were conducted preintervention and one year later with FP clients aged >=18 years. Primary outcomes were condom use at last sex and testing for HIV in last year. The quality of care scores were constructed to assess HIV and FP quality of care. Analysis of effectiveness used statistical methods for CRTs. Findings: A total of 1,111 CPOs and 1,111 EIs were completed at baseline and 1,223 CPOs and 1,264 EIs at follow-up. At follow-up 33.2% of women in the intervention arm had tested for HIV in the last year compared with 21.4% in the control arm; RR=1.56 (95%CI: 1.13– 2.15; p=0.01). Condom use at last sex was 43.7% in the intervention arm and 39.4% in the control arm; RR=1.10 (95%CI: 0.85–1.43; p=0.14). Interpretation There was strong evidence of higher frequency of HIV testing among FP clients at intervention clinics. However, condom use at last sex was similar across intervention and control arms. All QOC scores were higher in intervention clinics, but there was substantial variation across clinics and these differences were not significant.

The major birch allergen Betulae verrucosa 1, (Bet v 1), belongs to the proteinase 10 (PR-10) family of panallergens that are common to many fruits, nuts and vegetables. A high proportion of birch-sensitised individuals experience oral symptoms upon consumption of such foods. This has been termed ‘oral allergy syndrome (OAS),’ or ‘pollen-food syndrome.’ Birch pollen specific immunotherapy (BP-SIT) can successfully treat birchsensitive rhinitis; it has been postulated that BP-SIT might also reduce oral allergy symptoms. Previous studies have been small and contradictory, using differing methodology and primary outcome measures. We designed a placebo controlled, double blind, randomised study aiming to establish definitively whether BP-SIT can effectively treat the pollen-food syndrome; outcome measures include open and double blind placebo controlled food challenges (DBPCFC) after one and two years of treatment. To date 22 patients have been enrolled, 18 have undergone assessment at one year and ten at two years. Four patients are due to attend for final followup in Autumn 2015. Eight patients have dropped out. Of the ten who have completed the study: nine have an increased tolerance to fresh apple as evaluated by open challenge. When assessed with DBPCFC, six patients tolerated larger quantities of fresh apple at two years compared to baseline. However, a total of eight patients were noted to have an apple threshold of 100g or more at baseline, despite reacting to only 20g during screening. The clinical trial is on going and remains blinded; it is therefore impossible to draw any firm conclusions regarding the efficacy of BP-SIT to treat OAS at this time.

The Toll like receptors (TLRs) are a family of innate immune pattern recognition receptors that have been implicated in the pathogenesis of the autoimmune diseases rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Activation of these receptors by exogenous or endogenous ligands stimulates production of cytokines such as TNF and IL-6. Elevated serum cytokine levels correlate with disease activity and are targeted by modern therapeutics. Peripheral blood monocytes contribute to disease through extravasation into inflamed tissues and secretion of pro-inflammatory cytokines. This study sought to identify the expression of TLRs 2, 3, 4, 5, 7, 8 and 9 on monocytes from RA and SLE donors and healthy controls (HC). TLR-induced TNF, IL-1β, IL-6, IL-10 and IP-10 secretion from these cells was also assessed, as was the basal expression of signalling inhibitors which regulate the cascade downstream of TLRs. These results were correlated with each other and to the clinical data of the donors. Significant differences in the expression of TLRs relative to HC were determined for RA and SLE peripheral blood monocytes. However, none of the observed differences correlated with a functional effect in terms of cytokine secretion in response to stimulation. Instead, dysregulation of downstream TLR signalling inhibitors was identified in the disease groups that contributed to these responses. Firstly, in agreement with other reports expression of TNF-induced protein 3 (TNFAIP3, A20) was decreased in the majority RA and SLE monocytes. However, asubpopulation of donors existed with higher A20 expression. Secretion of TNF following TLR8 stimulation of RA and SLE monocytes was elevated in those donors with low A20 expression relative to both HC and RA/SLE donors that exhibited higher A20 expression. Secondly, expression of sterile-α and HEAT/Armadillo motifs containing protein (SARM) was decreased in RA monocytes. Decreased SARM correlated with disease activity and secretion of IL-1β in response to TLR2 stimulation. The mechanism of action was independent of SARMs documented role in the inhibition of the TLR signalling adaptor TIR domain containing adaptor inducing interferon-β (TRIF). Finally, Interleukin-receptor associated kinase-M (IRAK-M), another TLR signalling inhibitor, was observed to correlate with IL-6 production by SLE monocytes following TLR5 activation, which was elevated relative to HC. Following TLR5 stimulation, IRAK-M induction by SLE monocytes was lower than HC. Lower IRAK-M expression correlated with elevated serum IL-6 levels, potentially indicating a clinically significant role. These data suggest that TLR-induced cytokine secretion by monocytes is regulated at the level of signal transduction as opposed to the TLR expression level in RA and SLE. Failure to control aspects of the cascade downstream of TLRs may allow for elevated cytokine production, which could influence disease activity.

The recent relative success of the Thai Vaccine trial and failure of the STEP trial have prompted renewed calls for a 'back to basics' approach. This premise dictates a greater and clearer understanding of the various immune parameters at play in HIV infection. The overall aim of this project was to look at different immune parameters at play in our unique Chinese SM HIV-1 cohort, which has an unusually high proportion of slow progressors. The unique dynamics of the SM cohort make it ideal for studying immune parameters. As the HLA Class I region has been shown to have the strongest genetic association with durable control of HIV, we set out to investigate the HLA correlates for our unique SM cohort. We recruited a neighbouring Han village cohort for comparisons, and employed stringent statistical tests for our analysis. Our analysis revealed that there was no enrichment of any specific HLA alleles in the SM cohort. This may be due to the limited study size, or that the protective effect has waned over time. Next we were interested in the immune pressure exerted on Vpu protein, which has been shown recently to be critical in HIV release from the cell, and not many studies have focused on Vpu. We did not find any statistically significant associations in our analysis of HIV-HLA associated polymorphisms in Vpu, and our screening ELISPOTS using overlapping peptides did not reveal any T-cell responses to the protein. However, our analysis for NK-mediated pressure, identified a single amino acid position in the Vpu sequence which was significantly associated with the presence of a specific KIR gene (p = 0.0047): In amino acid position 14 of Vpu, HIV-1 sequences (59%) derived from KIR2DS1+ individuals encoded for a valine (V) whereas the consensus amino acid alanine (A) at this position is found in the vast majority (76%) of KIR2DS1- individuals. This is extremely interesting as Vpu position 14 has been shown to be critical for interaction with the host restriction factor, tetherin. We next explored further the role of NK cells in relation to antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cell-mediated virus inhibition (ADCVI). Using an adapted ADCC-ICS assay, we found ADCC responses to Env in 37% of the SM cohort, with five subjects also having responses to Vpu peptides. We devised an Enzyme-linked Immunosorbent Assay (ELISA) which allowed us to map the Vpu ADCC epitopes in the SM cohort. We also wanted to know whether the Env/Vpu ADCC responders also displayed ADCVI activity. We optimised the ADCVI assay in our lab using HIV NL4-3 as our infecting strain of virus. All 5 Env/Vpu responders had very similar inhibition of virus – nearing 95 %, in the ADCVI assay. We followed up on these 5 Env/Vpu ADCVI responders and tried to get more plasma - unfortunately we were only able to get plasma from one subject - SM 017 as the rest could not be traced. This patient - SM 017 2011 - demonstrated the prozone effect, and we hypothesise that the ADCC antibody titres have increased in SM 017 over the years, to augment the ADCC response in vivo. In conclusion, in this study, we analyse host genetic, virological, and immunologic factors to enrich our understanding of HIV/AIDS parameters in a unique Chinese slow progressors cohort. We build up on the HLA database for slow progressors, and demonstrate a KIR association with Vpu amino acid position 14, something which has not been reported before. We demonstrate ADCC responses in the SM cohort, and also optimised an ELISA-based assay to reduce amount of plasma used for the ADCC-ICS assay. We plan now to isolate the antibodies from a unique patient, and possibly, identify the first Vpu ADCC antibody.

Prior infection with hepatitis B virus (HBV) is a common occurrence in HIV infected subjects and an increasing cause of morbidity and mortality. The CD8 T cell response is crucial for the long-term control of the virus in patients resolving acute hepatitis B. I first examined the effect of HIV related immunodepletion on HBV-specific immune responses in patients who resolved HBV. A cross-sectional study showed a reduction in HBV-specific CD8 responses in HBV immune patients with HIV infection compared to those without. Longitudinal study of a subgroup of patients examined whether this attrition could be reversed by effective antiretroviral therapy. The introduction of highly active antiretroviral therapy (HAART) resulted in recovery of some HBV-specific CD8 and CD4 responses, in association with restoration of CD4 counts. These data provided a mechanism for the observed impairment of HBV control in the setting of HIV infection and support the ability of HAART to reconstitute functionally active responses. I also studied the HBV-specific cellular immune responses in HIV negative patients who resolved acute hepatitis B without symptoms, a group which has never been studied immunologically, but represents a significant proportion of hepatitis cases acquired in adulthood. In the last chapter I focused on the chronic hepatitis B carriers co-infected with HIV and the impact of HIV and HBV treatment. I showed that reconstitution of some HBV-specific T cell responses can also occur in HIV-positive patients after a reduction in HBV load. This potential to recover T cell responses provides support for the addition of anti-HBV therapy in the treatment of co-infected patients.

In clinical trials and cohort studies the event of interest is often not observable, and is known only to have occurred between the visit when the event was first observed and the previous visit such data are called interval-censored. This thesis develops three pieces of research that build upon published methods for interval-censored data. Novel methods are developed which can be applied via self-written macros in the standard packages, with the aim of increasing the use of appropriate methods in applied medical research. The non-parametric maximum likelihood estimator 1,2 (NPMLE) is the most common statistical method for estimating of the survivor function for interval-censored data. However, the choice of method for obtaining confidence intervals for the survivor function is unclear. Three methods are assessed and compared using simulated data and data from the MRC Delta trial 3. Non- or semi-parametric methods that correctly account for interval-censoring are not readily available in statistical packages. Typically the event time is taken to be the right endpoint of the censoring interval and standard methods (e.g. Kaplan-Meier) for the analysis of right-censored failure time data are used, giving biased estimates of the survival curve. A simulation study compared simple imputation using the right endpoint and interval midpoint to the NPMLE and a proposed smoothed version of the NPMLE that extends the work of Pan and Chappell 4. These methods were also applied to data from the CHIPS study 5. Different approaches to the estimation of a binary covariate are compared: (i) a proportional hazards model 6, (ii) a piecewise exponential model 7, (iii) a simpler proportional hazards model based on imputed event times, and (iv) a proposed approximation to the piecewise exponential model that is a more rigorous alternative to simple imputation methods whilst simple to fit using standard software.

Therapeutic options for patients with aspirin-sensitive, and aspirin tolerant nasal polyps include corticosteroids (topical and systemic), surgery to relieve nasal obstruction, or a combination of the two. Medical therapy i.e. corticosteroids aims to reduce the underlying inflammation and alleviates all nasal symptoms. The aim of surgery is to provide an adequate nasal airway for breathing. Intranasal lysine-aspirin has been used as an option in these patients, and trials have shown its beneficial effects. However, their design and interpretation have been open to scientific criticism. Therefore, we planned to study the effectiveness of intranasal lysine-aspirin in the two groups of polyp patients by conducting trials in a randomised, double blind, and placebo controlled manner. Diagnosis of aspirin-sensitivity or tolerance was confirmed by intranasal lysine-aspirin challenge prior to enrolment in the appropriate trials. Nasal biopsy and polyp tissue were collected from the patients for laboratory-based experiments. Analysis of their results was aimed at improving our understanding of aspirin-sensitivity, and to generate a hypothesis on its pathogenesis. Intranasal lysine-aspirin did not reduce polyp growth, or improve nasal symptoms in aspirin-sensitive or aspirin tolerant patients when compared to placebo. However, intranasal lysine-aspirin (16 mgs) did not have any deleterious effects in both groups. Immunohistochemistry revealed a significant increase in expression of CysLTj receptor on inflammatory cells in nasal biopsies from aspirin-sensitive compared to tolerant patients. This expression reduced following treatment with intranasal lysine-aspirin, which suggested a possible mechanism of desensitization. We also found significantly higher levels of iNOS activity in polyp tissue from aspirin-sensitive compared to tolerant patients. Enhanced expression of CysLTi receptor provides further evidence of the central role played by leukotrienes in aspirin-sensitivity. Also, we suggest that high iNOS activity in polyp tissue is secondary to increased leukotriene production, and that the latter is most likely to be confined to the respiratory mucosa of patients with aspirin-sensitive asthma and/or polyposis.

The introduction of highly active antiretroviral therapy (HAART) for the treatment of HIV infection has led to dramatic reductions in morbidity. However, these highly potent drugs have been associated with a number of side effects. It is important to accurately quantify the prevalence and incidence of these toxicities, and to be able to compare the impact of specific antiretrovirals on toxicity rates in an unbiased manner. As we have become aware of potential HAART-related toxicities, there has been an increase in the frequency of monitoring of associated laboratory markers. Thus, diagnoses are made more quickly, and randomly abnormal values are more likely to be observed. There have also been changes over time in the specific antiretrovirals prescribed in HAART regimens. Consequently, newer antiretrovirals may seem to be associated with greater toxicity, even if this is not the case. Data simulations performed suggest that, when comparing the impact of specific HAART regimens on the occurrence of toxicity, considering the first measurement in a specified window leads to the most unbiased results. The choice of most appropriate cut-off of a surrogate laboratory marker of toxicity can also lead to differences in prevlance estimates. My investigations suggest that the most appropriate cut-off for each toxicity and each laboratory marker must be considered individually, but that definitions in which a confirmatory measurement is required are often appropriate. Results of a simulation study investigating a method proposed to account for unmeasured confounding, sample selection models, found this method gives unbiased treatment estimates in the situations we investigated. However, this method can lead to a lack of precision, and requires identification of a variable that is associated with treatment allocation. Thus, the use of these models in real-life settings may be limited. Mis-specification of non-linear associations between variables in regression models can lead to biased estimates. I have found that the use of multi-fractional polynomials to systematically consider the most appropriate relationship between variables is a method that is easy to apply and leads to plausible results.

This thesis focuses mainly on the analysis of the restriction to HIV-1 infection using rabbit cells as a model. Rabbit cells are poorly permissive to HIV-1 infection and the nature of this block is not well understood. This work shows that the restriction is independent of the cell receptor used by the virus for entry, as shown by infection of cells with HIV-1 pseudotyped with different types of envelopes and that it occurs mainly at the level of reverse transcription. It cannot be effectively saturated with high doses of virus or virus-like particles and has a recessive phenotype in human-rabbit heterokaryons. These results point to the existence of a factor required for HIV-1 infection that is absent in SIRC cells but can be complemented by human cells. The reverse transcription complexes extracted from human and rabbit cells have been analysed biochemically and found to have different densities but to be competent for reverse transcription in both cases in an in vitro endogenous assay. Cell fractionation of infected cells showed that HIV-1 is trafficked in a different way in human and rabbit cells and that correct intracellular trafficking is related to efficient reverse transcription and high infectivity in vivo. It is shown as well that viral DNA accumulates in rabbit cell nuclei only at a later stage of infection and fails to associate with chromatin, suggesting a further block prior to integration in SIRC cells Finally, chimeric viruses are used to determine the viral components responsible for the block. Viral chimeras formed by HIV-1 and SIV or MLV are used to infect the human cell line HeLa and SIRC cells. It is found that HIV-1 capsid is the determinant of the block in SIRC cells. Our data point to the existence of cellular factors regulating the early stages of intracytoplasmic and possibly intranuclear HIV-1 trafficking.