RESUMO
Virus-like particles (VLPs) are being used for therapeutic developments such as vaccines and drug nanocarriers. Among these, plant virus capsids are gaining interest for the formation of VLPs because they can be safely handled and are noncytotoxic. A paradigm in virology, however, is that plant viruses cannot transfect and deliver directly their genetic material or other cargos into mammalian cells. In this work, we prepared VLPs with the CCMV capsid and the mRNA-EGFP as a cargo and reporter gene. We show, for the first time, that these plant virus-based VLPs are capable of directly transfecting different eukaryotic cell lines, without the aid of any transfecting adjuvant, and delivering their nucleic acid for translation as observed by the presence of fluorescent protein. Our results show that the CCMV capsid is a good noncytotoxic container for genome delivery into mammalian cells.
Assuntos
Bromovirus/genética , Técnicas de Transferência de Genes , Vírus de Plantas/genética , Vacinas de Partículas Semelhantes a Vírus/genética , Animais , Proteínas do Capsídeo/genética , Linhagem Celular , Células Eucarióticas/virologia , Genes Reporter/genética , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Transfecção/métodos , Montagem de Vírus/genéticaRESUMO
miRNAs are important immune regulators in the control of the CD4â¯+â¯T cells phenotype. miR-326 regulates the differentiation towards Th17 cells and the inhibition of miR-155 is associated with low levels of Treg cells. However, miRNAs expression and transcription factors associated with these lymphocyte subsets in obesity-induced adipose tissue inflammation is still unknown. The aim of this work was to identify Th17 cells in subcutaneous adipose tissue (SAT), proinflammatory cytokine production and their association with the miRNAs and transcription factors involved. We collected SAT samples obtained by lipoaspiration from individuals with normal weight, overweight and obesity. We obtained the stromal vascular fractions and then a Ficoll gradient was performed to obtain adipose tissue mononuclear cells (ATMC). Th17 cells were evaluated by flow cytometry and the expression of miR-326, miR-155, RORC2 and FOXP3 by qRT-PCR. We also analyzed cytokines from the supernatants of the ATMC culture and measured the FOXP3 methylation percentage by bisulfite conversion by PCR. According to the results, the frequency of Th17 cells and RORC2 expression was higher in individuals with obesity and associated with miR-326 expression. The ATMC from this group secreted a proinflammatory cytokine profile by in vitro assay. In contrast, lower levels of mRNA FOXP3 expression was detected in ATMC from individuals with obesity that correlated with methylation percentage of FOXP3 gene but no association with miR-155 was detected. Our results suggested that miR-326 participates in the polarization towards Th17 promoting the inflammatory state in the obesity-induced adipose tissue.
Assuntos
Tecido Adiposo/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Obesidade/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Adulto , Diferenciação Celular , Células Cultivadas , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Humanos , Ativação Linfocitária , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Obesidade/genética , Adulto JovemRESUMO
We assessed the possible association between several single nucleotide polymorphisms (SNP) of P2RX7 gene with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). We determined the function of P2X7 receptor and the frequency of the 489C>T, 1096C>G, and 1513A>C SNP of P2RX7 gene in 111 and 122 patients with SLE and RA, and 98 healthy subjects. We found no significant association between the SNPs studied and SLE or RA. We also detected that lymphocytes from SLE and RA patients with the 489C>T SNP showed a higher ethidium bromide uptake in response to ATP than wild type or 1096C>G/1513A>C subjects. In addition, cells from RA patients and the 489C>T genotype, showed higher [Ca(2+)]i responses to ATP. Our data indicate that the 489C>T SNP of P2RX7 gene confers an enhanced function of this receptor in patients with RA, which may contribute to the pathogenesis of this condition.