TNF-alpha and IL-17 cytokine levels in Brazilian patients with ankylosing spondylitis after anti-TNF therapy

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.

Lack of association of the KIR and HLA class I ligands with ZIKV infection in south and southeast of Brazil

BACKGROUND Zika virus (ZIKV) is an emerging arbovirus associated with foetal malformations and neurological complications. The infection is usually associated with mild symptoms. The comparison between the allelic frequency of polymorphic genes in symptomatic infected individuals in the population can clarify the pathogenic mechanisms of ZIKV. During ZIKV infection, cytokines are produced and natural killer (NK) cells are recruited, whose activation depends on signaling pathways activated by specific receptors, such as killer cell immunoglobulin-like receptors (KIR). These molecules interact with human leukocyte antigen (HLA) class I ligands and are encoded by polymorphic genes. OBJECTIVES This study aimed to evaluate the frequency of allelic variants of the genes encoding the KIR receptors and their HLA class I ligands in 139 symptomatic ZIKV-patients and 170 controls negative for the virus, and to evaluate the role of these variants for ZIKV susceptibility. METHODS KIR and HLA class I genes were genotyped using the polymerase chain reaction-sequence specific oligonucleotide (PCR-SSO) technique. FINDINGS No significant differences in the frequency distribution of KIRs and KIR-HLA in patients compared to controls were observed. MAIN CONCLUSIONS KIR and its HLA ligands might play a minor role in ZIKV infection in the south and southeast Brazilian individuals.

Optimization of HLA-B*27 ALLELE Genotyping by PCR-SSP

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.

Genotyping of Dombrock and Lutheran blood group systems in blood donors from the southwestern region of the state of Paraná, Southern Brazil

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.

Methods for blood group antigens detection: cost-effectiveness analysis of phenotyping and genotyping

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.

Frequências fenotípicas dos grupos sanguíneos Kell, Duffy e Kidd em doadores de sangue do Hemonúcleo de Apucarana, sul do Brasil / Phenotypic frequencies of the Kell, Duffy and Kidd blood groups in blood donors from Apucarana Blood Center, southern Brazil

Objetivo: O objetivo desse estudo foi determinar as frequências fenotípicas dos grupos sanguíneos Kell, Duffy e Kidd em uma população paranaense. Métodos: Foram avaliadas as frequências desses grupos sanguíneos em 1.759 doadores de sangue fenotipados no Hemonúcleo de Apucarana, sul do Brasil. A fenotipagem foi realizada pela aglutinação em gel-teste e os dados foram obtidos pelo sistema Report Smith-Access, da rede Hemepar. Resultados: Essa população apresentou uma distribuição das frequências fenotípicas de Kell, Kidd e Duffy compatível com populações caucasianas. Para averiguar esse fato, nós comparamos nossos dados com aqueles de uma população da mesma região do Paraná, composta principalmente por caucasianos. O fenótipo Fy(a+b-) foi mais frequente na população de Apucarana do que na população de Maringá (22,68 vs. 12,50%, P<0,001), enquanto que o fenótipo Fy (a+b+) foi menos frequente (37,24 vs. 48,0%, P<0,001). Conclusão: As frequências fenotípicas de três grupos sanguíneos foram determinadas e poderão ser utilizadas pelos Serviços de Hematologia e Hemoterapia na busca de unidades de concentrados de hemácias com fenótipos desejados e no cálculo da incidência de doadores compatíveis, em casos de receptores com múltiplos aloanticorpos, além de poderem ser utilizadas para comparações antropológicas e em estudos de associação com doenças.

Lack of association between Kidd blood group system and chronic kidney disease

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.

Evaluation of the association between the JAK2 46/1 haplotype and chronic myeloproliferative neoplasms in a Brazilian population

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.

Importance of immune response genes in hemophilia A

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.

Production of TNF-α, nitric oxide and hydrogen peroxide by macrophages from mice with paracoccidioidomycosis that were fed a linseed oil-enriched diet

Omega-3 polyunsaturated fatty acids (n-3 PUFA) can modulate the immune system and their primary effect is on macrophage function. Paracoccidioidomycosis (PCM) is an endemic systemic mycosis in Latin America that is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb). Macrophages are the main defence against this pathogen and have microbicidal activity that is dependent on interferon-Γ and tumour necrosis factor (TNF)-α. These cytokines stimulate the synthesis of nitric oxide (NO) and hydrogen peroxide (H2O2), leading to the death of the fungus. To study the effect of n-3 PUFA on the host immune response during experimental PCM, macrophages that were obtained from animals infected with Pb18 and fed a diet enriched by linseed (LIN) oil were cultured and challenged with the fungus in vitro. The macrophage function was analysed based on the concentrations of TNF-α, NO and H2O2. LIN oil seems to influence the production of TNF-α during the development of disease. A diet enriched with LIN oil influences the microbicidal activity of the macrophages by inducing the production of cytokines and metabolites such as NO and H2O2, predominantly in the chronic phase of infection.

Association between human leukocyte antigens and graft-versus-host disease occurrence after allogenic hematopoietic stem cell transplantation / Associação entre antígenos leucocitários humanos e a ocorrência da doença do enxerto contra o hospedeiro após o transplante alogênico de células-tronco hematopoiéticas

CONTEXT AND OBJECTIVE: Graft-versus-host disease (GVHD) is one of the complications following allogenic stem cell transplantation. This study investigated an association between human leukocyte antigen (HLA) and the occurrence of acute and chronic GVHD in patients who had received stem cell transplantations from HLA-identical siblings. DESIGN AND SETTING: Retrospective study at Hematology and Hemotherapy Center, Universidade Estadual de Campinas (Unicamp). METHODS: The participants were 176 patients whose first transplant was between 1997 and 2009. HLA genotyping was performed serologically and using the polymerase chain reaction with specific primer sequence. RESULTS: Acute GVHD was positively associated with HLA-A10 (P = 0.0007), HLA-A26 (P = 0.002), B55 (P = 0.001), DRB1*15 (P = 0.0211) and DQB1*05 (P = 0.038), while HLA-B16 (P = 0.0333) was more frequent in patients without acute GVHD. Chronic GVHD was positively associated with HLA-A9 (P = 0.01) and A23 (P = 0.0292) and negatively with HLA-A2 (P = 0.0031) and B53 (P = 0.0116). HLA-B35 (P = 0.0373), B49 (P = 0.0155) and B55 (P = 0.0024) were higher in patients with acute GVHD grade 3 or above, than in other patients. In patients with extensive chronic GVHD, HLA-A9 (P = 0.0004), A24 (P = 0.0059) and A26 (P = 0.0411) were higher than in other patients, while HLA-A2 was lower (P = 0.0097). CONCLUSION: This study suggests that HLA can influence the incidence and severity of acute and chronic GVHD. However, a study with a better design and more patients will be needed to confirm these results.

CONTEXTO E OBJETIVO: A doença do enxerto contra o hospedeiro (DECH) é uma das complicações pós-transplante alogênico de células progenitoras hematopoéticas. Este estudo investigou uma associação entre o antígeno leucocitário humano (HLA) e a ocorrência de DECH aguda e crônica, em pacientes que receberam transplantes de irmãos HLA-idênticos. TIPO DE ESTUDO E LOCAL: Estudo retrospectivo no Centro de Hematologia e Hemoterapia da Universidade Estadual de Campinas (Unicamp). MÉTODOS: Os participantes foram 176 pacientes cujo primeiro transplante foi entre 1997 e 2009. A tipagem HLA foi realizada por sorologia e reação em cadeia da polimerase (PCR) com sequência específica de primers. RESULTADOS: A DECH aguda foi associada positivamente com HLA-A10 (P = 0,0007), HLA-A26 (P = 0,002), B55 (P = 0,001), DRB1*15 (P = 0,0211) e DQB1*05 (P = 0,038), enquanto HLA-B16 (P = 0,0333) foi mais frequente em pacientes sem DECH aguda. A DECH crônica foi associada positivamente com HLA-A9 (P = 0,01) e A23 (P = 0,0292) e, negativamente, com HLA-A2 (P = 0,0031) e B53 (P = 0,0116). HLA-B35 (P = 0,0373), B49 (P = 0,0155) e B55 (P = 0,0024) estavam aumentados em pacientes com DECH aguda grau 3 ou maior, em comparação aos outros pacientes. Em pacientes com DECH crônica extensa, HLA-A9 (P = 0,0004), A24 (P = 0,0059) e A26 (P = 0,0411) estavam aumentados em comparação aos outros pacientes, enquanto HLA-A2 estava diminuído (P = 0,0097). CONCLUSÕES: Este estudo sugere que o HLA pode influenciar a ocorrência de DECH aguda e crônica e a sua gravidade. No entanto, um estudo com melhor desenho e com mais pacientes será necessário para confirmar esses resultados.

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.

Incorporation of n-3 fatty acids by the liver of mice fed linseed oil as a function of feeding duration

The objective of this study was to evaluate the incorporation of omega-3 fatty acids (n-3 FA) on male Swiss mice livers receiving diets based on linseed oil (LO) for up to 56 days. The mice were sacrificed at 7, 14, 28, 42, 56 days and FA concentration was analyzed by gas chromatography. A total of 13 FA were identified on LO feeds presenting alpha-linolenic acid (LNA) contents of 26.97 percent. A total 22 FA were identified in the livers of the mice. Part of the LNA was converted into n-3 FA (20:3n-3, eicosapentaenoic acid-EPA, 22:5n-3, and docosahexaenoic acid-DHA), and some of the LNA was stored in the liver. LNA, EPA and DHA concentrations (mg/g total lipids ) from 0 days for up 56 days increased from 1.29 to 18.90 (LNA), 0.20 to 18.90 (EPA) and 41.26 to 99.17 (DHA).The concentration of n-3 FA in the livers varied with the duration of the LO diet. During LO feed, n-6 FA concentration fell and n-3 FA concentration rose through the experimental period.

Genetic polymorphisms of Rh, Kell, Duffy and Kidd systems in a population from the State of Paraná, southern Brazil

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.

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.

Ácidos graxos poli-insaturados n-3 e n-6: metabolismo em mamíferos e resposta imune / Omega-3 and omega-6 polyunsaturated fatty acids: metabolism in mammals and immune response

A experimentação animal apresenta uma grande importância para o desenvolvimento da ciência. O uso de camundongos em experimentos ocorre devido à semelhança destes animais com os seres humanos, fácil criação e manutenção e resposta experimental bastante rápida. Esses animais possuem as mesmas enzimas dessaturases e elongases que os humanos, por isso são usados em pesquisas envolvendo incorporação e síntese de ácidos graxos em tecidos. Os ácidos graxos da família ômega-3 e ômega-6 são de suma importância na dieta humana, pois estes não são sintetizados pela síntese de novo e são precursores dos ácidos graxos poli-insaturados de cadeia muito longa, como os ácidos eicosapentaenóico, docosahexaenóico e araquidônico. Estes desempenham funções importantes no organismo, como a síntese de eicosanóides que estão envolvidos diretamente no sistema imune e nas respostas inflamatórias. A razão entre o consumo de ácidos graxos n-6 e n-3 na dieta é um importante fator para determinar a ingestão adequada de ácidos graxos bem como prevenir o aparecimento de doenças. Este artigo tem como objetivo avaliar a incorporação de ácidos graxos em tecidos de animais e discutir a importância dos ácidos da família n-3 e seus metabólitos no sistema imunológico.

Experiments with animals are very important for the improvement of science. The use of mice in experiments is due to their similarity with humans, the easy of raising and maintaining them and their very fast response. These animals have the same desaturase and elongase enzymes as humans and so they are used in research involving the incorporation and synthesis of fatty acids in tissues. The fatty acids omega-3 and omega-6 are extremely important in the human diet because they are not synthesized de novo and are precursors of very long-chain polyunsaturated fatty acids, such as the eicosapentaenoic, docosahexaenoic and arachidonic acids. These acids play important roles in animals, such as precursors of eicosanoids, which are directly involved in the immune system and inflammatory response. The dietary n-3:n-6 intake ratio is important for assessing proper fatty acid intake and for preventing the development of diseases. Thus, this article assessed the incorporation of fatty acids in animal tissues and discussed the importance of n-3 fatty acids and its metabolites for the immune system.

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.

Analysis of fatty acids in human plasma / Análise de ácidos graxos em plasma humano

Nesse fascículo da revista, o estudo de Morais et al. (2010) avaliou quatro metodologias clássicas de extração de lipídeos (métodos de Folch, Bligh-Dyer, Rose-Gottlieb e Gerber) e uma técnica alternativa, com o objetivo de avaliar a eficiência da extração e a composição em ácidos graxos de plasma humano. O método alternativo proposto pelos autores usou o forno de micro-ondas como ferramenta e foi considerado muito rápido na extração lipídica e adequado na identificação de ácidos graxos, mas não em sua quantificação. O método de extração mais indicado para quantificação de ácidos graxos em plasma humano foi o método de Folch.

In this issue of the journal, the study by Morais et al. (2010) evaluated four classical methodologies of lipid extraction (methods of Folch, Bligh-Dyer, Rose-Gottlieb and Gerber), and an alternative technique, in order to evaluate the efficiency of extraction and fatty acid composition of human plasma. The alternative method proposed by the authors used the microwave oven as a tool, and was considered very fast in lipid extraction and identification of fatty acids, but not in their quantification. The most suitable extraction method for quantification of fatty acids in human plasma was the method of Folch.

Evaluation of lipid extraction and fatty acid composition of human plasma

AIM: The objective of this study was to compare the efficiency of lipid extraction and to evaluate the fatty-acid composition in total lipids from human plasma, using a new technique and the established Folch, Lees and Stanley (FLS), Bligh and Dyer (BD), Rose-Gottlieb (RG), and Gerber (GM) methods. METHOD: A new technique, the alternative method, to extract total lipids using a microwave was proposed and evaluated. RESULTS: The total lipids extracted from human plasma varied between 0.19 percent and 0.41 percent; the highest total lipid extracted were obtained by the Folch, Lees and Stanley (0.41 percent), alternative method (0.37 percent) and Rose-Gottlieb (0.36 percent) methods. The Gerber method was ineffective to extract total lipids from human plasma. A total of 24 fatty-acid species were quantified by gas chromatography. Of the methods studied, the highest concentrations were found using the Folch, Lees and Stanley method (p < 0.05). CONCLUSION: The alternative method is a fast lipid-extraction technique that can be used for the identification of human plasma fatty acids, but it is not suitable for their measurement.

OBJETIVO: O objetivo desse estudo foi comparar a eficiência da extração lipídica e avaliar a composição em ácidos graxos nos lipídios totais (LT) no plasma humano usando uma nova técnica e os métodos já conhecidos: Folch, Lees e Stanley (FLS), Bligh e Dyer (BD), Rose-Gottlieb (RG) e Gerber (MG). MÉTODO: A nova técnica para extração de lipídios totais utiza o micro-ondas, método Alternativo (MA). RESULTADOS: Os lipídios totais (LT) extraídos do plasma variaram de 0,19 por cento a 0,41 por cento, os maiores teores foram obtidos por FLS (0,41 por cento), MA (0,37 por cento) e RG (0,36 por cento). O MG foi ineficaz na extração dos lipídios totais do plasma. Um total de 24 espécies de ácidos graxos foi quantificado no plasma por cromatografia em fase gasosa. Entre os métodos estudados, as maiores concentrações de ácidos graxos foram obtidas usando-se o método de FLS (p< 0,05). CONCLUSÃO: O método Alternativo foi considerado uma técnica rápida de extração lipídica, a qual poderá ser utilizada somente na identificação de ácidos graxos em plasma humano, mas não adequado para a quantificação dos mesmos.

Extração de DNA a partir de sangue humano coagulado para aplicação nas técnicas de genotipagem de antígenos leucocitários humanos e de receptores semelhantes à imunoglobulina / DNA extraction from coagulated human blood for application in genotyping techniques for human leukocyte antigen and immunoglobulin-like receptors

O objetivo deste estudo foi padronizar uma metodologia de extração de DNA de alta qualidade a partir de amostras de sangue coagulado. Quarenta e oito amostras de sangue humano coagulado foram utilizadas para a extração de DNA pelo kit comercial EZ-DNA® (Biological Industries, Beit Haemek, Israel), pelo kit de coluna Neoscience® (One Lambda Inc., San Diego, CA) e pelo método modificado de salting out. Apenas o método de salting out foi capaz de extrair altas concentrações de DNA (média, 180ng/µL), as quais foram medidas pelo detector de fluorescência Qubit® (Invitrogen, USA). Este método permitiu a amplificação dos genes HLA (human leukocyte antigens) pela tecnologia PCR-SSO (polymerase chain reaction - specific sequence of oligonucleotides) Luminex, a qual exige DNA de boa qualidade, e de genes KIR (killer cell immunoglobulin-like receptors) pela técnica made in house PCR-SSP (polymerase chain reaction-sequence specific of primers), a qual demanda uma concentração específica de DNA (10ng/µL). Concluímos que a técnica de salting out modificada foi muito eficiente, simples e rápida para a extração de DNA de amostras de sangue humano coagulado, com o objetivo de realizar a genotipagem de genes HLA e KIR.

The objective of this study was to standardize a method for extracting high-quality DNA from samples of coagulated blood. Forty-eight samples of human coagulated blood were used for DNA extraction by means of the EZ-DNA® commercial kit (Biological Industries, Beit Haemek, Israel), the Neoscience® column kit (One Lambda Inc., San Diego, CA, USA) and a modified salting-out method. Only the salting-out method was able to extract high concentrations of DNA (mean, 180 ng/»l), which were measured using the Qubit® fluorescence detector (Invitrogen, USA). This method enabled amplification of HLA (human leukocyte antigen) genes using the Luminex PCR-SSO (polymerase chain reaction - sequence-specific oligonucleotide) technology, which demands good quality DNA, and amplification of KIR (killer-cell immunoglobulin-like receptor) genes using an in-house PCR-SSP (polymerase chain reaction - sequence-specific primer) technique, which demands a specific concentration of DNA (10 ng/»l). We concluded that the modified salting-out technique was very efficient, simple and fast for DNA extraction from human coagulated blood samples, with the aim of genotyping the HLA and KIR genes.