Two novel HLA-DRB1 alleles, DRB1*11:261 and DRB1*13:286 identified by sequencing in Brazilian individuals.

Two novel HLA alleles DRB1*11:261 and DRB1*13:286 have nonsynonymous mutations in exon 2.

Genetic Polymorphisms of Toll-like receptors 2 and 9 as Susceptibility Factors for the Development of Ankylosing Spondylitis and Psoriatic Arthritis.

INTRODUCTION: Ankylosing spondylitis (AS) and psoriatic arthritis (PsA) are classified as spondyloarthritis (SpA), a group of inflammatory rheumatic diseases with complex genetic etiology. Toll-like receptors (TLRs) have an important role in the mechanism of innate immunity and may influence inflammatory responses. Polymorphisms in TLR genes that lead to changes in these receptors or that interfere with the transcription rates of mRNA TLR may be involved in the chronic inflammatory immune response observed in SpA. Currently, there is a lack of studies associating genetic polymorphisms in TLRs and SpA. OBJECTIVE: Therefore, this case-control study is aimed at analyzing the influence of the respective SNPs on TLR2 rs5743708, TLR6 rs5743810, and TLR9 rs5743836 and rs187084 in the immunopathogenesis of SpA. METHODS: The polymorphisms genotyped by PCR-RFLP were TLR2 rs5743708, TLR6 rs5743810, and TLR9 rs5743836 and rs187084. The HLA-B ∗ 27 was performed by PCR-SSP. RESULTS: Logistic regression analysis showed a strong association between SNPs in TLR2 and TLR9 and susceptibility to SpA (OR = 12.56; CI = 6.5-25.9 and OR = 1.62; CI = 1.20-2.21, respectively). No association was observed among HLA-B ∗ 27 and TLR polymorphisms (p = 0.72), nor among BASDAI and TLR polymorphisms (p = 0.85). DISCUSSION: Our findings suggest that polymorphisms in TLR2 and TLR9 genes may contribute to the immunopathogenesis of the SpA. The rs187084, rs5743836, and rs5743708 polymorphisms were associated with the risk of SpA development, in this study, and lead to significant changes in the innate and adaptive immune response profile, as well as the maintenance of the regulation of immunological mechanisms. CONCLUSION: The polymorphism rs5743708 for the TLR2 and the rs187084_rs5743836 TLR9 haplotypes appear to be involved in the development of clinical forms of SpA and can be a possible therapeutic target for the spondyloarthritis.

HLA-A, -B, -DRB1, -DQA1, and -DQB1 profile in a population from southern Brazil.

The aim of this study was to determine the allele and haplotype frequencies of HLA-A, -B, -DRB1, and -DQB1 in a self-declared White population from the north and northwestern state of Paraná, southern Brazil, and compare the data with populations worldwide. The genotyping was performed with a group of 641 individuals, based on PCR-SSO and -SSP methods, and allele and haplotype frequencies were estimated. Comparisons with European, African, Asian, and Amerindian populations were performed. The most frequent allelic groups, alleles and haplotypes were: HLA-A*02, HLA-B*35, HLA-DRB1*07:01, HLA-DQB1*03:01, and HLA-A*01/B*08/DRB1*03:01. The results reinforced a predominance of a European composition in the self-declared White population from the north and northwestern Paraná.

Influence of TNF and IL17 Gene Polymorphisms on the Spondyloarthritis Immunopathogenesis, Regardless of HLA-B27, in a Brazilian Population.

BACKGROUND AND OBJECTIVES: Spondyloarthritis (SpA) represents a heterogeneous group of immune-mediated inflammatory diseases that have overlapping clinical features, genetic predisposition, and pathogenic mechanisms. Hence, we investigated, through a case-control study, whether single-nucleotide polymorphisms of TNF and IL17 genes are associated with SpA, ankylosing spondylitis (AS), and psoriatic arthritis (PsA) in a mixed Brazilian population. METHODS: Genotyping of TNF-308 (rs1800629), TNF-238 (rs361525), IL17A (rs2275913), IL17F (rs763780), and HLA-B27 polymorphisms was performed in 243 patients with SpA and 210 controls from Southern Brazil using SSOP-Luminex (One Lambda) and PCR-SSP assays. RESULTS: Significant associations were confirmed between the HLA-B27 marker and SpA, AS, and PsA diseases. While TNF-308 (rs1800629) AA/GA, IL17A (rs2275913) AA/GA, and IL17F (rs763780) CC/TC genotype frequencies were associated, in the dominance inheritance model, with SpA and AS, regardless of gender, the presence of HLA-B27, TNF-238 (rs361525) GA/AA, IL17A (rs2275913) AA/GA, and IL17F (rs763780) genotypes was associated with PsA. CONCLUSION: In this Brazilian population, TNF and IL17 gene polymorphisms responsible for the expression of important inflammatory cytokines were associated with overall SpA, and, specifically, with AS and PsA, regardless of gender and HLA-B27. However, future larger studies with different ethnicities may be necessary to confirm these genetic associations.

Molecular matching for Rh and K reduces red blood cell alloimmunisation in patients with myelodysplastic syndrome.

BACKGROUND: Matching for Rh and K antigens has been used in an attempt to reduce antibody formation in patients receiving chronic transfusions but an extended phenotype matching including Fy(a) and Jk(a) antigens has also been recommended. The aim of this study was to identify an efficient transfusion protocol of genotype matching for patients with myelodysplastic syndrome (MDS) or chronic myelomonocytic leukaemia. We also examined a possible association of HLA class II alleles with red blood cell (RBC) alloimmunisation. MATERIALS AND METHODS: We evaluated 43 patients with MDS undergoing transfusion therapy with and without antibody formation. We investigated antigen-matched RBC units for ABO, D, C, c, E, e, K, Fy(a), Fy(b), Jk(a), Jk(b), S, s, Do(a), Do(b) and Di(a) on the patients' samples and on the donor units serologically matched for them based on their ABO, Rh and K phenotypes and presence of antibodies. We also determined the frequencies of HLA-DRB1 alleles in the alloimmunised and non-alloimmunised patients. RESULTS: Seventeen of the 43 patients had discrepancies or mismatches for multiple antigens between their genotype-predicted profile and the antigen profile of the units of blood serologically matched for them. We verified that 36.8% of patients had more than one RBC alloantibody and 10.5% of patients had autoantibodies. Although we were able to find a better match for the patients in our extended genotyped/phenotyped units, we verified that matching for Rh and K would be sufficient for most of the patients. We also observed an over-representation of the HLA-DRB1*13 allele in the non-alloimmunised group of patients with MDS. DISCUSSION: In our population molecular matching for C, c, E, e, K was able to reduce RBC alloimmunisation in MDS patients. An association of HLA-DRB1*13 and protection from RBC alloimmunisation should be confirmed.

HLA-DR and HLA-DQ alleles in patients from the south of Brazil: markers for leprosy susceptibility and resistance.

BACKGROUND: Many epidemiological studies have shown that the genetic factors of the host play a role in the variability of clinical response to infection caused by M. leprae. With the purpose of identifying genes of susceptibility, the present study investigated the possible role of HLA-DRB1 and DQA1/DQB1 alleles in susceptibility to leprosy, and whether they account for the heterogeneity in immune responses observed following infection in a Southern Brazilian population. METHODS: One hundred and sixty-nine leprosy patients and 217 healthy controls were analyzed by polymerase chain reaction amplification and reverse hybridization with sequence-specific oligonucleotide probes and sequence-specific primers (One Lambda, CA, USA). RESULTS: There was a positive association of HLA-DRB1*16 (*1601 and *1602) with leprosy per se (7.3% vs. 3.2%, P = 0.01, OR = 2.52, CI = 1.26-5.01), in accord with previous serological studies, which showed DR2 as a marker of leprosy. Although, HLA-DQA1*05 frequency (29.8% vs. 20.9%, P = 0.0424, OR = 1.61, CI = 1.09-2.39) was higher in patients, and HLA-DQA1*02 (3.0% vs. 7.5%, P = 0.0392, OR = 0.39, CI = 0.16 - 0.95) and HLA-DQA1*04 (4.0% vs. 9.1%, P = 0.0314, OR = 0.42, CI = 0.19 - 0.93) frequencies lower, P-values were not significant after the Bonferroni's correction. Furthermore, HLA-DRB1*1601 (9.0% vs. 1.8%; P = 0.0016; OR = 5.81; CI = 2.05-16.46) was associated with susceptibility to borderline leprosy compared to control group, and while HLA-DRB1*08 (11.2% vs. 1.2%; P = 0.0037; OR = 12.00; CI = 1.51 - 95.12) was associated with susceptibility to lepromatous leprosy, when compared to tuberculoid leprosy, DRB1*04 was associated to protection. CONCLUSION: These data confirm the positive association of HLA-DR2 (DRB1*16) with leprosy per se, and the protector effect of DRB1*04 against lepromatous leprosy in Brazilian patients.

Otimização de metodologia PCR-SSP para identificação de polimorfismos genéticos de TNF e IL2 / Optimization of the PCR-SSP methodology in the identification of TNF and IL2 genetic polymorphisms

A análise de polimorfismos únicos de nucleotídeos (SNPs) de citocinas pode ser útil em estudos de frequências alélicas e genotípicas em populações saudáveis de diversas regiões, em estudos de associação com doenças infecciosas ou autoimunes, em estudos antropológicos e na evolução pós-transplante. Estes SNPs podem ser avaliados por diferentes métodos moleculares. O objetivo deste estudo foi aperfeiçoar uma metodologia PCR-SSP simples e rápida para a genotipagem de três SNPs de citocinas usando um único teste laboratorial. Para a identificação de IL2-330T/G e IL2+166G/T foram utilizados dois procedimentos na mesma genotipagem, cada um baseado no uso de quatro iniciadores. Para a detecção de TNF-238G/A foram utilizados dois iniciadores que amplificam a guanina e adenina na posição -238. Este estudo permitiu aperfeiçoar um método simples e rápido para identificar três SNPs de citocinas num único teste, podendo ser utilizado em qualquer laboratório de biologia molecular, como alternativa ao uso de kits de alto custo.

The analysis of cytokine single nucleotide polymorphisms (SNPs) can be useful in studies of allelic and genotypic frequencies in healthy populations from different regions of Brazil, in association studies of infectious or auto-immune diseases, in anthropological studies and in studies on post-transplant evolution. These SNPs can be assessed by different molecular methods. The objective of this study was to improve a simple and fast methodology, PCR-SSP, for the genotyping of three cytokine SNPs using a single laboratorial test. To identify IL2-330T/G and IL2+166G/T, two procedures were used in the same genotyping assay, each one based on the use of 4 primers. To detect TNF-238G/A, two primers were used that amplify guanine and adenine at position -238. This study enabled the improvement of a simple and fast method for identifying three cytokine SNPs in a single test, which can be adopted in any Molecular Biology laboratory as an alternative to the use of expensive kits.

Correlation of IL-6 and IL-10 production following bone marrow transplantation with donor cytokine gene polymorphisms / Correlação da produção de IL-6 e IL-10 seguindo o transplante de medula óssea com os polimorfismos de genes de citocinas do doador

Several candidate gene studies have demonstrated that genetic polymorphisms in cytokine genes contribute to variations in the levels of cytokines produced and this variation may influence the occurrence and severity of complications after stem cell transplantation (HSCT). In this work we compared the serum concentrations of TNF-α, IFN-γ, IL-6, IL-10, and TGF-β1 in 13 recipients following HSCT with the TNF-308, IFNG+874, IL6-174, IL10-1082,-819,-592, and TGFB1+869,+915 polymorphisms. Serum cytokine levels were assessed using commercial ELISA kits for TNF-α, IFN-γ, IL-6, IL-10, and TGF-β1 (BioSource®, Nivelles, Belgium, Europe). Donor/recipient genotypes for these cytokine polymorphisms were analyzed by polymerase chain reaction-sequence-specific primer (PCR-SSP) with the Cytokine Genotyping Primers Kit (One Lambda , Canoga Park, CA, USA). We found correlation between the levels of IL-6 and IL-10 concentrations following HSCT and the IL6-174 and IL10-1082,-819,-592 polymorphisms, but not for other cytokines investigated in this study. Those with genotypes associated with low production of IL-6 and IL-10 produced lower levels of these cytokines than those with genotypes associated with high or intermediate production of these cytokines (P < 0.05).

Estudos de vários genes candidatos têm demonstrado que polimorfismos genéticos em genes de citocinas contribuem com variações nos níveis de citocinas produzidas e esta variação pode influenciar a ocorrência e gravidade de complicações após o transplante de células-tronco hematopoéticas (TCTH). Neste trabalho comparamos as concentrações séricas de TNF-α, IFN-γ, IL-6, IL-10 e TGF-β 1 em 13 receptores seguindo o TCTH com os polimorfismos TNF-308, IFNG+874, IL6-174, IL10-1082,-819,-592 e TGFB1+869,+915. Os níveis séricos de citocinas foram medidos usando-se kits comerciais de ELISA para TNF-α, IFN-γ, IL-6, IL-10 e TGF-β 1 (BioSource®, Nivelles, Belgium, Europe). Os genótipos de doadores/receptores para estes polimorfismos de citocinas foram analisados pela reação em cadeia da polimerase com sequências específicas de primer (PCR-SSP) com o kit Cytokine Genotyping Primers (One Lambda, Canoga Park, CA, USA). Encontramos correlação entre os níveis de IL-6 e IL-10 seguindo o TCTH e os polimorfismos IL6-174 e IL10-1082,-819,-592, mas não para outras citocinas investigadas neste estudo. Aqueles com genótipos relativos à baixa produção de IL-6 e IL-10 produziram mais baixos níveis destas citocinas que aqueles com genótipos relativos à produção alta e/ou intermediária destas citocinas (P < 0,05).

Papel das citocinas na imunopatogênese da doença do enxerto contra o hospedeiro / Role of cytokines in the immunopathogenesis of Graft-versus-Host Disease

O transplante de células progenitoras hematopoéticas é o tratamento de escolha para muitas doenças hematológicas e imunodeficiências primárias. A doença do enxerto contra o hospedeiro (DECH) é ainda uma grave complicação após o transplante alogênico e a principal causa de mortalidade e morbidade. O estudo da patogênese da DECH auxilia no desenvolvimento de medidas preventivas da doença, assim como na escolha de terapias imunossupressoras adequadas de tratamento. Este estudo discute os principais componentes imunológicos envolvidos na patogênese da DECH aguda e crônica, com ênfase à participação das citocinas e seu controle.

Stem cell transplantation is the first line treatment of many hematological diseases and primary immunodeficiencies. Graft-versus-host disease (GVHD) is still a severe complication after allogeneic transplantation and the main cause of mortality and morbidity. The study of the pathogenesis of GVHD may help to develop ways to prevent the disease, as well as to choose adequate immunosuppressant therapies. This study discusses the main immunological components involved in the pathogenesis of acute and chronic GVHD, with emphasis on the participation of cytokines and their control.

Associação dos níveis de citocinas no pós-transplante de células-tronco hematopoiéticas com a Doença do Enxerto Contra o Hospedeiro aguda / Association of cytokine levels with acute graft versus host disease following full match allogeneic stem cell transplantation

Este estudo foi realizado para investigar se os níveis séricos de sIL-2R, TNF-alfa, IFN-gama, IL-6, IL-10 e TGF-beta1 estavam associados com o desenvolvimento de DECH (Doença do Enxerto Contra o Hospedeiro) aguda. Os níveis de citocinas foram seqüencialmente mensurados por Elisa em 13 pacientes que haviam sido submetidos ao transplante alogênico de células progenitoras hematopoiéticas. Os níveis de sIL-2R e IL-10 da 1ª a 15ª semanas pós-transplante foram significativamente maiores no grupo que desenvolveu DECH aguda que naquele sem a doença. Os níveis de sIL-2R aumentaram em direta correlação com a pega do enxerto e ao tempo do DECH aguda, enquanto os níveis de IL-10 aumentaram transitoriamente pós-transplante. A média da concentração de TNF-alfa nas primeiras semanas após o transplante foi maior no grupo que desenvolveu DECH aguda. Além disso, uma queda dos níveis de TGF-beta1 após a pega esteve significativamente associada à DECH aguda. Nenhuma correlação foi encontrada entre DECH aguda e as outras citocinas investigadas. Estes resultados suportam a idéia de que um balanço entre as citocinas derivadas de linfócitos T auxiliadores do tipo 1 e 2 pode ser importante no desenvolvimento e controle da DECH aguda. Embora os níveis de sIL-2R, TNF-alfa, IL-10 e TGF-beta1 tenham sido correlacionados com a DECH aguda, os níveis de sIL-2R ao tempo da pega podem prover um melhor parâmetro para a detecção precoce de DECH aguda após o transplante alogênico.

This study was performed to investigate whether the serum levels of sIL-2R, TNF-alpha, IFN-gamma, IL-6, IL-10, and TGF-beta1 are associated with the development of acute GVHD. Serum cytokine levels were sequentially measured by sandwich Enzyme Linked-Immuno-Sorbent Assay (Elisa) in 13 patients who had received full match allogeneic stem cell transplantation. Serum sIL-2R and IL-10 levels from the 1st to the 15th week post transplantation were significantly higher in the group who developed acute GVHD than in the group without acute GVHD. Soluble IL-2R levels increased in direct correlation to engraftment and onset of acute GVHD, while IL-10 levels increased transiently following transplantation. The mean TNF-alpha concentration in the first weeks after transplantation was augmented in the group that developed acute GVHD. Furthermore, a drop in TGF-beta1 levels after the engraftment was significantly associated to acute GVHD. No correlation was found between acute GVHD and the other evaluated cytokines. These results support the idea that a balance between cytokines derived from type 1 and type 2 T-helper cells may be important in the development and control of acute GVHD. Although sIL-2R, TNF-alpha, IL-10, and TGF-beta1 levels, correlated with acute GVHD, sIL-2R levels at the engraftment may provide a better parameter for the early detection of acute GVHD after allogeneic stem cell transplantation.

Reconstituição imunológica após o transplante de medula óssea alogênico / Immunology reconstitution after allogeneic bone marrow transplantation

Após a transplantação de células progenitoras hematopoiéticas, todos os receptores desenvolvem um período de profunda neutropenia e imunodeficiência que é significativamente responsável pelas graves complicações infecciosas que ocorrem após o transplante. Devido ao intensivo regime de condicionamento pré-transplante, tanto a resposta celular quanto a humoral estão severamente comprometidas. A reconstituição imunológica é um importante componente para o sucesso do transplante, não somente porque os defeitos imunes estão relacionados à morbidade pós-transplante, mas também porque eles podem influenciar o risco de recaída e o desenvolvimento de malignidades secundárias após o transplante. Este estudo apresenta uma revisão sobre as principais variáveis envolvidas na reconstituição imunológica, após o transplante de medula óssea alogênico: uma falta de manutenção da imunidade antígeno-específica do doador, uma deficiência na reconstituição da ontogenia linfóide, as diferenças de histocompatibilidade entre doador e receptor, o efeito da GVHD e suas terapias e, a redução na função tímica do receptor.

After the hematopoietic progenitor cell transplantation, allreceptors experience a period of profound neutropenia andimmunodeficiency that is responsible for a significant number ofthe serious infectious complications that can occur after atransplant. Due to the intensive regimen of pre-transplantconditioning, both cellular and humoral responses are greatlyaffected. Immune reconstitution is an important component ofsuccessful bone marrow transplantation, not only because immunedefects are related to infectious post-transplant morbidity, but alsobecause they may influence the risk of relapse and the developmentof secondary malignancies after transplantation. This study givesa revision about the primary variables involved in immunologicreconstitution, after allogeneic bone marrow transplantation: alack of sustained transference of donor antigen-specific immunity,a deficiency in the lymphoid ontogeny reconstitution,histocompatibility differences between donor and receptor, the effectof GVHD and its therapy, and decreased thymic function of thereceptor.

Freqüência de anticorpos anti-Cysticercus cellulosae em indivíduos de cinco municípios da regiäo norte do Estado do Paraná - Brasil / Frequency of anti-cysticercus cellulosae antibodies in individuals from five counties in the southern region of Brazil

Realizou-se inquérito sorológico e epidemiológico para cisticercose em indivíduos de cinco municípios da regiäo Norte do Estado do Paraná, Brasil. De 2.180 indivíduos investigados através da reaçäo de imunofluorescência indireta, 69 (3,2 por cento) apresentaram títulos significativos de anticorpos anti-Cysticercus cellulosae. Os percentuais de indivíduos com títulos significativos encontrados em Sarandi (6,6 por cento) e Marialva (4,7 por cento) näo diferem estatisticamente (Z=1.319, P=0,0936), mas diferem dos percentuais encontrados em Mandaguaçu, Paiçandu e Maringá (P<0,01). Destes indivíduos, 47,9 por cento estavam na faixa etária de 21 a 49 anos e 79,4 por cento eram do sexo feminino. Foi comum o relato de queixas como "dores de cabeça" (70,6 por cento), "tonturas" (57,4 por cento) e "convulsöes" (7,4 por cento), além de história de teníase (22,1 por cento) e hábitos de ingestäo de carne crua bovina (41,2 por cento) ou suína 27,9 por cento) e carne com "canjiquinha' (25,0 por cento)