Expressão de CD1D na doença autoimune pênfigo foliáceo : quantificação e variantes de RNAm

Orientadora : Profª Drª Maria Luiza Petzl-ErlerDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Genética. Defesa: Curitiba, 25/09/2013Inclui referências : f. 35-38;50-59Área de concentraçãoResumo: O pênfigo foliáceo (PF) é uma doença autoimune da pele na qual há a formação de autoanticorpos contra a desmogleína 1, proteína importante na adesão entre queratinócitos. O PF se caracteriza pelo desprendimento da camada superior do epitélio, com formação de bolhas e erosões na pele. Como a maioria das doenças autoimunes, o pênfigo é uma doença complexa na qual atuam fatores ambientais e genéticos. O gene CD1D codifica a glicoproteína CD1d que participa da apresentação de antígenos glicolipídicos para células T natural killer (NKT). Além da importante função nas respostas imunes, o gene CD1D já foi reportado superexpresso em células TCD4+ de pacientes de pênfigo. O objetivo do presente trabalho foi comparar a expressão de CD1D e o padrão de expressão das suas variantes de RNAm em monócitos e linfócitos B IgG+ e IgG-, entre pacientes de pênfigo foliáceo e controles. Nossos resultados demonstraram que em monócitos CD1D está subexpresso em pacientes com lesão (P = 0,0079, diferença de expressão - DE = 0,44) e sem lesão (P = 0,0028, DE = 0,30) em relação aos indivíduos controle. Além disso, neste tipo celular o RNAm representado pela banda 1C (que possui os éxons 3 e 4 completos) é menos frequente em pacientes que em controles (P = 0,011). Em linfócitos B não foram encontradas diferenças estatisticamente significantes na expressão de CD1D entre pacientes e controles (células B IgG+ DE = 1,84 P = 0,18/ células B IgG- DE = 5,28 P = 0,15). Por meio desses resultados concluímos que a expressão de CD1D está alterada em pênfigo foliáceo, o que nos leva a sugerir que haja participação de células NKT e antígenos glicolipídicos na patogênese do pênfigo. O papel de CD1D e sua expressão em nível de RNA e proteína pode ser foco de estudos futuros tanto em pênfigo quanto em outras doenças autoimunes. Palavras chave: pênfigo foliáceo, expressão gênica, CD1D, variantes de RNAm, células NKT.Abstract: Pemphigus foliaceus is an epidermal autoimmune disease characterized by pathogenic autoantibodies against desmoglein 1, an important cell-cell adhesion protein. It is a blistering disease characterized by erosive lesions in the upper epidermal layers of the skin. Pemphigus foliaceus pathogenesis is not completely understood, yet, genetic and environmental factors generate and modulate the autoimmune response. The CD1D gene encodes an MHC class I like glycoprotein (CD1d) whose main function is presenting glycolipid antigens to natural killer T (NKT) cells. The CD1D mRNA was recently reported as overexpressed in T CD4+ cells of pemphigus patients when compared with healthy individuals. The aim of this work was compare the CD1D mRNA expression and the mRNA variants expression pattern in monocytes, IgG+ and IgG- B lymphocytes between patients and controls. We found that in monocytes CD1D mRNA is underexpressed in patients with (P = 0.0079 ED = 0.44) and without lesions (P = 0.0028 ED = 0.30). Furthermore the mRNA represented by 1C band (with complete exons 3 and 4) was significantly less frequent in patients than in controls (P = 0.011). For B cells there were not found statistically significant differences in the level of mRNA of CD1d between pemphigus patients and controls (IgG+ B cells ED = 1.84 P = 0.1829/ IgG- B cells ED = 5.28 P = 0.1508). These results led us to conclude that the expression of CD1D is altered in pemphigus foliaceus and point to participation of NKT cells and glycolipid antigens in pemphigus. The role of CD1D and its expression at the RNA and protein levels in pemphigus and other autoimmune diseases should be focused in future studies. Key-words: pemphigus foliaceus, CD1D, gene expression, mRNA variants, NKT cells

Genetic Associations and Differential mRNA Expression Levels of Host Genes Suggest a Viral Trigger for Endemic Pemphigus Foliaceus

The long search for the environmental trigger of the endemic pemphigus foliaceus (EPF, fogo selvagem) has not yet resulted in any tangible findings. Here, we searched for genetic asso ciations and the differential expression of host genes involved in early viral infections and innate antiviral defense. Genetic variants could alter the structure, expression sites, or levels of the gene products, impacting their functions. By analyzing 3063 variants of 166 candidate genes in 227 EPF patients and 194 controls, we found 12 variants within 11 genes associated with differential suscepti bility (p < 0.005) to EPF. The products of genes TRIM5, TPCN2, EIF4E, EIF4E3, NUP37, NUP50, NUP88, TPR, USP15, IRF8, and JAK1 are involved in different mechanisms of viral control, for example, the regulation of viral entry into the host cell or recognition of viral nucleic acids and proteins. Only two of nine variants were also associated in an independent German cohort of sporadic PF (75 patients, 150 controls), aligning with our hypothesis that antiviral host genes play a major role in EPF due to a specific virus–human interaction in the endemic region. Moreover, CCL5, P4HB, and APOBEC3G mRNA levels were increased (p < 0.001) in CD4+ T lymphocytes of EPF patients. Because there is limited or no evidence that these genes are involved in autoimmunity, their crucial role in antiviral responses and the associations that we observed support the hypothesis of a viral trigger for EPF, presumably a still unnoticed flavivirus. This work opens new frontiers in searching for the trigger of EPF, with the potential to advance translational research that aims for disease prevention and treatment

Evaluation Of The Overload Of Care In Families Of Psychiatric Patients In Psychosocial Care Center

Introduction: The burden of care in family refers to the weight caused by the primary caregiver role to psychiatric patients and the difficulties encountered in performing this function in daily life. Objectives: Assessing the objective and subjective overload of family members who live with the reality of psychiatric disorder in a child day-care psychosocial care center. Methods: Cross-sectional study, descriptive-exploratory, of quantitative approach, with non-probabilistic samples of accidental type with 80 families of psychiatric patients held in a Psychosocial Care Center. For overload evaluation, the subscales "B" and "D" of the Family Overload Rating Scale (FBIS-BR) were used. Results: The study was conducted with 80 families of psychiatric patients. The average age of female caregivers was 39,6 years old, and 40,7 years old for male caregivers, with female predominance (87,5%) compared to men (12,5%), with low education for both genres. Family caregivers presented high objective burden due to excessive demand attention (p&lt;0,001), heteroaggressiveness (p&lt;0,001) and perplexing behavior of psychiatric patients regarding the supervision of problematic behaviors (p&lt;0,001). The items on the impact on the family's daily routine have not helped to generate objective overload for the family members. On subjective overload, it was clear to observe familiar members with high degree of disturbance in all the dimensions assessed (p &lt; 0,001). Conclusion: The high degree of care overload observed in family members indicates the need to develop contacts with the family of the psychiatric patient to answer questions, offer support and assistance to the family caregiver. Keywords: Caregivers. Patients. Mental Health Services

Perfil de expressão de RNAs e polimorfismo de IncRNAs no pênfigo foliáceo endêmico

Orientadora: Profaª Drª Maria Luiza Petzl-Erler.Coorientadores: Dr. Rodrigo Coutinho de Almeida e Prof. Dr. Saleh Ibrahim.Tese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Genética. Defesa : Curitiba, 26/04/2019.Inclui referências: p. 91-100.Resumo: Nas últimas décadas, o uso de técnicas de sequenciamento em larga escala adicionou informações valiosas sobre a complexidade do genoma humano e de sequências não codificantes nele presentes. Por muito tempo uma porção considerável do genoma foi considerada DNA lixo por não codificar proteína, porém, estudos demonstraram que parte significativa do DNA é transcrita, mas não traduzida, e que muitos desses transcritos são importantes em processos essenciais às células. Entre eles estão os RNAs longos não codificadores (lncRNAs) que têm se destacado funcionalmente na regulação da expressão gênica. Compreender melhor a multiplicidade funcional do genoma contribui para o avanço no entendimento de doenças complexas nas quais fatores genéticos têm efeito. O pênfigo foliáceo (PF) é uma doença autoimune da pele na qual há a formação de autoanticorpos contra a desmogleína 1, uma proteína constituinte dos desmossomos responsáveis pela adesão entre queratinócitos. O PF se caracteriza pelo desprendimento da camada superior do epitélio, com formação de bolhas e erosões na pele. É uma doença em cuja patogênese atuam fatores ambientais e genéticos, e é endêmica em certas regiões geográficas do mundo, sendo o Brasil o país em que atinge sua maior incidência. Neste trabalho utilizamos duas abordagens principais com o objetivo de contribuir para uma melhor compreensão da patogênese do pênfigo foliáceo endêmico (PFE): uma, de análise de associação genética com polimorfismos de RNAs longos não codificadores (lncRNAs) e outra, de expressão diferencial em nível de RNA. Primeiramente investigamos a existência de associação entre 2.080 polimorfismos de um único nucleotídeo (SNPs) localizados em genes de lncRNAs e o PFE, a fim de descrevermos variantes de predisposição à doença. Encontramos um SNP associado, rs7144332 localizado no lncRNA AL110292.1, e outros cinco SNPs sugestivos de associação, os quais podem alterar a função de lncRNAs e influenciar a susceptibilidade ao PFE. Na segunda abordagem, realizamos o sequenciamento de RNA (RNA-seq) de células T CD4+ de pacientes de PFE e de indivíduos sem a doença com o objetivo de descrever o perfil de mRNAs e lncRNAs com expressão alterada na condição de doença. Identificamos 324 genes diferencialmente expressos, entre esses, 32 genes de lncRNAs. Vários desses genes têm funções na resposta imune e inflamatória, e alguns deles já haviam sido encontrados diferencialmente expressos em PF em uma análise de microarranjo feita anteriormente. Por meio da técnica de PCR quantitiva (qPCR) validamos a diferença de expressão encontrada para os genes LEF1-AS1, IL18RAP, GNLY e S1PR5 em células T CD4+. Também avaliamos a expressão dos mesmos genes em células B de pacientes e controles, sendo que IL18RAP, GNLY e S1PR5 foram detectados diferencialmente expressos. Desta maneira, foi possível corroborar a participação de diversos genes codificadores e não codificadores de proteína no desencadeamento e/ou manutenção do PFE, além de apontar outros genes nunca antes implicados no pênfigo. Tais genes se destacam como candidatos interessantes para estudos posteriores, e podem tornar-se potenciais alvos terapêuticos em PF.Abstract: In recent decades, the use of large-scale sequencing techniques has added valuable insights into the complexity of the human genome and the non-coding sequences present in it. For a long time, a considerable portion of the genome was considered junk DNA because it did not encode protein. However, studies have shown that a significant part of DNA is transcribed but not translated, and many of these transcripts are important in processes essential to cells. Among them are the long non-coding RNAs (lncRNAs) that have been functionally prominent in the regulation of gene expression. Better understanding of the functional multiplicity of the genome contributes to advancing the understanding of complex diseases in which genetic factors have an effect. Pemphigus foliaceus (PF) is an autoimmune disease of the skin in which there is the formation of autoantibodies against desmoglein 1, a protein constituent of the desmosomes responsible for adhesion between keratinocytes. PF is characterized by the detachment of the upper epithelial layer, with formation of blisters and erosions on the skin. It is a disease whose pathogenesis affects environmental and genetic factors and is endemic in certain geographic regions of the world. Brazil is the country where it reaches its highest incidence. In this work we used two main approaches with the aim of contributing to a better understanding of the pathogenesis of endemic pemphigus foliaceus (EPF): genetic association analysis with polymorphisms of long non-coding RNAs (lncRNAs) and differential expression analysis at the RNA level. We first investigated the association between 2,080 single nucleotide polymorphisms (SNPs) located in lncRNAs genes and EPF, in order to describe variants of predisposition to the disease. We found one associated SNP, rs7144332 located in the lncRNA AL110292.1, and other five SNPs suggestive of association, which may alter the function of lncRNAs and influence the susceptibility to EPF. In the second approach, we performed RNA sequencing (RNAseq) of CD4+ T cells from EPF patients and individuals without the disease aiming to describe the profile of mRNAs and lncRNAs whose expression is altered in the disease condition. We identified 324 differentially expressed genes, among them, 32 lncRNA genes. Several of these genes have functions in the immune and inflammatory response and some of them had previously been found differentially expressed in EPF by microarray analysis. Using the quantitative PCR technique (qPCR) we validate the expression difference found for the LEF1-AS1, IL18RAP, GNLY and S1PR5 genes in CD4+ T cells. We also evaluated the expression of the same genes in patients and controls B cells, and IL18RAP, GNLY and S1PR5 were detected differentially expressed. In this way, it was possible to corroborate the participation of several protein-coding and non-coding genes of in the onset and/or maintenance of EPF, and to point out genes never previously implicated in pemphigus. Such genes stand out as interesting candidates for further studies, and may become potential therapeutic targets in PF

Besides Pathology: Long Non-Coding RNA in Cell and Tissue Homeostasis

A significant proportion of mammalian genomes corresponds to genes that transcribe long non-coding RNAs (lncRNAs). Throughout the last decade, the number of studies concerning the roles played by lncRNAs in different biological processes has increased considerably. This intense interest in lncRNAs has produced a major shift in our understanding of gene and genome regulation and structure. It became apparent that lncRNAs regulate gene expression through several mechanisms. These RNAs function as transcriptional or post-transcriptional regulators through binding to histone-modifying complexes, to DNA, to transcription factors and other DNA binding proteins, to RNA polymerase II, to mRNA, or through the modulation of microRNA or enzyme function. Often, the lncRNA transcription itself rather than the lncRNA product appears to be regulatory. In this review, we highlight studies identifying lncRNAs in the homeostasis of various cell and tissue types or demonstrating their effects in the expression of protein-coding or other non-coding RNA genes

Long non-coding RNA polymorphisms influence susceptibility to endemic pemphigus foliaceus

Barreto, Mauricio Lima. Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil. “Documento produzido em parceria ou por autor vinculado à Fiocruz, mas não consta à informação no documento”.Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2019-02-21T12:59:53Z No. of bitstreams: 1 Lobo Alves S.C. Long non-coding RNA... 2019.pdf: 369633 bytes, checksum: e2ad6cce856e57dca065c5368ad9ca21 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2019-02-21T13:21:27Z (GMT) No. of bitstreams: 1 Lobo Alves S.C. Long non-coding RNA... 2019.pdf: 369633 bytes, checksum: e2ad6cce856e57dca065c5368ad9ca21 (MD5)Made available in DSpace on 2019-02-21T13:21:27Z (GMT). No. of bitstreams: 1 Lobo Alves S.C. Long non-coding RNA... 2019.pdf: 369633 bytes, checksum: e2ad6cce856e57dca065c5368ad9ca21 (MD5) Previous issue date: 2019Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Araucária, and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).Universidade Federal do Paraná. Laboratório de Genética Molecular Humana. Departamento de Genética. Curitiba, PR, Brasil.Universidade Federal do Paraná. Laboratório de Genética Molecular Humana. Departamento de Genética. Curitiba, PR, Brasil / Universidade Estadual de Santa Cruz. Departamento de Ciências Biológicas. Santa Cruz, Ilhéus, Brasil.Instituto Mário Penna. Núcleo de Ensino e Pesquisa. Belo Horizonte, MG, Brasil / Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Biologia. Belo Horizonte, MG, Brazil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Biologia. Belo Horizonte, MG, Brazil.Fundação Oswaldo Cruz. Instituto Rene Rachou. Belo Horizonte, MG, Brasil.Universidade Federal da Bahia. Instituto de Saúde Coletiva. Salvador, BA, Brasil.Universidade Federal de Pelotas. Pós-Graduação em Epidemiologia. Pelotas, RS, Brasil.Leiden University Medical Center. Department of Biomedical Data Sciences. Section Molecular Epidemiology. Leiden, The Netherlands.Universidade Federal do Paraná. Laboratório de Genética Molecular Humana. Departamento de Genética. Curitiba, PR, Brasil.Pemphigus foliaceus (PF) is an epidermal autoimmune disease, characterized by the presence of autoantibodies against the desmosomal protein desmoglein 1. Genetic and environmental factors contribute to PF, a complex disease that is endemic in Brazil and Colombia and neighbouring countries, and in Tunisia. Long non-coding RNAs (lncRNAs) may participate in gene regulation by interacting with DNA, proteins, and other RNAs. Dysregulation of lncRNAs has recently been recognized as an important co-player in the onset or progression of complex diseases. In addition, single-nucleotide polymorphisms (SNP) located in lncRNA genes have been associated with differential risk to cancer, autoimmunity and infection

Genetic Association and Differential RNA Expression of Histone (De)Acetylation-Related Genes in Pemphigus Foliaceus—A Possible Epigenetic Effect in the Autoimmune Response

Pemphigus foliaceus (PF) is an autoimmune skin blistering disease characterized by antidesmoglein-1 IgG production, with an endemic form (EPF) in Brazil. Genetic and epigenetic factors have been associated with EPF, but its etiology is still not fully understood. To evaluate the genetic association of histone (de)acetylation-related genes with EPF susceptibility, we evaluated 785 polymorphisms from 144 genes, for 227 EPF patients and 194 controls. Carriers of HDAC4_rs4852054*A were more susceptible (OR = 1.79, p = 0.0038), whereas those with GSE1_rs13339618*A (OR = 0.57, p = 0.0011) and homozygotes for PHF21A_rs4756055*A (OR = 0.39, p = 0.0006) were less susceptible to EPF. These variants were not associated with sporadic PF (SPF) in German samples of 75 SPF patients and 150 controls, possibly reflecting differences in SPF and EPF pathophysiology. We further evaluated the expression of histone (de)acetylation-related genes in CD4+ T lymphocytes, using RNAseq. In these cells, we found a higher expression of KAT2B, PHF20, and ZEB2 and lower expression of KAT14 and JAD1 in patients with active EPF without treatment compared to controls from endemic regions. The encoded proteins cause epigenetic modifications related to immune cell differentiation and cell death, possibly affecting the immune response in patients with PF

Consensus of the Brazilian Association of Hematology, Hemotherapy and Cellular Therapy (ABHH) and the Brazilian Ministry of Health - General management of blood and blood products on the tests necessary for the release of exceptional medicines for sickle cell disease

To date, hydroxyurea is the only effective and safe drug that significantly reduces morbidity and mortality of individuals with Sickle cell disease. Twenty years of real-life experience has demonstrated that hydroxyurea reduces pain attacks, vaso-occlusive events, including acute chest syndrome, the number and duration of hospitalizations and the need for transfusion. The therapeutic success of hydroxyurea is directly linked to access to the drug, the dose used and adherence to treatment which, in part, is correlated to the availability of hydroxyurea. This consensus aims to reduce the number of mandatory exams needed to access the drug, prioritizing the requesting physician's report, without affecting patient safety