Diversidade de genes KIR em populações de diferentes ancestralidades e possível associação do polimorfismo de KIR3DS1 com a doença auto-imune pênfigo foliáceo endêmico

Orientador: Maria Luiza Petzl-ErlerMonografia (Bacharelado) - Universidade Federal do Paraná. Setor de Ciências Biológicas. Curso de Graduação em Ciências BiológicasResumo : Os genes KIR (killer cell immunoglobulin-like receptors) são responsáveis por codificar um grupo de receptores semelhantes às imunoglobulinas que interagem com moléculas HLA de classe I na superfície das células natural killers (NK ou assassinas naturais), uma subpopulação de linfócitos que participam principalmente da resposta imune inata, tendo a função de reconhecer e destruir células infectadas por vírus ou células tumorais. Estes genes estão contidos no Leukocyte Receptor Complex (LRC), na posição 19q13.4, e apresentam um nível incomum de diversidade. Estudos de genética de populações e de associação com doenças complexas têm cada vez mais envolvido os genes KIR. O Pênfigo Foliáceo Endêmico (PFE ou Fogo Selvagem) é uma doença auto-imune restrita a países da América do Sul e caracterizada por uma resposta de linfócitos T, principalmente Th2, e pela ocorrência de autoanticorpos de classe IgG, subclasse IgG4, que atuam sobre as desmogleínas tipo 1, glicoproteínas pertencentes à família das caderinas e fundamentais para a adesão celular de células de superfície. Nosso objetivo foi caracterizar três amostras populacionais (uma ameríndia Kaingang da reserva do Ivaí, uma afro-brasileira e outra de ascendência principalmente japonesa) quanto às freqüências (gênica e de presença) de sete genes KIR e verificar se o polimorfismo de ausência e presença dos genes KIR3DL1 e KIR3DS1 apresenta associação com o PFE. Utilizamos a técnica de PCR-SSOP para a genotipagem dos quatro genes moldura e de KIR2DS3, KIR3DL1 e KIR3DS1. A amostra ameríndia de Kaingang do Ivaí diferiu estatisticamente das amostras afrodescendente e de origem predominantemente japonesa em relação aos três genes não moldura. Estas duas últimas amostras apresentaram distribuição semelhante entre si e em relação a populações de origem predominantemente européia. O gene KIR2DS3 apresentou freqüência nula apenas na amostra ameríndia, o que pode sugerir que este gene tenha desaparecido por deriva genética e efeito fundador nesta população. Os genes moldura foram detectados em todos indivíduos analisados, com exceção de um indivíduo da amostra afro-brasileira que não apresentou KIR3DL3. Encontramos associação negativa do gene KIR3DS1 (OR = 0,56; IC(95%) 0,32-0,97) com o PFE, a partir da análise de uma sub-amostra de pacientes e controles de origem predominantemente européia, levando nos a sugerir que a presença deste gene ofereça aos seus portadores menor predisposição à doença

Análise de expressão de MicroRNA candidatos à regulação da resposta imune adaptativa em Pênfigo Foliáceo

Orientadora : Profa. Dra. Maria Luiza Petzl-ErlerDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Genética. Defesa: Curitiba, 17/02/2012Bibliografia: fls. 63-70Resumo: Células T reguladoras naturais expressam constitutivamente a molécula CD25 e o fator de transcrição Foxp3 e representam uma importante subpopulação de linfócitos T CD4 com atividade supressora. Estas células têm sido estudadas em várias doenças autoimunes, incluindo pênfigo vulgar, mas até o momento não foram estudadas em pênfigo foliáceo (PF). MicroRNA constituem uma classe de pequenos RNA nãocodificantes, de aproximadamente 22 nucleotídeos, que atuam póstranscricionalmente, participando da sintonia fina da expressão gênica através do pareamento com a região 3' não traduzida de certos RNA mensageiros. MicroRNA são importantes para o desenvolvimento e função normais do sistema imune, sendo que alterações quantitativas estão frequentemente associadas à doenças autoimunes. PF trata-se de uma doença autoimune caracterizada pela produção de autoanticorpos IgG dependente de células Th2 e direcionados contra proteínas de adesão entre células da pele. Considerando esta importância de células Th2 e B autorreativas, bem como o evidente papel de células T reguladoras na perda de autotolerância, o objetivo deste trabalho foi verificar o padrão de expressão de cinco microRNA candidatos à regulação da resposta (auto)imune adaptativa do PF (miR-145, miR-148a, miR-155, miR-338-5p, and miR-1321) nas três subpopulações de linfócitos acima mencionadas, bem como analisar a proporção de células T reguladoras CD4+CD25+. Os resultados principais foram: (1) em células B de memória expressando IgG de superfície (B IgG+) e Th2, miR-155 esteve 2 e 3 vezes superexpresso, respectivamente, em pacientes sem lesão em relação aos controles sadios (P = 0,02 and P = 0,03, respectivamente); (2) miR- 1321 apresentou tendência de superexpressão em células Th2 do conjunto total de pacientes comparados aos controles sadios (P = 0,10) e há evidências de que este microRNA regule a expressão de BLyS, um estimulador de células B, uma vez que os seus níveis mostraram-se inversamente proporcionais aos de BLyS; (3) miR-148a não foi detectado em células B IgG+ de pacientes sem lesão, mas foi consistentemente expresso por células de pacientes com a doença ativa; e (4) células T reguladoras CD4+CD25+ estiveram significativamente reduzidas em pacientes com a doença ativa em relação aos controles sadios (P = 0,02) e aos pacientes sem lesão (P = 0,03). Além disso, foi detectada uma tendência de menor expressão de Foxp3 em pacientes com a doença ativa, sugerindo que células T reguladoras sejam ainda menos frequentes no PF. Curiosamente, células Th2 e B IgG+, potenciais alvos de células T reguladoras, mostraram-se sutilmente mais frequentes em pacientes com lesões ativas. Estes resultados colocam miR-148a, miR-155 e miR-1321 como potenciais alvos para terapias alternativas em PF. Além disso, sugere-se que uma redução da frequência de células T reguladoras esteja associada ao PF, destacando a importância destas células para a manutenção da autotolerância.Abstract: Natural regulatory T cells express the CD25 molecule and Foxp3 transcription factor constitutively and represent an important CD4+ T cell subpopulation with suppressive functions. These cells have been implicated in many autoimmune diseases, including pemphigus vulgaris, but have not yet been studied in pemphigus foliaceus (PF). MicroRNAs are a class of small non-coding RNAs of approximately 22 nucleotides that act at the posttranscriptional level as important gene expression fine-tuners, through binding to the 3' UTR of mRNAs. MicroRNAs have been increasingly implicated in normal immune function and their misregulation is frequently associated to autoimmune diseases. PF is an autoimmune disease characterized by a Th2-dependet production of IgG autoantibodies directed against skin adhesion proteins. Considering this importance of autoreactive Th2 and B cells, as well as the evidence of the role of regulatory T cells in loss of self-tolerance, we aimed to verify the expression patterns of five microRNAs candidates of PF adaptive (auto)immune regulation (miR-145, miR-148a, miR-155, miR- 338-5p, and miR-1321) in the three mentioned lymphocytes subpopulations, as well as to analyze the proportion of regulatory CD4+CD25+ T cells in the disease. Among our findings are: (1) in IgG+ B cells and Th2 cells, miR-155 is 2- and 3-fold upregulated, respectively, in patients with inactive disease compared to healthy controls (P = 0.02 and P = 0.03, respectively); (2) miR-1321 tends to be upregulated in Th2 cells of PF patients compared to healthy controls (P = 0.10) and is believed to target BLyS, a B cell stimulator, as its levels are inversely proportional to those of BLyS; (3) miR-148a was not detected in IgG+ B cells of patients with inactive disease, but was consistently expressed by these cells in patients with active disease; and (4) the CD4+CD25+ T cell subpopulation is significantly reduced in patients with active disease in relation to healthy controls (P = 0.02) and patients with inactive disease (P = 0.03). Moreover, we detected a tendency for lower expression of Foxp3 in patients with active disease, suggesting that this regulatory T cell subpopulation must be even less frequent in the disease. Interestingly, Th2 cells and IgG expressing memory B cells, potential targets of regulatory T cells, were slightly more frequent in patients with active disease. Together, these results place miR-148a, miR-155 and miR-1321 as potential targets for new therapeutic approaches in PF. In addition, we suggest that a quantitative impairment of regulatory T cells is associated with PF pathogenesis, highlighting the importance of these cells for maintenance of self-tolerance

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

A non-canonical landscape of the microRNA system

Microribonucleic acids, best known as microRNAs or miRNAs, are small, non-coding RNAs with important regulatory roles in eukaryotic cells. Here, I present a broad review about highly relevant but generally non-depicted features of miRNAs, among which stand out the non-conventional miRNA seed sites, the unusual messenger RNA (mRNA) target regions, the non-canonical miRNA-guided mechanisms of gene expression regulation and the recently identified new class of miRNA ligands. Furthermore, I address the miRNA uncommon genomic location, transcription, and subcellular localization. Altogether, these unusual features and roles place the miRNA system as a very diverse, complex and intriguing biological mechanism

Sparking Fire Under the Skin? Answers From the Association of Complement Genes With Pemphigus Foliaceus

Skin blisters of pemphigus foliaceus (PF) present concomitant deposition of autoantibodies and components of the complement system (CS), whose gene polymorphisms are associated with susceptibility to different autoimmune diseases. To investigate these in PF, we evaluated 992 single-nucleotide polymorphisms (SNPs) of 44 CS genes, genotyped through microarray hybridization in 229 PF patients and 194 controls. After excluding SNPs with minor allele frequency &lt;1%, out of Hardy–Weinberg equilibrium in controls or in strong linkage disequilibrium (r2 ≥ 0.8), 201 SNPs remained for logistic regression. Polymorphisms of 11 genes were associated with PF. MASP1 encodes a crucial serine protease of the lectin pathway (rs13094773: OR = 0.5, p = 0.0316; rs850309: OR = 0.23, p = 0.03; rs3864098: OR = 1.53, p = 0.0383; rs698104: OR = 1.52, p = 0.0424; rs72549154: OR = 0.55, p = 0.0453). C9 (rs187875: OR = 1.46, p = 0.0189; rs700218: OR = 0.12, p = 0.0471) and C8A (rs11206934: OR = 4.02, p = 0.0323) encode proteins of the membrane attack complex (MAC) and C5AR1 (rs10404456: OR = 1.43, p = 0.0155), a potent anaphylatoxin-receptor. Two encode complement regulators: MAC-blocking CD59 (rs1047581: OR = 0.62, p = 0.0152) and alternative pathway-blocking CFH (rs34388368: OR = 2.57, p = 0.0195). One encodes opsonin: C3 (rs4807895: OR = 2.52, p = 0.0239), whereas four encode receptors for C3 fragments: CR1 (haplotype with rs6656401: OR = 1.37, p = 0.0382), CR2 (rs2182911: OR = 0.23, p = 0.0263), ITGAM (CR3, rs12928810: OR = 0.66, p = 0.0435), and ITGAX (CR4, rs11574637: OR = 0.63, p = 0.0056). Associations reinforced former findings, regarding differential gene expression, serum levels, C3, and MAC deposition on lesions. Deregulation of previously barely noticed processes, e.g., the lectin and alternative pathways and opsonization-mediated phagocytosis, also modulate PF susceptibility. The results open new crucial avenues for understanding disease etiology and may improve PF treatment through additional therapeutic targets

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<p>Skin blisters of pemphigus foliaceus (PF) present concomitant deposition of autoantibodies and components of the complement system (CS), whose gene polymorphisms are associated with susceptibility to different autoimmune diseases. To investigate these in PF, we evaluated 992 single-nucleotide polymorphisms (SNPs) of 44 CS genes, genotyped through microarray hybridization in 229 PF patients and 194 controls. After excluding SNPs with minor allele frequency <1%, out of Hardy–Weinberg equilibrium in controls or in strong linkage disequilibrium (r² ≥ 0.8), 201 SNPs remained for logistic regression. Polymorphisms of 11 genes were associated with PF. MASP1 encodes a crucial serine protease of the lectin pathway (rs13094773: OR = 0.5, p = 0.0316; rs850309: OR = 0.23, p = 0.03; rs3864098: OR = 1.53, p = 0.0383; rs698104: OR = 1.52, p = 0.0424; rs72549154: OR = 0.55, p = 0.0453). C9 (rs187875: OR = 1.46, p = 0.0189; rs700218: OR = 0.12, p = 0.0471) and C8A (rs11206934: OR = 4.02, p = 0.0323) encode proteins of the membrane attack complex (MAC) and C5AR1 (rs10404456: OR = 1.43, p = 0.0155), a potent anaphylatoxin-receptor. Two encode complement regulators: MAC-blocking CD59 (rs1047581: OR = 0.62, p = 0.0152) and alternative pathway-blocking CFH (rs34388368: OR = 2.57, p = 0.0195). One encodes opsonin: C3 (rs4807895: OR = 2.52, p = 0.0239), whereas four encode receptors for C3 fragments: CR1 (haplotype with rs6656401: OR = 1.37, p = 0.0382), CR2 (rs2182911: OR = 0.23, p = 0.0263), ITGAM (CR3, rs12928810: OR = 0.66, p = 0.0435), and ITGAX (CR4, rs11574637: OR = 0.63, p = 0.0056). Associations reinforced former findings, regarding differential gene expression, serum levels, C3, and MAC deposition on lesions. Deregulation of previously barely noticed processes, e.g., the lectin and alternative pathways and opsonization-mediated phagocytosis, also modulate PF susceptibility. The results open new crucial avenues for understanding disease etiology and may improve PF treatment through additional therapeutic targets.</p

Oncogenic Role of Exosomal Circular and Long Noncoding RNAs in Gastrointestinal Cancers

Circular RNAs (circRNAs) and long noncoding RNAs (lncRNAs) are differentially expressed in gastrointestinal cancers. These noncoding RNAs (ncRNAs) regulate a variety of cellular activities by physically interacting with microRNAs and proteins and altering their activity. It has also been suggested that exosomes encapsulate circRNAs and lncRNAs in cancer cells. Exosomes are then discharged into the extracellular environment, where they are taken up by other cells. As a result, exosomal ncRNA cargo is critical for cell&ndash;cell communication within the cancer microenvironment. Exosomal ncRNAs can regulate a range of events, such as angiogenesis, metastasis, immune evasion, drug resistance, and epithelial-to-mesenchymal transition. To set the groundwork for developing novel therapeutic strategies against gastrointestinal malignancies, a thorough understanding of circRNAs and lncRNAs is required. In this review, we discuss the function and intrinsic features of oncogenic circRNAs and lncRNAs that are enriched within exosomes