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HIV-1 persists to be a major health problem worldwide. A prophylactic or therapeutic vaccine offers the best hope to restrain the HIV-1 epidemic, however a consistent correlate of immune protection is yet to be found. HLA class I expression and their restricting HIV-1 specific CD8+ T cell responses have been shown to play a vital role in the control of HIV-1 infection. The interactions between HIV-1 and CD8⁺ T cell responses are complex and the mechanisms involved in the success or failure to control viraemia remain uncertain. Thus, the aim of these studies was to help define what CD8+ T cell responses a vaccine needs to induce to achieve durable immune control of HIV-1 infection. Focusing initially on HLA-B*35, an allele that has consistently been associated with rapid HIV-1 disease progression in the context of B clade infection, this study shows substantial differences in markers of HIV-1 disease outcome associated with different HLA-B*35 subtypes. Preliminary data suggest that effective targeting of a single epitope in Gag may be associated with HLA-B*35 mediated control of HIV-1 disease progression. Increased breadth of the Gag- specific CD8⁺ T cell responses is found to be associated with decreasing viral loads. These data therefore support the Gag hypothesis, and suggest that targeting of certain regions of the HIV-1 genome may have a positive effect in disease outcome, even for individuals carrying “detrimental” alleles. The extensive diversity of the HIV-1 genome and rapid viral adaptation are the main chal- lenges to vaccine design. Previous studies have suggested that effective CD8⁺ T cell responses drive selection of escape mutations that reduce viral replication capacity (VRC). There is also evidence that certain escape mutations can be transmitted from one host to another allow- ing for its accumulation in a population. The second study looked at the impact of HLA driven evolution of HIV-1 in VRC at a population level. This study compared two ART-naïve HIV-1 B clade infected cohorts, in Mexico and Barbados, in which protective HLA alleles (HLA- B*27/57/58:01/81:01) are expressed at 10% and 35% respectively, to analyze differences in VRC at a population level. Viral loads (VL) were found to be significantly higher in Mexico compared to Barbados and median CD4⁺ T cell counts significantly lower. Analysis of VRC in a subset of subjects in each cohort matched by CD4⁺ T cell counts between 300-500 cells/μl revealed that VL and VRC was significantly higher in the Mexican subset. This VRC difference was associated with accumulations in Barbados of eight previously described Gag escape mutation where fitness cost has previously been implicated. Accumulation remained significant in mismatched subjects. These data suggest that VLs and disease progression rates may differ between distinct populations as a result of the frequency of protective alleles in the respective populations, and that CD4⁺ T cell count-based guidelines to initiate antiretroviral therapy (ART) may need to be modified accordingly, to optimize the effectiveness of treatment-for-prevention strategies and reduce HIV-1 transmission rates to the absolute minimum. The final project aimed to improve the HIV-1 replication fitness assays currently used in the context of C clade infection. In order to achieve this, we attempted to design a clade C infectious molecular clone for the testing of gal-pol gene regions. However, the clones produced were not replication competent. Sequence analysis showed a large quantity of stop codons, most located within env which may explain the lack of infectivity. Chapter 5 describes the methodology used in the construction of the clade C isolate and suggests future work. Although we were unsuccessful in producing a replication competent virus, the construction of a C clade backbone which replicates efficiently remain an aim due to its importance for research directed to the analysis of genetic determinants of C clade virus. Data presented in this thesis suggest that vaccine-induced immune responses should aim to focus on vulnerable regions of the virus. These are conserved regions that can not escape without a high fitness cost and with a complex and difficult selection of compensatory mutations. Although much work remains to be done to achieve an effective CD8⁺ T cell based vaccine, hope remains that the induction of HIV control may be possible.

MicroRNAs (miRNAs) have now been identified in a vast array of organisms and a great deal of research has been carried out to elucidate the role they play. The dysregulation of miRNA expression has been implicated in a number of disease states and their importance has been highlighted by the beginning of their utilisation as therapeutics. The focus of this study was to identify the role played by miRNAs encoded by the Marek’s disease vaccine viruses, Marek’s disease virus serotype 2 (MDV-2) and Herpesvirus of turkeys (HVT). In order to better understand the functions of these miRNAs we wanted to identify their targets within the host cell. Using a combination of bioinformatic and biochemical approaches we were able to build up a library of potential targets. Three viral miRNA targets; AKT3, RAP1A and DEK, were further validated using dual-luciferase assays to highlight the exact site of miRNA targeting, and western blots to demonstrate an effect of miRNA targeting on protein abundance. An attempt at using label-free proteomics to observe the viral miRNA mediated changes in the host proteome is also described, however this proved to be unsuccessful. Additionally the function of one particular MDV-2 miRNA, mdv2-miR-M21, was explored in more detail, describing its role as a potential ortholog of the host miRNA; gga-miR-29b. By using the observation that the viral miRNA contained an identical 'seed' region to the host miRNA, we were able to use the data collected from existing studies on miR-29b to search for targets of mdv2-miR-M21. We demonstrated that mdv2-miR-M21 targeted DNMT3B, crucial for epigenetic modification of the genome. The final part of this study aimed to understand the wider context the viral miRNAs played in the viral biology and protective ability of the vaccine viruses. The miRNAs were deleted from the viruses, and then the miRNA-deletion viruses were used to vaccinate birds before challenge with the oncogenic Marek's disease virus serotype 1 (MDV-1), survival rates to the 'wild-type' MDV-2 and HVT vaccine viruses were then compared.

Studies indicate that overweight and obesity protect against HIV-disease progression in antiretroviral therapy (ART)-naïve patients. We examined retrospectively the relationship of overweight/obesity with HIV-disease progression in ART-naïve HIV+ adults in Botswana in a case-control study with 18-month follow-up, which included 217 participants, 139 with BMI 18.0-24.9 kg/m2 and 78 with BMI ≥25 kg/m2. Archived plasma samples were used to determine inflammatory markers: leptin and bacterial endotoxin lipopolysaccharide (LPS), and genotype single nucleotide polymorphisms (SNPs) of the Fat Mass and Obesity Associated Gene (FTO). At baseline, BMI was inversely associated with risk for AIDS-defining conditions (HR=0.218; 95%CI=0.068, 0.701, P=0.011), and higher fat mass was associated with reduced risk of the combined outcome of CD4+cell count ≤250/µL and AIDS-defining conditions, whichever occurred earlier (HR=0.918; 95%CI=0.847, 0.994, P=0.036) over 18 months, adjusting for age, gender, marriage, children, and baseline CD4+cell count and HIV-viral load. FTO-SNP rs17817449 was associated with BMI (OR=1.082; 95%CI=1.001, 1.169; P=0.047). Fat mass was associated with the risk alleles of rs1121980 (OR=1.065; 95%CI=1.009, 1.125, P=0.021), rs8050136 (OR=1.078; 95%CI=1.021, 1.140; P=0.007), and rs17817449 (OR=1.086; 95%CI=1.031, 1.145; P=0.002), controlling for age, gender, tribe, total energy intake, and activity. There were no associations of SNPs with markers of disease progression. Leptin levels were positively associated with BMI (β=1.764; 95%CI=0.788, 2.739; P=0.022) and fat mass (β=0.112; 95%CI=0.090, 0.135; P<0.001), but inversely with viral load (β=-0.305; 95%CI=-0.579, -.031; P=0.030). LPS levels were inversely associated with BMI (OR=0.790, 95%CI=0.630, 0.990; P=0.041), and fat mass (OR=0.852, 95%CI=0.757, 0.958; P=0.007) and directly with viral load (OR=2.608, 95%CI=1.111, 6.124; P=0.028), adjusting for age, gender, smoking and %fat mass. In this cohort, overweight/obesity predicted slower HIV-disease progression. Obesity may confer an advantage in maintaining fat stores to support the overactive immune system. FTO-SNPs may contribute to the variation in fat mass; however, they were not associated with HIV-disease progression. Our findings suggest that the obesity paradox may be explained by the association of increased LPS with lower BMI and higher viral load; while viral load decreased with increasing leptin levels. Studies in African populations are needed to clarify whether genetic variation and inflammation mediate the obesity paradox in HIV-disease progression.

Public health planning for influenza is based on morbidity and mortality estimates derived from statistical models. Lower than anticipated 2009 H1N1 pandemic death estimates have raised questions about the method. Examining the statistical method is important for future policy and program development. We compared the main methods of estimating influenza burden through a systematic literature review and by comparing statistical estimates of influenza-attributable burden at the Ottawa Hospital (TOH) to clinical estimates validated through chart review. We identified heterogeneity in methods used to estimate influenza-attributable mortality in the literature which resulted in within-season estimate variation by study. We found statistical estimates of influenza burden at TOH to be 4-8 times greater than clinically validated data. We also found no significant association between the outcomes examined and epidemic periods at TOH. The findings of this study suggest discordance between model estimates by model approach and between model estimates and validated findings. Examining reasons for these discordances should be pursued.

Orientador: Helaine Maria Besteti Pires Mayer Milanez / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T05:11:32Z (GMT). No. of bitstreams: 1 Candido_ElaineCristina_M.pdf: 1043425 bytes, checksum: 4797b5103af38ecbe53b5dd29b496856 (MD5) Previous issue date: 2013 / Resumo: Objetivos: avaliar a transmissão vertical (TV) em gestantes portadoras de hepatite B crônica, em um serviço universitário. Sujeitos e Método: foram analisadas as sorologias para hepatite B de todas as gestantes atendidas no serviço entre 2000 e 2005, identificando-se as HbsAg +; nessas foi realizado levantamento de prontuários, avaliando a presença do marcador de replicação viral (HbeAg positivo), imunoprofilaxia neonatal e taxa de TV. Análise de dados: foi avaliada a proporção de casos com HbsAg+ e nessas a presença do HbeAg. Para as portadoras de hepatite B, analisaram-se características clínicas e epidemiológicas através de frequências simples e a presença de TV. Resultados: entre 2000 e 2005 foram rastreadas para hepatite B no CAISM 5638 mulheres; dessas 28 (0,5%) apresentavam HbsAg+, definindo-se como portadoras crônicas. Não se encontrou nenhuma com replicação viral (HbeAg+). A idade média foi de 25 anos, com escolaridade média de sete anos, sendo 57% de brancas. O número de gestações médio foi de dois, sendo 52% de nulíparas. A categoria de exposição foi ignorada em 20; em quatro a via foi a sexual, em duas por TV e em duas por uso de drogas. A média de Idade gestacional ao parto foi de 38 semanas, com uma taxa de cesárea de 42%. O peso médio ao nascimento foi de 3094g e todos os recém-nascidos apresentaram boas condições de vitalidade e receberam imunoprofilaxia neonatal (vacina e imunoglobulina específica) nas primeiras horas de vida. Não houve TV. Conclusões: Nas gestantes atendidas no período, a prevalência de hepatite B crônica foi de 0,5%. Todas as crianças receberam imunoprofilaxia neonatal nas primeiras horas de vida e não ocorreu nenhum caso de TV, reforçando que para as gestantes sem replicação viral, as medidas de imunoprofilaxia neonatal protegeram a totalidade de seus recém-nascidos / Abstract: The purpose of this paper is to evaluate mother-to-child transmission of chronic hepatitis B in a university hospital. Subjects and methods: Hepatitis B serologic studies were pooled from all pregnant women referred to this prenatal service from 2000 to 2005. HBsAg positive patients were selected and, for those, clinical, laboratory and epidemiologic data were analyzed, including presence of HBeAg marker, immunoprophylactic procedures for the newborn and mother-to-child transmission rates. Data analysis: HBsAg carriers were characterized for clinical and epidemiologic factors associated with mother-to-child transmission. Results: Between 2000 and 2005, 5638 pregnant women were referred to high-risk prenatal care at our facility; of these, 28 women (0,5%) were HbsAg+ ¿ defined as chronic Hepatitis B virus (HBV) carriers. None of these were seropositive for HBeAg. Mean age was 25 years with a mean of 7 years of formal education and 57% were white; 52% were nulliparous. Exposure to hepatitis B virus was ignored in 20 women, sexual in 4, from mother-to-child transmission in 2 and associated with drug use in 2. Mean gestational age at delivery was 38 weeks with cesarean delivery in 42% of women. Mean weight at birth was 3094g and all newborns presented with good vitality and received immunoprophylactic procedures. There were no cases of mother-to-child transmission. Conclusion: Among all pregnant women seen at this tertiary high risk prenatal care facility between 2000 and 2005, chronic HBV infection was detected in 0,5% of patients. All newborns received immunoprophylaxis during the first hours after delivery and no case of mother-to-child transmission was detected. Our findings support that, among pregnant chronic HBV carriers without serologic evidence of active viral replication, immunoprophylactic measures are effective in preventing mother-to-child transmission in all instances / Mestrado / Saúde Materna e Perinatal / Mestra em Ciências da Saúde

orionpizango@gmail.com / Case report: A 43-year-old female presented with decreased visual acuity in the right eye.“Snowball-like” retinal lesions were found in both eyes on examination. Due to a lackof improvement with intravitreal antifungal empirical treatment, vitreous culture wasperformed and Candida glabrata was isolated. The patient then received intravitreal ampho-tericin B, as well as systemic treatment with caspofungin and amphotericin B lipid complex.Discussion: Endogenous fungal endophthalmitis is a sight-threatening condition. There arefew reports of C. glabrata endogenous endophthalmitis. Treatment regimens for Candidaendophthalmitis include combinations of systemic and/or intravitreal antifungals, as wellas vitrectomy.

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Billions of cells die everyday as a result of normal tissue turnover, infection, trauma or injury. These dead cells are taken up, processed and presented to T cells by antigen presenting cells resulting in tolerance or immunity. Apoptotic cells induce tolerance; however, the precise mechanisms are still unknown. Previous studies have shown that direct infusion of apoptotic cells induce tolerance mediated by TRAIL-expressing CD8+ T cells. We hypothesized that immunologic tolerance induced by apoptotic cells is dependent on the activation status of apoptotic cells and mediated by direct killing of target cells by TRAIL-expressing CD8+ T cells. Three different experimental systems were used to elucidate the mechanisms by which apoptotic cells regulate immune responses. Using a classical system of tolerance induction, we examined the immunological consequence of intravenous (i.v.) delivery of ex vivo-generated naïve or activated apoptotic cells. Naïve apoptotic cells induced tolerance when injected i.v.; however, previously activated apoptotic cells induced immunity. Further analysis revealed a key role for CD154 in the tolerogenic or immunogenic nature of the naïve or activated apoptotic cells, respectively, as tolerance resulted after i.v. injection of either naïve or activated apoptotic CD154-/- cells, while co-injection of an agonistic anti-CD40 mAb with naïve apoptotic T cells induced robust immunity. The infusion of large numbers of apoptotic cells has limited physiological relevance, so the investigation of the influence of apoptotic cells on the immune system turned to another experimental tolerance model where soluble peptide antigen is injected systemically to induce the peripheral deletion of a population of antigen-specific T cells. Using this system, we investigated how apoptotic cells generated in vivo leads to T cell tolerance. Following adoptive transfer of OT-II cells, wild-type mice injected with soluble OVA323-339 became unresponsive to subsequent CFA/OVA immunization. Interestingly, Trail-/- or Dr5-/- mice developed robust immunity; even though all strains displayed peripheral deletion of OVA-specific T cells. Subsequent investigation found the mechanism of action of the CD8+ T cells was TRAIL-mediated deletion of the OVA-responsive T cells in a TCR-specific manner. The experimental systems used above have some clinical relevance but are still not physiologic. To study the impact of apoptotic cells in a physiologic setting, we took advantage of the medical condition sepsis, which is accompanied by massive apoptosis of multiple immune cell populations. Thus, the final set of experiments in this thesis examined the tolerance induced during sepsis using a clinically-relevant cecal-ligation and puncture (CLP) model that included a secondary bacterial infection. CLP-treated WT mice had a reduced ability to control the secondary bacterial infection, which was paralleled by suppressed T cell responses, compared to sham-treated WT mice. In contrast, CLP- and sham-treated Trail-/- and Dr5-/- mice were able to similarly control the bacterial infection and generated bacterial antigen-specific T cell responses. The ability of CLP-treated wild-type mice to control the secondary infection and generate T cell immunity could be restored by the administration of a blocking anti-TRAIL mAb. These results suggest the importance of TRAIL in the induction of sepsis-induced immune suppression, such that TRAIL neutralization may be a potential therapeutic target to restore cellular immunity in septic patients.

T cells are an essential component of the adaptive immune system, which evolved to facilitate development of long-term, effective protection against infectious diseases. Upon activation, T cells play an important role in clearing infections, and especially, in preventing establishment of subsequent infections with the same pathogen. Because this is such a powerful response, it must be tightly regulated. Our lab has long been interested in how signaling molecules regulate the function of T and B lymphocytes. Our prior studies stimulated an interest in the signaling adapter molecule, Tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3). Our group previously produced a T cell-conditional (CD4-Cre) TRAF3-/- mouse, which demonstrated that TRAF3 unexpectedly plays an important positive role in T cell functions, including providing help for B cell responses, protection from infectious pathogens, cytokine production and proliferation. After TCR engagement, TRAF3 associates with the T Cell Receptor (TCR)/CD28 complex. These data identified a new role for TRAF3 in T cell activation. There are three signals that are required for full T cell activation. The three types of receptors that deliver these signals are the TCR, co-stimulatory receptors and cytokine receptors. This dissertation explores the regulatory role of TRAF3 in the 3 signals required for T cellsactivation. In signal 1, TRAF3 enhances TCR signaling by regulating the localization of the TCR inhibitors, PTPase non-receptor type 22 (PTPN22) and the c-Src kinase (Csk). Our lab previously reported that recruitment of TRAF3 to the TCR complex requires co-stimulation of CD28, the primary receptor for signal 2. In this dissertation, we show that TRAF3 associates with the Linker of Activated T cells (LAT) complex, demonstrating preference for distinct LAT-associated proteins. For delivery of signal 3, T cells require stimulation of a cytokine receptor, such as IFNαR, for differentiation of a T cell to an effector cell. Upon IFN stimulation, TRAF3 inhibits IFNαR-induced early molecular events, which results in the regulation of both canonical and non-canonical IFNαR signaling pathways. The results presented in this dissertation highlight the dynamic roles of TRAF3 as a regulator of T cell activation, by regulating multiple T cell signaling pathways.