Sofia Rocha Lieber

Micronucleus monitoring to assess human occupational exposure to organochlorides

Health surveillance for hazardous situations due to chemical exposure, in particular those which are carcinogenic, requires sensitive monitoring tests. Although experimental studies have shown the genotoxic and carcinogenic effect of several organochlorides, the lack of epidemiologic studies prevents their classification as carcinogenic to human beings. In this context, genotoxicity tests of short duration in human cells gain importance. The relation between the clastogenic effects (chromosome breaks) and cancer induction is already known to the scientific literature. The micronucleus test has been proposed as a good indicator of clastogenesis. In the present study, we evaluated, by means of the micronucleus test, 41 workers of a chemical industry in the state of São Paulo, southeast region of Brazil, who had been exposed to a mixture of chlorinated solvents (carbon tetrachloride, perchloroethylene, and hexachlorobenzene) and 28 workers who had not been exposed. Peripheral lymphocytes stimulated by phytohemagglutinin and with cytokinesis blocked by cytochalasin B were used. The results showed that the exposed workers presented a statistically significant higher frequency of micronuclei than the group which had not been exposed. Environ. Mol. Mutagen. 29: 46–52, 1997

IL2 and TNFA Gene Polymorphisms and the Risk of Graft-versus-Host Disease after Allogeneic Haematopoietic Stem Cell Transplantation

This study aimed to analyse the association of gene polymorphisms with the outcome of allogeneic haematopoietic stem cell transplantation. We studied 122 donor/recipient pairs who received HLA‐identical transplants from siblings at the Universidade Estadual de Campinas, Brazil, between June 1996 and June 2006. Donor/recipient alleles for TNFA−238 and IL2−330/+166 single‐nucleotide polymorphisms (SNP) were analysed by PCR‐SSP. No association was observed between the risk of acute graft‐versus‐host disease (GVHD) and these SNP. However, our findings suggest that the polymorphism of promoter gene TNFA−238GA is associated with the occurrence and severity of chronic GVHD. The probability of chronic GVHD in patients with GA genotype at position −238 of TNFA gene is 91.7% in contrast to 59.4% in patients with GG genotype (P = 0.038). In patients with donor GA genotype the probability of chronic GVHD is 90.8%, and 57.9% in patients with donor GG genotype (P = 0.038). The probability of extensive chronic GVHD in patients with TNFA−238GA is 91.7% compared with 46.3% in patients with TNFA−238GG (P = 0.0046). In patients with donor GA genotype at position −238 of the TNFA gene, it is 81.7%, compared with 44.5% in patients with donor GG genotype (P = 0.016). However, further studies with more patients are required to identify cytokine gene polymorphisms and their association with transplant‐related complication in Brazil, particularly due to ethnic background, the relatively low power of detection of genetic markers of this study, and the complexity of the MHC region.

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 Fishers 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 Joinvilles, Santa Catarina, and Rio de Janeiros, Rio de Janeiro (RJ), Euro‐Brazilian populations than to those of Salvadors, Bahia, and Rio de Janeiros, 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.

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.

Correlation of mixed lymphocyte culture with chronic graft-versus-host disease following allogeneic stem cell transplantation

The purpose of the present study was to evaluate the mixed lymphocyte culture as a predictive assay of acute and chronic graft-versus-host disease (GVHD). We studied 153 patients who received a first bone marrow transplantation from human leukocyte antigen-identical siblings. Acute GVHD was observed in 26 of 128 (20.3%) patients evaluated and chronic GVHD occurred in 60 of 114 (52.6%). One-way mixed lymphocyte culture (MLC) assays were performed by the standard method. MLC results are reported as the relative response (RR) from donor against patient cells. The responses ranged from -47.0 to 40.7%, with a median of 0.5%. The Kaplan-Meier probability of developing GVHD was determined for patients with positive and negative MLC. There was no significant difference in incidence of acute GVHD between the groups studied. However, the incidence of chronic GVHD was higher in recipients with RR >4.5% than in those with RR < or =4.5%. The Cox Proportional Hazards model was used to examine the effect of MLC levels on incidence of chronic GVHD, while adjusting for the potential confounding effect of others suspected or observed risk factors. The relative risk of chronic GVHD was 2.5 for patients with positive MLC (RR >4.5%), 2.9 for those who received peripheral blood progenitor cells as a graft, and 2.2 for patients who developed previous acute GVHD. MLC was not useful for predicting acute GVHD, but MLC with RR >4.5% associated with other risk factors could predict the development of chronic GVHD, being of help for the prevention and/or treatment of this late complication.

Papel das citocinas na imunopatogênese da doença do enxerto contra o hospedeiro

O transplante de celulas progenitoras hematopoeticas e o tratamento de escolha para muitas doencas hematologicas e imunodeficiencias primarias. A doenca do enxerto contra o hospedeiro (DECH) e ainda uma grave complicacao apos o transplante alogenico e a principal causa de mortalidade e morbidade. O estudo da patogenese da DECH auxilia no desenvolvimento de medidas preventivas da doenca, assim como na escolha de terapias imunossupressoras adequadas de tratamento. Este estudo discute os principais componentes imunologicos envolvidos na patogenese da DECH aguda e cronica, com enfase a participacao das citocinas e seu controle.Stem cell transplantation is the first line treatment of many hematological diseases and primary immunodeficiencies. Graftversus-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. Rev. bras. hematol. hemoter. 2008; 30(2):142-152.

Correlation of IL-6 and IL-10 production following bone marrow transplantation with donor cytokine gene polymorphisms

Estudos de varios genes candidatos tem demonstrado que polimorfismos geneticos em genes de citocinas contribuem com variacoes nos niveis de citocinas produzidas e esta variacao pode influenciar a ocorrencia e gravidade de complicacoes apos o transplante de celulas-tronco hematopoeticas (TCTH). Neste trabalho comparamos as concentracoes sericas de TNF-α, IFN-γ, IL-6, IL-10 e TGF-β 1 em 13 receptores seguindo o TCTH com os polimorfismos TNF-308, IFNG+874, IL6-174, IL10-1082,-819,-592 e TGFB1+869,+915. Os niveis sericos de citocinas foram medidos usando-se kits comerciais de ELISA para TNF-α, IFN-γ, IL-6, IL-10 e TGF-β 1 (BioSource®, Nivelles, Belgium, Europe). Os genotipos de doadores/receptores para estes polimorfismos de citocinas foram analisados pela reacao em cadeia da polimerase com sequencias especificas de primer (PCR-SSP) com o kit Cytokine Genotyping Primers (One Lambda, Canoga Park, CA, USA). Encontramos correlacao entre os niveis de IL-6 e IL-10 seguindo o TCTH e os polimorfismos IL6-174 e IL10-1082,-819,-592, mas nao para outras citocinas investigadas neste estudo. Aqueles com genotipos relativos a baixa producao de IL-6 e IL-10 produziram mais baixos niveis destas citocinas que aqueles com genotipos relativos a producao alta e/ou intermediaria destas citocinas (P < 0,05).

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.

Associação dos níveis de citocinas no pós-transplante de células-tronco hematopoiéticas com a Doença do Enxerto Contra o Hospedeiro aguda

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

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.