RESUMO
Influenza A virus has caused a number of pandemics in past decades, including the recent H1N1-2009 pandemic. Viperin is an interferon (IFN)-inducible protein of innate immunity, and acts as a broad-spectrum antiviral protein. We explored the antiviral activities and mechanisms of viperin during influenza virus (IFV) infection in vitro and in vivo. Wild-type (WT) HeLa and viperin-expressing HeLa cells were infected with influenza A/WSN/33/H1N1 (WSN33) virus, and subjected to virological, light and electron microscopic analyses. Viperin expression reduced virus replication and titres, and restricted viral budding. Young and old viperin-knockout (KO) mice and WT control animals were challenged with influenza WSN33 at lethal doses of 10(3) and 10(4) p.f.u. via the intratracheal route. Lungs were subjected to histopathological, virological and molecular studies. Upon lethal IFV challenge, both WT and KO mice revealed similar trends of infection and recovery with similar mortality rates. Viral quantification assay and histopathological evaluation of lungs from different time points showed no significant difference in viral loads and lung damage scores between the two groups of mice. Although the in vitro studies demonstrated the ability of viperin to restrict influenza H1N1 virus replication, the viperin-deficient mouse model indicated that absence of viperin enhanced neither the viral load nor pulmonary damage in the lungs of infected mice. This may be due to the compensation of IFN-stimulated genes in the lungs and/or the influenza non-structural protein 1-mediated IFN antagonism dampening the IFN response, thereby rendering the loss of viperin insignificant. Nevertheless, further investigations that exploit the antiviral mechanisms of viperin as prophylaxis are still warranted.
Assuntos
Antivirais/metabolismo , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/metabolismo , Proteínas/metabolismo , Animais , Linhagem Celular , Feminino , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/genética , Influenza Humana/virologia , Pulmão/metabolismo , Pulmão/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Proteínas/genética , Carga Viral , Replicação ViralRESUMO
Adherence of Helicobacter pylori to inflamed gastric mucosa is dependent on the sialic acid-binding adhesin (SabA) and cognate sialylated/fucosylated glycans on the host cell surface. By in situ hybridization, H. pylori bacteria were observed in close association with erythrocytes in capillaries and post-capillary venules of the lamina propria of gastric mucosa in both infected humans and Rhesus monkeys. In vivo adherence of H. pylori to erythrocytes may require molecular mechanisms similar to the sialic acid-dependent in vitro agglutination of erythrocytes (i.e., sialic acid-dependent hemagglutination). In this context, the SabA adhesin was identified as the sialic acid-dependent hemagglutinin based on sialidase-sensitive hemagglutination, binding assays with sialylated glycoconjugates, and analysis of a series of isogenic sabA deletion mutants. The topographic presentation of binding sites for SabA on the erythrocyte membrane was mapped to gangliosides with extended core chains. However, receptor mapping revealed that the NeuAcalpha2-3Gal-disaccharide constitutes the minimal sialylated binding epitope required for SabA binding. Furthermore, clinical isolates demonstrated polymorphism in sialyl binding and complementation analysis of sabA mutants demonstrated that polymorphism in sialyl binding is an inherent property of the SabA protein itself. Gastric inflammation is associated with periodic changes in the composition of mucosal sialylation patterns. We suggest that dynamic adaptation in sialyl-binding properties during persistent infection specializes H. pylori both for individual variation in mucosal glycosylation and tropism for local areas of inflamed and/or dysplastic tissue.
Assuntos
Adesinas Bacterianas/metabolismo , Helicobacter pylori/fisiologia , Hemaglutininas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/metabolismo , Adesinas Bacterianas/genética , Adsorção , Animais , Antígenos de Bactérias/metabolismo , Aderência Bacteriana , Sítios de Ligação , Ligação Competitiva , Capilares , Eritrócitos/metabolismo , Eritrócitos/microbiologia , Gangliosídeos/metabolismo , Mucosa Gástrica/irrigação sanguínea , Mucosa Gástrica/microbiologia , Deleção de Genes , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/metabolismo , Hemaglutinação , Humanos , Técnicas In Vitro , Macaca mulatta , Oligossacarídeos/metabolismo , Antígeno Sialil Lewis X , VênulasRESUMO
Helicobacter pylori strains harboring the vacAs1, cagA and babA2 have been associated with ulcer disease (UD). We compared the prevalence of these different genotypes and adhesive properties in H. pylori infected patients with UD in four European countries. Genomic DNA was isolated from 314 H. pylori strains: Germany (GER; n=92), Sweden (SWE, n=74), Portugal (POR, n=91) and Finland (FIN, n=57). The frequencies of babA2 genotype varied from 35% to 60%. Triple-positive strains (vacAs1+, cagA+ and babA2+) were significantly associated with UD in GER and POR and were closely correlated with UD in FIN, but not in SWE. Classification as triple-positive strains had a higher specificity for detection of UD in GER, POR and FIN than type1 or cagA+ strains. In vitro adhesion assays revealed that Swedish strains showed high adhesion properties and were thus correlated with the diagnosis of UD, although PCR detected the babA2 gene at lower frequencies and failed to show a correlation with UD. This finding appears to reflect allelic variations of the babA2 gene in SWE, although adhesive properties of the strains are retained.
Assuntos
Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Úlcera Duodenal/microbiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/patogenicidade , Adesinas Bacterianas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Úlcera Duodenal/fisiopatologia , Europa (Continente) , Feminino , Variação Genética , Genótipo , Infecções por Helicobacter/fisiopatologia , Helicobacter pylori/classificação , Helicobacter pylori/genética , Helicobacter pylori/fisiologia , Humanos , Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
Influenza A virus has caused significant pandemics in the past decades, including the H1N12009 pandemic. Viperin is an interferoninducible protein that acts as a broadspectrum antiviral protein via the inhibition of farnesyl pyrophosphate synthase (FPPS). To mimic this activity of viperin, the present study investigated the effectiveness of a commercially available FPPS inhibitor (pamidronate) as an inhibitor of influenza virus infection in vitro and in vivo. HeLaM cells were treated with pamidronate to determine its effect on the replication of influenza virus A/H1N1/WSN/1933. C57BL/6 mice were also subjected to intratracheal pamidronate treatment regimes prior to and following lethal influenza challenge. Treatment with the FPPS inhibitor in vitro resulted in a considerable reduction in the viral titer of ~1 log and diminished lipid raft formation without cellular toxicity, thus mimicking the antiviral effect of viperin. However, pamidronate lacked efficacy in vivo and was associated with increased pulmonary damage, most likely due to the complexity of drughost interactions in the infected mice. Further studies are warranted on pamidronate treatment in other infectious diseases that are more susceptible to FPPS inhibition.
Assuntos
Antivirais/farmacologia , Difosfonatos/farmacologia , Geraniltranstransferase/antagonistas & inibidores , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Proteínas Virais/antagonistas & inibidores , Animais , Antivirais/uso terapêutico , Difosfonatos/uso terapêutico , Geraniltranstransferase/metabolismo , Células HeLa , Humanos , Vírus da Influenza A Subtipo H1N1/enzimologia , Microdomínios da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/metabolismo , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/mortalidade , Pamidronato , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Despite the seriousness of dengue-related disease, with an estimated 50-100 million cases of dengue fever and 250,000-500,000 cases of dengue hemorrhagic fever/dengue shock syndrome each year, a clear understanding of dengue pathogenesis remains elusive. Because of the lack of a disease model in animals and the complex immune interaction in dengue infection, the study of host response and immunopathogenesis is difficult. The development of genomics technology, microarray and high throughput quantitative PCR have allowed researchers to study gene expression changes on a much broader scale. We therefore used this approach to investigate the host response in dengue virus-infected cell lines and in patients developing dengue fever. METHODOLOGY/PRINCIPAL FINDINGS: Using microarray and high throughput quantitative PCR method to monitor the host response to dengue viral replication in cell line infection models and in dengue patient blood samples, we identified differentially expressed genes along three major pathways; NF-kappaB initiated immune responses, type I interferon (IFN) and the ubiquitin proteasome pathway. Among the most highly upregulated genes were the chemokines IP-10 and I-TAC, both ligands of the CXCR3 receptor. Increased expression of IP-10 and I-TAC in the peripheral blood of ten patients at the early onset of fever was confirmed by ELISA. A highly upregulated gene in the IFN pathway, viperin, was overexpressed in A549 cells resulting in a significant reduction in viral replication. The upregulation of genes in the ubiquitin-proteasome pathway prompted the testing of proteasome inhibitors MG-132 and ALLN, both of which reduced viral replication. CONCLUSION/SIGNIFICANCE: Unbiased gene expression analysis has identified new host genes associated with dengue infection, which we have validated in functional studies. We showed that some parts of the host response can be used as potential biomarkers for the disease while others can be used to control dengue viral replication, thus representing viable targets for drug therapy.
Assuntos
Linhagem Celular/metabolismo , Linhagem Celular/virologia , Vírus da Dengue/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Animais , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/virologia , Quimiocina CXCL10/metabolismo , Quimiocina CXCL11 , Cricetinae , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/fisiologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Células HeLa/metabolismo , Células HeLa/virologia , Células Hep G2/metabolismo , Células Hep G2/virologia , Humanos , Interferon beta/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Proteínas/genética , Proteínas/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The human pathogen Helicobacter pylori expresses two dominant adhesins; the Lewis b blood group antigen binding adhesin, BabA, and the sialic acid-binding adhesin, SabA. These adhesins recognize specific carbohydrate moieties of the gastric epithelium, i.e. the Lewis b antigen, Le(b), and the sialyl-Lewis x antigen, sLe(x), respectively, which promote infection and inflammatory processes in the gastroduodenal tract. To assess the contribution of each of BabA, SabA and the neutrophil activating protein (HP-NAP) in a local inflammation, we investigated the traits of H. pylori mutants in their capacity to interact with and stimulate human neutrophils. We thence found that the SabA adhesin was not only the key inducer of oxidative metabolism (Unemo et al. J Biol Chem 280:15390-15397, 2005), but also essential in phagocytosis induction, as evaluated by flow cytometry, fluorescence microscopy and luminol-enhanced chemiluminescence. The napA deletion resulted in enhanced generation of reactive oxygen species and impaired adherence to the host cells. In conclusion, the SabA adhesin stimulates human neutrophils through selectin-mimicry. Interestingly, HP-NAP modulates the oxidative burst, which could tune the impact of the H. pylori infection for establishment of balanced and chronic inflammation of the gastric mucosa.
Assuntos
Adesinas Bacterianas/toxicidade , Proteínas de Bactérias/farmacologia , Helicobacter pylori/química , Inflamação/induzido quimicamente , Neutrófilos/efeitos dos fármacos , Regulação para Baixo , Humanos , Neutrófilos/metabolismoRESUMO
The unique environment of the human stomach makes it difficult to establish representative in vitro models for Helicobacter pylori that mimic the natural infection. The in vitro explant culture (IVEC) technique is based on coculture of human gastric explants with H. pylori, where bacteria-host interaction is studied on the basis of interleukin (IL)-8 secretion of the explant tissue in response to infection. In this study, it was shown that H. pylori attachment to gastric epithelial tissue was required for induction of representative inflammatory responses, assessed here by IL-8 production. Furthermore, IL-8 production by the explant tissue in response to H. pylori infection demonstrated that the explants were adequately responsive. The IVEC technique for studies of the interplay between H. pylori and the human gastric mucosa during conditions of experimental infections in vitro could add knowledge to our understanding of the complex bacteria-host cross-talk in vivo.
Assuntos
Mucosa Gástrica/microbiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/crescimento & desenvolvimento , Gastropatias/microbiologia , Adulto , Biópsia , Contagem de Colônia Microbiana , Técnicas de Cultura , Feminino , Fucosiltransferases/farmacologia , Mucosa Gástrica/imunologia , Mucosa Gástrica/metabolismo , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/metabolismo , Helicobacter pylori/imunologia , Humanos , Imuno-Histoquímica , Interleucina-8/análise , Interleucina-8/biossíntese , Gastropatias/imunologia , Gastropatias/metabolismoRESUMO
Helicobacter pylori adherence in the human gastric mucosa involves specific bacterial adhesins and cognate host receptors. Here, we identify sialyl-dimeric-Lewis x glycosphingolipid as a receptor for H. pylori and show that H. pylori infection induced formation of sialyl-Lewis x antigens in gastric epithelium in humans and in a Rhesus monkey. The corresponding sialic acid-binding adhesin (SabA) was isolated with the "retagging" method, and the underlying sabA gene (JHP662/HP0725) was identified. The ability of many H. pylori strains to adhere to sialylated glycoconjugates expressed during chronic inflammation might thus contribute to virulence and the extraordinary chronicity of H. pylori infection.