RESUMO
Amyloid deposition of human islet amyloid polypeptide (hIAPP) within the islets of Langerhans is a pathological feature of type 2 diabetes mellitus. Substantial evidence indicates that the membrane-mediated aggregation and subsequent deposition of hIAPP are linked to dysfunction and death of pancreatic ß-cells, but the molecular processes of hIAPP deposition are poorly understood. In this study, we examined the membrane-mediated aggregation and deposition of hIAPP at supported planar lipid bilayers with and without raft components (i.e. cholesterol and sphingomyelin) to provide insight into hIAPP-induced membrane dysfunction. The adsorption of hIAPP onto the bilayers was studied using a quartz crystal microbalance with dissipation monitoring, which showed enhanced accumulation of the peptide onto the bilayer containing raft components. Microscope observations demonstrated the growth of the aggregates formed from the membrane-adsorbed hIAPP. The examination of the membrane interfaces revealed that hIAPP aggregates retained the ability to associate with the membranes during the aggregation process, resulting in insertion of the aggregates into the bilayers. We also report the inhibitory effect of insulin on the hIAPP deposition. These findings demonstrate the aggregation of hIAPP at the membrane interfaces leading to amyloid deposits associated with the membrane and suggest a role for insulin in hIAPP deposition. A presumed mechanism regulating hIAPP deposition at the membrane interfaces is discussed.
Assuntos
Membrana Celular/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Amiloide/metabolismo , Humanos , Insulina/metabolismoRESUMO
Viral pneumonia has been frequently reported during early stages of influenza virus pandemics and in many human cases of highly pathogenic avian influenza (HPAI) H5N1 virus infection. To better understand the pathogenesis of this disease, we produced nonlethal viral pneumonia in rhesus macaques by using an HPAI H5N1 virus (A/Anhui/2/2005; referred to as Anhui/2). Infected macaques were monitored for 14 days, and tissue samples were collected at 6 time points for virologic, histopathologic, and transcriptomic analyses. Anhui/2 efficiently replicated in the lung from 12 h to 3 days postinfection (p.i.) and caused temporal but severe pneumonia that began to resolve by day 14. Lung transcriptional changes were first observed at 6 h, and increased expression of vascular permeability regulators and neutrophil chemoattractants correlated with increased serum leakage and neutrophil infiltration in situ. Additional inflammatory, antiviral, and apoptotic genes were upregulated from 12 h, concurrent with viral antigen detection and increasing immune cell populations. A shift toward upregulation of acquired immunity was apparent after day 6. Expression levels of established immune cell molecular markers revealed remarkable similarity with pathological findings, indicating early and robust neutrophil infiltration, a slight delay in macrophage accumulation, and abundant late populations of T lymphocytes. We also characterized the putative mechanisms regulating a unique, pneumonia-associated biphasic fever pattern. Thus, this study is the first to use a comprehensive and integrative approach to delineate specific molecular mechanisms regulating influenza virus-induced pneumonia in nonhuman primates, an important first step toward better management of human influenza virus disease.
Assuntos
Virus da Influenza A Subtipo H5N1/patogenicidade , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Transcriptoma , Animais , Modelos Animais de Doenças , Feminino , Histocitoquímica , Pulmão/patologia , Pulmão/virologia , Macaca mulatta , Macrófagos/imunologia , Masculino , Neutrófilos/imunologia , Linfócitos T/imunologia , Fatores de TempoRESUMO
Type A human seasonal influenza (FluA) virus infection causes exacerbations of bronchial asthma and chronic obstructive pulmonary disease (COPD). l-carbocisteine, a mucolytic agent, reduces the frequency of common colds and exacerbations in COPD. However, the inhibitory effects of l-carbocisteine on FluA virus infection are uncertain. We studied the effects of l-carbocisteine on FluA virus infection in airway epithelial cells. Human tracheal epithelial cells were pretreated with l-carbocisteine and infected with FluA virus (H(3)N(2)). Viral titers in supernatant fluids, RNA of FluA virus in the cells, and concentrations of proinflammatory cytokines in supernatant fluids, including IL-6, increased with time after infection. l-carbocisteine reduced viral titers in supernatant fluids, RNA of FluA virus in the cells, the susceptibility to FluA virus infection, and concentrations of cytokines induced by virus infection. The epithelial cells expressed sialic acid with an alpha2,6-linkage (SAalpha2,6Gal), a receptor for human influenza virus on the cells, and l-carbocisteine reduced the expression of SAalpha2,6Gal. l-carbocisteine reduced the number of acidic endosomes from which FluA viral RNA enters into the cytoplasm and reduced the fluorescence intensity from acidic endosomes. Furthermore, l-carbocisteine reduced NF-kappaB proteins including p50 and p65 in the nuclear extracts of the cells. These findings suggest that l-carbocisteine may inhibit FluA virus infection, partly through the reduced expression of the receptor for human influenza virus in the human airway epithelial cells via the inhibition of NF-kappaB and through increasing pH in endosomes. l-carbocisteine may reduce airway inflammation in influenza virus infection.
Assuntos
Carbocisteína/uso terapêutico , Expectorantes/uso terapêutico , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Influenza Humana/tratamento farmacológico , Idoso , Animais , Células Cultivadas , Cães , Endossomos/efeitos dos fármacos , Células Epiteliais/virologia , Feminino , Humanos , Concentração de Íons de Hidrogênio , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Interleucinas/biossíntese , Masculino , Glicoproteínas de Membrana/biossíntese , NF-kappa B/biossíntese , Doença Pulmonar Obstrutiva Crônica/patologia , Doença Pulmonar Obstrutiva Crônica/virologia , RNA Viral/efeitos dos fármacos , RNA Viral/metabolismo , Receptores Virais/biossíntese , Traqueia/citologiaRESUMO
Human influenza viruses attach to sialic acid with an alpha2,6linkage (SAalpha2,6Gal) on the airway epithelial cells, and the entry of the viruses into the cells and uncoating of the viruses require low pH of endosomes. Bafilomycin A(1), a macrolide antibiotic and a specific inhibitor of vacuolar H(+)-ATPase, inhibits growth of type A and type B human influenza viruses in Madin-Darby canine kidney cells. However, the inhibitory effects of clinically used macrolide antibiotics on influenza virus infection in human airways have not been studied. To examine the effects of clarithromycin on seasonal human influenza virus infection, cultured human tracheal epithelial cells were infected with type A influenza virus (H3N2). Influenza virus infection increased viral titers and the content of cytokines, including interleukin (IL)-1beta and IL-6, in supernatant fluids, and viral RNA in the cells. Clarithromycin reduced viral titers and the content of cytokines in supernatant fluids, viral RNA in the cells, and the susceptibility to virus infection. Clarithromycin reduced the expression of SAalpha2,6Gal, a receptor for human influenza virus, on the mucosal surface of human tracheae, and the number and fluorescence intensity of acidic endosomes in the cells from which viral ribonucleoproteins enter into the cytoplasm. Furthermore, clarithromycin reduced nuclear factor-kappaB (NF-kappaB) proteins, including p50 and p65, in the nuclear extracts. These results suggest that clarithromycin may inhibit seasonal human influenza virus infection by reducing SAalpha2,6Gal partly through the inhibition of NF-kappaB, and increasing pH in endosomes in airway epithelial cells. Clarithromycin may modulate airway inflammation in influenza virus infection.
Assuntos
Antivirais/farmacologia , Claritromicina/farmacologia , Células Epiteliais/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Mucosa Respiratória/efeitos dos fármacos , Idoso , Animais , Células Cultivadas , Citocinas/metabolismo , Cães , Endossomos/química , Endossomos/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Feminino , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Vírus da Influenza A Subtipo H3N2/fisiologia , Masculino , NF-kappa B/metabolismo , RNA Viral/efeitos dos fármacos , Receptores Virais/biossíntese , Mucosa Respiratória/citologia , Mucosa Respiratória/virologia , Traqueia/citologia , Traqueia/efeitos dos fármacos , Traqueia/virologiaRESUMO
A six-year-old female Labrador retriever dog was suffering from osteomyelitis in her hindlimb. A puncture wound caused by a rotted bamboo stick was presumed as the source of infection. The dog suffered from pre-existing aortic stenosis, but otherwise exhibited no significant abnormality in her systemic conditions excluding claudication of the left hindlimb. The results of cytology and pathological examinations of biopsy samples revealed the diagnosis of mycotic osteomyelitis in this dog. Mycological and DNA tests showed the pathogen as the mushroom Schizophyllum commune. Antibiotic sensitivity testing also revealed susceptibility to itraconazole, which was used to successfully treat the dog. This is a rare case of canine basidiomycosis with S. commune as the etiologic agent.