RESUMO
IL-10 is an immunomodulatory cytokine that plays an obligate role in preventing spontaneous enterocolitis in mice. However, little is known about IL-10 function in the human intestinal mucosa. We showed here that IL-10 was constitutively expressed and secreted by the human normal colonic mucosa, including epithelial cells. Depletion of IL-10 in mucosal explants induced both downregulation of the IL-10-inducible, immunosuppressive gene BCL3 and upregulation of IFN-gamma, TNF-alpha, and IL-17. Interestingly, TGF-beta blockade also strongly induced IFN-gamma production. In addition, the high levels of IFN-gamma produced upon IL-10 depletion were responsible for surface epithelium damage and crypt loss, mainly by apoptosis. Polymyxin B, used as a scavenger of endogenous LPS, abolished both IFN-gamma production and epithelial barrier disruption. Finally, adding a commensal bacteria strain to mucosa explant cultures depleted of both IL-10 and LPS reproduced the ability of endogenous LPS to induce IFN-gamma secretion. These findings demonstrate that IL-10 ablation leads to an endogenous IFN-gamma-mediated inflammatory response via LPS from commensal bacteria in the human colonic mucosa. We also found that both IL-10 and TGF-beta play crucial roles in maintaining human colonic mucosa homeostasis.
Assuntos
Colo/efeitos dos fármacos , Interferon gama/fisiologia , Interleucina-10/fisiologia , Mucosa Intestinal/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Fator de Crescimento Transformador beta/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Colo/metabolismo , Colo/patologia , Feminino , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Lipopolissacarídeos/antagonistas & inibidores , Masculino , Pessoa de Meia-IdadeRESUMO
To understand the regulation of the MexAB OprM efflux system in a clinical strain of Pseudomonas aeruginosa presenting a decreased susceptibility to ticarcillin and aztreonam, the mexR repressor gene was amplified by polymerase chain reaction (PCR) and was shown to be disrupted by an insertion sequence of more than 2 kb, with characteristic direct and inverted repeat sequences. Sequencing revealed a 2131-bp IS21 insertion sequence. A reverse transcription PCR method was used to quantify mexA transcripts and showed an increased transcription rate of mexA in this strain, compared with a PAO1 control strain. The nalB phenotype in P. aeruginosa may be due to point mutations, but also to the presence of an insertion sequence in the mexR regulator gene.
Assuntos
Proteínas de Bactérias , Elementos de DNA Transponíveis/genética , Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa/efeitos dos fármacos , Proteínas Repressoras/genética , Resistência beta-Lactâmica/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Humanos , Lactente , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Óperon , Reação em Cadeia da Polimerase/métodos , Proteínas Repressoras/metabolismo , Transcrição Gênica , beta-Lactamas/farmacologiaRESUMO
In Escherichia coli, beta-lactam resistance usually depends on beta-lactamase production. AmpC chromosomal cephalosporinase hyperproduction is generally due to mutations in the ampC gene promoter. In order to study ampC expression in E. coli clinical strains, we have compared two methods: conventional and real-time reverse transcription-polymerase chain reaction (RT-PCR). With both methods, ampC mRNA was found to be greatly increased in strains presenting -42 or -32 mutations in the ampC promoter, and moderately increased when a -11 mutation was present in the Pribnow box. Real-time RT-PCR represents a powerful tool combining amplification, fluorescent detection and analysis.
Assuntos
Proteínas de Bactérias , Escherichia coli/genética , Escherichia coli/metabolismo , Perfilação da Expressão Gênica/métodos , Resistência beta-Lactâmica , beta-Lactamases/biossíntese , beta-Lactamases/genética , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Genes Bacterianos , Testes de Sensibilidade Microbiana , Mutação Puntual , Regiões Promotoras Genéticas , RNA Bacteriano/análise , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transcrição Gênica , beta-Lactamas/farmacologiaRESUMO
OBJECTIVE: To compare the genetic environments of ampC genes in different Acinetobacter baumannii isolates showing different levels of beta-lactam resistance. METHODS: The patterns of beta-lactam resistance and beta-lactamase production were investigated for 42 A. baumannii clinical strains. The MICs of various beta-lactams were determined in the presence or absence of the class C cephalosporinase inhibitor, cloxacillin (500 mg/L). The ampC gene and its 5' adjacent sequence were analysed by PCR and DNA sequencing. An RT-PCR method was developed to evaluate ampC transcript levels. RESULTS: Strains fell into three resistance groups: first, strains with a ceftazidime MIC < or =8 mg/L (20 strains, 47.6%); secondly, strains with a ceftazidime MIC 32 mg/L, which was reduced four-fold in the presence of cloxacillin (eight strains, 19%); and thirdly, strains with a ceftazidime MIC > or =256 mg/L, which did not decrease in the presence of cloxacillin (14 strains, 33.4%). In all of the resistant isolates (groups II and III), but not in any of the ceftazidime-susceptible isolates (group I), a 1180 bp insert showing all the characteristics of an insertion sequence was detected upstream from the ampC gene. Isolates having this insert overexpress ampC, according to RT-PCR experiments. CONCLUSION: Presence of an insertion sequence upstream of ampC in A. baumannii clinical isolates, possibly including a strong promoter, has the potential to cause over-expression of AmpC, resulting in high-level ceftazidime resistance.
