Abstract The aim of this study was to evaluate tumor necrosis factor alpha (TNF-α), interleukin (IL)- 17A/F levels in the serum of ankylosing spondylitis (AS) patients after anti-TNF therapy, in order to understand how these cytokines are involved in this therapeutic response. Forty-four AS patients were included in the study: thirty using anti-TNF therapy were classified according to their therapy response as responders (15) and non-responders (15) and 14 without anti-TNF therapy were classified as AS control. Fifteen healthy individuals formed the control group. Serum levels of TNF-α were determined using Luminex technology and for IL-17A and IL-17F using ELISA. The non-responder patients presented higher serum levels of TNF-α than the responders and AS control; the same results were found when HLA-B*27 positive or negative patients were separately analyzed. IL-17A and IL17F serum levels were similar for all groups. According to the clinical disease activity, AS patients with BASDAI ≥4 had higher serum levels of TNF-α than AS patients with BASDAI <4. Positive correlation was found between TNF-α levels and BASDAI. In AS patients, TNF-α serum levels were associated with anti-TNF therapy and disease activity independently of HLA-B*27, and IL-17A and IL-17F were not related to anti-TNF treatment.

OBJECTIVES: HLA-B27 is strongly associated with ankylosing spondylitis (AS) and its presence helps to confirm AS diagnosis. Due to the high HLA polymorphism and the differentiated contribution of alleles and molecules encoded by them, HLA-B*27 allele identification is relevant in the clinical follow-up, diagnosis, and treatment of this spondyloarthropathy. Inexpensive genotyping techniques with high specificity and sensitivity are of great interest in histocompatibility laboratories. This work aimed to optimize HLA-B*27 genotyping by Polymerase Chain Reaction Sequence-specific Primer (PCR-SSP), which is an accessible and inexpensive technique. METHODS: The PCR-SSP was standardized using 26 HLA-B*27 positive and 3 HLA-B*27 negative samples previously defined by Polymerase Chain Reaction Sequence-specific Oligonucleotide Probes (PCR-SSOP) (medium resolution, One Lambda®) and primers described by Duangchanchot et al. (2009). For validating the technique, 397 samples were genotyped using PCR-SSP as well as PCR-SSOP. RESULTS: The PCR-SSP technique was standardized for identifying the alleles HLA-B*27:02, HLA-B*27:CAFRW (05/13/16/17/28/37/38/39/42), HLA-B*27:CAFRZ (08/26/40), HLA-B*27:09 and HLA-B*27:12, which were found in 90 positive samples (22.67%). There was 100% agreement between the two techniques for heterozygous samples; however, two homozygous samples could not be detected by PCR-SSP. CONCLUSION: The HLA-B*27 genotyping using PCR-SSP, an easy-to-use, specific, and affordable technique, was optimized for heterozygous samples. This technique may contribute to AS diagnosis.

Abstract Background Lutheran and Dombrock are two blood group systems with low immunogenic antigens; they can cause mild-to-moderate transfusion reactions. For both, immunophenotyping is not performed in the pretransfusion routine in Brazil. In addition, the distribution of their antigenic frequencies is an important marker of ethnicity. Thus, the goal of this study was to carry out the genotyping of the LU*01, LU*02, DO*01 and DO*02 alleles of the Lutheran and Dombrock blood group systems in blood donors from the southwestern region of the state of Paraná, Southern Brazil. Method Genotyping was performed for 251 blood donors by specific allele-polymerase chain reaction. The genotype and allele frequencies were obtained through direct counting and compared with other Brazilian populations using the chi-square test with Yates correction. Results The distribution of genotype frequencies for LU were 0.4% for LU*01/LU*01, 6.8% for LU*01/LU*02 and 92.8% for LU*02/LU*02 and for DO, they were 19.9% for DO*01/DO*01, 44.6% for DO*01/DO*02 and 35.5% for DO*02/DO*02. The allele and genotype frequencies of LU and DO were similar to those expected for Caucasians, but the DO*01/DO*01 genotype frequency was different to other Brazilian populations. The rare LU*01/LU*01 genotype was found in a loyal blood donor. Conclusion The genotyping techniques allowed the evaluation of the LU*01, LU*02, DO*01 and DO*02 alleles in blood donors registered in the Hemotherapy Center of the southwestern region of Paraná, Southern Brazil, and contributed to a genotyped blood donor database.

Abstract Background Alloimmunization is a major problem in transfusion practice due to the clinical complications of the patients and the difficulty of choosing a unit of compatible blood product. Serological methods are widely used in blood banks, but they not always determine the phenotype. Thus, genotyping is an important complement to the serology tool as it allows one to predict the phenotype from deoxyribonucleic acid (DNA) with high accuracy. Objective To compare the centrifugation gel, microarray, Restriction Fragment Length Polymorphismone PCR (PCR-RFLP) and Sequence-Specific Primer PCR (PCR-SSP) techniques, in terms of cost, reaction time and reliability of the results. Methods The RHCE, Kidd, Kell and Duffy blood group systems were chosen to determine the approximate cost of each technique, considering the reagents used in both methods and considering only one sample. The time required for the development of each reaction was obtained at the Maringa Regional Blood Center and Immunogenetics Laboratory at the State University of Maringa. Data from Microarray reactions were obtained at the Campinas Blood Center. The results of phenotyping and genotyping of the 16 samples were compiled in a spreadsheet and compared. Results The PCR-SSP was more economical compared to other methods, and the serological method was faster than the molecular methods. However, all methods proved to be effective and safe in the detection of erythrocyte antigens. Conclusion Analyzing the advantages and limitations of the molecular and serological methods tested in this study, we note that both are important and complementary. However, the choice of a methodology depends on the reality and needs of each health service.

Abstract Background: The Kidd blood group system has three antigens, Jka, Jkb and Jk3, found on red blood cells and on endothelial cells of the inner lining of blood vessels in the renal medulla. These are known as urea transporter B (UT-B). Researchers have found that individuals carrying the Jk(a − b−) or Jk-null (UT-B null) phenotypes have a lower urine-concentrating capability and risk of severe renal impairment. This study evaluated the distribution of the Kidd phenotypes in patients with chronic kidney disease and a possible association of Kidd antigens with the development of renal disease. Methods: Jka and Jkb antigens were phenotyped using the gel column agglutination test (ID-cards Bio-RAD) in 197 patients with chronic kidney disease and 444 blood donors, as the control group. The phenotype and antigen frequencies between patients and controls were evaluated using the Chi-square method with Yates correction and logistic regression after adjustments for gender and age. Results: No differences were observed between the Kidd phenotypes frequency distribution between patients with chronic kidney disease and blood donors [Jk(a − b+) = 22.3% and 27.2%; Jk(a + b−) = 30.5% and 24.3%; Jk(a + b+) = 47.25% and 48.4%, respectively]. Conclusion: The distribution of Kidd phenotypes found in the studied population is expected for Caucasians; Jka and Jkb antigens and phenotypes were not found to be related to susceptibility for chronic kidney disease.

Resumo O sucesso do reimplante dentário depende da condição apresentada pelo ligamento periodontal cementário pós-avulsão que pode ser influenciado pelo meio de estocagem. O Sangue de Dragão (Croton lechleri) é sugerido como um meio promissor por auxiliar na formação de novo colágeno e apresentar propriedades cicatrizante, anti-inflamatória, antimicrobiana. Assim, o objetivo deste trabalho foi avaliar a eficácia da seiva Sangue de Dragão como meio de estocagem para dentes avulsionados por meio da aferição da viabilidade funcional e metabólica celular. Este estudo in vitro comparou a eficácia dos meios para a manutenção da viabilidade das células mononucleares de sangue periférico humano e do ligamento periodontal mantidas em cultura. Foi testada a diluição a 10% da seiva Sangue de Dragão enquanto que a PBS foi selecionada como seu controle. O leite ultrapasteurizado integral foi utilizado como meio comparativo por ser tradicionalmente utilizado como meio de estocagem. O DMEM e a água destilada foram os controles positivos e negativos, respectivamente. A avaliação da viabilidade foi feita por meio dos testes de exclusão por Azul de Tripan e colorimétrica a base de tetrazolato (MTT), após 1, 3, 6, 10 e 24 h de incubação. A seiva Sangue de Dragão apresentou resultados promissores devido à sua considerável manutenção da viabilidade celular. Para a metodologia com o Azul de Tripan, a seiva Sangue de Dragão foi semelhante ao leite.

Abstract Tooth replantation success depends on the condition of cementum periodontal ligament after tooth avulsion; which is influenced by storage medium. The dragon's blood (Croton lechleri) sap has been suggested as a promising medium because it supports collagen formation and exhibits healing, anti-inflammatory and antimicrobial properties. Thus, the aim of this study was to evaluate the efficacy of dragon's blood sap as a storage medium for avulsed teeth through evaluation of functional and metabolic cell viability. This in vitro study compared the efficacy of different storage media to maintain the viability of human peripheral blood mononuclear and periodontal ligament cells. A 10% dragon's blood sap was tested while PBS was selected as its control. Ultra pasteurized whole milk was used for comparison as a commonly used storage medium. DMEM and distilled water were the positive and negative controls, respectively. The viability was assessed through trypan blue exclusion test and colorimetric MTT assay after 1, 3, 6, 10 and 24 h of incubation. The dragon's blood sap showed promising results due to its considerable maintenance of cell viability. For trypan blue test, the dragon's blood sap was similar to milk (p<0.05) and both presented the highest viability values. For MTT, the dragon's blood sap showed better results than all storage media, even better than milk (p<0.05). It was concluded that the dragon's blood sap was as effective as milk, the gold standard for storage medium. The experimental sap preserved the membrane of all cells and the functional viability of periodontal ligament cells.

OBJECTIVE: The JAK2 46/1 haplotype has recently been described as a major contributing factor to the development of myeloproliferative neoplasm, whether positive or negative forthe JAK2 V617F mutation. The G allele, identified by a single-nucleotide polymorphism known as JAK2 rs10974944, is part of the JAK2 46/1 haplotype. The aim of this study was to verify the association between the presence of the G allele and the development of BCR-ABL-negative chronic myeloproliferative neoplasms in our population. METHODS: Blood and oral mucosa swab samples were obtained from 56 patients of two local Brazilian hospitals who had previously been diagnosed with BCR-ABL-negative chronic myeloproliferative neoplasms. Blood samples from 90 local blood donors were used as controls. The presence of the G allele was assessed using a PCR-RFLP assay after extracting DNA from the samples. RESULTS: The presence of the G allele was strongly associated with the presence of BCR-ABL-negative chronic myeloproliferative neoplasms (p = 0.0001; OR = 2.674; 95% CI = 1.630-4.385) in the studied population. CONCLUSION: In agreement with previous reports, the JAK2 46/1 haplotype, represented in this study by the presence of the G allele, is an important predisposing factor in the oncogenetic development of these neoplasms in our population.

Hemophilia A is a disease caused by a deficiency of coagulation factor VIII resulting from genetic inheritance linked to chromosome X. One treatment option is the administration of plasma or recombinant FVIII. However, some patients develop inhibitors or antibodies against this factor. Inhibitors are alloantibodies that bind to the epitope of factor VIII causing it to be recognized by the immune system as a foreign peptide. This is the most serious complication in hemophilia patients in respect to replacement therapy. Some studies have suggested that genetic factors influence the development of factor VIII inhibitors such as ethnicity, family history, mutations in the factor VIII gene and in genes of the immune system. The aim of this study was to conduct a literature review to assess the influence of genetic factors of immune response genes, especially genes of the major histocompatibility complex and cytokines, which may be related to the development of factor VIII inhibitors in hemophilia A patients. Understanding these risk factors will help to determine future differential treatment in the control and prevention of the development of inhibitors.

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% vs 4,1%; p = 0,0345; OR = 1,72; CI = 1,05 - 2,81), HLA-B*38 (2,7% vs 1,1; p = 0,0402; OR = 2,44; CI 95% = 1,05-5,69), HLA-C*12 (9,4% vs 5,4%; p = 0,01; OR = 1,82; CI 95% = 1,17-2,82) e HLA-C*16 (3,1 vs 6,5%; p = 0,0124; OR = 0,47; CI 95% = 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.

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.

BACKGROUND: Red blood group genes are highly polymorphic and the distribution of alleles varies among different populations and ethnic groups. AIM: To evaluate allele polymorphisms of the Rh, Kell, Duffy and Kidd blood group systems in a population of the State of Paraná METHODS: Rh, Kell, Duffy and Kidd blood group polymorphisms were evaluated in 400 unrelated blood or bone marrow donors from the northwestern region of Paraná State between September 2008 and October 2009. The following techniques were used: multiplex-polymerase chain reaction genotyping for the identification of the RHD gene and RHCE*C/c genotype; allele-specific polymerase chain reaction for the RHDΨ and restriction fragment length polymorphism polymerase chain reaction for the RHCE*E/e, KEL, FY-GATA and JK alleles. RESULTS: These techniques enabled the evaluation of the frequencies of Rh, Kell, Duffy and Kidd polymorphisms in the population studied, which were compared to frequencies in two populations from the eastern region of São Paulo State. CONCLUSION: The RHCE*c/c, FY*A/FY*B, GATA-33 T/T, JK*B/JK*B genotypes were more prevalent in the population from Paraná, while RHCE*C/c, FY*B/FY*B, GATA-33 C/C, JK*A/JK*B genotypes were more common in the populations from São Paulo.

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.

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% 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% 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.

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.

A compatibilidade genética HLA entre doador e receptor é um fator importante para o sucesso do transplante de células progenitoras hematopoiéticas (TCPH). No entanto, outros genes não-HLA estão sendo investigados em relação ao seu papel na incidência e gravidade da doença do enxerto contra o hospedeiro e na sobrevida, por modularem a intensidade da inflamação e os danos teciduais. Estes genes, não-HLA, incluem os genes de citocinas com polimorfismos dentro das seqüências 5' ou 3' regulatórias dos genes. Os polimorfismos ou microssatélites podem alterar a ligação dos fatores de transcrição aos sítios dentro dos genes promotores e a quantidade de citocina produzida. Este estudo revisa o papel potencial destes polimorfismos genéticos relativos às citocinas em prever o curso do TCPH.

HLA genetic matching of donor and recipient is an important requirement for optimizing outcome following hematopoietic stem cell transplantation (HSCT). However, other non-HLA genes are being investigated for their role in graft-versus-host disease incidence and severity and in survival, by modulating the intensity of inflammation and tissue injury. These non-HLA-encoded genes include cytokine genes with polymorphisms within the 5' or 3' regulatory sequences of the genes. The polymorphisms or microsatellites may alter the transcription factor binding sites within the gene promoters and the amount of cytokine produced. This chapter summarizes the potential role of these genetic polymorphisms regarding the cytokines in predicting outcome of HSCT.

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.