Assuntos
Antibacterianos/farmacologia , Cefalosporinas/farmacologia , Corantes Fluorescentes/química , Bactérias Gram-Negativas/efeitos dos fármacos , Resistência beta-Lactâmica , beta-Lactamas/farmacologia , Animais , Enterobacter cloacae/efeitos dos fármacos , Enterobacter cloacae/enzimologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Bactérias Gram-Negativas/enzimologia , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Ovinos , Espectrometria de FluorescênciaRESUMO
Nfatc2 and Tob1 are intrinsic negative regulators of T cell activation. Nfatc2-deficient and Tob1-deficient T cells show reduced thresholds of activation; however, whether these factors have independent or overlapping roles in negative regulation of T cell responses has not been previously examined. Here, we show that Nfatc2 knockout (KO) but not Tob1 KO mice have age-associated accumulation of persistently activated T cells in vivo and expansion of the CD44+ memory cell compartment and age-associated lymphocytic infiltrates in visceral organs, without significant changes in numbers of CD4+CD25+Foxp3+ regulatory T cells (Treg). In vitro, CD4+CD25- "conventional" T cells (Tconvs) from both KO strains showed greater proliferation than wild type (WT) Tconvs. However, while Tregs from Nfatc2 KO mice retained normal suppressive function, Tregs from Tob1 KOs had enhanced suppressive activity. Nfatc2 KO Tconvs expanded somewhat more rapidly than WT Tconvs under conditions of homeostatic proliferation, but their accelerated growth capacity was negated, at least acutely, in a lymphoreplete environment. Finally, Nfatc2 KO mice developed a previously uncharacterized increase in B-cell malignancies, which was not accelerated by the absence of Tob1. The data thus support the prevailing hypothesis that Nfatc2 and Tob1 are non-redundant regulators of lymphocyte homeostasis.
Assuntos
Linfócitos B/metabolismo , Carcinogênese/genética , Proteínas de Transporte/genética , Regulação Neoplásica da Expressão Gênica , Linfoma/genética , Fatores de Transcrição NFATC/genética , Linfócitos T Reguladores/metabolismo , Animais , Linfócitos B/patologia , Antígenos CD4/genética , Antígenos CD4/metabolismo , Carcinogênese/metabolismo , Carcinogênese/patologia , Proteínas de Transporte/metabolismo , Transformação Celular Neoplásica , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Deleção de Genes , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Linfoma/metabolismo , Linfoma/patologia , Masculino , Camundongos , Camundongos Knockout , Fatores de Transcrição NFATC/deficiência , Transdução de Sinais , Linfócitos T Reguladores/patologiaRESUMO
In bacterial biofilms, high molecular weight, secreted exopolysaccharides can serve as a scaffold to which additional carbohydrates, proteins, lipids, and nucleic acids adhere, forming the matrix of the developing biofilm. Here we report methods to extract and purify high molecular weight (>15 kDa) exopolysaccharides from biofilms of eight human pathogens, including species of Staphylcococcus, Klebsiella, Acinetobacter, Pseudomonas, and a toxigenic strain of Escherichia coli O157:H7. Glycosyl composition analysis indicated a high total mannose content across all strains with P. aeruginosa and A. baumannii exopolysaccharides comprised of 80-90% mannose, K. pneumoniae and S. epidermidis strains containing 40-50% mannose, and E. coli with â¼10% mannose. Galactose and glucose were also present in all eight strains, usually as the second and third most abundant carbohydrates. N-acetyl-glucosamine and galacturonic acid were found in 6 of 8 strains, while arabinose, fucose, rhamnose, and xylose were found in 5 of 8 strains. For linkage analysis, 33 distinct residue-linkage combinations were detected with the most abundant being mannose-linked moieties, in line with the composition analysis. The exopolysaccharides of two P. aeruginosa strains analyzed were consistent with the Psl carbohydrate, but not Pel or alginate. The S. epidermidis strain had a composition rich in mannose and glucose, which is consistent with the previously described slime associated antigen (SAA) and the extracellular slime substance (ESS), respectively, but no polysaccharide intracellular adhesion (PIA) was detected. The high molecular weight exopolysaccharides from E. coli, K. pneumoniae, and A. baumannii appear to be novel, based on composition and/or ratio analysis of carbohydrates.
Assuntos
Biofilmes , Polissacarídeos Bacterianos/metabolismo , Acinetobacter/fisiologia , Escherichia coli/fisiologia , Cromatografia Gasosa-Espectrometria de Massas , Klebsiella/fisiologia , Manose/análise , Microscopia de Fluorescência , Polissacarídeos Bacterianos/análise , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/isolamento & purificação , Pseudomonas aeruginosa/fisiologia , Staphylococcus/fisiologiaRESUMO
Natural killer cell (NK) receptors for major histocompatibility complex (MHC) class I influence engraftment and graft-versus-tumor effects after allogeneic bone marrow transplantation. We find that SH2-containing inositol phosphatase (SHIP) influences the repertoire of NK receptors. In adult SHIP-/- mice, the NK compartment is dominated by cells that express two inhibitory receptors capable of binding either self or allogeneic MHC ligands. This promiscuous repertoire has significant functional consequences, because SHIP-/- mice fail to reject fully mismatched allogeneic marrow grafts and show enhanced survival after such transplants. Thus, SHIP plays an important role in two processes that limit the success of allogeneic marrow transplantation: graft rejection and graft-versus-host disease.