Role of the gene Slc11a1 and selected genotypes for minimum and maximum inflammatory reactivity in the process of alveolar bone healing in mice / Papel do gene Slc11a1 e de genótipos selecionados para mínima e máxima reatividade inflamatória no processo de reparo ósseo alveolar em camundongos

The process of alveolar bone healing can be influenced by several local and systemic factors, which include the immune system and healing related genes. However, the exact role of host inflammatory responsiveness and genetic background in bone healing process remains unclear. In this context, we evaluated the influence inflammation in alveolar bone healing taking advantage of mice strains genetically selected for maximum (AIRmax) or minimum (AIRmin) acute inflammatory response, as well AIR strains homozygous for RR/SS Slc11a1 genotypes. Experimental groups (N=5/time/group) comprised 8-week-old male or female AIRmax and AIRmin; and substrains AIRminRR, AIRminSS and AIRmaxRR and AIRmaxSS; submitted to extraction of upper right incisor and evaluated at 0, 3, 7, 14 and 21 days after upper incision extraction by micro-computed tomography (CT), histomorphometry, birefringence, immunohistochemistry and molecular (PCRArray) analysis. Initially, our results demonstrated that AIRmin mice presented an early increase (p<0.05) in bone volume, hyperdense regions, density of bone matrix and osteoblasts, increased (p<0.05) expressed of BMP4, BMP7 and RUNX2 when compared to AIRmax strain. AIRmin mice also presented lower counts of GR1+ and CD80+ cells, and higher counts of F4/80+ and CD206+ cells, in parallel with higher mRNA expression of CX3CL1, CCL5, CCR5 and ARG when compared to AIRmax animals. In late repair stages, the AIRmin strain presented a decreased (p<0.05) density of osteoclast and blood vessels than AIRmax, along lower RANKL and Catepk and higher PHEX and SOST mRNA expression, but the healing outcome at the endpoint was similar in AIRmin and AIRmax strains. When analyzed the effect of RR/SS Slc11a1 genotypes was evaluated in parallel with the influence AIRmin/AIRmax background, we initially observed that the AIRmax strain, associated with both RR and SS Slc11a1 genotypes, presented a more effective bone healing, characterized by increased (p<0.05) of bone volume and predominance of red fiber in analysis in contrast to AIRmin strains. AIRmaxRR presented increased (p<0.05) F4/80+ and decreased CD80+ e CD206+ cells count, while AIRmaxSS presented increased (p<0.05) GR1+, F4/80+ and CD80+ and decreased CD206+ cells. When the analysis was performed in order to address the influence Slc11a1 variants, AIRmaxSS strain presented a bone healing delay when compared to AIRmaxRR; characterized by decreased (p<0.05) of bone volume, trabecular number and red collagen fibers, increased (p<0.05) GR1+ and CD80+ and decreased F4/80+ and CD206+. Conversely, AIRminSS presented a more effective healing when compared with AIRminRR mice; characterized by increased (p<0.05) of bone volume, trabecular number/separation and red birefringence, increased GR1+ and decreased CD206+ cells count. In conclusion, while AIRmin and AIRmax strains presents a similar healing outcome at the endpoint, the early repair in AIRmin strain was associated with decreased presence of neutrophils and M1 macrophages, and increased M2 macrophages. Additionally, our while results showed that AIRmax inflammatory background was associated to a more effective bone healing process irrespective of the presence of RR/SS Slc11a1 genotypes, RR genotype favors the healing in AIRmax background and SS genotype was found to favor the healing in the AIRmin background.(AU)

O processo de reparo ósseo alveolar pode ser influenciado por vários fatores locais e sistêmicos, que incluem o sistema imunológico e os genes relacionados ao reparo. No entanto, o exato papel da resposta inflamatória do hospedeiro e genético background no processo de reparo ósseo ainda não está claro. Neste contexto, avaliamos a influência da inflamação no reparo óssea alveolar, em camundongos selecionadas geneticamente para uma resposta inflamatória aguda máxima (AIRmax) ou mínima (AIRmin), como também em camundongos AIR homozigoto para os alelos RR/SS do gene Slc11a1.Neste estudo foram utilizados camundongos machos e fêmeas (N=5/tempo/grupo), das linhagens selecionados para máxima e mínima (AIRmax e AIRmin) reação inflamatória, e também as sublinhagens AIRminRR, AIRminSS, AIRmaxRR e AIRmaxSS com idade aproximada de 8 semanas. Todos foram submetidos à extração do incisivo superior direito e avaliados nos períodos de 0, 3, 7, 14 e 21 dias pós extração, seguido pela análise tomografia computadorizada (CT), análise histomorfometria, análise de birrefringência, análise imuno-histoquímica e análise molecular (PCRArray). Inicialmente, nossos resultados demonstraram que a linhagem AIRmin, no período inicial, apresentou um aumento (p<0.05) no volume ósseo, nas regiões hiperdensas, na densidade de matriz óssea e osteoblastos, seguido pelo aumento (p<0.05) na expressão de BMP4, BMP7 e RUNX2 quando comparado a linhagem AIRmax. Camundongos AIRmin também apresentou uma menor contagem de células GR1+ e CD80+ e aumento da contagem de células F4/80+ e CD206+, em paralelo com aumento da expressão de mRNA de CX3CL1, CCL5, CCR5 e ARG quando comparado aos camundongos AIRmax. Nos períodos tardios, a linhagem AIRmin apresentou uma diminuição (p<0.05) na densidade de osteoclastos e vasos sanguíneos em comparação AIRmax, seguido por uma diminuição na expressão de mRNA de RANKL e Catepk e aumento de PHEX e SOST, mas o processo de reparo ósseo alveolar, no período final foi semelhante entres as linhagens AIRmin e AIRmax. Quando analisamos o efeito dos alelos RR/SS do gene Slc11a1 em paralelo com a influência do background AIRmin/AIRmax, nós inicialmente observamos que a linhagem AIRmax associada com ambos os alelos RR/SS do gene Slc11a1 apresentaram um processo de reparo mais efetivo, caracterizado pelo aumento (p<0.05) volume ósseo e predominância de fibras vermelhas em comparação com a linhagem AIRmin. Camundongos AIRmaxRR apresentaram aumento (p<0.05) na contagem de células F4/80+ e diminuição na contagem de células CD80+ e CD206+, enquanto, camundongos AIRmaxSS apresentou um aumento (p<0.05) na contagem de células GR1+, F4/80+, CD80+ e diminuição na contagem de células CD206+. Quando analisamos a influência dos alelos do gene Slc11a1, a linhagem AIRmaxSS apresentaram um atraso no reparo óssea quando comparado ao AIRmaxRR; caracterizado pela diminuição (p<0.05) do volume ósseo, número trabecular e fibras colágenas vermelhas, seguido pelo aumento (p<0.05) da contagem de células GR1+ e CD80+ e diminuição de células F4/80+ e CD206+. Por outro lado, camundongos AIRminSS apresentaram um reparo ósseo mais efetivo quando comparada com AIRminRR; caracterizada pelo aumento (p<0.05) do volume ósseo, número / separação trabecular e birrefringência das fibras colágenas no espectro vermelho, seguido pelo aumento da contagem de células GR1+ e diminuição das células CD206+. Diante disso, os nossos resultados demonstraram que as linhagens AIRmin e AIRmax apresentaram um processo de reparo ósseo alveolar semelhantes no período final do reparo, enquanto no reparo inicial a linhagem AIRmin estava associada com a diminuição de neutrófilos e macrófagos M1 e aumento dos macrófagos M2. Além disso, nossos resultados demonstraram que background AIRmax estava associado a um processo de reparo mais efetivo, independentemente da presença de genótipos RR/SS Slc11a1, o genótipo RR favorece o reparo no background AIRmax e o genótipo SS favoreceu a reparo no background AIRmin.(AU)

Association of the solute carrier family 11 member 1 gene polymorphisms with susceptibility to leprosy in a Brazilian sample

Natural resistance-associated macrophage protein 1/solute carrier family 11 member 1 gene (Nramp1/Slc11a1) is a gene that controls the susceptibility of inbred mice to intracellular pathogens. Polymorphisms in the human Slc11a1/Nramp1 gene have been associated with host susceptibility to leprosy. This study has evaluated nine polymorphisms of the Slc11a1/Nramp1 gene [(GT)n, 274C/T, 469+14G/C, 577-18G/A, 823C/T, 1029 C/T, 1465-85G/A, 1703G/A, and 1729+55del4] in 86 leprosy patients (67 and 19 patients had the multibacillary and the paucibacillary clinical forms of the disease, respectively), and 239 healthy controls matched by age, gender, and ethnicity. The frequency of allele 2 of the (GT)n polymorphism was higher in leprosy patients [p = 0.04, odds ratio (OR) = 1.49], whereas the frequency of allele 3 was higher in the control group (p = 0.03; OR = 0.66). Patients carrying the 274T allele (p = 0.04; OR = 1.49) and TT homozygosis (p = 0.02; OR = 2.46), such as the 469+14C allele (p = 0.03; OR = 1.53) of the 274C/T and 469+14G/C polymorphisms, respectively, were more frequent in the leprosy group. The leprosy and control groups had similar frequency of the 577-18G/A, 823C/T, 1029C/T, 1465-85G/A, 1703G/A, and 1729+55del4 polymorphisms. The 274C/T polymorphism in exon 3 and the 469+14G/C polymorphism in intron 4 were associated with susceptibility to leprosy, while the allele 2 and 3 of the (GT)n polymorphism in the promoter region were associated with susceptibility and protection to leprosy, respectively.

Contexto sociodemográfico de los homicidios en México D.F.: un análisis espacial / Sociodemographic context of homicide in Mexico City: a spatial analysis

OBJETIVO:Investigar el patrón de distribución espacial de la tasa de homicidios y su relación con las características sociodemográficas en las delegaciones de Benito Juárez, Coyoacán y Cuauhtémoc de la Ciudad de México en el año 2010. MÉTODOS: Estudio inferencial de corte transversal que usa métodos de análisis espacial para estudiar la asociación espacial de la tasa de homicidios y las características demográficas. La asociación espacial fue determinada a través del cociente de localización, análisis de regresión múltiple y el uso de la regresión geográficamente ponderada. RESULTADOS: Los homicidios muestran un patrón de localización heterogéneo con altas tasas en zonas con uso del suelo no residencial, con baja densidad de población y baja marginación. CONCLUSIONES: El uso de herramientas de análisis espacial son instrumentos poderosos para el diseño de políticas de seguridad pública preventiva y recreativa que busquen reducir la mortalidad por causas externas como homicidios.

OBJECTIVE:Investigate the spatial distribution pattern of the homicide rate and its relation to sociodemographic features in the Benito Juárez, Coyoacán, and Cuauhtémoc districts of Mexico City in 2010. METHODS: Inferential cross-sectional study that uses spatial analysis methods to study the spatial association of the homicide rate and demographic features. Spatial association was determined through the location quotient, multiple regression analysis, and the use of geographically weighted regression. RESULTS: Homicides show a heterogeneous location pattern with high rates in areas with non-residential land use, low population density, and low marginalization. CONCLUSIONS: Spatial analysis tools are powerful instruments for the design of prevention- and recreation-focused public safety policies that aim to reduce mortality from external causes such as homicides.

Menkes disease: importance of diagnosis with molecular analysis in the neonatal period / Doença de Menkes: importância do diagnóstico com análise molecular no período neonatal

Summary Menkes disease is a congenital disorder caused by changes in copper metabolism derived from mutations in the ATP7A gene. It is characterized by physical and neurological alterations. In the neonatal period, these alterations can be nonspecific, which makes early diagnosis a challenge. Diagnosis can be suspected when there are low levels of ceruloplasmin and serum copper. Molecular analysis confirms the diagnosis. Treatment is parenteral administration of copper histidine. We report a familial case with molecular confirmation. The proband had clinical and biochemical suspicious. Treatment with copper histidine was indicated, but initiated at the age of 2 months and 27 days only. He did not present improvements and died at 6 months. The mother became pregnant again, a male fetus was identified and copper histidine was manufactured during pregnancy. He was born healthy, biochemical markers were reduced and treatment was indicated. Molecular analysis was performed confirming mutation in both the mother and the proband, while the other son did not have mutation, so treatment was discontinued. We support the clinical relevance of molecular confirmation for the correct diagnosis and genetic counseling, once clinical findings in the neonatal period are nonspecific and early treatment with parenteral copper histidine must be indicated.

Resumo A doença de Menkes é causada por uma alteração genética no metabolismo do cobre, por mutações no gene ATP7A. Caracteriza-se por alterações neurológicas e no exame físico. No período neonatal, essas alterações podem ser inespecíficas, o que torna o diagnóstico precoce um desafio. O diagnóstico pode ser suspeitado quando há baixos níveis séricos de cobre e ceruloplasmina. A análise molecular confirma o diagnóstico, e o tratamento deve ser feito com histidina de cobre. Nós relatamos um caso familial de doença de Menkes. O probando apresentava quadro clínico e alterações bioquímicas compatíveis com a doença de Menkes, em consulta com 1 mês de vida. O tratamento foi indicado, mas apenas iniciado com 2 meses e 27 dias. Ele não apresentou melhora clínica e veio a óbito com 6 meses. A mãe teve uma nova gestação, foi identificado um feto do sexo masculino e foi solicitada a manipulação da histidina de cobre ainda durante a gestação. O bebê nasceu saudável, os marcadores bioquímicos estavam diminuídos e o tratamento com histidina de cobre foi indicado. Realizamos a análise molecular, que confirmou mutação no gene ATP7A na mãe e no probando; porém, o outro filho não apresentava mutação e o tratamento foi interrompido. Nós defendemos a importância clínica da confirmação molecular para o correto diagnóstico e o aconselhamento genético da doença de Menkes, uma vez que os achados clínicos e as alterações bioquímicas no período neonatal são inespecíficos, e o tratamento com histidina de cobre parenteral deve ser rapidamente instituído.

Limites e possibilidades do ensino à distância (EaD) na educação permanente em saúde: revisão integrativa / Limits and possibilities of distance learning in continuing education in health: integrative review

Trata-se de um estudo sobre o uso do ensino a distância (EaD) como uma estratégia de ensino na educação permanente em saúde (EPS), que teve como objetivo identificar e analisar os limites e possibilidades do uso da EaD na EPS. Estudo de revisão integrativa. O resultado aponta que a EaD é uma estratégia inovadora possível e potencial para a EPS, facilitando o desenvolvimento da aprendizagem dentro ou fora da instituição de saúde, porém é evidente a escassez de pesquisas na área. As limitações para a realização dos programas estão relacionadas à variável tempo, preparação para lidar com as tecnologias e importância do tutor como facilitador da aprendizagem. Conclui-se que o uso da EaD tem tido uma importante contribuição para o desenvolvimento dos recursos humanos em saúde, seja no processo de formação e/ou no processo contínuo de conhecimento.

This is a study on the use of distance learning (EaD, in Portuguese) as a teaching strategy in continuing health education (EPS, in Portuguese), which aimed to identify and analyze the limits and posibilities of using EaD in the EPS. Integrative Review Study. The result shows that EaD is an innovative, possible and potential strategy for EPS, facilitating the development of learning within or outside the health institution, although is evident the lack of research in the area. The limitations for the implementation of the programs are related to the time variable, preparation for dealing with the technologies and the importance of the tutor as a facilitator of learning. It concludes that the use of EaD has an important contribution to the development of human resources in health, is in the process of training and/or in the continuous knowledge process.

Zinc transporter 8 autoantibodies in patients with type 1 diabetes from a multiethnic population and their first degree relatives / Autoanticorpos transportadores de zinco 8 em pacientes com diabetes tipo 1 de uma população multiétnica e seus parentes de primeiro grau

Objective Zinc transporter 8 autoantibodies (ZnT8A) have been poorly studied in non-Caucasian individuals. We aimed to investigate the prevalence of ZnT8 autoantibodies in patients with T1D and their first degree relatives (FDR) from a multiethnic population, as well as its relation with the insulin (INS) or the protein tyrosine phosphatase non-receptor 22 (PTPN22) gene polymorphisms. Subjects and methods ZnT8A were analyzed in sera from T1D patients (n = 72, mean age of 30.3 ± 11.4 years) of variable duration (15.7 ± 11.8 years) and their FDR (n = 78, mean age of 18.3 ± 9.1 years) by a triple mix Radioligand Binding Assay (RBA) for the ZnT8 autoantibody (ZnT8-RWQ) variants. SNP (single nucleotide polymorphism) for INS and PTPN22 were genotyped. Results The prevalence of ZnT8A was higher in T1D patients than FDR, for ZnT8TripleA (24% vs. 4%,p = 0.001), ZnT8RA (24% vs. 4%, p < 0.001) and ZnT8QA (15% vs. 3%, p = 0.004). All FDR with ZnT8A (n = 3) had at least another positive antibody. Heterozygosis for PTPN22 was associated with a higher frequency of ZnT8TripleA (p = 0.039) and ZnT8RA (p = 0.038). Conclusions ZnT8A is observed in non-Caucasian patients with T1D, even years after the disease onset, as well as in their FDR. In those, there was an overlap between ZnT8A and other T1D antibodies. ZnT8A was associated with PTPN22 polymorphisms. Further longitudinal studies are necessary to elucidate the importance of these findings in the natural history of T1D patients with multiethnic background. .

Objetivo Os autoanticorpos transportadores de zinco 8 (ZnT8A) foram pouco estudados em indivíduos não caucasianos. Nosso objetivo foi investigar a prevalência de autoanticorpos ZnT8 em pacientes com T1D e seus parentes de primeiro grau (PPG) em uma população multiétnica, assim como a sua relação com os polimorfismos genéticos da insulina (INS) ou proteína tirosina fosfatase não receptora tipo 22 (PTPN22). Sujeitos e métodos ZnT8A foram analisados no soro de pacientes com T1D (n = 72, idade média de 30,3 ± 11,4 anos) de duração variável (15,7 ± 11,8 anos) e seus PPG (n = 72, idade média de 30,3 ± 11,4 anos) usando-se um ensaio de competição com radioligantes (RBA) para variantes dos autoanticorpos ZnT8 (ZnT8-RWQ). Os polimorfismos de nucleotídeo único para a INS e PTPN22 foram genotipados. Resultados A prevalência de ZnT8A foi mais alta em pacientes T1D do que nos PPG, para ZnT8TriploA (24% contra 4%, p = 0,001), ZnT8RA (24% contra 4%, p < 0,001) e ZnT8QA (15% contra 3%, p = 0,004). Todos os PPG com ZnT8A (n = 3) apresentaram positividade para pelo menos outro anticorpo. A heterozigose para PTPN22 foi associada a uma frequência mais alta de ZnT8TriploA (p = 0,039) e de ZnT8RA (p = 0,038). Conclusões Os ZnT8A foram observados em pacientes não caucasianos com T1D, mesmo depois de anos do início da doença, assim como em seus PPG. Nos parentes, houve uma sobreposição entre os ZnT8A e outros anticorpos para T1D. Os ZnT8A mostraram-se associados aos polimorfismos PTPN22. São necessários outros estudos longitudinais para se elucidar a importância desses achados na história natural de pacientes com T1D com antecedentes étnicos variados. .

Importância da detecção de mutações do gene ATP7B para o diagnóstico da doença de Wilson / The importance of detecting ATP7B gene mutations for the diagnosis of Wilson's disease

O diagnóstico da doença de Wilson (DW) é realizado por exames clínicos, laboratoriais, anatomopatológicos e de imagem. Mais de 500 mutações no gene ATP7B foram descritas como causadoras da DW. Para avaliar a importância da detecção de mutações no diagnóstico da DW em nosso meio, analisamos 35 pacientes com DW, 20 familiares de wilsonianos a partir de rastreamento familiar, 18 com hepatite crônica criptogênica e sete com insuficiência hepática aguda grave. Para o diagnóstico da DW foi utilizado o sistema de escore sugerido pela Sociedade Europeia para o Estudo do Fígado de 2012. Os dados demográficos, clínicos, laboratoriais e histológicos foram obtidos retrospectivamente. Obteve-se o DNA genômico de cada paciente a partir de sangue periférico e realizou-se o sequenciamento direto dos 21 éxons e suas bordas intrônicas do gene ATP7B. Todos os pacientes com DW apresentavam no mínimo quatro pontos. No grupo de rastreamento familiar o sequenciamento foi importante para o diagnóstico de DW em 14 familiares; no grupo de hepatite crônica criptogênica em oito pacientes e no grupo de insuficiência hepática aguda grave em três pacientes. Foi caracterizada uma família com cinco genótipos diferentes (dois homozigotos p.A1135Qfs/p.A1135Qfs e p.M645R/p.M645R), um heterozigoto composto (p.A1135Qfs/p.M645R) e dois heterozigotos simples (p.A1135Qfs/0 e p.M645R/0) com fenótipos variados. Foram detectadas duas mutações em heterozigose simples em pacientes com insuficiência hepática aguda grave. A mutação p.A1135Qfs e p.L708P foram as mais frequentes em todos os grupos. Foi identificada pela primeira vez a mutação p.M645R em homozigose. Concluímos que os resultados confirmaram que o sequenciamento do gene ATP7B foi útil: 1) para confirmar que as mutações p.A1135Qfs e p.L708P são as mais importantes na população brasileira; 2) para demonstrar que a mutação tida como a mais frequente na Europa, a p.H1069Q, tem bem menor importância em nosso meio, embora mais...

Wilson's disease (WD) is an autosomal recessive disorder secondary to mutations in the ATP7B gene resulting in toxic accumulation of copper in various tissues. The diagnosis of WD is made by the analysis of clinical, laboratory, histological findings and imaging tests. More than 500 mutations have been described in the ATP7B gene as the cause of WD. In order to expand the knowledge of the importance of mutation detection in the diagnosis of WD, we analyzed 36 patients with WD, 20 individuals from family screening, 18 with cryptogenic chronic hepatitis and seven with severe acute liver failure. For the diagnosis of WD the International Scoring System suggested by the European Association for the Study of the Liver (EASL) in 2012 was used. Demographic, clinical, laboratory and histological data were obtained retrospectively. Direct sequencing of 21 exons and intron boundaries of ATP7B gene was performed in genomic DNA extracted from peripheral blood leucocytes of all subjects. All patients with WD have at least four points of the scoring system without considering the DPA challenge test. In the family screening group, sequencing was important for the diagnosis of DW in fourteen patients; eight patients in the group of cryptogenic chronic hepatitis, and three patients in the group of severe acute liver failure. Five different genotypes were identified in one family (two homozygous, p.A1135Qfs/p.A1135Qfs and p.M645R/p.M645R, one compound heterozygous p.A1135Qfs/p.M645R, and two simple heterozygous p.A1135Qfs/0 and p.M645R/0). Two patients with acute liver failure were detected as simple heterozygous. The p.A1135Qfs and p.L708P were the most frequent mutations in all groups. It is the first time p.M645R mutation was detected in homozygosity. The ATP7B gene sequencing was useful: 1) to confirm that p.A1135Qfs and p.L708P mutations are the most frequent in the Brazilian population; 2) to confirm that the most common mutation in Europe, p.H1069Q has lower frequency...

Wilson's disease in Southern Brazil: genotype-phenotype correlation and description of two novel mutations in ATP7B gene / Doenca de Wilson no sul do Brasil: correlacao genotipica-fenotipica e a descricao de duas novas mutacoes no gene ATP7B

OBJECTIVE: Wilson's disease (WD) is an inborn error of metabolism caused by abnormalities of the copper-transporting protein encoding gene ATP7B. In this study, we examined ATP7B for mutations in a group of patients living in southern Brazil. METHODS: 36 WD subjects were studied and classified according to their clinical and epidemiological data. In 23 subjects the ATP7B gene was analyzed. RESULTS: Fourteen distinct mutations were detected in at least one of the alleles. The c.3207C>A substitution at exon 14 was the most common mutation (allelic frequency=37.1%) followed by the c.3402delC at exon 15 (allelic frequency=11.4%). The mutations c.2018-2030del13 at exon 7 and c.4093InsT at exon 20 are being reported for the first time. CONCLUSION: The c.3207C>A substitution at exon 14, was the most common mutation, with an allelic frequency of 37.1%. This mutation is the most common mutation described in Europe. .

OBJETIVO: A doença de Wilson (DW) é um erro inato do metabolismo causado por abnormalidades no gene ATP7B, que codifica uma proteína transportadora de cobre. Neste estudo, avaliamos as mutações do gene ATP7B em um grupo de pacientes do sul do Brasil. MÉTODOS: Foram estudados 36 pacientes com DW e classificados do ponto de vista clínico e epidemiológico. Em 23 pacientes, o gene ATP7B foi analisado. RESULTADOS: A substituição c.3207C>A no éxon 14 foi a mutação mais comum seguida pela mutação c.3402delC no éxon 15 . A mutação c.2018-2030del13 no éxon 7 e a c.4093InsT no éxon 20 são relatadas pela primeira vez na literatura. CONCLUSÃO: A mutação do gene ATP7B, com a substituição c.3207C>A no éxon 14 foi a mais frequente. Esta mutação é a mais comumente encontrada em pacientes europeus. .

Comparação entre microssatélites e o gene Ml MntH como alvos para a identificação do Mycobacterium leprae por PCR na hanseníase / Comparison between microsatellites and Ml MntH gene as targets to identify Mycobacterium leprae by PCR in leprosy

FUNDAMENTOS: PCR tem sido frequentemente utilizada no diagnóstico molecular da hanseníase. OBJETIVOS: comparar os resultados da PCR com 4 pares de primers específicos para Mycobacterium leprae, bem como os resultados da PCR à classificação operacional, segundo a OMS, de multibacilar (MB) e paucibacilar (PB) da hanseníase. MÉTODO: Vinte e oito amostras de DNA, extraído de biópsias congeladas de pele e de imprint de biópsias em papel de filtro de 23 pacientes (14 MB e 9 PB), foram utilizadas na PCR com primers que amplificam 131pb, 151pb e 168pb de regiões de microssatélites, e um fragmento de 336pb do gene Ml MntH (ML2098) do bacilo. RESULTADOS: O bacilo pôde ser detectado em 22 (78,6 por cento) das 28 amostras. Nove (45 por cento) das 20 amostras de biópsia e 6 (75 por cento) das 8 amostras de imprints foram positivas para TTC. Sete (35,5 por cento) amostras de biópsias e 5 (62,5 por cento) imprints foram positivos para AGT, e 11 (55 por cento) biópsias e 4 (50 por cento) imprints foram positivos para AT. Oito (38 por cento) amostras de biópsias e 5 (62,5 por cento) imprints foram positivos para o gene Ml MntH. Dentre o grupo MB, os microssatélites detectaram o bacilo em 78,5 por cento das amostras, e o gene Ml MntH, em 57,1 por cento das amostras, independentemente do material clínico. No grupo PB, 55,5 por cento das amostras foram positivas para os microssatélites, enquanto que 22,2 por cento o foram para o gene Ml MntH. CONCLUSÕES: Estes resultados mostram que, tanto as regiões específicas de microssatélites quanto o gene Ml MntH, podem representar ferramentas úteis na detecção do Ml MntH por PCR em amostras de biópsias e imprint de biópsias.

BACKGROUND: The Polymerase Chain Reaction (PCR) technique has been frequently used in the molecular diagnosis of leprosy. OBJECTIVES: To compare the results of PCR with four pairs of Mycobacterium leprae specific primers as well as to compare these results to multibacillary (MB) and paucibacillary (PB) leprosy according to the WHO operational classification. METHOD: 28 DNA samples, collected from the frozen skin biopsies and biopsy imprints on filter paper of 23 patients (14 MB and PB 9), were examined for PCR using primers which amplify 131, 151 and 168bp of specific microsatellite regions and a 336 fragment of the Ml MntH (ML2098) gene. RESULTS: M.leprae bacillus could be detected in 22 (78.6 percent) of the 28 samples. 9 (45 percent) of the 20 biopsy samples and 6 (75 percent) of the 8 imprints were positive to TTC. 7 (35.5 percent) skin biopsy specimens and 5 (62.5 percent) imprints were positive to AGT, and 11 (55 percent) biopsies and 4 (50 percent) were positive to AGT. 11 (55 percent) skin biopsies and 4 (50 percent) imprints were positive to AT. 8(38 percent) skin biopsies and 5 (62.5 percent) imprints were positive to the Ml MntH gene. In the MB group, the microsatellites detected the bacillus in 78.5 percent of the samples, and the Ml MntH gene in 57.1 percent of the samples, independent of the clinical material. In the PB group 55.5 percent of samples were positive to the microsatellite primers, while 22.2 percent were positive to the Ml MntH gene. CONCLUSIONS: These results show that both the specific regions of microsatellites, as well as the Ml MntH gene fragment can be useful tools for detecting the M. leprae DNA by PCR in frozen skin biopsy samples and filter paper biopsy imprints.

Polimorfismos do gene NRAMP1 em indivíduos com reações hansênicas, atendidos em dois Centros de Referência no Recife, nordeste do Brasil / NRAMP1 gene polymorphisms in individuals with leprosy reactions attended at two reference centers in Recife, northeastern Brazil

INTRODUÇÃO: Para investigar susceptibilidade às reações hansênicas, três polimorfismos do gene natural resistance-associated macrophage protein (NRAMP1), foram determinados em 201 indivíduos, atendidos em dois centros de referência no Recife, entre 2007 e 2008, sendo 100 paucibacilares e 101 multibacilares. MÉTODOS: A determinação dos polimorfismos 274C/T, D543N e 1729+55del4 do gene NRAMP1 foi realizada utilizando a técnica do polimorfismo de fragmento de restrição em DNA extraído de sangue periférico e as estimativas das freqüências alélicas e genotípicas foram feitas por contagem direta. RESULTADOS: Os genótipos predominantes foram: CC (51,8 por cento) para 274C/T, GG (86,6 por cento) para D543N e +-TGTG (59,9 por cento) para 1729+55del4. O genótipo mutante 274 TT predominou na negatividade da reação reversa (p=0,03) e na positividade do eritema nodoso (p=0,04). CONCLUSÕES: Nossos resultados sugerem que o polimorfismo 274 C/T do gene NRAMP1 pode auxiliar na determinação da susceptibilidade à reação tipo II em indivíduos com hanseníase.

INTRODUCTION: To investigate susceptibility to leprosy reactions, three polymorphisms of the natural resistance-associated macrophage protein (NRAMP1) gene were determined in 201 individuals who were attended at two reference centers in Recife, between 2007 and 2008. Of these, 100 were paucibacillary and 101 were multibacillary. METHODS: The 274C/T, D543N and 1729+55del4 polymorphisms of the NRAMP1 gene were determined using the technique of restriction fragment polymorphism on DNA extracted from peripheral blood. Allelic and genotypic frequencies were estimated by direct counting. RESULTS: The predominant genotypes were: CC (51.8 percent) for 274C/T; GG (86.6 percent) for D543N; and +-TGTG (59.9 percent) for 1729+55del4. The mutant genotype 274 TT predominated in negativity of the reverse reaction (p = 0.03) and in positivity of erythema nodosum leprosum (p = 0.04). CONCLUSIONS: Our results suggest that 274 C/T polymorphism of the NRAMP1 gene may aid in determining the susceptibility to type II reactions among leprosy patients.

Suplementación con cobre entre comidas no tiene efecto sobre la nutrición de hierro en hombres / Copper supplementation between meals has no effect on iron status of men

Iron deficiency is prevalent in most of the developing world where it coexists with other micronutrient deficiencies such as copper. Combined supplementation with iron and copper is one of the strategies that can be used to improve the iron and copper status of a population. However, there is concern about potential negative interactions between these two micronutrients due to a competitive binding to the divalent metal transporter 1 (DMTl), a proton-coupled transporter of a variety of divalent metals including copper. The aim of this study was to measure the effect of daily supplementation with 8 mg of copper, as copper sulfate during 6 months on the iron status. Sixty healthy male adults were randomized to receive a copper supplement or a placebo. Fasting blood samples were obtained before and after copper supplementation to evaluate the iron and copper nutritional status. Copper supplementation did not change significantly iron and copper status parameters. In conclusion, daily supplementation with 8 mg of copper during 6 months does not deteriorate iron nutrition in adult men.

La deficiencia de hierro coexiste con otras carencias, entre ellas de cobre. La suplementación combinada con estos nutrientes es una de las estrategias utilizadas en su prevención. Sin embargo, existe la posibilidad de interacciones negativas, ya que el DMT1, principal transportador de hierro no hem a nivel intestinal, también transporta cobre. El propósito del estudio fue medir el efecto de la suplementación con 8 mg diarios de cobre, como sulfato de cobre, durante 6 meses, sobre la nutrición de hierro. Sesenta hombres adultos, aparentemente sanos, fueron seleccionados al azar para recibir el suplemento de cobre o un placebo. Se tomaron muestras de sangre en ayunas antes y después de finalizada la suplementacion para evaluar la nutrición de hierro y de cobre. La suplementacion con cobre no determinó cambios significativos en los indicadores de nutrición de cobre y de hierro. En conclusión, la suplementacion con 8 mg diarios de Cu administrado entre comidas durante 6 meses no deterioró la nutrición de hierro en hombres adultos.

The NRAMPI, VDR and TNF-¦Á gene polymorphisms in Iranian tuberculosis patients: the study on host susceptibility

The natural resistance-associated macrophage protein (NRAMP1), Vitamin-D receptor (VDR) and Tumor necrosis factor (TNF-¦Á) have been associated in susceptibility to tuberculosis, but the results have been inconsistent. This study aimed to determine the association of NRAMP1, VDR, and TNF-¨¢ variant with development of pulmonary tuberculosis (PTB) among Iranian patients. The single nucleotide polymorphisms (SNPs) at INT4, D543, 3'UTR of NRAMP1 gene, SNPs in restriction sites of BsmI, and FokI of the VDR gene and SNPs of TNF-¦Á at -238,-308, -244,857,-863 positions were analyzed by PCR-RFLP among two groups of individual; patients with PTB (n=117) and healthy controls (n=60). Thereafter, the frequencies of extended haplotypes and diplotypes were estimated. No statistically significant differences were observed in allele frequencies of INT4, D543, 3'UTR of NRAMPI, FokI of VDR and TNF-¦Á at -238, -244,-863 and -857 position. Although, the frequency of b allele of BsmI [ORs: 0.24 CI95 percent (0.07-0.67 (p=0.001)] and -308 A variant in TNF-¦Á promoter region [ORs:0.26 CI95 percent( 0.07-0.77) (p=0.006)] were significantly more in PTB patients than healthy controls. The frequency of extended diplotypes of NRAMP [GG TGTG++GA; 0.02(0.001-0.0035)], VDR [FFBB; 0.2(0.6-0.6] and TNF-¦Á [CCCCGGGGGG; 0.49(0.25-0.97)] were statistically different in patients and control subjects (p<0.05). This study confirmed the association of SNPs in BsmI (B/b + b/b) of VDR and SNPs in -308A (G/A +G/G) of TNF-¦Á genes with susceptibility to tuberculosis in Iranian PTB patients. Furthermore, the extended haplotypes and diplotypes analysis can be considered as an alternative way to determine the host susceptibility to TB.

Analysis of HFE and non-HFE gene mutations in Brazilian patients with hemochromatosis

BACKGROUND: Approximately one-half of Brazilian patients with hereditary hemochromatosis (HH) are neither homozygous for the C282Y mutation nor compound heterozygous for the H63D and C282Y mutations that are associated with HH in Caucasians. Other mutations have been described in the HFE gene as well as in genes involved in iron metabolism, such as transferrin receptor 2 (TfR2) and ferroportin 1 (SCL40A1). AIMS: To evaluate the role of HFE, TfR2 and SCL40A1 mutations in Brazilian subjects with HH. PATIENTS AND METHODS: Nineteen male subjects (median age 42 [range: 20-72] years) with HH were evaluated using the Haemochromatosis StripAssay A®. This assay is capable of detecting twelve HFE mutations, which are V53M, V59M, H63D, H63H, S65C, Q127H, P160delC, E168Q, E168X, W169X, C282Y and Q283, four TfR2 mutations, which are E60X, M172K, Y250X, AVAQ594-597del, and two SCL40A1 mutations, which are N144H and V162del. RESULTS: In our cohort, nine (47 percent) patients were homozygous for the C282Y mutation, two (11 percent) were heterozygous for the H63D mutation, and one each (5 percent) was either heterozygous for C282Y or compound heterozygous for C282Y and H63D. No other mutations in the HFE, TfR2 or SCL40A1 genes were observed in the studied patients. CONCLUSIONS: One-third of Brazilian subjects with the classical phenotype of HH do not carry HFE or other mutations that are currently associated with the disease in Caucasians. This observation suggests a role for other yet unknown mutations in the aforementioned genes or in other genes involved in iron homeostasis in the pathogenesis of HH in Brazil.

DMT1: Which metals does it transport?

DMT1 _ Divalent Metal (Ion) Transporter 1 or SLC11A2/DCT1/Nramp2 _ transports Fe2+ into the duodenum and out of the endosome during the transferrin cycle. DMT1 also is important in non-transferrin bound iron uptake. It plays similar roles in Mn2+ trafficking. Voltage clamping showed that six other metals evoked currents, but it is unclear if these metals are substrates for DMT1. This report summarizes progress on which metals DMT1 transports, focusing on results from the authors' labs. We recently cloned 1A/+IRE and 2/-IRE DMT1 isoforms to generate HEK293 cell lines that express them in a tetracycline-inducible fashion, then compared induced expression to uninduced expression and to endogenous DMT1 expression. Induced expression increases about 50x over endogenous expression and about 10x over uninduced levels. Fe2+, Mn2+, Ni2+ and Cu1+ or Cu2+ are transported. We also explored competition between metal ions using this system because incorporation essentially represents DMT1 transport and find this order for transport affinity: Mn>?Cd>?Fe>Pb Co Ni>Zn. The effects of decreased DMT1 also could be examined. The Belgrade rat has diminished DMT1 function and thus provides ways of testing. A series of DNA constructs that generate siRNAs specific for DMT1 or certain DMT1 isoforms yield another way to test DMT1-based transport.

Cop-like operon: Structure and organization in species of the Lactobacillale order

Copper is an essential and toxic trace metal for bacteria and, therefore, must be tightly regulated in the cell. Enterococcus hirae is a broadly studied model for copper homeostasis. The intracellular copper levels in E. hirae are regulated by the cop operon, which is formed by four genes: copA and copB that encode ATPases for influx and efflux of copper, respectively; copZ that encodes a copper chaperone; and copY, a copper responsive repressor. Since the complete genome sequence for E. hirae is not available, it is possible that other genes may encode proteins involved in copper homeostasis. Here, we identified a cop-like operon in nine species of Lactobacillale order with a known genome sequence. All of them always encoded a CopY-like repressor and a copper ATPase. The alignment of the cop-like operon promoter region revealed two CopY binding sites, one of which was conserved in all strains, and the second was only present in species of Streptococcus genus and L. johnsonii. Additional proteins associated to copper metabolism, CutC and Cupredoxin, also were detected. This study allowed for the description of the structure and organization of the cop operon and discussion of a phylogenetic hypothesis based on the differences observed in this operon's organization and its regulation in Lactobacillale order.

Hereditary hemochromatosis: An opportunity for gene therapy

Levels of body iron should be tightly controlled to prevent the formation of oxygen radicals, lipoperoxidation, genotoxicity, and the production of cytotoxic cytokines, which result in damage to a number of organs. Enterocytes in the intestinal villae are involved in the apical uptake of iron from the intestinal lumen; iron is further exported from the cells into the circulation. The apical divalent metal transporter-1 (DMT1) transports ferrous iron from the lumen into the cells, while the basolateral transporter ferroportin extrudes iron from the enterocytes into the circulation. Patients with hereditary hemochromatosis display an accelerated transepithelial uptake of iron, which leads to body iron accumulation that results in cirrhosis, hepatocellular carcinoma, pancreatitis, and cardiomyopathy. Hereditary hemochromatosis, a recessive genetic condition, is the most prevalent genetic disease in Caucasians, with a prevalence of one in 300 subjects. The majority of patients with hereditary hemochromatosis display mutations in the gene coding for HFE, a protein that normally acts as an inhibitor of transepithelial iron transport. We discuss the different control points in the homeostasis of iron and the different mutations that exist in patients with hereditary hemochromatosis. These control sites may be influenced by gene therapeutic approaches; one general therapy for hemochromatosis of different etiologies is the inhibition of DMT1 synthesis by antisense-generating genes, which has been shown to markedly inhibit apical iron uptake by intestinal epithelial cells. We further discuss the most promising strategies to develop gene vectors and deliver them into enterocytes.

Vesicular transport of Fe and interaction with other metal ions in polarized Caco2 Cell monolayers

Two aspects of the mechanisms by which iron is absorbed by the intestine were studied in the Caco2 cell model, using 59Fe(II)-ascorbate. Data showing the importance of vesicular processes and cycling of apotransferrin (apoTf) to uptake and overall transport of Caco2 cell monolayers (or basolateral 59Fe release) were obtained by comparing effects of: a) adding apoTf to the basal chamber; b) adding vesicular transport inhibitors; or c) cooling to 4°C. These showed that apoTf may be involved in as much as half of Fe transfer across the basolateral membrane, and that vesicular processes may also play a role in non-apoTf-dependent Fe transport. Studies were initiated to examine potential interactions of other metal ions with Fe(II) via DMT1. Kinetic data showed a single, saturable process for uptake of Fe(II) that was pH dependent and had a Km of 7 ìM. An excess of Mn(II) and Cu(I) over Fe(II) of 200: 1 (ìM: ìM) in 1 mM ascorbate markedly inhibited Fe uptake. The kinetics were not competitive. Km increased and Vmax decreased. We conclude that vesicular transport, involving endo- and exocytosis at both ends of the enterocyte, is a fundamental aspect of intestinal iron absorption and that DMT1 may function as a transporter not just for divalent but also for monovalent metal ions.

Iron and glutathione at the crossroad of redox metabolism in neurons

Neurons, as non-dividing cells, encounter a myriad of stressful conditions throughout their lifespan. In particular, there is increasing evidence that iron progressively accumulates in the brain with age and that iron-induced oxidative stress is the cause of several forms of neurodegeneration. Here, we review recent evidence that gives support to the following notions: 1) neuronal iron accumulation leads to oxidative stress and cell death; 2) neuronal survival to iron accumulation associates with decreased expression of the iron import transporter DMT1 and increased expression of the efflux transporter IREG1; and 3) the adaptive process of neurons towards iron-induced oxidative stress includes a marked increase in both the expression of the catalytic subunit of gamma glutamate-cysteine ligase and glutathione. These findings may help to understand aging-related neurodegeneration hallmarks: oxidative damage, functional impairment and cell death.

Regulation of transepithelial transport of iron by hepcidin

Hepcidin (Hepc) is a 25 amino acid cationic peptide with broad antibacterial and antifungal actions. A likely role for Hepc in iron metabolism was suggested by the observation that mice having disruption of the gene encoding the transcription factor USF2 failed to produce Hepc mRNA and developed spontaneous visceral iron overload. Lately, Hepc has been considered the stores regulator, a putative factor that signals the iron content of the body to intestinal cells. In this work, we characterized the effect of Hepc produced by hepatoma cells on iron absorption by intestinal cells. To that end, human Hepc cDNA was cloned and overexpressed in HepG2 cells and conditioned media from Hepc-overexpressing cells was used to study the effects of Hepc on intestinal Caco-2 cells grown in bicameral inserts. The results indicate that Hepc released by HepG2 inhibited apical iron uptake by Caco-2 cells, probably by inhibiting the expression of the apical transporter DMT1. These results support a model in which Hepc released by the liver negatively regulates the expression of transporter DMT1 in the enterocyte.