Association between human leukocyte antigens and graft-versus-host disease occurrence after allogenic hematopoietic stem cell transplantation.

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.

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.

Importância de polimorfismos de genes reguladores de citocinas em transplantes de células progenitoras hematopoiéticas / Importance of regulatory cytokine gene polymorphisms in hematopoietic stem cell transplantation

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.

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.

[Association between HLA and leukemia in a mixed Brazilian population]. / Associação entre HLA e leucemia em uma população brasileira de etnia mista.

OBJECTIVE: The main purpose of this study was to investigate the class I HLA antigens and class II HLA allele frequencies in 164 patients with leukemia: 35 patients with ALL (acute lymphoid leukemia), 50 with AML (acute myeloid leukemia) and 78 with CML (chronic myeloid leukemia). METHODS: The genotyping of class I HLA was performed by microlymphocytotoxicity and of class II by PCR-SSP (polymerase chain reaction - sequence specific of primers) (One Lambda, Canoga Park, CA, USA). RESULTS: In patients with LLA, frequencies of HLA-B45 and HLA-B56 were higher (P = 0.02; OR = 3.13; 95%IC = 0.94-10.44; P = 0.03; OR = 3.61; 95%IC = 0.47-27.64, respectively), than in controls. In patients with AML, the frequency of HLA-B7 (P = 0.01; OR = 2.41; 95%IC = 1.25-4.67) was higher than in controls. The presence of HLA-B45 (P= 0.01; OR = 3.29; 95%IC = 1.46-7.40), HLA-DRB1*04 (P = 0.002; OR = 2.17; 95%IC = 1.36-3.46) and HLA-DRB1*08 (P = 0.004; OR = 2.36; 95%IC = 1.34-4.16) was associated to increased risk of CML developing. CONCLUSION: Our results suggest that variants of HLA confer susceptibility to the same forms of leukemia, and could provide new tools for the investigation of genetics and etiology of this disease.

Associação entre HLA e leucemia em uma população brasileira de etnia mista / Association between HLA and leukemia in a mixed Brazilian population

OBJETIVOS: O objetivo deste estudo foi investigar a freqüência de antígenos HLA Classe I e de alelos HLA Classe II em 164 pacientes com vários tipos de leucemias: 35 pacientes com LLA (leucemia linfóide aguda), 50 com LMA (leucemia mielóide aguda) e 78 com LMC (leucemia mielóide crônica). MÉTODOS: A tipagem HLA Classe I foi realizada por microlinfocitotoxicidade e a de Classe II por PCR-SSP (polymerase chain reaction - sequence specific of primers), ambas da One Lambda (Canoga Park, CA, US). RESULTADOS: Em pacientes com LLA, as freqüências das variantes HLA-B45 e HLA-B56 foram maiores (P = 0,02; OR = 3,13; 95 por centoIC = 0,94-10,44; P = 0,03; OR = 3,61; 95 por centoIC = 0,47-27,64, respectivamente), quando comparadas com controles. Nos pacientes com LMA, a freqüência de HLA-B7 (P = 0,01; OR = 2,41; 95 por centoIC = 1,25-4,67) foi maior que em controles. A presença de HLA-B45 (P= 0,01; OR = 3,29; 95 por centoIC = 1,46-7,40) e de HLA-DRB1*04 (P = 0,002; OR = 2,17; 95 por centoIC = 1,36-3,46) e HLA-DRB1*08 (P = 0,004; OR = 2,36; 95 por centoIC = 1,34-4,16) foi associada ao maior risco de desenvolver LMC. CONCLUSÃO: Nossos resultados sugerem que variantes HLA conferem susceptibilidade a algumas formas de leucemia e podem prover novas ferramentas para a investigação da genética e etiologia desta doença.

OBJECTIVE: The main purpose of this study was to investigate the class I HLA antigens and class II HLA allele frequencies in 164 patients with leukemia: 35 patients with ALL (acute lymphoid leukemia), 50 with AML (acute myeloid leukemia) and 78 with CML (chronic myeloid leukemia). METHODS: The genotyping of class I HLA was performed by microlymphocytotoxicity and of class II by PCR-SSP (polymerase chain reaction - sequence specific of primers) (One Lambda, Canoga Park, CA, USA). RESULTS: In patients with LLA, frequencies of HLA-B45 and HLA-B56 were higher (P = 0.02; OR = 3.13; 95 percentIC = 0.94-10.44; P = 0.03; OR = 3.61; 95 percentIC = 0.47-27.64, respectively), than in controls. In patients with AML, the frequency of HLA-B7 (P = 0.01; OR = 2.41; 95 percentIC = 1.25-4.67) was higher than in controls. The presence of HLA-B45 (P= 0.01; OR = 3.29; 95 percentIC = 1.46-7.40), HLA-DRB1*04 (P = 0.002; OR = 2.17; 95 percentIC = 1.36-3.46) and HLA-DRB1*08 (P = 0.004; OR = 2.36; 95 percentIC = 1.34-4.16) was associated to increased risk of CML developing. CONCLUSION: Our results suggest that variants of HLA confer susceptibility to the same forms of leukemia, and could provide new tools for the investigation of genetics and etiology of this disease.

Relationship between cytokine gene polymorphisms and graft-versus-host disease after allogeneic stem cell transplantation in a Brazilian population.

Graft-versus-host disease (GVHD) is the major complication of allogeneic hematopoietic stem cell transplantation (HSCT), and cytokines are recognized as important mediators of GVHD. Polymorphisms in the regulatory regions of several cytokine genes have been associated with a number of immune diseases as well as organ transplant complications. In this study we have investigated the role of tumor necrosis factor-alpha(-308), interleukin (IL)-6(-174), IL-10(-1082, -819, -592), Interferon-gamma(-874), and transforming growth factor-beta1(+869, +915) polymorphisms on HSCT outcome. Donor/recipient genotypes were analyzed by polymerase chain reaction with sequence specific primers (PCR-SSP). Although we have found a small number of low IL-6, a polymorphism at position -174 of the recipient and donor IL-6 gene was associated with the increased incidence of chronic GVHD. Therefore, this study emphasizes the probable potential role of genetic variability of donor and recipient in determining outcome after transplantation.

Serum cytokine levels and acute graft-versus-host disease after HLA-identical hematopoietic stem cell transplantation.

OBJECTIVE: The aim of this study was to investigate whether the serum levels of soluble interleukin-2R (sIL-2R), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), IL-6, IL-10, and transforming growth factor-beta(1) (TGF-beta(1)) were associated with the development of acute graft-vs-host disease (aGVHD). PATIENTS AND METHODS: Serum cytokine levels were sequentially measured by sandwich enzyme-linked immunosorbent assay in 13 patients who had received full-match allogeneic hematopoietic stem cell transplantation (HSCT). RESULTS: Serum sIL-2R and IL-10 levels from the 1st to the 15th week post transplantation were significantly higher in the group that developed aGVHD than in the group without aGVHD. sIL-2R levels increased in direct correlation to engraftment and at onset of aGVHD, whereas IL-10 levels increased transiently following HSCT. The mean TNF-alpha concentration in the first weeks after transplantation was augmented in the group that developed aGVHD. Furthermore, a decrease in TGF-beta(1) levels after engraftment was significantly associated with aGVHD. No correlation was found between aGVHD and the other cytokines. CONCLUSIONS: 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 aGVHD. Although sIL-2R, TNF-alpha, IL-10, and TGF-beta(1) levels have been correlated with aGVHD, sIL-2R levels at engraftment may provide a better parameter for early detection of aGVHD after allogeneic HSCT.

Addition of exogenous cytokines in mixed lymphocyte culture for selecting related donors for bone marrow transplantation.

CONTEXT: Mixed lymphocyte culturing has led to conflicting opinions regarding the selection of donors for bone marrow transplantation. The association between a positive mixed lymphocyte culture and the development of graft-versus-host disease (GVHD) is unclear. The use of exogenous cytokines in mixed lymphocyte cultures could be an alternative for increasing the sensitivity of culture tests. OBJECTIVE: To increase the sensitivity of mixed lymphocyte cultures between donor and recipient human leukocyte antigen (HLA) identical siblings, using exogenous cytokines, in order to predict post-transplantation GVHD and/or rejection. TYPE OF STUDY: Prospective study. SETTING: Bone Marrow Transplantation Unit, Universidade Estadual de Campinas. PARTICIPANTS: Seventeen patients with hematological malignancies and their respective donors selected for bone marrow transplantation procedures. PROCEDURES: Standard and modified mixed lymphocyte culturing by cytokine supplementation was carried out using donor and recipient cells typed for HLA. MAIN MEASUREMENTS: Autologous and allogenic responses in mixed lymphocyte cultures after the addition of IL-4 or IL-2. RESULTS: In comparison with the standard method, average responses in the modified mixed lymphocyte cultures increased by a factor of 2.0 using IL-4 (p < 0.001) and 6.4 using IL-2 (p < 0.001), for autologous donor culture responses. For donor-versus-recipient culture responses, the increase was by a factor of 1.9 using IL-4 (p < 0.001) and 4.1 using IL-2 (p < 0.001). For donor-versus-unrelated culture responses, no significant increase was observed using IL-4, and a mean response inhibition of 20% was observed using IL-2 (p < 0.001). Neither of the cytokines produced a significant difference in the unrelated control versus recipient cell responses. CONCLUSION: IL-4 supplementation was the best for increasing the mixed lymphocyte culture sensitivity. However, IL-4 also increased autologous responses, albeit less intensively than IL-2. Thus, with this loss of specificity we believe that it is not worth modifying the traditional mixed lymphocyte culture method, even with IL-4 addition.

Addition of exogenous cytokines in mixed lymphocyte culture for selecting related donors for bone marrow transplantation

CONTEXT: Mixed lymphocyte culturing has led to conflicting opinions regarding the selection of donors for bone marrow transplantation. The association between a positive mixed lymphocyte culture and the development of graft-versus-host disease (GVHD) is unclear. The use of exogenous cytokines in mixed lymphocyte cultures could be an alternative for increasing the sensitivity of culture tests. OBJECTIVE: To increase the sensitivity of mixed lymphocyte cultures between donor and recipient human leukocyte antigen (HLA) identical siblings, using exogenous cytokines, in order to predict post-transplantation GVHD and/or rejection. TYPE OF STUDY: Prospective study. SETTING: Bone Marrow Transplantation Unit, Universidade Estadual de Campinas. PARTICIPANTS: Seventeen patients with hematological malignancies and their respective donors selected for bone marrow transplantation procedures. PROCEDURES: Standard and modified mixed lymphocyte culturing by cytokine supplementation was carried out using donor and recipient cells typed for HLA. MAIN MEASUREMENTS: Autologous and allogenic responses in mixed lymphocyte cultures after the addition of IL-4 or IL-2. RESULTS: In comparison with the standard method, average responses in the modified mixed lymphocyte cultures increased by a factor of 2.0 using IL-4 (p < 0.001) and 6.4 using IL-2 (p < 0.001), for autologous donor culture responses. For donor-versus-recipient culture responses, the increase was by a factor of 1.9 using IL-4 (p < 0.001) and 4.1 using IL-2 (p < 0.001). For donor-versus-unrelated culture responses, no significant increase was observed using IL-4, and a mean response inhibition of 20 percent was observed using IL-2 (p < 0.001). Neither of the cytokines produced a significant difference in the unrelated control versus recipient cell responses. CONCLUSION: IL-4 supplementation was the best for increasing the mixed lymphocyte culture sensitivity. However, IL-4 also increased autologous responses, albeit less intensively than IL-2. Thus, with this loss of specificity we believe that it is not worth modifying the traditional mixed lymphocyte culture method, even with IL-4 addition