Thesis (M.Sc.)--Memorial University of Newfoundland, 2002. / Restricted until June 2003. Bibliography: leaves 189-214.

Thesis (M.Sc.)--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 Master of Science in Experimental Medicine, Department of Medicine. Title from pdf file main screen (viewed on April 24 2010). Includes bibliographical references.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 115-153.

Τα MHC τάξης I πεπτίδια περιορισμένα μόνο σε καρκινικό ιστό, τα οποία παράγονται από πρωτεΐνες του ίδιου του οργανισμού υπερεκφραζόμενες από μία μεγάλη ποικιλία ανθρώπινων κυττάρων νεοπλασιών, είναι δυνητικοί στόχοι για CTL (κυτταροτοξικά Τ λεμφοκύτταρα - Cytotoxic T lymphocyte), στα οποία βασίζεται η ανοσοθεραπεία πολλών ειδών καρκίνου. Ωστόσο, δεδομένου ότι παρουσιάζονται και από τα φυσιολογικά κύτταρα μπορεί να αναπτυχθεί επίσης θυμική και / ή περιφερική ανεκτικότητα. Η αυτό-ανοχή αφορά Τ λεμφοκύτταρα ειδικά για πεπτίδια υψηλής συγγένειας προς το MHC. Αντίθετα, τα Τ κύτταρα που αντιδρούν με όγκους, κατευθύνεται εναντίον πεπτιδίων όγκων με χαμηλή συγγένεια προς το MHC. Το μεγαλύτερο εμπόδιο για τη χρήση τέτοιων πεπτιδίων για ανοσο¬θεραπεία, είναι ότι αυτά είναι ασθενώς ή καθόλου ανοσογόνα. Αυτό οφείλεται στην αδυναμία τους να σχηματίζουν μεγάλο αριθμό σταθερών συμπλοκών MHC/πεπτιδίων στην επιφάνεια των ACP (κύτταρα που παρουσιάζουν αντιγόνα, Antigen Presenting Cells). Στην παρούσα ερ¬γασία περιγράφουμε μία προσέγγιση η οποία επιτρέπει την επαγωγή CTL απόκρισης έναντι χαμηλής συγγένειας HLA Α2.1 περιορισμένων πεπτιδίων. Αυτή συνίσταται στην τροποποίηση της πεπτιδικής αλληλουχίας με την εισαγωγή στη θέση 1 μίας Tyr, η οποία ευνοεί την αλληλεπίδρα¬ση πεπτιδίου/HLA Α2.1. Χρησιμοποιήθηκαν 25 πεπτίδια με διάφορες συγγένειες και ικανότητες σταθεροποίησης με το HLA Α2.1. Η αξιολόγηση της ικανότητα τους να επάγουν CTL απόκριση έδειξε μία αυστηρή συσχέτιση μεταξύ της συγγένειας σύζευξης/ικανότητας σταθεροποίησης και της ανοσογονικότητας. Τα ανάλογά τους με Ρ1Tyr παρουσίασαν υψηλότερη συγγένεια 1,5 έως 55,5 φορές και σταθεροποίησαν το HLA Α2.1 για τουλάχιστον 2 ώρες περισσότερο από τα α¬ντίστοιχα φυσικά πεπτίδια. Επιπλέον, η Ρ1Tyr τροποποίηση δεν μετέβαλε την αντιγονική ειδικό¬τητα των πεπτιδίων, αφού τα ανάλογα με Ρ1Tyr πάντα αναγνωρίζονταν από ειδικά CTL για τα φυσικά πεπτίδια και αντιστρόφως, ειδικά CTL για τα Ρ1Tyr ανάλογα αναγνώρισαν τα φυσικά πεπτίδια. Τελικά, όπως ήταν αναμενόμενο, τα Ρ1Tyr ανάλογα των χαμηλής συγγένειας με το HLA Α2.1 πεπτιδίων νεοπλασιών ενεργοποίησαν αποτελεσματικά in vivo απόκριση ειδικών CTL για τα φυσικά πεπτίδια. / Induction of CTL response to HLA A2.1 restricted tumor peptides exhibiting a low MHC binding affinity and a weak immunogenicity. MHC class I restricted peptides derived from self-proteins overexpressed by a wide variety of human tumor cells are potential targets for a broad spectrum CTL based immunotherapy in cancer. However, since they are presented by normal cells they may also represent thymic and/or peripheral tolerogens. Self-tolerance principally concerns high MHC/affinity peptide specific T cells and the tumor reactive T cells repertoire that is available to be mobilized by peptide based tumor vaccination protocols is directed against low MHC/affinity tumor peptides. A major barrier to the use of low MHC/affinity tumor peptide for immunotherapy is that they are weakly or even not immunogenic. This is due to their inability to form a high number of stable MHC /peptide complexes on the surface of antigen presenting cells. In the present work we describe an approach that allows the induction of a CTL response against low affinity HLA A2.1 restricted tumor peptides. It consisted in modifying the peptide sequence by introducing a Y in the first position known to have a favorable effect in the peptide/HLA A2.1 interaction. Twenty five peptides with variable HLA A2.1 stabilization capacity are included in this study and the evaluation of their capacity to induce a CTL response in a HLA A2.1 transgenic, Db-/-, mb2m-/- mouse showed a strict correlation between MHC binding affinity/MHC stabilization capacity and immunogenicity. Their P1Y variants exhibited a 1.5 to > 55 fold higher affinity and stabilized the HLA A2.1 for at least 2 hrs more than the corresponding native peptides. These effects were more pronounced for the low affinity native peptides. Moreover, P1Y modification did not alter the antigenic specificity of peptides since P1Y variants were always recognized by the native peptide specific CTL and, inversely P1Y specific CTL recognized native peptides. Finally, as expected, P1Y variants of low HLA A2.1 affinity tumor peptides efficiently triggered in vivo a native peptide specific CTL response.

There is an urgent need for novel preventive and therapeutic strategies for graft versus host disease (GvHD) occurring after allogeneic hematopoietic cell transplantation (allo-HCT). T-cell-based immunotherapies have been developed, however there are still some hurdles for the use of currently availably regulatory T-cells in clinical practice (naturally occurring FOXP3 + nTregs and inducible regulatory T cells), mainly owing to the lack of specific cell surface markers. The hypomethylating agent azacytidine (5-aza-dC) has been shown to generate immunoregulatory T-cells ex vivo. Interestingly, it has been shown that genes other than FOXP3 are responsible for the suppressor function of 5-aza-dC induced T-regs. HLA-G is a surface molecule with potent immunoregulatory functions which is normally expressed during pregnancy protecting the “semi-allogeneic” fetus from maternal immune attack and then is epigenetically repressed. The aim of this study was the induction of HLA-G expression in T-lymphocytes with the use of the demethylating agent 5-Aza-dC and investigation of their possible immunoregulatory properties. Our results showed that short in vitro treatment of peripheral blood T-cells with 5-aza-dC induces HLA-G expression and, more importantly, these induced HLA-G + T-cells could suppress lymphoproliferation when added as third party cells in mixed lymphocyte cultures. This suppression seems to be reduced after HLA-G neutralization and cell-to-cell contact independent. Furthermore, these induced HLA-G + T-cells show a reduced proliferation to allogeneic stimuli. Taken together, our results indicate the ex vivo production of HLA-Gpos T-lymphocytes with immunoregulatory properties. Our long term goal is the use of this population as adoptive cellular therapy for GvHD and other T-cell mediated diseases. / -

Orientadora : Profª Drª Maria da Graça Bicalho / Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Genética. Defesa: Curitiba, 27/02/2012 / Inclui bibliografia / Resumo: O cancer cervical (CC) e o segundo cancer que mais afeta mulheres no mundo todo, ele progride a partir de lesoes no cervix uterino causadas pelo virus HPV, estas lesoes sao denominadas neoplasias intraepiteliais cervicais (NIC). As NICs são classificadas em lesao e baixo grau (NIC I) e lesoes de alto grau (NIC II e III). O desenvolvimento do CC depende da persistencia do virus no organismo e esta persistencia esta diretamente relacionada com a eficiencia da resposta imune contra as celulas infectadas. O antigeno leucocitario humano (HLA)-G e um gene HLA de classe I nao classico que apresenta um papel imunorregulatorio. Ele e capaz de inibir a resposta imune quando interage com receptores inibitorios presentes nas celulas Natural Killer e em Linfocitos T. A proteina HLA-G pode ser expressa na forma ligada a membrana celular, ou soluvel (sHLA-G). A expressao de HLA-G ja foi relacionada com diversas patologias e diferentes tipos de canceres como uma estrategia de escape do sistema imune. Neste estudo o objetivo foi investigar a associacao de polimorfismos do gene HLA-G e sua expressao soluvel com o desenvolvimento de lesoes cervicais. A amostra constitui de 100 mulheres diagnosticadas com NIC II, 101 NIC III, e 106 mulheres saudaveis, negativas para NIC. Foi feito a tipagem alelicas para o gene HLA-G e para o polimorfismo de 14 pb presente no exon 8 deste gene; alem disso foi realizada a quantificacao serica de sHLA-G. Nao foi registrada associacao entre a frequencia dos alelos identificados na amostra, ou do polimorfismo de 14 pb e a quantificacao de sHLA-G com predisposicao ou protecao ao desenvolvimento de lesoes cervicais. Tambem nao foi possivel definir associacoes entre os niveis sericos de sHLA-G com os alelos, ou com a presenca ou ausencia dos 14 pb. Palavras chave: HLA-G, sHLA-G, HPV, NIC e cancer cervical. / Abstract: Cervical cancer (CC) is the second cancer that most affects women worldwide, it progresses from lesions in the uterine cervix caused by HPV virus, these lesions are called cervical intraepithelial neoplasia (CIN). CINs are classified in low-grade (CIN I) and high-grade lesions (CIN II and III). The development of the CC depends on the persistence of the virus in the body and this persistence is directly related to the efficiency of the immune response against infected cells. Human leukocyte antigen (HLA)-G is an HLA class I non-classic that presents an immunoregulatory role. It is able to inhibit the immune response when interacting with inhibitory receptors present on natural killer cells and T lymphocytes The HLA-G protein can be expressed in the membrane bound form, or soluble (sHLA-G). The expression of HLA-G has been associated with several diseases and different types of cancers as a strategy to escape from the immune system. In this study the aim was to investigate the association of polymorphisms of the HLA-G gene and its soluble expression with the development of cervical lesions. The sample consisted of 100 women diagnosed with CIN II, 101 with CIN III, and 106 healthy women negative for CIN. Allele typing for the gene HLA-G and for the 14 bp polymorphism present in exon 8 of this gene was done, also serum sHLA-G was quantified. No association was recorded between the frequency of the alleles identified in the sample, or 14 bp polymorphism and quantification of sHLA-G with predisposition or protection to the development of cervical lesions. Nor was it possible to define associations between serum levels of sHLA-G with the alleles, or the presence or absence of 14 bp. Keywords: HLA-G, sHLA-G, HPV, NIC and cervical cancer.

Introdução e Objetivos: A maior prevalência de doença celíaca (DC) em indivíduos com diabetes mellitus tipo I (DM1) já é reconhecida. Ambas as doenças tem causa autoimune, em que os genes HLA classe 2 representam o principal fator genético de risco. Porém, existe uma considerável parcela da população que não manifesta tais doenças e são portadores desses genes. Estudo de associação genômica (GWAS) identificaram polimorfismos de susceptibilidade às duas doenças em genes diferentes do sistema HLA, que poderão auxiliar na compreensão da causa e das suas variabilidades clínicas. Os objetivos desse estudo foram avaliar as frequências dos polimorfismos HLA e não HLA em pessoas como DM1 e com DC e relacionar esses dados com a ocorrência de sintomas gastrointestinais, com a idade do diagnóstico da DM1 e com história alimentar. Métodos: Delineamento transversal, com avaliações retrospectivas e prospectivas, em pessoas com DM1 com e sem DC. Foram realizadas entrevista e revisão de prontuário dos pessoas, seguido de coleta de sangue ou saliva. A pesquisa dos genes RGS1, IL2-IL21, BACH2, TLR7/TLR8 e IL18RAP foi realizada por PCR Real-Time. Os alelos DQA1* 0501 e DQB1* 0201 para DQ2.5 e o alelo DQB1*0302 para DQ8 foram identificados a partir da técnica de genotipagem de HLA Tag-single-nuleotide polymorphism (Tag SNP). Resultados: As frequências alélicas e genotípicas entre 273 pessoas com DM1 sem DC e 39 pessoas com DM1 e DC não apresentaram diferença significativa. A presença de sintoma gastrointestinal foi mais frequente nos portadores dos polimorfismos dos genes RGS1 e IL18RAP. O tempo de aleitamento materno, a idade de introdução do glúten e a idade do diagnóstico da DM1 foram semelhantes entre os grupos. A comparação dos cinco polimorfismos com a combinação dos haplótipos para DQ2.5 e DQ8 não apresentou diferença significativa. Nos 312 indivíduos, com DM1 com e sem DC e nos 66 indivíduos portadores de DC sem DM1 foi identificado alelos DQ2.5 e ou DQ8 em 97% dos casos, enquanto que nos indivíduos com DC sem DM1 identificou-se em 76% dos casos. DQ2.5 foi mais frequente entre pessoascom DC e DQ8 foi mais frequentes entre pessoas com DM1. Conclusões: A presença dos polimorfismos dos genes estudados não modificou a chance do indivíduo com DM1 ter ou não DC. Houve associação dos genes RGS1 e IL18RAP com sintomas gastrointestinais. A pesquisa dos alelos DQ2.5 e DQ8, pela técnica Tag-SNP, permitiu determinar um alto valor preditivo negativo no diagnóstico de DC na população com DM1 e com DC, semelhante ao descrito na literatura com a técnica convencional. / Introduction and Objectives: The higher prevalence of celiac disease (CD) in individuals with diabetes mellitus type I (T1D) is already recognized. Both diseases have autoimmune cause, where HLA genes class 2 represent the major genetic risk factor. However, there is a considerable portion of the population that does not manifest such diseases and are carriers of these genes. Genome-wide association studies (GWAS) have identified susceptibility polymorphisms to both diseases in different genes of the HLA system that may assist in understanding the etiology and in its clinical variabilities. The objectives of this study were to evaluate the frequencies of HLA and non-HLA polymorphisms in patients with T1D and CD, related to the occurrence of gastrointestinal symptoms, the age of diagnosis of T1D and food history. Methods: Mixed design with retrospective and prospective evaluations in patients with T1D with and without DC. They were conducted interview and review of medical records of patients, followed by collecting blood or saliva. The search for genes RGS1, IL21-IL2, BACH2, TLR7 / TLR8 and IL18RAP was performed by Real-Time PCR. The alleles DQA1 * 0501 and DQB1 * 0201 for DQ2.5 and DQB1 * 0302 for DQ8 were identified from the Tag-single-nucleotide polymorphism (tag SNP) genotyping HLA technique Results: The allelic and genotypic frequencies between 273 T1D patients without CD and 39 patients with T1D and CD showed no significant difference. The presence of gastrointestinal symptoms were more frequent in patients with polymorphisms of genes RGS1 and IL18RAP. The duration of breastfeeding, the age of introduction of gluten and the age of diagnosis of T1D were similar between the groups. The comparison of the five polymorphisms with the combination of haplotypes for DQ2.5 and DQ8 showed no significant difference. In 312 individuals with DM1 with and without CD and 66 individuals with CD without T1D was identified alleles DQ2.5 and/or DQ8 in 97% of cases, whereas in individuals with CD without T1D was identified in 76% of cases . DQ2.5 was more frequent among patients with CD and DQ8 was more frequent among patients with T1D Conclusions: The presence of polymorphisms of genes studied did not modify the chance of T1D whether or not DC. There was an association of RGS1 and IL18RAP genes with gastrointestinal symptoms. The survey of DQ2.5 and DQ8 alleles by Tag-SNP technique allowed determining a high negative predictive value in the diagnosis of CD in the population of patients with T1D and DC, similar to that described in the literature with the conventional technique.

A person is exposed to many pathogens throughout their lifetime, and with the resolution of each infection, there remains a pool of pathogen-specific immune cells that protect that person from re-infection with the same pathogen. However, there is a great deal of evidence to suggest that the pool of pathogen-specific memory cells can also participate in the immune response to future infections with unrelated pathogens. Many believe T cells to be cross-reactive in nature because of their interaction with self antigens during development in the thymus and their interaction with foreign antigens once in the periphery. There are many features of the interaction between a T cell and its ligand that facilitate this cross-reactive nature. Based on solved crystal structures, relatively few contacts are required for a stable interaction, and that interaction is often mediated by the flexible CDR3 loops of the T cell receptor that accommodate ligands of various structure. There is also evidence in the murine and human systems that subsets of virus-specific memory CD8 T cells take on an activated phenotype upon infection with an unrelated virus. In murine models, these memory T cell subsets could kill target cells, secrete several cytokines, and proliferate in response to a cross-reactive stimulation, suggesting that a cross-reactive T cell response could impact the outcome of a viral infection. In fact, upon heterologous infection, mice immune to a previous virus were often protected, having lower titers of the second unrelated virus, their epitope-specific and T cell receptor repertoires were often skewed, and they were more prone to immune-mediated pathologies. All of these observations coincided with the presence of cross-reactive T cell responses. Thus, we define heterologous immunity as changes in viral replication and the disease pathology associated with that viral infection as a result of the host's history of infection, and this can be mediated, in part, by cross-reactive CD8 T cell responses. Since many human viral infections are associated with a wide range of disease states, we questioned whether cross-reactive CD8 T cell responses occurred as commonly as they appeared to occur in the murine models and whether they influenced the outcome of such infections. Epstein-Barr virus (EBV) infects over 90% of the U. S. population and has a large genome with the capacity to encode a multitude of T cell epitopes. The first part of this thesis research focuses on the identification of cross-reactive CD8 T cell responses with specificity for known epitopes derived from EBV, a common human virus. We directed our study to HLA-A2-restricted responses because of the common expression of this MHC Class I allele in the U. S. population. This study resulted in the detection of cross-reactive responses with five different specificities that involved either the immunodominant lytic EBV-BMLF1280 epitope or the latent EBNA 3A596epitope. Three of the cross-reactive responses had specificity for epitopes derived from another unrelated, but common, human virus, influenza A virus (IV). Each of these cross- reactive responses had the potential to participate in the collective immune response to acute EBV infection. EBV is also well-suited as a model system to study heterologous immunity in humans, as infection at an early age is frequently asymptomatic, while the same infection during adolescence often results in an immune-mediated syndrome, infectious mononucleosis (IM). Since older individuals have presumably been exposed to more pathogens in their lifetime and, therefore, would have memory CD8 T cell pools with more extensive specificities, we hypothesized that acute EBV infection activated cross-reactive memory CD8 T cell responses that promoted the development of IM. In order to determine if the cross-reactive responses we identified above contributed to the immune response to acute EBV infection, we first screened the blood of IM patients for cross-reactive T cells with specificity for EBV-BMLFl280 and IV-M158. The total number of M1-specific T cells of 5 of 8 patients was increased at presentation with IM, which was suggestive of their specific activation during the EBV infection since a bystander mechanism would have resulted in 8 out of 8 patients having increased numbers of M1-specific T cells. Our hypothesis was further supported by the fact that we clearly detected cross-reactive T cells capable of recognizing both BMLF1 and M1 epitopes in the blood of 2 of the 5 IM patients with an augmented M1-specific T cell frequency. Furthermore, the M1-specific TCR repertoires of those two patients were dramatically skewed, which was an indication of cross-reactive M1-specific T cell expansions and, therefore, participation in the lymphoproliferation characteristic of IM. In addition, T cell lines derived from 3 out of 8 healthy donors with previous exposure to both viruses contained a subset of T cells that responded to both BMLF1 and M1 epitopes, suggesting that these cross-reactive cells are often maintained in memory. These cross-reactive T cells were cytotoxic and produced MIP-1β, IFNγ, and TNFα, functions which could potentially promote the symptoms of IM and, indeed, may have been contributed to the severe case of IM noted in one patient. The final part of this thesis research focused on defining the structure of the cross-reactive TCR that recognized both BMLF1 and M1 epitopes, which have only 33% sequence similarity. In addition, we examined the cross-reactive TCR repertoire organization of multiple individuals to determine the breath and, therefore, the likelihood that this cross-reactive T cell response will occur. These studies revealed that a wide range of Vα and Vβ families can mediate interaction with both epitopes and that the cross-reactive TCR repertoire was unique to each individual, relying heavily on the T cell clones present in that individual's private BMLF1- and M1-specific repertoires. We also observed an increased frequency of TCRs with longer CDR3 regions within the cross-reactive repertoire, which were often extended by non-bulky amino acid residues that could provide these TCRs with more flexibility in order. to accommodate the two different epitope structures. Given that we detected a cross-reactive T cell response with specificity for two immunodominant epitopes derived from two of the most common human viruses among people that share one of the most common MHC Class I alleles in the U. S. population, we predict that cross-reactive T cells are common components of human immune responses. The variability in the magnitude and specificity of each cross-reactive T cell response is dependent on each individual's unique history of infection and th,eir unique TCR repertoire, and such responses likely represent one of many factors that could explain the individual variability in disease severity associated with EBV and many other human viral infections.

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Belo Horizonte, MG, Brasil / A Duffy binding protein do Plasmodium vivax (PvDBP) é uma proteína essencial para o processo de invasão do Plasmodium vivax em eritrócitos humanos Duffy/DARC positivos, sendo consequentemente uma forte candidata à vacina antimalárica. Apesar disso, estudos desenvolvidos pelo nosso grupo de pesquisa mostraram que a maioria dos indivíduos expostos à malária na Amazônia brasileira não desenvolvem anticorpos anti-PvDBP. Isto pode estar relacionado tanto a características do parasito quanto do hospedeiro vertebrado. Entre as características do parasito estão a baixa imunogenicidade da PvDBP ao sistema imune e o alto polimorfismo na região do ligante (região II). Entretanto, estas características do parasito não explicam o fato de que a maioria dos indivíduos expostos a diferentes variantes do parasito, por um longo período de tempo, não desenvolvem anticorpos bloqueadores da interação ligante-receptor. Diante disso, faz-se necessário avaliar características do hospedeiro vertebrado que poderiam influenciar nessa baixa resposta de anticorpos. Portanto, o presente trabalho teve como objetivo avaliar a influência de polimorfismos do receptor DARC e do HLA classe II (antígeno leucocitário humano) na resposta imune anti-PvDBP. O estudo foi do tipo coorte aberta de base populacional realizado em área de assentamento agrícola da Amazônia brasileira onde os indivíduos foram acompanhados por cerca de 12 meses. A abordagem metodológica envolveu: (i) genotipar o receptor Duffy/DARC (Real time PCR) e o HLA de classe II (locis HLA-DRB1, HLA-DQB1 e HLA-DQA1, por PCR-SSO) na população estudada (n=620); (ii) realizar ensaios sorológicos, convencionais (ELISA) e funcionais (bloqueio da interação DBPII-DARC) no plasma dos indivíduos estudados. Os resultados mostram que, na área estudada, o genótipo de DARC mais frequente foi FY*A/FY*B, o que foi consistente com o que tem sido descrito para as populações residentes na Amazônia brasileira. Em relação à resposta de anticorpos, nenhuma associação significativa foi encontrada entre os genótipos de DARC e as respostas de anticorpos IgG no ELISA (região II ou regiões II-IV da PVDBP). Contudo, a resposta de anticorpos que inibem a interação ligante (DBPII) - receptor (DARC) foi significativamente mais frequente em indivíduos heterozigotos carreadores de um alelo DARC positivo (genótipos FY*A/FY*BES e FY*B/FY*BES). Em geral, a distribuição de frequência dos alelos de HLA classe II na população estudada corrobora com as frequências já descritas em estudos anteriores realizados no Brasil e na América Latina. De interesse, o estudo permitiu identificar 11 alelos de HLA classe II associados positivamente com a resposta de anticorpos anti-PvDBP detectados no ELISA, com destaque para os alelos DQA1*01:03 e HLA-DRB1*02:02. Por outro lado, o estudo permitiu identificar 4 alelos associados negativamente com a resposta de anticorpos. Além disso, quatro alelos - HLADQA1* 02:01, HLA-DRB1*07:01, HLA-DQB1*02:02 e HLA-DQA1*02:01 - parecem influenciar positivamente na resposta de anticorpos inibitórios da interação DBPII-DARC, enquanto que o alelo HLA-DRB1*16:02 influenciou negativamente nesta resposta. Estes achados são de grande importância, pois, em áreas hiperendêmicas de malária, a resposta de anticorpos inibitórios tem sido associada com proteção clínica. Em conjunto, os resultados do presente estudo permitiram demonstrar, pela primeira vez, que polimorfismos do receptor DARC e do HLA classe II podem influenciar na resposta imune protetora contra a PvDBP. / The Duffy binding protein of Plasmodium vivax (PvDBP) is a critical adhesion ligand that participates in merozoite invasion of human Duffy/DARC positive erythrocytes, being considered, therefore, a target for vaccine-mediated immunity. Although anti-PvDBP immune responses have been well characterized, few studies have investigated the influence of host variability on the development of this immune response. In the present study, we examined whether host genetic polymorphisms might affect humoral immunity against PvDBP. More specifically, we investigated polymorphisms on the DARC receptor and in the human leucocyte antigen (HLA) class II. For that, we carried out a 12-months follow-up study in an agricultural settlement of the Brazilian Amazon region (n=620 volunteers). The methodological approach included (i) to genotype the DARC receptor (Real-time PCR), and the HLA class II (loci HLA-DRB1, HLA-DQB1 and HLADQA1 by PCR-SSO); (ii) to analyze the plasma levels of PvDBP antibodies through conventional serology (ELISA-detected IgG antibodies) and inhibitory binding assays (PvDBP binding inhibitory antibodies, BIAbs). The results showed no association between DARC polymorphisms and ELISA–detected antibodies. However, the follow-up study demonstrated that BIAbs targeting PvDBP towards to be more frequent in heterozygous carrying a DARC-silent allele (genotypes FY*A/FY*BES and FY*B/FY*BES), suggesting a gene-dosage effect. Regarding the HLA class II polymorphisms, we identified 11 alleles positively associated with the response of PvDBP ELISA-detected antibodies. In addition, the study revealed four alleles negatively associated with the antibody response (HLA-DRB1*10:01, HLA-DRB*14:02, DRB1*14:02 e DQA1*05:01 citar os 4 aqui). Of relevance, four alleles (HLA-DQA1*02:01, HLA-DRB1*07:01, HLADQB1* 02:02 and HLA-DQA1*02:01) were positively associated with the PvDBP BIAb, whereas a single allele (HLA-DRB1*16:02) was negatively associated with this protective response. Together, the results of the present study demonstrate, for the first time, that polymorphisms on the DARC receptor and in the HLA class II may influence the protective immune response against PvDBP.

Introdução e Objetivos: A maior prevalência de doença celíaca (DC) em indivíduos com diabetes mellitus tipo I (DM1) já é reconhecida. Ambas as doenças tem causa autoimune, em que os genes HLA classe 2 representam o principal fator genético de risco. Porém, existe uma considerável parcela da população que não manifesta tais doenças e são portadores desses genes. Estudo de associação genômica (GWAS) identificaram polimorfismos de susceptibilidade às duas doenças em genes diferentes do sistema HLA, que poderão auxiliar na compreensão da causa e das suas variabilidades clínicas. Os objetivos desse estudo foram avaliar as frequências dos polimorfismos HLA e não HLA em pessoas como DM1 e com DC e relacionar esses dados com a ocorrência de sintomas gastrointestinais, com a idade do diagnóstico da DM1 e com história alimentar. Métodos: Delineamento transversal, com avaliações retrospectivas e prospectivas, em pessoas com DM1 com e sem DC. Foram realizadas entrevista e revisão de prontuário dos pessoas, seguido de coleta de sangue ou saliva. A pesquisa dos genes RGS1, IL2-IL21, BACH2, TLR7/TLR8 e IL18RAP foi realizada por PCR Real-Time. Os alelos DQA1* 0501 e DQB1* 0201 para DQ2.5 e o alelo DQB1*0302 para DQ8 foram identificados a partir da técnica de genotipagem de HLA Tag-single-nuleotide polymorphism (Tag SNP). Resultados: As frequências alélicas e genotípicas entre 273 pessoas com DM1 sem DC e 39 pessoas com DM1 e DC não apresentaram diferença significativa. A presença de sintoma gastrointestinal foi mais frequente nos portadores dos polimorfismos dos genes RGS1 e IL18RAP. O tempo de aleitamento materno, a idade de introdução do glúten e a idade do diagnóstico da DM1 foram semelhantes entre os grupos. A comparação dos cinco polimorfismos com a combinação dos haplótipos para DQ2.5 e DQ8 não apresentou diferença significativa. Nos 312 indivíduos, com DM1 com e sem DC e nos 66 indivíduos portadores de DC sem DM1 foi identificado alelos DQ2.5 e ou DQ8 em 97% dos casos, enquanto que nos indivíduos com DC sem DM1 identificou-se em 76% dos casos. DQ2.5 foi mais frequente entre pessoascom DC e DQ8 foi mais frequentes entre pessoas com DM1. Conclusões: A presença dos polimorfismos dos genes estudados não modificou a chance do indivíduo com DM1 ter ou não DC. Houve associação dos genes RGS1 e IL18RAP com sintomas gastrointestinais. A pesquisa dos alelos DQ2.5 e DQ8, pela técnica Tag-SNP, permitiu determinar um alto valor preditivo negativo no diagnóstico de DC na população com DM1 e com DC, semelhante ao descrito na literatura com a técnica convencional. / Introduction and Objectives: The higher prevalence of celiac disease (CD) in individuals with diabetes mellitus type I (T1D) is already recognized. Both diseases have autoimmune cause, where HLA genes class 2 represent the major genetic risk factor. However, there is a considerable portion of the population that does not manifest such diseases and are carriers of these genes. Genome-wide association studies (GWAS) have identified susceptibility polymorphisms to both diseases in different genes of the HLA system that may assist in understanding the etiology and in its clinical variabilities. The objectives of this study were to evaluate the frequencies of HLA and non-HLA polymorphisms in patients with T1D and CD, related to the occurrence of gastrointestinal symptoms, the age of diagnosis of T1D and food history. Methods: Mixed design with retrospective and prospective evaluations in patients with T1D with and without DC. They were conducted interview and review of medical records of patients, followed by collecting blood or saliva. The search for genes RGS1, IL21-IL2, BACH2, TLR7 / TLR8 and IL18RAP was performed by Real-Time PCR. The alleles DQA1 * 0501 and DQB1 * 0201 for DQ2.5 and DQB1 * 0302 for DQ8 were identified from the Tag-single-nucleotide polymorphism (tag SNP) genotyping HLA technique Results: The allelic and genotypic frequencies between 273 T1D patients without CD and 39 patients with T1D and CD showed no significant difference. The presence of gastrointestinal symptoms were more frequent in patients with polymorphisms of genes RGS1 and IL18RAP. The duration of breastfeeding, the age of introduction of gluten and the age of diagnosis of T1D were similar between the groups. The comparison of the five polymorphisms with the combination of haplotypes for DQ2.5 and DQ8 showed no significant difference. In 312 individuals with DM1 with and without CD and 66 individuals with CD without T1D was identified alleles DQ2.5 and/or DQ8 in 97% of cases, whereas in individuals with CD without T1D was identified in 76% of cases . DQ2.5 was more frequent among patients with CD and DQ8 was more frequent among patients with T1D Conclusions: The presence of polymorphisms of genes studied did not modify the chance of T1D whether or not DC. There was an association of RGS1 and IL18RAP genes with gastrointestinal symptoms. The survey of DQ2.5 and DQ8 alleles by Tag-SNP technique allowed determining a high negative predictive value in the diagnosis of CD in the population of patients with T1D and DC, similar to that described in the literature with the conventional technique.