Class-I human leukocyte alleles in leprosy patients from Southern Brazil.

INTRODUCTION: The present study was designed to investigate a possible role of HLA (histocompatibility leucocyte antigen) class-I alleles (HLA-A, -B, and -C) in leprosy patients from Southern Brazil. METHODS: Two hundred and twenty-five patients with leprosy and 450 individuals for the control group were involved in this research. HLA genotyping was performed through PCR-SSO protocols (One Lambda, USA); the frequency of these alleles was calculated in each group by direct counting, and the frequencies were then compared. RESULTS: There was an association between HLA-A*11 (6.9% vs 4.1%, p=0.0345, OR=1.72, 95% CI=1.05-2.81), HLA-B*38 (2.7% vs. 1.1%, p=0.0402, OR=2.44, 95% CI=1.05-5.69), HLA-C*12 (9.4% vs. 5.4%, p=0.01, OR=1.82, 95% CI=1.17-2.82), and HLA-C*16 (3.1% vs. 6.5%, p=0.0124, OR=0.47, 95% CI=0.26-0.85) and leprosy per se. In addition, HLA-B*35, HLA-C*04, and HLA-C*07 frequencies were different between lepromatous (LL) and tuberculoid (TT) patients. However, after adjusting for the number of alleles compared, Pc values became nonsignificant. CONCLUSIONS: Although our results do not support the previous findings that HLA class-I alleles play a role in leprosy pathogenesis, we suggest new studies because of the importance of the association between the HLA and KIR in the innate immune response to leprosy.

Class-I human leukocyte alleles in leprosy patients from Southern Brazil / Alelos leucocitários humanos em pacientes com hanseníase do sul do Brasil

INTRODUCTION: The present study was designed to investigate a possible role of HLA (histocompatibility leucocyte antigen) class-I alleles (HLA-A, -B, and -C) in leprosy patients from Southern Brazil. METHODS: Two hundred and twenty-five patients with leprosy and 450 individuals for the control group were involved in this research. HLA genotyping was performed through PCR-SSO protocols (One Lambda, USA); the frequency of these alleles was calculated in each group by direct counting, and the frequencies were then compared. RESULTS: There was an association between HLA-A*11 (6.9 percent vs 4.1 percent, p=0.0345, OR=1.72, 95 percent CI=1.05-2.81), HLA-B*38 (2.7 percent vs. 1.1 percent, p=0.0402, OR=2.44, 95 percent CI=1.05-5.69), HLA-C*12 (9.4 percent vs. 5.4 percent, p=0.01, OR=1.82, 95 percent CI=1.17-2.82), and HLA-C*16 (3.1 percent vs. 6.5 percent, p=0.0124, OR=0.47, 95 percent CI=0.26-0.85) and leprosy per se. In addition, HLA-B*35, HLA-C*04, and HLA-C*07 frequencies were different between lepromatous (LL) and tuberculoid (TT) patients. However, after adjusting for the number of alleles compared, Pc values became nonsignificant. CONCLUSIONS: Although our results do not support the previous findings that HLA class-I alleles play a role in leprosy pathogenesis, we suggest new studies because of the importance of the association between the HLA and KIR in the innate immune response to leprosy.

INTRODUÇÃO: O presente estudo foi desenhado para investigar um possível papel para os alelos HLA (histocompatibility leucocyte antigen) de classe I (HLA-A, -B, and -C) em pacientes com hanseníase do sul do Brasil. MÉTODOS: Duzentos e vinte e cinco pacientes com hanseníase e 450 indivíduos para o grupo-controle foram envolvidos nesse estudo. O genótipo HLA foi determinado por protocolos PCR-SSO (One Lambda, USA) e, a frequência desses alelos foi calculada em cada grupo por contagem direta e, após, comparadas. RESULTADOS: Houve associação entre HLA-A*11 (6,9 por cento vs 4,1 por cento; p = 0,0345; OR = 1,72; CI = 1,05 - 2,81), HLA-B*38 (2,7 por cento vs 1,1; p = 0,0402; OR = 2,44; CI 95 por cento = 1,05-5,69), HLA-C*12 (9,4 por cento vs 5,4 por cento; p = 0,01; OR = 1,82; CI 95 por cento = 1,17-2,82) e HLA-C*16 (3,1 vs 6,5 por cento; p = 0,0124; OR = 0,47; CI 95 por cento = 0,26-0,85) e hanseníase per se. Além disso, as frequências de HLA-B*35, HLA-C*04 e HLA-C*07 foram diferentes entre os pacientes com as formas lepromatosa (LL) e tuberculoide (TT). Contudo, após o ajuste para o número de alelos comparados, os valores de p se tornaram não significativos. CONCLUSÕES: Embora nossos resultados não sustentem as conclusões anteriores de que os alelos HLA de classe I desempenham um papel na associação com a patogênese da hanseníase, sugerimos novos estudos devido à importância da associação entre HLA e KIR na resposta imune inata à hanseníase.

Importance of killer immunoglobulin-like receptors in allogeneic hematopoietic stem cell transplantation.

Hematopoietic stem cell transplantation is the treatment of choice for many hematologic diseases, such as multiple myeloma, bone marrow aplasia and leukemia. Human leukocyte antigen (HLA) compatibility is an important tool to prevent post-transplant complications such as graft rejection and graft-versus-host disease, but the high rates of relapse limit the survival of transplant patients. Natural Killer cells, a type of lymphocyte that is a key element in the defense against tumor cells, cells infected with viruses and intracellular microbes, have different receptors on their surfaces that regulate their cytotoxicity. Killer immunoglobulin-like receptors are the most important, interacting consistently with human leukocyte antigen class I molecules present in other cells and thus controlling the activation of natural killer cells. Several studies have shown that certain combinations of killer immunoglobulin-like receptors and human leukocyte antigens (in both donors and recipients) can affect the chances of survival of transplant patients, particularly in relation to the graft-versusleukemia effect, which may be associated to decreased relapse rates in certain groups. This review aims to shed light on the mechanisms and effects of killer immunoglobulin-like receptors - human leukocyte antigen associations and their implications following hematopoietic stem cell transplantation, and to critically analyze the results obtained by the studies presented herein.

Importance of killer immunoglobulin-like receptors in allogeneic hematopoietic stem cell transplantation

Hematopoietic stem cell transplantation is the treatment of choice for many hematologic diseases, such as multiple myeloma, bone marrow aplasia and leukemia. Human leukocyte antigen (HLA) compatibility is an important tool to prevent post-transplant complications such as graft rejection and graft-versus-host disease, but the high rates of relapse limit the survival of transplant patients. Natural Killer cells, a type of lymphocyte that is a key element in the defense against tumor cells, cells infected with viruses and intracellular microbes, have different receptors on their surfaces that regulate their cytotoxicity. Killer immunoglobulin-like receptors are the most important, interacting consistently with human leukocyte antigen class I molecules present in other cells and thus controlling the activation of natural killer cells. Several studies have shown that certain combinations of killer immunoglobulin-like receptors and human leukocyte antigens (in both donors and recipients) can affect the chances of survival of transplant patients, particularly in relation to the graft-versusleukemia effect, which may be associated to decreased relapse rates in certain groups. This review aims to shed light on the mechanisms and effects of killer immunoglobulin-like receptors - human leukocyte antigen associations and their implications following hematopoietic stem cell transplantation, and to critically analyze the results obtained by the studies presented herein.

Otimização de metodologia para o estudo de genes KIR / Methodology optimization for KIR genotyping

Receptores killer cell immunoglobulin-like (KIRs) são moléculas localizadas na superfície de células natural killer (NK) e em subpopulações de linfócitos T codificadas por genes do cromossomo 19q13.4. A interação entre receptores KIR e moléculas antígeno leucocitário humano (HLA) de classe I determina se células NK exercerão ou não sua função citotóxica e/ou secretora de citocinas ou se esta será inibida. Este trabalho teve por finalidade otimizar a metodologia para a genotipagem KIR, baseando-se nas condições descritas por Martin (2004). A técnica utilizada foi a reação em cadeia da polimerase com primers de sequência específica (PCR-SSP) com iniciadores sintetizados pela Invitrogen® e visualização do produto amplificado em gel de agarose a 2 por cento com brometo de etídio. Adaptações foram realizadas e a concentração de alguns reagentes foi alterada, como a do controle interno de 100 nM para 150 nM, iniciadores específicos senso e antissenso de KIR12.5/12.3, KIR13.5/13.3, KIR14.5/14.3, KIR22.5/22.3 e KIR36.5/36.3 de 500 nM para 750 nM e da solução de MgCl2 de 1,5 mM para 2 mM. As concentrações dos demais reagentes e temperaturas de amplificação foram mantidas. Nessas condições, o uso da Taq DNA polimerase recombinante (Invitrogen®) foi satisfatório. Os resultados das genotipagens de 70 indivíduos foram confirmados por rSSO-Luminex® (One Lambda, Canoga Park, CA, EUA). A tipagem de genes KIR por essa técnica apresentou sensibilidade, especificidade, reprodutibilidade e baixo custo.

The killer cell immunoglobulin-like receptors (KIRs) are molecules expressed on natural killer (NK) cells surface and in T-cell subsets encoded by genes located in chromosome 19q13.4. The interaction between KIR receptors and HLA class I molecules determines if the NK cells will fulfill their cytotoxic function and/or cytokine secretion or if this function will be inhibited. The objective of this work was to optimize KIR genotyping method described by Martin (2004). It was used PCR-SSP (polymerase chain reaction-sequence-specific primers) with primers synthesized by Invitrogen® and visualization of the amplified products on 2 percent agarose gel electrophoresis, containing ethidium bromide. Some adaptations were made and the reagents had their concentrations increased: the internal control from 100 nM to 150 nM, forward and reverse specific primers KIR12.5/12.3, KIR13.5/13.3, KIR14.5/14.3, KIR22.5/22.3 and KIR36.5/36.3 from 500 nM to 750 nM, and MgCl2 solution from 1.5 mM to 2 mM. Other reagent concentrations and amplification temperatures were maintained. Satisfactory results were obtained with Taq DNA Polymerase Recombinant (Invitrogen®). The results of seventy samples were confirmed by rSSO-Luminex® (One Lambda, Canoga Park, CA, USA). This KIR typing method proved to be accurate, specific, reproducible and cost effective.

Influence of TNF and IL10 gene polymorphisms in the immunopathogenesis of leprosy in the south of Brazil.

OBJECTIVE: To determine whether cytokine polymorphisms are associated with leprosy and/or their subtypes in a Brazilian population. METHODS: Genotyping using polymerase chain reaction with sequence-specific primers (PCR-SSP) was performed for: TNF(-308/-238), IL2(-330/+166), IL6(-174), IFNG(+874), TGFB1(+869/+915), and IL10(-592/-819/-1082) in 240 healthy controls and 167 patients with leprosy. RESULTS: For TNF(-308), a higher frequency of GG genotype (85.5% vs. 74.1% in healthy controls, p = 0.009), along with a decreased frequency of GA/AA genotypes was observed among leprosy patients as compared to the control group (14.5% vs. 25.9%, p = 0.009). The GG genotype was particularly higher in patients with tuberculoid (TT) and borderline (BB) leprosy (90.5% and 89.8%, respectively). Analysis of IL10 genotypes revealed a lower frequency of GCC/GCC haplotype in lepromatous leprosy (LL) patients (6.2%) in comparison to controls (15.4%). CONCLUSION: It is suggested that the G-->A substitution at position -308 in the TNF promoter region plays an important role in leprosy patients.

Killer cell immunoglobulin-like receptor gene diversity in a Southern Brazilian population from the state of Paraná.

Killer cell immunoglobulin-like receptors (KIR) are encoded by polymorphic genes and have as binding human leukocyte antigen (HLA) class I molecules. The aim of this study was to investigate the distribution of KIR genes and inhibitory KIR/HLA pairs in a population from Southern Brazil, in the state of Paraná, and to compare the results with results from other populations. The genotyping of 16 KIR genes and HLA class I alleles of 289 unrelated individuals was accomplished by reverse sequence-specific oligonucleotide Luminex (One Lambda, Inc., Canoga Park, CA). This Brazilian population demonstrated several similarities to Caucasian populations with regard to the frequency of KIR genes. Thirty-eight genotypes were defined in which the most frequent was the homozygous haplotype A (33.2%). Therefore, it was possible to define two new genotypes. Most of the individuals demonstrated at least one inhibitory KIR/HLA pair. Two pairs were the most frequent (40.4%), followed by three pairs (38.2%), one pair (14.6%), and four pairs (6.4%). The KIR2DL2/3 + HLA-C1 pair was the most frequent (79.9%) and the least frequent pair was KIR3DL2 + HLA-A3/11 (25.0%). This study demonstrated the diversity of KIR genes in a population of Paraná, as well as the characteristic pattern of Caucasians with racial admixture, which enabled the definition of two new genotypes and the identification of one individual without the inhibitory KIR/HLA pair.

TNF, IFNG, IL6, IL10 and TGFB1 gene polymorphisms in South and Southeast Brazil.

This study attempted to establish single nucleotide polymorphism frequencies of TNF, IL6, IFNG, IL10 and TGFB1 genes among healthy individuals from South and Southeast Brazil. The sample included 108 healthy individuals from South and 106 from Southeast Brazil. Polymerase chain reaction using sequence-specific primers genotyping was performed for these gene cytokines with Cytokine Genotyping Primers (One Lambda, Canoga Park, CA, USA). Differences in genotypic and allelic frequencies between the populations were assessed by chi-square with either Yates' correction or Fisher's exact test. Our investigations showed that there were not any significant differences between these two Brazilian populations for these polymorphisms. A statistically significant difference in the distribution of alleles and genotypes for both IL6 and IL10 genes was observed between the Brazilian population and the African-derived populations. IL6-174GG genotype and allele G and IL10-819CT/-592CA genotypes are more frequent in African-derived populations than in this mixed Brazilian population, while IL10-1082GG genotype is more frequent in our population. This mixed Brazilian population is closer to those of Joinville's, Santa Catarina, and Rio de Janeiro's, Rio de Janeiro (RJ), Euro-Brazilian populations than to those of Salvador's, Bahia, and Rio de Janeiro's, RJ, African-Brazilian populations. These findings have an enormous importance for experimental design and empowering future linkage and association mapping studies of the role of cytokines in human diseases and allotransplantation outcome in Brazil.

Receptores Kir de células Natural Killer / Kir receptors of natural killer cells

As células NK (natural killer) são uma subpopulação de linfócitos que desempenham função essencial na resposta imune inata. As moléculas KIR (killer immunoglobulin-like receptor) são receptores expressos na superfície dessas células com função inibitória ou ativatória e contribuem para a regulação da função dascélulas NK. Os genes KIR fazem parte do Complexo de Receptores Leucocitários, localizado no cromossomo 19q13 e apresentam alto polimorfismo. Os ligantes de KIR são moléculas HLA de classe I, e a regulação dascélulas NK está relacionada à variação da expressão dessas moléculas na superfície das células-alvo, principalmente células infectadas, tumorais e alogênicas. O objetivo desse trabalho foi proceder a uma revisão bibliográfica sobre os receptores KIR. O levantamento foi realizado nos sites Pubmed/medline e ScienceDirect,e foram utilizadas como palavras-chave receptor, NK e KIR. A estrutura molecular desses receptores, a nomenclatura e classificação de KIR, a variabilidade gênica, alélica e haplotípica e os ligantes foram apresentados. Ênfase foi dada à regulação da expressão dos genes KIR e sua relação com a função das célulasNK.

NKC (natural killer cells) are a population of lymphocytes that play an essential role in innate immunity. KIR molecules are receptors expressed on the surface of these cells with an inhibitory or activating function that contributes to the regulation of NK cells. The KIR genes are located on chromosome 19q13 at the Leukocyte Receptor Complex, and exhibit high polymorphism. The KIR ligands are HLA class I molecules. NK cell functionsare related to the variation of the expression of these molecules on the surface of the target cells – especially infected, allogeneic and tumor cells. The aim of this work was to make a review about KIR receptors. The Pubmed/medline and ScienceDirect online databases were accessed, using receptor, NK and KIR as keywords. KIR molecular structure, nomenclature and classification, gene diversity, allelic and haplotypic variability and itsligands were described. Regulation of KIR genes expression and NK cell function were also presented.

Las células NK (natural killer) son una subpoblación de linfocitos que desempeñan una función esencial en la respuesta inmune innata. Las moléculas KIR (killer immunoglobulin-like receptor) son receptores expresos en la superficie de esas células con función inhibidora o activadora y contribuyen para la regulación de la función delas células NK. Los genes KIR hacen parte del Complejo de Receptores Leucocitarios, localizado en el cromosoma 19q13 y presentan alto polimorfismo. Los ligantes de KIR son moléculas HLA de clase I, y laregulación de las células NK está relacionada a la variación de la expresión de esas moléculas en la superficiede las células blanco, principalmente células infectadas, tumorosos y alogénicas. El objetivo de ese trabajo fue proceder una revisión bibliográfica sobre los receptores KIR. La pesquisa fue realizada en los sites Pubmed/medline y ScienceDirect, y fueron utilizadas como palabras clave receptor, NK y KIR. La estructura molecular de esos receptores, la nomenclatura y clasificación de KIR, la variabilidad génica, alélica y haplotípicay los ligantes fueron presentados. Énfasis fue dada a la regulación de la expresión de los genes KIR y surelación con la función de las células NK.