<p>Amb a 1 is the major allergen found in ragweed. Our observations have suggested that Amb a 1 may bind lipopolysaccharide (LPS), which would likely contribute to the allergenicity of Amb a 1. In order to assess whether Amb a 1 can bind LPS, peptide sequences from Amb a 1 were assayed for their ability to bind to LPS using an ELISA based LPS binding assay. A 15 amino acid sequence in the β- chain of Amb a 1 demonstrated affinity for biotin labeled <em>E. coli </em>LPS. The sequence also bound to <em>P.</em> <em>aeruginosa</em> LPS, which is structurally disparate in the lipid A region, indicating that the sequence has flexibility in recognizing different lipid A moieties, or that the binding site may not include the lipid A portion of the LPS molecule. An IL-10 ELISA was also used to determine whether the LPS bound to the peptides induced an immunological response in leukocytes. Peptides containing the LPS-binding sequence were able to bind to LPS and induce IL-10 production, suggesting the interaction between Amb a 1 and LPS may have immunological consequences. We have identified a sequence within the major ragweed allergen Amb a 1 that has the potential to bind to LPS. This indicates that the allergen may provide its own adjuvant when encountered by the immune system, leading to an enhanced immunological response to an otherwise innocuous environmental protein.</p> / Master of Science (MSc)

Type 2 (TH2) immune responses evolved to respond to helminth parasite infections by the production of TH2 cytokines, which stimulate anti-helminth immunity. Macrophage migration inhibitor factor (MIF) is a pleiotropic cytokine, which is produced by many cell types. We demonstrate that mice deficient in MIF have enhanced clearance of a helminth parasite. MIF deficiency in CD4+ T cells was found to be the most important for mediating parasite clearance. We mimicked MIF deficiency by administering an inhibitor of the MIF tautomerase activity, sulforaphane, and this also increased parasite clearance (Section I). TH2 immune responses underlie allergy and allergic asthma, in which the same cytokines that help expel parasites are released in response to innocuous substances. Integral to the initiation of adaptive TH2 immunity are dendritic cells (DCs), which take up antigen and stimulate antigen-specific CD4+ T cell responses. We found that DC expression of ADAM10, a zinc-dependent metalloproteinase, is critical for the development of TH2 immune responses and IgE production from B cells. This effect is demonstrated in both allergic airway inflammation and anaphylaxis models. ADAM10-deficient DCs are unable to cleave Notch1 receptors, resulting in reduced IL-6 production and this ultimately results in decreased TH2 activity. ADAM17 is closely related to ADAM10 in both structure and function. Interestingly, mice from which ADAM10 and 17 are removed from DCs (ADAM10/17DC-/-) have a distinct phenotype from both ADAM10DC-/- and ADAM17DC-/- mice in models of allergic airway inflammation (Section I). We also examined another effect of TH2 cytokines on the interaction between mast cells and myeloid derived suppressor cells (MDSCs). We sought to understand how histamine and IL-13, mediators made by mast cells, affect the immunoregulatory function of MDSCs. MDSCs in IL-13-deficient mice with tumor are more prevalent in circulation rather than in tumor or organs, which could be due to changes in CCL2/CCR2 chemotaxis. In addition, MDSC function after treatment with the DNA methyltransferase inhibitor, decitabine was examined. This treatment reduced their suppressive function and increased the expression of molecules needed for antigen presentation. Overall, TH2 immunity has multifaceted roles in anti-parasite immunity, allergic asthma, and MDSC function (Section II).

Pediatric Acute-Onset Neuropsychiatric Syndrome (PANS) is a clinical diagnosis characterized by sudden onset of obsessive compulsive disorder and is considered a type of Autoimmune Encephalitis. Pediatric Autoimmune Neuropsychiatric Disorder associated with Streptococcal Infection (PANDAS) is a subset of PANS characterized by a similar presentation but specifically results from infection by Group A β-hemolytic streptococcus. Early and accurate diagnosis is essential, as PANS can become a chronic condition. PANS and PANDAS are frequently misdiagnosed. There are a variety of differential diagnoses. The intent of this thesis is to evaluate differences in symptoms between PANDAS patients and those with a differential diagnosis and to synthesize existing knowledge to evaluate research areas that need improvement and reduce the rate of misdiagnosis. A review of clinical studies on the PubMed database was done using the key terms: "pediatric autoimmune encephalitis," "pediatric acute-onset neuropsychiatric syndrome," "pediatric autoimmune neuropsychiatric disorder associated with streptococcal infection," and "clinical study." A literature review was done to examine research articles and case reports to compare symptom presentation between PANDAS Patients and Differential Diagnosis Patients. The results of this thesis show that clinical studies only make up 2.73% of the articles and references on PubMed, revealing a need for increased clinical research. 16 symptoms were compared between PANDAS patients and Differential Diagnosis Patients. A One-Way ANOVA test was done, and 12 symptoms were found to be significantly higher in the PANDAS Patients compared to the Differential Diagnosis Patients. Symptom overlap between PANDAS Patients and Differential Diagnosis Patients and the results of the One-Way ANOVA test were compiled into a PANS Diagnostic Form for clinician use.

Professional antigen presenting cells (APCs) represent an important link between the innate and adaptive immune system. Macrophages (MΦs) and dendritic cells (DCs) serve as sentinels in the periphery collecting samples from their environment and processing this information. These cells then present antigenic fragments to T cells in the context of self-MHC molecules. Although a clear role for both of these APCs in the stimulation of already activated or memory T cells has been established, the ability of MΦs to activate naive T cells is still unknown. In this thesis the ability of bone marrow-derived MΦs and DCs to prime naive CD8+ and CD4+ T cells was investigated. Using adoptively transferred transgenic CFSE-Iabeled P-14 T cells, specific for gp33 from lymphocytic choriomeningitis virus in the context of Db, we were able to demonstrate the ability of both MΦs and DCs to induce naive CD8+ T cells proliferation. Once primed by MΦs these T cells gained effector function as shown by interferon- γ (IFN-γ) production and in vivo cytolysis. In addition, immunization of wild type animals with gp33-pulsed MΦs, as well as DCs, led to greater than a 95% reduction in lymphocytic choriomeningitis virus titers. To rule out the role of cross-presentation in the observed priming, two models were used. In the first model, lethally irradiated F1 bxs chimeras reconstituted with either H-2s or H-2b bone marrow were used as host for the adoptive transfer experiments. Since the gp33 peptide binds to Db, the H-2s reconstituted animals should be unable to cross-present the peptide to the P-14 T cells. Using this model, we were able to clearly demonstrate the ability of MΦs to activate naive P-14 T cells to undergo division. Additional experiments, demonstrated that these MΦ primed T cells went on to develop into effector cells. Finally, the ability of the MΦ primed T cells to develop into functional memory cells was demonstrated. To confirm the chimera results, these experiments were repeated using β2 microglobulin deficient animals (whose cells don't express MHC I) as host in adoptive experiments. MΦs were able to stimulate the naive P-14 T cells to divide and gain effector function as demonstrated by the ability to produce IFN-γ. In contrast to the CD8 system, MΦ were poor stimulators of D011.10 CD4+ T cell proliferation. Additionally, D011.10 T cells stimulated by DCs were able to produce interleukin-2 (IL-2), IL-4, tumor necrosis factor and granulocyte-macrophage colony stimulating factor where as MΦ stimulated D011.10 T cells were only able to produce IL-2. In conclusion this body of work clearly demonstrates the in vivo ability of MΦ to stimulate CD8+ T cell proliferation, effector function, as well as the formation of functional CD8+ T cell memory. Whether or not the nature of the memory pools stimulated by the two APCs is exactly the same is still unknown and needs further investigation. The ability of APCs other than DCs to stimulate functional protective memory needs to be considered in the quest to design vaccines that offer broad-spectrum protection.

Key Points: (1) Nutrition management for individuals infected with HIV can be helpful in maintaining lean body weight, combating oxidative stress, reducing complications from hyperglycemia and hyperlipidemia, and managing gastrointestinal function. (2) Patients may need to be individualized to meet each individual's unique requirements. (3) Consideration should be given to including the expertise of a registered dietitian/nutritionist s part of the health care team to promote wellness in the individuals infected with HIV.

Pentraxin 3 (PTX3) is a vital regulator of innate immune function that has been shown to counterregulate pro-inflammatory signaling and protect against the development of cardiovascular disease (CVD). Less is known about how PTX3 may mitigate against CVD risk by regulating the pro-inflammatory response at the cellular level. Therefore, this dissertation details four manuscripts which aimed to examine the capacity of PTX3 to regulate the innate immune response of peripheral blood mononuclear cells (PBMCs) isolated from healthy adults. Manuscript 1 examined the capacity of PTX3 to alter the inflammatory milieu following in vitro stimulation of isolated PBMCs with the pro-inflammatory lipid palmitate. In addition, Manuscript 2 sought to examine how participation in acute exercise, a powerful anti-inflammatory behavior that reduces CVD risk, alters the inflammatory phenotype and response of mononuclear cells following ex vivo stimulation with lipopolysaccharide (LPS). Manuscript 3 aimed to further elucidate the potential impact of cardiorespiratory fitness on the capacity of PTX3 to stimulate an innate immune response prior to and immediately following acute exercise in aerobically trained and untrained individuals. Finally, Manuscript 4 investigated the impact of healthy aging on plasma PTX3 concentrations and its relationship with telomere length in middle-aged compared to young adults. The capacity of isolated PBMCs to express a key cellular mechanism involved in maintaining longer telomere lengths, human telomerase reverse transcriptase (hTERT), following cellular stimulation with LPS, PTX3, and PTX3+LPS was also examined to address a mechanism that might explain how persistent exposure of circulating immune cells to the age-related pro-inflammatory milieu contributes to the shortening of telomere lengths.

Currently, the Caribbean has the second highest new cases of HIV infection, only after Sub-Saharan Africa. Women are becoming disproportionally more at risk for HIV/AIDS, mainly through heterosexual contact. The purpose of this dissertation study was to evaluate HIV knowledge, attitudes, and sexual risk behaviors among Trinidadian women. A sample of 113 participants was recruited for this study. The Theory of Planned Behavior (TPB) and Purnell Model of Cultural Competence were used to guide this study. Data were gathered using the HIV Knowledge Questionnaire (HIV-KQ-18), Condom Attitude Scale (CAS), Safe Sex Behavior Questionnaire (SSBQ), and a demographic questionnaire. Data were analyzed using statistical analysis software package (SPSS) version 22. Descriptive and Frequencies, Pearson product-moment correlation coefficient (r), one-way between groups ANOVA, and Multiple Regression analyses were implemented to assess HIV knowledge, attitudes about condom use, religious beliefs, level of education, and substance use among Trinidadian women. The results of this study indicated that level of education and race/ethnic backgrounds were associated with HIV knowledge among Trinidadian women. Religious beliefs had a negative correlation with attitudes about condom use. Also, there was a positive correlation between attitudes about condom use and safer sexual behaviors. The empirical knowledge obtained from this study can be used to provide a baseline for healthcare providers and policy makers to develop culturally aware, gender-relevant interventions to decrease the rate of HIV infection among Trinidadian women.

Infection with human immunodeficiency virus type 1 (HIV-1) results in a wide range of immunologic and hematopoietic abnormalities. The overall goal of this dissertation was directed toward obtaining a better understanding of the interactions of HIV-1 and myeloid cells in relation to the pathogenesis of AIDS. The human myelomonocytic cell line, HL-60, was used as a model system to determine if HIV-1 infects myeloid progenitor cells and subsequently, if infection affects their differentiation. HL-60 cells and the human prototypic T cell line, H9 were infected with three different HIV-l isolates (IIIB, PM213, and NL4-3) which are known to infect T cells. All three isolates productively infected both H9 and HL-60 cells; however, HIV-1 antigen expression and cytopathicity was delayed by approximately 15 days in infected HL-60 cells compared H9 cells. To examine the effect of HIV-l infection on myeloid differentiation, chronically infected HL-60 cells and clonal lines derived from them were induced to differentiate into either granulocytes by treatment with dimethyl formamide (DMF) or into monocytes by treatment with phorbol l2-myristate 13 acetate (PMA). By both cellular morphology and function, approximately the same percentage of treated, HIV-infected HL-60 cells differentiated into either granulocytes or monocytes as treated, control HL-60 cells. Taken together, these results indicate that HIV-1 infection does not affect the morphological or functional differentiation of HL-60 cells. In an effort to understand the differences in the regulation of HIV-l infection in myeloid versus T cells, the life cycle of NL4-3 was examined in HL-60 cells and H9 cells. Initially, NL4-3 replication was restricted in HL-60 cells compared to H9 cells. This restriction was overcome 15 days after infection by the generation of a viral isolate, NL4-3(M). NL4-3(M), harvested during the lytic phase of NL4-3 infection of HL-60 cells, caused cell death approximately 8 days after infection in both H9 and HL-60 cells. Although measurements of viral entry kinetics demonstrated that the timing of entry of NL4-3 and NL4-3(M) in HL-60 cells and NL4-3 in H9 cells was similar, a quantitative polymerase chain reaction (PCR) analysis of newly reverse transcribed NL4-3 DNA in H9 and HL-60 cells revealed that NL4-3 infected H9 cells and NL4-3(M) infected HL-60 cells contain consistently higher amounts of newly reverse transcribed DNA than NL4-3 infected HL-60 cells. The delay in NL4-3 replication in HL-60 cells was further amplified by inefficient spread of the virus throughout the HL-60 culture as measured by RNA production and DNA integration suggesting that another step in the viral life cycle after reverse transcription was also restricted. These results suggest that the efficiency of NL43 replication in HL-60 cells is restricted at several steps in the viral life cycle. Further, these restrictions are overcome by the generation of a viral variant, NL4-3(M), which efficiently replicates in myeloid cells. The tropism of NL4-3(M) was further characterized by testing its growth in monocyte-derived macrophages (MDM). Unlike NL4-3, NL4-3(M) productively infected MDM cultures. The ability of NL4-3(M) to infect macrophages was conferred by the envelope gene. This was demonstrated by the ability of the recombinant virus, NL4-3envA, which contains the envelope of NL4-3(M) in the context of the NL4-3 genome, to infect and replicate in MDM cultures. The envelope gene of NL4-3(M), however, did not confer ability to rapidly kill HL-60 cells. Together, these findings demonstrate that viral determinants controlling entry into MDM are different trom the determinants controlling the cytopathic phenotype in HL-60 cells.

Submitted by Claudinei Pracidelli (cpracide@ipen.br) on 2015-04-10T16:49:05Z No. of bitstreams: 0 / Made available in DSpace on 2015-04-10T16:49:05Z (GMT). No. of bitstreams: 0 / Tese (Doutorado em Tecnologia Nuclear) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Submitted by Claudinei Pracidelli (cpracide@ipen.br) on 2015-04-10T16:49:05Z No. of bitstreams: 0 / Made available in DSpace on 2015-04-10T16:49:05Z (GMT). No. of bitstreams: 0 / Durante e após o tratamento de câncer, de portadores de HIV ou transplantes, a alimentação pode oferecer um bem estar ao paciente, pois o ato de se alimentar corretamente auxilia as pessoas a se sentirem fortalecidas normalmente. Pessoas saudáveis possuem o seu sistema imunológico funcionando de maneira adequada, podendo combater pequenas quantidades de bactérias. Entretanto, pessoas imunocomprometidas podem não conseguir combater esta pequena quantidade de microrganismos e necessitam de uma dieta com baixa contagem microbiológica para evitar o contato com bactérias potencialmente danosas à saúde. Esta dieta é denominada dieta neutropênica. Esses pacientes são suscetíveis à contaminação alimentar, não sendo aconselhável a ingestão de produtos crus. A irradiação em vegetais, com baixas doses, tem a finalidade de reduzir a carga microbiana. Dessa forma a proposta deste trabalho foi obter dados de aspectos microbiológicos, sensoriais e físico-químicos em Mix Primavera minimamente processados submetidos a diferentes doses de radiação ionizante em dietas destinadas a pacientes imunocomprometidos ou imunossuprimidos. Foram utilizadas doses de 1,0kGy, 2,0kGy, 3,0kGy e 4,0kGy irradiadas no Irradiador Multipropósito 60Co localizado no Centro de Tecnologia das Radiações (CTR) IPEN-CNEN/SP. Foram realizadas análises microbiológicas (n=25), utilizando Petrifilm, análises sensoriais utilizando o teste sensorial de aceitação (n=30) e triangular (n=15) e análise de textura (n=90) no Laboratório de Microbiologia de Alimentos no Centro de Tecnologia das Radiações. As análises de textura foram realizadas com o auxílio de um texturômetro T.A.XT. Plus (Stable Micro System) equipado com a célula de carga de 50kg utilizando a sonda (probe) de corte triangular com lâmina Warner-Bratzler Knife com velocidade de descida de 2mm/s. Todos os resultados foram expressos em Newtons (N). Os resultados obtidos demostraram que para as análises microbiológicas os padrões foram seguidos de acordo com a RDC nº 12/01 ANVISA. A análise sensorial não apresentou diferença significativa entre as amostras, entretanto no teste triangular com as doses de 4,0kGy e controle houve diferença significativa entre as amostras, demonstrando que a amostra de 3,0kGy seria a mais indicada para o público específico deste trabalho. Pode-se concluir que para uma dieta neutropênica sugere-se uma dose de 2,0kGy. / Tese (Doutorado em Tecnologia Nuclear) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP