RESUMEN
Dermatofibrosarcoma protuberans (DFSP) is an uncommon, slow growing, sarcoma of dermal and subcutaneous tissue with an infiltrative growth pattern. Although DFSP has a high rate of local recurrence, it rarely metastasizes. DFSP is characterized by a chromosomal translocation involving the collagen type I a 1 (COL1A1) gene on chromosome 17 and the platelet-derived growth factor B-chain (PDGFB) gene on chromosome 22. Various exons of the COL1A1 gene have been reported to be involved in the fusion with exon 2 of the PDGFB gene. In this study, we examined the COL1A1-PDGFB fusion transcripts using frozen specimens from three patients with DFSP. The molecular biology study with reverse transcriptase polymerase chain reaction (RT- PCR) and sequencing showed that the ends of exons 25, 31, and 45 in the COL1A1 gene were fused with PDGFB. The exon 2 of the PDGFB gene fused with exon 31 of the COL1A1 gene was a novel fusion gene.
Asunto(s)
Colágeno Tipo I/genética , Dermatofibrosarcoma/genética , Fusión Génica , Proteínas Proto-Oncogénicas c-sis/genética , Neoplasias Cutáneas/genética , Adulto , Cromosomas Humanos Par 17 , Cromosomas Humanos Par 22 , Cadena alfa 1 del Colágeno Tipo I , Dermatofibrosarcoma/patología , Exones , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Neoplasias Cutáneas/patología , Translocación GenéticaRESUMEN
Interleukin 8 (IL8) is usually produced in both epithelial and monocytic cells during bacterial infections, causing inflammation. Helicobacter pylori induces production of IL8 from gastric epithelial cells via its cag pathogenicity island (cag PAI) system, LPS and outer-membrane protein. In some bacteria, heat-shock protein 60 (HSP60) also elicits a strong pro-inflammatory response in cells of the innate immune system. Three recombinant H. pylori HSP60 (rHSP60) proteins of different sizes were produced and one of these was used to raise two monoclonal antibodies (2E7 and 7B5). IL8 production was found to be induced in cultured monocytic cells treated with H. pylori cells or rHSP60 proteins, as measured by ELISA, and the amount of IL8 produced was dose-dependent. Pre-incubation of H. pylori cells or rHSP60 preparations with the antibody 2E7 significantly inhibited IL8 production from monocytic cells. These results indicated that HSP60 is closely associated with IL8 production in monocytic cells.
Asunto(s)
Chaperonina 60/metabolismo , Helicobacter pylori/metabolismo , Interleucina-8/biosíntesis , Monocitos/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Línea Celular , Chaperonina 60/genética , Chaperonina 60/inmunología , Femenino , Helicobacter pylori/inmunología , Humanos , Interleucina-8/antagonistas & inhibidores , Interleucina-8/inmunología , Ratones , Ratones Endogámicos BALB C , Monocitos/inmunología , Monocitos/microbiología , Péptidos/síntesis química , Péptidos/inmunología , ARN Mensajero/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células U937RESUMEN
Contact between Helicobacter pylori and gastric epithelial cells results in activation of NF-kappaB followed by secretion of interleukin (IL)-8. However, host-cell receptor(s) and their ligands involved in H. pylori-related IL-8 production have yet to be fully defined. In this study, the interaction between Toll-like receptors (TLRs), which are host receptors for pathogens involved in the innate immune response, and heat-shock protein (HSP) 60, an immune-potent antigen of H. pylori, was examined during H. pylori-induced IL-8 secretion in vitro. Recombinant H. pylori HSP60 (rHpHSP60) was prepared and added to cultured KATO III human gastric epithelial cells with or without pre-incubation with mouse monoclonal anti-TLR2 or anti-TLR4 antibodies. IL-8 mRNA expression and IL-8 protein release were analysed by Northern blotting and immunoassay. Involvement of NF-kappaB activation was analysed immunocytochemically by anti-NF-kappaB p65 antibody and ammonium pyrrolidinedithiocarbamate (PDTC), an inhibitor of NF-kappaB-mediated transcriptional activation. rHpHSP60 induced IL-8 mRNA expression and IL-8 secretion in a dose-dependent manner in KATO III cells. Anti-TLR2 antibody inhibited rHpHSP60-induced IL-8 secretion by 75 %, and anti-TLR4 antibody inhibited it by 30 %. rHpHSP60 induced nuclear translocation of NF-kappaB p65, which was inhibited by pretreatment with anti-TLR2 antibody. Treatment with PDTC significantly decreased the secretion of IL-8 induced by rHpHSP60. These findings suggest that H. pylori HSP60 activates NF-kappaB and induces IL-8 production through TLR-triggered pathways in gastric epithelial cells. Thus, it is possible that H. pylori HSP60 and TLR interaction in host cells contributes to the development of gastric inflammation caused by H. pylori infection.
Asunto(s)
Chaperonina 60/metabolismo , Células Epiteliales/microbiología , Mucosa Gástrica/microbiología , Helicobacter pylori/patogenicidad , Interleucina-8/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Línea Celular , Chaperonina 60/genética , Mucosa Gástrica/citología , Helicobacter pylori/inmunología , Humanos , Inmunohistoquímica , Inflamación/etiología , Ratones , FN-kappa B/metabolismo , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-LikeRESUMEN
Mice and ducks were subcutaneously immunized with recombinant whole heavy (H) chains of Clostridium botulinum type C and D neurotoxins, which were expressed as glutathione S-transferase fusion proteins. In the case of mice, it was confirmed that two immunizations with type C- and D-H chains, 10 microg each time, significantly increased the specific antibodies against 100-kDa H chains of type C and D neurotoxins in an immunoblot analysis and an enzyme-linked immunosorbent assay, respectively. The mice immunized with type C- and D-H chains showed no symptoms of botulism when they were challenged with C- and D-16 S toxins at doses, given intraperitoneally, of up to 10(5) and 10(6) minmum lethal doses (MLD), respectively, per mouse. Ducks were immunized with a total of 100 microg of type C-H chain. The ducks also developed specific antibodies to the type C-H chain and showed significant protection against a challenge with 10(3) duck MLD of C-16 S toxin given intravenously. These results indicate that recombinant whole H chains can be used as an effective and safe vaccine for type C and D botulism in domestic animals.