Background: Type 1 diabetes mellitus is a multifactorial disease caused by beta cell destruction of Langerhans pancreatic islets. From the genetic aspect the main predisposition lays on HLA class II genes (40 - 50%), molecules of which present exogenous peptides to CD4+ T lymphocytes. Enviromental factors play a crucial role in the etiopathogenesis of T1DM. Through epigenetic regulation (e.g. DNA methylation) the genetic and enviromental factors communicate. The level of methylation in the regulatory regions can significantly affect expression of these genes. Aims: The aim of the diploma thesis was to define methylation profile of HLA DQB1 alleles in type 1 diabetes mellitus patients and determine their expression. Methods: The genotyping of HLA class II genes (HLA-DRB1, HLA-DQA1, HLA-DQB1) was performed using sequence specific primers. DNA was treated with sodium bisulfite, regulatory region of HLA DQB1 was amplified and cloned into E.coli, strain DH5α/XL1-Blue. Positive clones were sent for sequencing and results analyzed. RNA was transcribed to cDNA by reverse transcription and the level of expression was analyzed by quantitative PCR. Results: Statistically significant differences were found in total methylation of DQB1*0201 and *0302 alleles in the B section of DQB1 gene. Difference in...

Introduction: Glycoproteins of the major histocompatibility complex (MHC) are an irreplaceable part of immune response regulation and immune homeostasis maintenance. The regulation of the expression plays an important role in adaptive immune response. Recently, DNA methylation in regulatory areas, crucial for DNA availability to transcription factors, is one of the most researched mechanisms of this type of regulation. The DNA methylation is, among others, related to the aging processes. Increased predisposition age-related immunosenescence in higher age could result from the changes in methylation status of regulatory areas of MHC class II genes. Aims: The aim of this thesis is to analyze the methylation status of regulatory areas of DQB1 gene and to compare the differences between generations and specific alleles. The differences in the levels of DQB1 gene mRNA transcription between generations and specific alleles is also compared. Methods: Both DNA and RNA were isolated from blood samples obtained from donors of three different age groups. DNA was genotypized and modified by bisulfite conversion. The regulatory areas of DQB1 genes were then amplified and subcloned into bacteria. The positive clones were selected and subjected to DNA methylation analysis. RNA was reverse transcribed into cDNA...

Transplantation is based on a satisfactory matching of the patient and donor genes for Human Leukocyte Antigen (HLA), which increases the chance of a successful transplantation. HLA gives individual cell surface markers. The Major Histocompatibility Complex (MHC) region, encoding HLA in humans, is the most polymorphic in the human genome. The genes are located on chromosome six and consists of 200 genes. Those genes encode protein products essential for the acquired immune system. MHC molecule’s role is to represent foreign substance for B- and T-lymphocytes. MHC is an important system as it contributes to the activation of the immune system to combat viruses, bacteria, parasites and cancer cells. HLA-typing is determined through certain antigens in the HLA system. The classical transplantation antigens are HLA-A, -B, -C, -DR, -DQ and -DP. By amplifying the DNA with sequence specific primers in the Polymerase Chain Reaction (PCR), the amplicons can be detected and alleles present in the patient genome can be determined. The purpose of this study was to compare occurrence of non-specific DNA binding using master mix where Ampli Taq DNA polymerase is added and master mix with polymerase included in the PCR. Samples from 16 patients were tested with both master mix- solutions. The analyses were performed with primer plates for HLA-A, HLA-B and HLA-DRP1. The results showed that the master mix with Taq polymerase included should be applied, because it gave clearer specific band, better image quality and gave weaker and approximately 30% fewer non- specific DNA binding compared to the master mix with added Taq polymerase.

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The human Class Ib gene, HLA-E inhibits both Natural Killer Cells and a subset of CD8+ cytotoxic T lymphocytes by engaging the CD94/NKG2A inhibitory receptor. IFN-γ induces the expression of HLA-E as well as Class Ia molecules, which are required for the killing of target cells. Since HLA-E has negative effects on immune killing of target cells, we have sought to identify locus specific mechanisms of IFN-γ induction in order to identify molecular targets for selective activation of Class Ia genes, but not HLA-E. We have previously identified a unique upstream IFN-γ response region in the HLA-E promoter and showed that GATA-1 is required for its function in the K562 leukemic cell line. We have now examined the effect of GATA family members on IFN-γ induction of HLA-E in other cell types. HLA-E CAT reporter gene assays demonstrate that tumor cells that express GATA factors as determined by western blot and quantitative PCR, mediate a 2.4 to 4.0 fold enhanced response to IFN-γ stimulation. Functional constructs containing mutations of the core nucleotides in the GATA binding site had a 4.8 fold decreased response to IFN-γ in A2780 cells and a 8.5 to 14.0 fold decreased response to IFN-γ in SKOV3 cells. Knockdown of GATA-6 using siRNA resulted in a 40% decrease in HLA-E induction in Seg1 cells and a 30% decrease in HLA-E induction in HCT116 cells. Tetracycline regulated shRNA knockdown of GATA-6 expression in the SKOV3 cell line revealed a 3 fold decrease in the IFN-γ response of HLA-E reporter driven constructs. Additionally we observed a decreased IFN-γ response in SKOV3 cells transfected with siRNA specific for CBP and IRF-9. We conclude that GATA factors play a tissue specific role in regulation of IFN-γ mediated HLA-E expression and that IRF-9 may be a target for the differential manipulation of classical MHC and HLA-E.

Being a dissertation presented in fulfilment of the requirements governing the degree of Masters of Science in the Faoulty of Medicine, University of the Witwatersrand / Recent investigations have shown enhanced or aberrant expression of major histocompatibility system (MHC) antigens on cells lines derived from human hepatocellular carcinoma (HCC) in vitro and HCC in vivo. ( Abbreviation abstract ) / AC2017

Embora existam vários mecanismos propostos, o papel das moléculas de histocompatibilidade na susceptibilidade ao DM1 ainda não está totalmente esclarecido. Existem várias evidências de que o número de células apresentadoras de antígenos e a densidade de expressão das moléculas de histocompatibilidade nessas células pode influenciar o resultado da resposta imune. Assim, neste estudo, foram avaliadas as porcentagens das células CD3+, CD4+, CD8+, CD19+ e CD14+, coexpressando as moléculas de histocompatibilidade de classe I e II, a densidade de expressão das moléculas de histocompatibilidade de classe I e II nessas populações linfomonocitárias e a correlação entre densidade de expressão dessas moléculas com o perfil imunogenético do DM1. Para esse fim, foram avaliados 20 pacientes com DM1, recentemente diagnosticados, metabolicamente compensados, sendo 12 do sexo masculino. Como controles, foram avaliados 20 indivíduos saudáveis, pareados com os pacientes em termos de idade, sexo e raça, procedentes da mesma região geográfica dos pacientes. A densidade de expressão das moléculas HLA nas diversas subpopulações linfomonocitárias foi avaliada por citometria de fluxo. Os marcadores imunogenéticos foram tipados utilizando-se iniciadores de oligonucleotídeos seqüência específicos. Os resultados foram analisados usando o teste não paramétrico de Mann Whitney U. Foi observado aumento da densidade de expressão das moléculas de histocompatibilidade de classe I em linfócitos T CD3+, CD4+ e CD8+ de pacientes com DM1 quando comparados aos controles. Em relação às moléculas HLA de classe II, o número e a porcentagem dos linfócitos T CD4+, coexpressando as moléculas HLA-DQ de pacientes estavam diminuídos em relação aos controles. Os resultados referentes à correlação do perfil genotípico dos pacientes revelam que pacientes portadores dos alelos HLA-DQB1*02 apresentaram diminuição no número e porcentagem das células CD3+, CD4+, CD8+, CD19+ e CD14+ coexpressando as moléculas HLA-DQ, e ainda, o aumento da densidade de expressão da molécula HLA-DQ nas células CD19+, em relação aos pacientes sem esses alelos. Pacientes com o alelo HLA-DQB1*0302 apresentaram aumento do número de células CD14+ e CD19+ coexpressando as moléculas HLA-DQ, e ainda, o aumento da densidade de expressão dessas molécula nas células CD14+ em relação aos pacientes negativos para esse alelo. Além da instabilidade de ligação dos peptídeos com as moléculas de susceptibilidade ao DM1, este estudo reafirma a importância da densidade de expressão das moléculas de classe II na susceptibilidade ao DM1. / Althougth the role of MHC molecules in the susceptibility to DM1 has not been elucidated, the density of MHC molecules on cell surface may influence the outcome of the immune response. In this study, the number of CD3+, CD4+, CD8+, CD19+ and CD14+ cells coexpressing MHC class I and II, and the correlation between the density of MHC molecules and the immunogenetic profile of DM1 patients were studied. A total of 20 recently diagnosed patients (12 males) and 20 control individuals matched to the patients in terms of age, sex and race were studied. MHC molecules on cell sufaces were evaluated using flow cytometry. MHC aleles were typed using sequence specific probes. Statistical analysis was performed by the non-parametric Mann Whitney-U test. Increased expression of MHC class I molecules was observed in patients T CD3+, CD4+ and CD8+ cells in relation to controls. The number and porcentage of double-positive CD4+/HLA-DQ+ cells were significantly decreased in patients. Compared to DM1 patients who were not typed as HLA-DQB1*02, DM1 patients typed as HLA-DQB1*02 exhibited decreased numbers of CD3+, CD4+, CD8+, CD19+ and CD14+ cells expressing HLA-DQ molecules, whereas the density of HLA-molecules was increased in CD19+ cells. Compared to non-HLA-DQB1*0302 patients, those typed as HLA-DQB1*0302 presented increased number of CD14+ and CD19+ cells expressing HLA-DQ molecules. Besides the instability of peptide ligation with susceptibility molecules, this study stresses the relevance of the density of MHC class II molecules on the susceptibility to DM1.

O presente estudo avaliou a ocorrência de imunoparalisia e sua associação com pior prognóstico em pacientes pediátricos internados em uma UTI de hospital terciário. Para determinar a presença de imunoparalisia procedeu-se a dosagem da expressão de mHLA-DR usando o QuantiBRITE TM Anti HLA-DR/ Anti- Monocyte, um novo reagente que padroniza os valores da citometria de fluxo para o mHLA-DR. Determinamos a expressão de mHLA-DR em 30 pacientes com sepse grave ou choque sépticos admitidos na UTI Pediátrica no período do estudo, mHLA-DR foi quantificado por duas vezes: entre os dias 3 a 5 (mHLA-DR1) e 5 a 7 (mHLA-DR2) após o inicio do quadro séptico. Também foi calculado o deltamHLA-DR (mHLA-DR2 - mHLA-DR1). Dosamos, ainda, o mHLA-DR em vinte e um controles hígidos. O objetivo do estudo foi determinar se a expressão de mHLA-DR correlaciona-se com a mortalidade em pacientes sépticos pediátricos. Os resultados mostram que o mHLA-DR foi significativamente menor nos pacientes sépticos do que nos controles (p = 0.0001). A mortalidade foi de 46% nos pacientes com valores negativos ou < 1000 mAb/cell de deltaHLA-DR, e 7% em pacientes com valores positivos ou > 1000 mAb/cell de deltaHLADR. O deltamHLA-DR médio foi significativamente diferente entre sobreviventes e pacientes que foram a óbito (p = 0.023). Dessa forma, após a análise estatística dos resultados concluímos que o deltaHLA-DR correlaciona-se com a mortalidade em pacientes pediátricos com sepse grave e choque séptico / This study analysis the presence of Immunoparalysis and its association with prognosis in pediatric septic patients of a Tertiary Intensive Care Unit. To determine the presence of immunoparalysis we performed the mHLA-DR dosage using the QuantiBRITE TM Anti HLA-DR/ Anti- Monocyte, a novel reagent that standardizes flow cytometry values. We determined mHLA-DR expression in 30 patients with severe sepsis or septic shock admitted to PICU, mHLA-DR expression was quantified between days 3-5 and 5-7 after the onset of sepsis and calculated the deltamHLA-DR (mHLA-DR2 - mHLADR1). We also measured mHLA-DR levels in twenty-one healthy patients. The objective of this study was to determine if mHLA-DR values correlate with mortality in pediatric septic patients. The results showed that the mean mHLA-DR expression was significantly lower in septic patients compared with controls (p = 0.0001). Mortality was 46% in patients with negative deltaHLA-DR or < 1000 mAb/cell and 7% in patients with positive deltaHLA-DR or > 1000 mAb/cell. Mean deltamHLA-DR levels were significantly different between survivors and non-survivors (p = 0.023). After statistical analysis we concluded that deltaHLA-DR correlates with mortality in pediatric patients with septic shock or severe sepsis

Orientador: Sofia Rocha Lieber / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-23T05:57:47Z (GMT). No. of bitstreams: 1 Beck_SandraTrevisan_M.pdf: 8682564 bytes, checksum: 567844e2e230e3234538dc17848ea7b0 (MD5) Previous issue date: 1998 / Resumo: Convencionalmente, a sensibilização aos antígenos HLA é definida pelo nível de reatividade do soro do paciente contra painel de linfócitos de pelo menos 30 indivíduos HLA distintos, empregando-se o método de citotoxicidade dependente de complemento (COC). A necessidade de obtenção de linfócitos viáveis torna o método difícil de ser processado num curto espaço de tempo. O presente trabalho teve por objetivos: a padronização do método CELlSA, passível de automação, utilizando-se suspensões desidratadas de linfócitos T, armazenadas em microplacas a -20°C; e a avaliação de sua sensibilidade e especificidade em relação ao método de COC e potencializado com antigamaglobulina humana (COC-AGH). Partindo de soros controles HLA-positivos e HLA-negativos, foram avaliados os seguintes pontos: tipo de placas poliestireno disponíveis no mercado; tampões bloqueadores de sítios inespecíficos; concentração do conjugado e métodos de tratamento do soro, descritos a seguir. Com o uso de solução bloqueadora constituída de tampão TRIS/HCL (pH 7,6), 0,1% de Triton-X-100, 1% de soro de coelho e 3% de leite em pó desnatado, os três tipos de placas avaliadas mostraram resultados semelhantes. Recomenda-se portanto, o uso de SX104 linfócitos por escavação (SOµ I de 1X106/PBS), devido ao fato de que concentrações maiores resultaram em aumento de reações inespecíficas e concentrações menores diminuíram a sensibilidade do teste. Antes da realização dos testes, as amostras de soro devem ser previamente congeladas, posteriormente tratadas com trombina e centrifugadas, pois soros de alguns pacientes com desordem de coagulação, apresentam restos de fibrina, mesmo após a retração do coágulo, levando a resultados falsos-positivos. A comparação de 226 provas cruzadas revelou 75,2 % de concordância entre os métodos CELlSA e COC e 81,4% entre os métodos CELlSA e COC-AGH, utilizando-se soros de gestantes no primeiro trimestre de gestação e pacientes renais crônicos politransfundidos. Em relação à boa ou à má evolução do enxerto renal, não foi possível avaliar a relevância de aloanticorpos detectados pelos métodos CELlSA ou COC-AGH, devido ao pequeno número de pacientes transplantados, incluídos no estudo. Finalmente conclui-se que o método de CELlSA se mostrou útil para a pesquisa de anticorpos anti-linfocitários, com sensibilidade compatível com o método COC potencializado por AGH. Entretanto, anticorpos da classe IgM ou de baixa avidade não seriam detectados pelo método CELlSA. Este fato, porém, não invalida o objetivo proposto, de determinação do nível de RCP na amostra pré-transplante. O método também pode ser empregado, rotineiramente, para acompanhar a evolução da RCP em amostras de soro de pacientes renais crônicos, colhidas ao longo do tempo. Quando não for possível a caracterização das especificidades, recomenda-se a realização da RCP pelo método COC-AGH, que, embora mais trabalhoso e dependente de células viáveis, é, como o CELlSA, nitidamente mais sensível que o método COC clássico / Abstract: Conventionally, HLA allosensitization is defined by the reactivity levei of the patient's serum against a panel of Iymphocytes of at least 30 distinct HLA individuais (RCP), by the classic complement Iymphocytotoxicity assay (COC). This method is difficult to process in a short period of time due to the need of obtainig viable Iymphocytes. The aim of the presente work was to standardize the CELlSA method, with possible automation, using dehydrated suspensions of T Iymphocytes, stored on microplates at -200 and to evaluate their sensitivity and specificity for COC and potencialized with human antiglobulin (COC-AHG). Based on HLA positive and HLA negative control sera, the following topics were evaluated: types of microtiter plates available on market, blocking buffers for undetermined sites, conjugate concentration and serum treatment methods. When a blocking solution composed of TRIS-HCL(pH 7,6), 0,1% Triton-x-100, 1% rabbit serum and 3% non fat milk powder, was used the tree types of plates which were evaluated presented similar results. We recommend the use of 5x104 Iymphocytes per well (SOµ of 1x1061PBS), as higher concentration resulted in an increase in non specific reactions and lower concentration a decrease the sensibility of the test. Sefore the tests are carried out, the serum samples should be frozen, and afterwards treated with thrombin and centrifuged, due to the fact that serum of some patients with coagulation disorders may present fibrin clots even after clot retraction, and this could lead to false positive results. The comparison of 226 crosmatches tests revealed that there was a concordance of 75,2% between CELlSA and COC methods and a of 81,4% between CELlSA and COC-AHG methods, using the serum of pegnant women in the first trimester and of poli-transfused chronic renal patients. It was not possible to evaluate the relevance of the all oantibodies detected by CELlSA and COG.AGH methods in graft outcome due to the small number of transplanted patients included in this stud. Finally we conclude that the CELlSA method was useful in the detection of Iymphocyte alloantibodies, presenting an equivalent sensitivity to the potencilized COC method (AHG protocol). However IgM class antibody or low affinity antibodies would not be detected by the CELlSA method, this fact though, does not invalidate the purpose of this work, which was the determination of the RCP level in pre-transplantation samples. The method can be used, routinely, to follow-up the evolution of RCP in the serum samples of chronic renal patients, collected at different times. When the identification of HLA specificities is not possible, we recommend to perform RCP using the COG.AHG method, which in spite of being laborious and dependent on viable cells, is, as CELlSA , clearly more sensitive than the classic COC method / Mestrado / Fisiologia / Mestre em Ciências Biológicas