Asunto(s)
Programas de Optimización del Uso de los Antimicrobianos/organización & administración , Carbapenémicos/administración & dosificación , Prescripciones de Medicamentos/estadística & datos numéricos , Algoritmos , Antibacterianos/administración & dosificación , Antibacterianos/economía , Prescripciones de Medicamentos/economía , Hospitales Comunitarios/economía , Hospitales Comunitarios/métodos , HumanosRESUMEN
Progranulin (PGRN) is a secreted glycoprotein expressed in neurons and glia that is implicated in neuronal survival on the basis that mutations in the GRN gene causing haploinsufficiency result in a familial form of frontotemporal dementia (FTD). Recently, a direct interaction between PGRN and tumor necrosis factor receptors (TNFR I/II) was reported and proposed to be a mechanism by which PGRN exerts anti-inflammatory activity, raising the possibility that aberrant PGRN-TNFR interactions underlie the molecular basis for neuroinflammation in frontotemporal lobar degeneration pathogenesis. Here, we report that we find no evidence for a direct physical or functional interaction between PGRN and TNFRs. Using coimmunoprecipitation and surface plasmon resonance (SPR) we replicated the interaction between PGRN and sortilin and that between TNF and TNFRI/II, but not the interaction between PGRN and TNFRs. Recombinant PGRN or transfection of a cDNA encoding PGRN did not antagonize TNF-dependent NFκB, Akt, and Erk1/2 pathway activation; inflammatory gene expression; or secretion of inflammatory factors in BV2 microglia and bone marrow-derived macrophages (BMDMs). Moreover, PGRN did not antagonize TNF-induced cytotoxicity on dopaminergic neuroblastoma cells. Last, co-addition or pre-incubation with various N- or C-terminal-tagged recombinant PGRNs did not alter lipopolysaccharide-induced inflammatory gene expression or cytokine secretion in any cell type examined, including BMDMs from Grn+/- or Grn-/- mice. Therefore, the neuroinflammatory phenotype associated with PGRN deficiency in the CNS is not a direct consequence of the loss of TNF antagonism by PGRN, but may be a secondary response by glia to disrupted interactions between PGRN and Sortilin and/or other binding partners yet to be identified.
Asunto(s)
Citocinas/metabolismo , Regulación de la Expresión Génica/inmunología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal/fisiología , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Análisis de Varianza , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Granulinas , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Péptidos y Proteínas de Señalización Intercelular/genética , Isoquinolinas/metabolismo , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/efectos de los fármacos , Microglía/metabolismo , FN-kappa B/metabolismo , Progranulinas , Unión Proteica/genética , Receptores del Factor de Necrosis Tumoral/genética , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Resonancia por Plasmón de Superficie , TransfecciónRESUMEN
We describe a bioinformatic analysis of germline and rearranged immunoglobulin kappa chain (IGK) gene sequences, performed in order to assess the completeness and reliability of the reported IGK repertoire. In contrast to the reported heavy-chain gene repertoire, which includes many dubious sequences, only five IGK variable gene (IGKV) alleles appear to have been reported in error. There was, however, insufficient evidence to justify removing these IGKV genes from the germline repertoire. Bioinformatic analysis of apparent mismatches between reported germline genes and 1,863 expressed IGK sequences suggested the existence of two unreported IGKV polymorphisms. Genomic screening of 12 individuals led to the confirmation of both of these polymorphisms, IGKV1-16*02 and IGKV2-30*02. We also show that in contrast to the heavy chain, the IGK repertoire is dominated by sequences that use just a handful of kappa variable (IGKV) and junction (IGKJ) gene pairs. There is also little modification of IGKV and IGKJ genes by the processes of exonuclease removal and N nucleotide addition. The expressed IGK repertoire therefore lacks diversity and the junction region is particularly constrained. Remarkably, the analysis of a dataset of 435 relatively unmutated rearranged kappa genes showed that ten amino acid sequences account for almost 10% of the rearrangements, with identical sequences being derived from as many as seven independent sources. Such dominant sequences are likely to have important roles in the operation of the humoral immune response.