Thesis (Ph.D.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Department of Medical Microbiology and Immunology. Title from pdf file main screen (viewed on April 24, 2010). Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2009 Apr 26

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 108-129).

Thesis (Ph.D. in Immunology) -- University of Colorado, 2005. / Typescript. Includes bibliographical references (leaves 133-151). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

The overall emphasis in this thesis is the study of the regulation of virus infections by natural killer (NK) cells. In initial analyses, vaccinia virus (VV)-infected cells were found to be more sensitive to NK cell-mediated lysis during a discrete period of time post-infection. This enhanced susceptibility to lysis correlated with enhanced triggering (but not binding) of the effector cells and a concomitant decrease in target cell H-2 class I antigen expression. Furthermore, VV-infected cells became resistant to lysis by allospecific cytotoxic T lymphocytes (CTL) at a time when they were very sensitive to killing by NK cells or VV-specific CTL. This suggested that alterations in class I MHC antigens may affect target cell sensitivity to lysis by NK cells. The hypothesis that viral peptide charging of H-2 class I molecules can modulate target cell sensitivity to NK cell-mediated lysis was tested by treating target cells with synthetic viral peptides corresponding to the natural or minimal immunodominant epitopes defined for virus-specific CTL, and then target cell susceptibility to NK cell-mediated lysis was assessed. None of the 12 synthetic viral peptides used were able to significantly alter target cell lysis by NK cells under any of the conditions tested. In order to determine if H-2 class I molecules were required in the regulation of a virus infection by NK cells in vivo, intact or NK depleted (treated with anti-asialo GM1 antiserum) β2-microglobulin-deficient [β2m (-/-)] mice, which possess a defect in H-2 class I antigen expression, were infected with the prototypic NK-sensitive virus, murine cytomegalovirus (MCMV). In anti-asialo GM1-treated β2m (-/-) mice, as well as in β2m + (H-2 class I normal) control mice also treated with anti-asialo GM1 a significant enhancement in splenic MCMV titers as compared to NK-intact animals, was observed. When thymocyte expression of H-2 class I molecules (H-2Db) in normal mice was analyzed, it was found that following MCMV infection, H-2Db expression was significantly greater than the low level of expression found in uninfected thymocytes. In marked contrast, thymocytes from β2m (-/-) mice did not display any detectable H-2Db before or after infection. These in vivoresults demonstrate that NK cells can regulate a virus infection, at least in the case of MCMV, independent of H-2 class I molecule expression. Thymocytes from uninfected normal mice were found to be very sensitive to NK cell-mediated lysis, whereas those from MCMV-infected animals were completely resistant, presumably due to the protective effects of MCMV-induced interferon (IFN). However, thymocytes from MCMV-infected β2m (-/-) mice were only slightly protected from lysis by NK cells, consistent with the inverse correlation between MHC class I antigen expression and sensitivity to NK cell-mediated lysis. These results provide in vivoevidence suggesting a requirement for MHC class I molecules in IFN-mediated protection from lysis by NK cells. In addition to the analysis of H-2 class I molecules on target cells, the identity of a molecule present on the surface of all NK cells and other cytotoxic effector cells, which is recognized by a monoclonal antibody (mAb) generated in this laboratory designated CZ-1, and can also modulate NK cell triggering, was also of interest. This laboratory has previously reported that this antigen is upregulated on cytotoxic (and other) lymphocytes following a virus infection in vivo, or upon activation in vitro. Using competitive FACS analysis and fibroblasts transfected with various isoforms of CD45, it was found that mAb CZ-1 recognizes a sialic acid-dependent epitope associated with a subpopulation of CD45RB molecules.

Base teórica: A sepse é uma síndrome heterogênea, definida como disfunção orgânica que ameaça à vida, causada por uma resposta desregulada do hospedeiro à infecção. É um problema de saúde mundial, graças à sua alta prevalência, morbimortalidade associada, além de custos para seu tratamento. As células Natural Killer (NK) fazem parte do sistema imune inato reconhecendo moléculas de HLA de classe I em células alvo, através de seus receptores de membrana killer cell immunoglobulin-like receptors (KIR). A intensidade da resposta à infecção pode variar entre indivíduos, logo pode-se considerar que esta seja determinada por bases genéticas, e estas influenciem na ocorrência de sepse e variabilidade nos desfechos. Objetivos: Avaliar a associação entre os genes KIR e os ligantes HLA em pacientes críticos, comparando pacientes com sepse e controles não sépticos internados na mesma UTI. Métodos: Foi examinado o polimorfismo de 16 genes KIR e seus ligantes HLA em 271 pacientes críticos, caucasóides, sendo 211 pacientes com sepse e 60 controles, pela técnica de PCR-SSO e PCR-SSP, respectivamente. Resultados: Os genes ativadores KIR2DS1 e KIR3DS1 foram mais frequentes nos controles que nos pacientes com sepse (41,23% versus 55,00%, e 36,49% versus 51,67%; p = 0.041 e 0,025, respectivamente). Estes resultados fornecem informação inicial sobre o papel de polimorfismos de KIR na sepse, sugerindo que este possa ser um potencial marcador diagnóstico ou prognóstico da doença. / Background: Sepsis is a heterogeneous syndrome, defined a life-threatening organic dysfunction caused by a dysregulated host response to infection. Sepsis is a global health problem, due to its high prevalence, associated morbidity and mortality, and costs for its treatment. Cells Natural Killer (NK) cells are part of the innate immune system that recognize HLA class I molecules on target cells via membrane receptors called killer cell immunoglobulin-like receptors (KIR). The intensity of the response to an infection may vary among individuals and might be influenced genetic features affecting sepsis occurrence and variability in outcomes. Objectives: To evaluate the association between KIR genes and HLA ligands in critically ill patients, comparing patients with sepsis and without sepsis admitted to the same ICU. Methods: We examined the polymorphism of 16 KIR genes and their HLA ligands in 271 critically ill patients, Caucasians, and 211 patients with sepsis and 60 controls by PCR-SSO and PCR-SSP, respectively. Results: Activating KIR2DS1 and KIR3DS1 genes were more common in controls than in patients with sepsis (41.23% versus 55.00% and 36.49% versus 51.67%, p = 0.041 and 0.025, respectively). These results provide initial information on the role of polymorphism of KIR in sepsis, suggesting that this may be a potential diagnostic or prognostic marker of the disease.

A transformação do mosto de uva em vinho envolve uma série de ações combinadas de diferentes gêneros e espécies de microrganismos. A espécie Saccharomyces cerevisiae domina a fase intermediária e a fase final da fermentação alcoólica. De modo geral, as leveduras enológicas podem ser caracterizadas pela capacidade fermentativa, produção de H2S (sulfeto de hidrogênio) e seu comportamento killer. A Embrapa uva e vinho possui em sua Coleção, diversas leveduras autóctones isoladas de bagas de uvas oriundas de diversas regiões do Brasil. Entretanto, a diversidade genética destes isolados não é conhecida. Neste estudo foram avaliados a capacidade fermentativa, formação de H2S, fator killer e sensibilidade ao fator killer de 150 leveduras provenientes das cultivares Malvasia Bianca (FMB14), Moscato Alexandria (FMA14) e Moscato Tradicional (MBTF14) todas oriundas da região de Farroupilha- RS. A capacidade fermentativa foi avaliada juntamente com a formação de H2S, inoculando as leveduras em meio mosto sulfito. Os testes ao fator killer e sensibilidade ao fator killer foram avaliados através do meio Lorena/ELNC (80:20). As linhagens com perfil para elaboração de vinhos e produtoras da toxina killer foram identificadas por amplificação da região ITS1- 5.S- ITS2 por PCR e por PCR-RFLP. Foi avaliada também a diversidade genética de 23 linhagens da espécie de Saccharomyces cerevisiae da Coleção da Embrapa Uva e Vinho, usando a técnica de PCR-RAPD. Foram empregados para detectar a variabilidade genética das leveduras os oligonucleotídeos iniciadores: (GTG)5, (GAC)5, (GACA)4 e M13. Os resultados mostraram que a maioria das linhagens apresentaram baixa velocidade fermentativa aliada à diferentes níveis de produção de H2S. Somente 3 linhagens apresentaram capacidade fermentativa adequada quando comparadas com as linhagens de referencia 1vvt/97 e K1, quais sejam, 29MBF14, 39MBTF14 e 50MBF14. Apenas a linhagem 29MBTF4 formou pequenas quantidades de H2S. Verificou-se que 64% das linhagens isoladas mostraram-se metabolicamente capazes de biossintetizar H2S. Somente 9,33% apresentaram comportamento killer e apenas 6,66% mostraram sensibilidade à proteína killer. Os resultados apresentados sugerem ter relação com as cultivares utilizadas no isolamento. Verificou-se a existência de diferenças genéticas entre as linhagens de Saccharomyces cerevisiae estudadas com todos os iniciadores utilizados. Os iniciadores que mais discriminaram linhagens de Saccharomyces cerevisiae foram (GTG)5 e (GAC)5. / Grape must conversion into wine involve combined actions of different genus and species of microorganism. The species Saccharomyces cerevisiae dominates intermediate and final stages of alcoholic fermentation. Generally, the oenological yeasts are characterized by their fermentative capacity, production of H2S (hydrogen sulfide) and killer behavior. Embrapa Grape and Wine has a Yeast Collection that encompasses many autochthonous strains isolated of grape berries from different regions of Brazil. However, the genetic diversity of these yeasts are stilling known. This study has evaluated the fermentative capacity, production of H2S, killer factor and killer factor sensibility of 150 yeasts isolated from the cultivars Malvasia Bianca (FMB14), Moscato Alexandria (FMA14) and Moscato Tradicional (MBTF14) all belonging from Farroupilha commune in Rio Grande do Sul State. The fermentative capacity has been tested along with the evaluation of H2S production by the inoculation of the yeasts in sulfite must medium. The production and detection of factor killer and the evaluation of sensitive characteristics have been measured in Lorena/ELNC (80:20) solid medium. Yeasts with optimal fermentative characteristics and the ones producing killer toxin have been identified by amplification of ITS1-5.8S-ITS2 region by PCR -RFPL. This study also has evaluated the genetic diversity of 23 yeasts strains of Saccharomyces cerevisiae, belonging to the Yeast Collection of Embrapa Grape and Wine, employing PCRRAPD technique. The primers (GTG)5, (GAC)5, (GACA)4 and M13 have been used to detect the yeasts genetic diversity. The results showed that the majority of the yeasts analyzed have demonstrated low fermentative velocity combined with different levels of H2S production. From the three cultivars analyzed, only Moscato Tradicional showed yeasts with a suitable fermentative capacity when compared to the reference yeasts EMBRAPA 1vvt97 e K1, and they were named as 29MBF14, 39MBTF14 and 50MBF14. It was verified that 84%, 76% and 36% of the isolated strains from Malvasia Bianca, Moscato Tradicional and Moscato Alexandria, respectively, were capable to biosynthesize H2S. Concerning to killer behavior, 14%, 12% and 2% of the isolated strains from Moscato Tradicional, Moscato Alexandria and Malvasia Bianca, respectively, were capable of producing killer factor. These outcomes suggest the influence of the cultivar into the microflora biodiversity. Genetic differences were also demonstrated between the strains of Saccharomyces cerevisiae for all the primers tested. Primers GTG5 and GAC5 were the most discriminative.

Base teórica: A sepse é uma síndrome heterogênea, definida como disfunção orgânica que ameaça à vida, causada por uma resposta desregulada do hospedeiro à infecção. É um problema de saúde mundial, graças à sua alta prevalência, morbimortalidade associada, além de custos para seu tratamento. As células Natural Killer (NK) fazem parte do sistema imune inato reconhecendo moléculas de HLA de classe I em células alvo, através de seus receptores de membrana killer cell immunoglobulin-like receptors (KIR). A intensidade da resposta à infecção pode variar entre indivíduos, logo pode-se considerar que esta seja determinada por bases genéticas, e estas influenciem na ocorrência de sepse e variabilidade nos desfechos. Objetivos: Avaliar a associação entre os genes KIR e os ligantes HLA em pacientes críticos, comparando pacientes com sepse e controles não sépticos internados na mesma UTI. Métodos: Foi examinado o polimorfismo de 16 genes KIR e seus ligantes HLA em 271 pacientes críticos, caucasóides, sendo 211 pacientes com sepse e 60 controles, pela técnica de PCR-SSO e PCR-SSP, respectivamente. Resultados: Os genes ativadores KIR2DS1 e KIR3DS1 foram mais frequentes nos controles que nos pacientes com sepse (41,23% versus 55,00%, e 36,49% versus 51,67%; p = 0.041 e 0,025, respectivamente). Estes resultados fornecem informação inicial sobre o papel de polimorfismos de KIR na sepse, sugerindo que este possa ser um potencial marcador diagnóstico ou prognóstico da doença. / Background: Sepsis is a heterogeneous syndrome, defined a life-threatening organic dysfunction caused by a dysregulated host response to infection. Sepsis is a global health problem, due to its high prevalence, associated morbidity and mortality, and costs for its treatment. Cells Natural Killer (NK) cells are part of the innate immune system that recognize HLA class I molecules on target cells via membrane receptors called killer cell immunoglobulin-like receptors (KIR). The intensity of the response to an infection may vary among individuals and might be influenced genetic features affecting sepsis occurrence and variability in outcomes. Objectives: To evaluate the association between KIR genes and HLA ligands in critically ill patients, comparing patients with sepsis and without sepsis admitted to the same ICU. Methods: We examined the polymorphism of 16 KIR genes and their HLA ligands in 271 critically ill patients, Caucasians, and 211 patients with sepsis and 60 controls by PCR-SSO and PCR-SSP, respectively. Results: Activating KIR2DS1 and KIR3DS1 genes were more common in controls than in patients with sepsis (41.23% versus 55.00% and 36.49% versus 51.67%, p = 0.041 and 0.025, respectively). These results provide initial information on the role of polymorphism of KIR in sepsis, suggesting that this may be a potential diagnostic or prognostic marker of the disease.

A transformação do mosto de uva em vinho envolve uma série de ações combinadas de diferentes gêneros e espécies de microrganismos. A espécie Saccharomyces cerevisiae domina a fase intermediária e a fase final da fermentação alcoólica. De modo geral, as leveduras enológicas podem ser caracterizadas pela capacidade fermentativa, produção de H2S (sulfeto de hidrogênio) e seu comportamento killer. A Embrapa uva e vinho possui em sua Coleção, diversas leveduras autóctones isoladas de bagas de uvas oriundas de diversas regiões do Brasil. Entretanto, a diversidade genética destes isolados não é conhecida. Neste estudo foram avaliados a capacidade fermentativa, formação de H2S, fator killer e sensibilidade ao fator killer de 150 leveduras provenientes das cultivares Malvasia Bianca (FMB14), Moscato Alexandria (FMA14) e Moscato Tradicional (MBTF14) todas oriundas da região de Farroupilha- RS. A capacidade fermentativa foi avaliada juntamente com a formação de H2S, inoculando as leveduras em meio mosto sulfito. Os testes ao fator killer e sensibilidade ao fator killer foram avaliados através do meio Lorena/ELNC (80:20). As linhagens com perfil para elaboração de vinhos e produtoras da toxina killer foram identificadas por amplificação da região ITS1- 5.S- ITS2 por PCR e por PCR-RFLP. Foi avaliada também a diversidade genética de 23 linhagens da espécie de Saccharomyces cerevisiae da Coleção da Embrapa Uva e Vinho, usando a técnica de PCR-RAPD. Foram empregados para detectar a variabilidade genética das leveduras os oligonucleotídeos iniciadores: (GTG)5, (GAC)5, (GACA)4 e M13. Os resultados mostraram que a maioria das linhagens apresentaram baixa velocidade fermentativa aliada à diferentes níveis de produção de H2S. Somente 3 linhagens apresentaram capacidade fermentativa adequada quando comparadas com as linhagens de referencia 1vvt/97 e K1, quais sejam, 29MBF14, 39MBTF14 e 50MBF14. Apenas a linhagem 29MBTF4 formou pequenas quantidades de H2S. Verificou-se que 64% das linhagens isoladas mostraram-se metabolicamente capazes de biossintetizar H2S. Somente 9,33% apresentaram comportamento killer e apenas 6,66% mostraram sensibilidade à proteína killer. Os resultados apresentados sugerem ter relação com as cultivares utilizadas no isolamento. Verificou-se a existência de diferenças genéticas entre as linhagens de Saccharomyces cerevisiae estudadas com todos os iniciadores utilizados. Os iniciadores que mais discriminaram linhagens de Saccharomyces cerevisiae foram (GTG)5 e (GAC)5. / Grape must conversion into wine involve combined actions of different genus and species of microorganism. The species Saccharomyces cerevisiae dominates intermediate and final stages of alcoholic fermentation. Generally, the oenological yeasts are characterized by their fermentative capacity, production of H2S (hydrogen sulfide) and killer behavior. Embrapa Grape and Wine has a Yeast Collection that encompasses many autochthonous strains isolated of grape berries from different regions of Brazil. However, the genetic diversity of these yeasts are stilling known. This study has evaluated the fermentative capacity, production of H2S, killer factor and killer factor sensibility of 150 yeasts isolated from the cultivars Malvasia Bianca (FMB14), Moscato Alexandria (FMA14) and Moscato Tradicional (MBTF14) all belonging from Farroupilha commune in Rio Grande do Sul State. The fermentative capacity has been tested along with the evaluation of H2S production by the inoculation of the yeasts in sulfite must medium. The production and detection of factor killer and the evaluation of sensitive characteristics have been measured in Lorena/ELNC (80:20) solid medium. Yeasts with optimal fermentative characteristics and the ones producing killer toxin have been identified by amplification of ITS1-5.8S-ITS2 region by PCR -RFPL. This study also has evaluated the genetic diversity of 23 yeasts strains of Saccharomyces cerevisiae, belonging to the Yeast Collection of Embrapa Grape and Wine, employing PCRRAPD technique. The primers (GTG)5, (GAC)5, (GACA)4 and M13 have been used to detect the yeasts genetic diversity. The results showed that the majority of the yeasts analyzed have demonstrated low fermentative velocity combined with different levels of H2S production. From the three cultivars analyzed, only Moscato Tradicional showed yeasts with a suitable fermentative capacity when compared to the reference yeasts EMBRAPA 1vvt97 e K1, and they were named as 29MBF14, 39MBTF14 and 50MBF14. It was verified that 84%, 76% and 36% of the isolated strains from Malvasia Bianca, Moscato Tradicional and Moscato Alexandria, respectively, were capable to biosynthesize H2S. Concerning to killer behavior, 14%, 12% and 2% of the isolated strains from Moscato Tradicional, Moscato Alexandria and Malvasia Bianca, respectively, were capable of producing killer factor. These outcomes suggest the influence of the cultivar into the microflora biodiversity. Genetic differences were also demonstrated between the strains of Saccharomyces cerevisiae for all the primers tested. Primers GTG5 and GAC5 were the most discriminative.

OBJETIVOS: Avaliar o perfil e o número absoluto e percentual de células NK verdadeiras (CD56+CD16+CD3-) e de células NK e NKT (CD56+) no sangue periférico de uma coorte de pacientes com artrite reumatóide (AR) antes e durante o tratamento com rituximabe (RTX). MÉTODOS: Foram analisados dez pacientes do grupo controle (doadores de sangue) e dez pacientes com AR que receberam duas infusões de RTX 1g separadas por intervalo de 14 dias. As análises imunofenotípicas para avaliação do perfil e quantificação de células NK foram realizadas pré e após a infusão ou até a recaída clínica. Pacientes respondedores e não respondedores foram classificados de acordo com os critérios do Colégio Americano de Reumatologia (ACR) em 6 meses. RESULTADOS: A quantidade de células NK verdadeiras não demonstrou variação significativa pré e após o tratamento com RTX. Contudo, houve aumento percentual de células CD56+ entre o primeiro e o segundo mês após a infusão com RTX. Além disso, os pacientes respondedores apresentaram uma tendência de aumento do número absoluto de células NK verdadeiras após dois meses de tratamento. Já em relação ao grupo controle, observou-se um aumento significativo do número de células NK basais nos pacientes com AR (p<0,05). CONCLUSÕES: Foi identificada uma tendência de aumento nos valores absolutos de células NK verdadeiras entre os pacientes respondedores no segundo mês após a infusão com RTX. Não foi identificada uma variação significativa no perfil e quantidade de células NK nos pacientes com AR pré e após o tratamento com RTX. Contudo, foi observado que os pacientes com AR possuem uma quantidade maior de células NK do que os controles, sugerindo um possível envolvimento destas células na AR. / OBJECTIVES: To assess the profile as well as the absolute number and percentage of true NK cells (CD56+CD16+CD3-), and NK and NKT cells (CD56+) in the peripheral blood of a cohort of patients with rheumatoid arthritis (RA) before and during rituximab (RTX) therapy. METHODS: Ten control patients (blood donors) and ten patients with RA were assessed. The latter group received two intravenous infusions of 1g RTX, separated by a 14 day interval. Immunophenotypic analyses of NK cells were conducted before and after infusion, or until clinical relapse. After six months, respondents and nonrespondents were reassessed according to American Rheumatology Criteria (ARC). RESULTS: The number of true NK cells did not significantly change after treatment with RTX. However, an increase in the percentage of CD56+ cells was observed between the first and second month after RTX infusion. Respondents also displayed a tendency toward an increased number of true NK cells after two months of treatment. At baseline, the number of NK cells was also found to be significantly higher in patients with RA than in control individuals (p<0.05). CONCLUSIONS: Respondents displayed a tendency toward an increase in the absolute number of true NK cells in the second month after RTX infusion. No significant changes in the profile and frequency of NK cells were found between preand post-RTX treatment assessments of patients with RA. However, it was found that patients with RA have a higher number of NK cells than control partcipants, suggesting a possible role of these cells in RA.