RESUMO
Intravenous human immunoglobulin G (IVIG) may have therapeutic benefit in neuromyelitis optica spectrum disorders (herein called NMO), in part because of the anti-inflammatory properties of the IgG Fc region. Here, we evaluated recombinant Fc hexamers consisting of the IgM µ-tailpiece fused with the Fc region of human IgG1. In vitro, the Fc hexamers prevented cytotoxicity in aquaporin-4 (AQP4) expressing cells and in rat spinal cord slice cultures exposed to NMO anti-AQP4 autoantibody (AQP4-IgG) and complement, with >500-fold greater potency than IVIG or monomeric Fc fragments. Fc hexamers at low concentration also prevented antibody-dependent cellular cytotoxicity produced by AQP4-IgG and natural killer cells. Serum from rats administered a single intravenous dose of Fc hexamers at 50â¯mg/kg taken at 8â¯h did not produce complement-dependent cytotoxicity when added to AQP4-IgG-treated AQP4-expressing cell cultures. In an experimental rat model of NMO produced by intracerebral injection of AQP4-IgG, Fc hexamers at 50â¯mg/kg administered before and at 12â¯h after AQP4-IgG fully prevented astrocyte injury, complement activation, inflammation and demyelination. These results support the potential therapeutic utility of recombinant IgG1 Fc hexamers in AQP4-IgG seropositive NMO.
Assuntos
Fragmentos Fc das Imunoglobulinas/uso terapêutico , Imunoglobulina G/uso terapêutico , Neuromielite Óptica/terapia , Administração Intravenosa , Animais , Aquaporina 4/genética , Aquaporina 4/imunologia , Aquaporina 4/metabolismo , Aquaporina 4/toxicidade , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Autoanticorpos/uso terapêutico , Células CHO , Complemento C1q/metabolismo , Cricetulus , Nucleotídeos de Desoxiuracil/imunologia , Nucleotídeos de Desoxiuracil/metabolismo , Nucleotídeos de Desoxiuracil/uso terapêutico , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/química , Técnicas In Vitro , Mutação/genética , Neuromielite Óptica/imunologia , Neuromielite Óptica/patologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Medula Espinal/patologia , Estatísticas não Paramétricas , Fatores de Tempo , TransfecçãoRESUMO
AIMS: A simple DNA macroarray system was developed for detection of antibiotic resistance and other marker genes associated with the multidrug-resistant food pathogen Salmonella enterica subsp. enterica serotype Typhimurium DT104. METHODS AND RESULTS: A multiplex polymerase chain reaction (PCR) incorporating digoxigenin-dUTP was used to simultaneously amplify seven marker sequences, with subsequent rapid detection of the amplicons by hybridization with an array of probes immobilized on polyester cloth and immunoenzymatic assay of the bound label. This system provided sensitive detection of the different genetic markers in the S. Typhimurium DT104 genome, giving positive reactions with as few as 10 CFU, and the hybridizations were highly specific, with no reactions of amplicons with heterologous probes on the array. CONCLUSIONS: This cloth-based hybridization array system (CHAS) provides a simple, cost-effective tool for monitoring S. Typhimurium DT104 in foods and their production environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The CHAS is a simple and cost-effective tool for the simultaneous detection of amplicons generated in a multiplex PCR, and the concept is broadly applicable to the detection and characterization of food pathogens.
Assuntos
Digoxigenina/análogos & derivados , Farmacorresistência Bacteriana Múltipla/genética , Hibridização de Ácido Nucleico/métodos , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Proteínas de Bactérias/genética , Resistência ao Cloranfenicol/genética , Sondas de DNA , DNA Bacteriano/análise , DNA Bacteriano/isolamento & purificação , Nucleotídeos de Desoxiuracil/imunologia , Nucleotídeos de Desoxiuracil/metabolismo , Digoxigenina/imunologia , Digoxigenina/metabolismo , Microbiologia de Alimentos , Genes Bacterianos , Técnicas Imunoenzimáticas , Integrases/genética , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Resistência a Tetraciclina/genética , Fatores de Virulência/análise , Fatores de Virulência/genética , Resistência beta-Lactâmica/genéticaRESUMO
Human aminoacyl-tRNA synthetases (ARSs) are normally located in cytoplasm and are involved in protein synthesis. In the present work, we found that human methionyl-tRNA synthetase (MRS) was translocated to nucleolus in proliferative cells, but disappeared in quiescent cells. The nucleolar localization of MRS was triggered by various growth factors such as insulin, PDGF, and EGF. The presence of MRS in nucleoli depended on the integrity of RNA and the activity of RNA polymerase I in the nucleolus. The ribosomal RNA synthesis was specifically decreased by the treatment of anti-MRS antibody as determined by nuclear run-on assay and immunostaining with anti-Br antibody after incorporating Br-UTP into nascent RNA. Thus, human MRS plays a role in the biogenesis of rRNA in nucleoli, while it is catalytically involved in protein synthesis in cytoplasm.