RESUMO
The aim of this study was to assess the immune response to parainfluenza virus type 3 (PIV3), rhinovirus 1B (RV1B) and intracellular Toll-like receptors (TLR) agonists in nasal epithelial cells (NECs) from patients with allergic rhinitis and healthy controls. NECs were obtained from eight patients with allergic rhinitis (AR) and 11 non-atopic healthy controls (HC) by nasal scraping, grown to confluence and exposed to PIV3, RV1B infection or TLR-3 and TLR-7/8 agonists. Interferon (IFN)-λ1, IFN-α, IFN-ß and regulated on activation, normal T expressed and secreted (RANTES) release into the cell culture supernatants was assessed at 8, 24 and 48 h upon infection or 8 and 24 h after stimulation with poly(I:C) and R848. mRNA levels of IFNs, RANTES, interferon regulatory transcription factor (IRF)3, IRF7 and viral gene copy number were determined using real-time polymerase chain reaction (RT-PCR). PIV3 but not RV1B replication 48 h after infection was significantly lower (P < 0·01) in NECs from AR patients compared to HC. PIV3 infection induced significantly less IFN-λ1 (both protein and mRNA) in NECs from AR compared to HC. IFN-ß mRNA expression and RANTES protein release and mRNA expression tended to be smaller in AR compared HC cells in response to both viruses. Stimulation with TLR-3 agonist [poly (I:C)] induced similar IFN-λ1 and RANTES generation in AR and HC subjects. Viral infections in NECs induced IRF7 expression, which correlated with IFN and RANTES expression. These data suggest that virus proliferation rates and the immune response profile are different in nasal epithelial cells from patients with allergic rhinitis compared to healthy individuals.
Assuntos
Resfriado Comum/imunologia , Células Epiteliais/imunologia , Imunidade Inata , Vírus da Parainfluenza 3 Humana/fisiologia , Infecções por Respirovirus/imunologia , Rinite Alérgica/imunologia , Rhinovirus/fisiologia , Replicação Viral , Adulto , Células Cultivadas , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Feminino , Humanos , Imidazóis/farmacologia , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , Interferons/genética , Interferons/metabolismo , Masculino , Pessoa de Meia-Idade , Nariz/patologia , Poli I-C/farmacologia , Rinite Alérgica/virologia , Receptor 3 Toll-Like/agonistas , Receptor 7 Toll-Like/agonistas , Adulto JovemRESUMO
The plasmid profiles of virulent Rhodococcus equi strains isolated on three horse-breeding farms located in different parts of Hungary were investigated. From 49 soil samples collected on the three farms, 490 R. equi isolates (10 from each sample) were obtained and tested for the presence of 15- to 17-kDa antigens (VapA) by immunoblotting and PCR. Ninety-eight VapA-positive isolates were detected from 30 of the 49 culture-positive samples with a prevalence ranging from 13.1% to 23.2%. Of the 98 virulent isolates, 70 contained an 85-kb type I plasmid, 13 contained an 87-kb type I plasmid, and 15 contained an 85-kb type III plasmid which had been uniquely isolated from soil isolates in the United States. This study demonstrates that the virulent form of R. equi is very widespread in the soil environment of these stud farms in Hungary and the plasmid pattern is different from farm to farm.
Assuntos
Infecções por Actinomycetales/veterinária , Doenças dos Cavalos/epidemiologia , Doenças dos Cavalos/virologia , Rhodococcus equi/isolamento & purificação , Microbiologia do Solo , Infecções por Actinomycetales/epidemiologia , Infecções por Actinomycetales/virologia , Animais , Cruzamento , DNA Bacteriano/análise , Cavalos , Hungria/epidemiologia , Plasmídeos/análise , Reação em Cadeia da Polimerase/veterinária , Rhodococcus equi/classificação , Rhodococcus equi/genética , Rhodococcus equi/patogenicidadeRESUMO
The proinflammatory cytokine interleukin-17A (IL-17A) is known to mediate antimicrobial activity, but its role during rhinovirus (RV) infections and in asthma needs further investigation. Therefore, we addressed the role of IL-17A during allergic asthma and antiviral immune response in human and murine immunocompetent cells. In this study we found that asthmatic children with a RV infection in their upper airways have upregulated mRNA levels of the antiviral cytokine interferon type I (IFN)-ß and the transcription factor T-box 21 (TBX21) and reduced levels of IL-17A protein in their peripheral blood mononuclear cells (PBMCs). We also found that IL-17A inhibited RV1b replication in infected human lung epithelial cells A549. Furthermore, by using gene array analysis we discovered that targeted deletion of Il17a in murine lung CD4(+) T cells impaired Oas1g mRNA downstream of Ifnß, independently from RV infection. Additionally, in PBMCs of children with a RV infection in their nasalpharyngeal fluid OAS1 gene expression was found downregulated. Finally RV1b inhibited IL-17A production in lung CD4(+) T cells in a setting of experimental asthma. These results indicate that the RV1b inhibits IL-17A in T helper type 17 cells and IL-17A clears RV1b infection in epithelial cells. In both cases IL-17A contributes to fend off RV1b infection by inducing genes downstream of interferon type I pathway.
Assuntos
Asma/imunologia , Linfócitos T CD4-Positivos/imunologia , Hipersensibilidade a Drogas/imunologia , Interleucina-17/imunologia , Infecções por Picornaviridae/imunologia , Rhinovirus/imunologia , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/imunologia , Células A549 , Animais , Asma/genética , Asma/virologia , Linfócitos T CD4-Positivos/virologia , Criança , Pré-Escolar , Hipersensibilidade a Drogas/genética , Hipersensibilidade a Drogas/virologia , Feminino , Regulação da Expressão Gênica , Humanos , Interferon beta/genética , Interferon beta/imunologia , Interleucina-17/genética , Pulmão/imunologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Knockout , Ovalbumina/administração & dosagem , Infecções por Picornaviridae/genética , Infecções por Picornaviridae/virologia , Cultura Primária de Células , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Rhinovirus/crescimento & desenvolvimento , Transdução de Sinais , Proteínas com Domínio T/genética , Proteínas com Domínio T/imunologiaRESUMO
The study aimed to investigate whether the genetic polymorphisms in the 3'UTR of the caprine SLC11A1 gene are functional, and to assess the role of MAP as a regulatory parameter in gene expression. To this goal we constructed plasmids expressing the Luciferase reporter gene in transient transfections of a mouse (Balb/c) macrophage cell line (RAW264.7), incorporating those polymorphisms that our previous work indicated as more prominent in terms of SLC11A1 expression and responsiveness to MAP infection. Gene expression variation was recorded on the average of the respective measurements after exposure to Mycobacterium avium subsp. paratuberculosis (MAP) combined with microbial antigens and cytokines. In silico analysis of the region under study allowed identification of one cis-acting RNA element, five putative transcriptional regulatory elements and 85 3'end microRNA binding sites. The two polymorphic regions (regions A and B) of the 3'UTR of the caprine SLC11A1 gene were recognized as regulators of its activity, at transcriptional and post-transcriptional level. The GT16 polymorphism at region A, combined with the GT8 polymorphism at region B, results in up-regulation of the SLC11A1 gene. The specific genotype was also found to be more responsive to MAP exposure at a statistically significant level.
Assuntos
Regiões 3' não Traduzidas , Proteínas de Transporte de Cátions/genética , Cabras/genética , Mycobacterium avium subsp. paratuberculosis/patogenicidade , Polimorfismo Genético , Animais , Proteínas de Transporte de Cátions/imunologia , Regulação da Expressão Gênica , Doenças das Cabras/genética , Doenças das Cabras/imunologia , Cabras/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Mycobacterium avium subsp. paratuberculosis/imunologia , Paratuberculose/genética , Paratuberculose/imunologia , Células RAW 264.7RESUMO
Johne's disease or paratuberculosis is a chronic, progressive intestinal disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). One of the genes that have been targeted with regard to resistance or sensitivity to paratuberculosis is the SLC11A1 (solute carrier family 11 member A1). Here we extend our previous work to the sequence and structure analysis of the caprine SLC11A1 gene and we assess the functional impact of the most frequent polymorphisms of the 3' UTR region of the SLC11A1 gene to its expression in goat macrophages exposed in vitro to MAP. The role of these polymorphisms in primary immune response is also investigated with connection to gene expression of two interleukins (IL), one of which pro (IL-1a), and the other anti-inflammatory (IL-10). In order to assess gene response, quantitative detection of the SLC11A1, IL-10 and IL1a mRNA was performed by real time PCR before, and at 1, 3 and 24h after exposure of primary cultures of peripheral blood monocyte-derived macrophages to MAP, collected from 54 goats of the Greek native goat breed. Sequence analysis of the 3' UTR end of the caprine SLC11A1 gene determined its full length to be 522 bases. Structure analysis confirmed the presence of two microsatellites consisted of a variable number of guanine-thymine repeats (regions A and B). The homozygous B7 genotype [B(GTn)7/7] was associated at a statistically significant level with increased expression of the SLC11A1 and IL-1α genes indicating increased in vitro responsiveness and therefore resistance of mononuclear derived macrophages to MAP infection.