RESUMO
BACKGROUND: Pediatricians sometimes see patients with severe aseptic meningitis and prolonged fever or severe headache, or both. This condition generally has a good prognosis and is usually treated with supportive therapy. However, there is neither guideline nor consensus for the treatment of patients with severe aseptic meningitis. Here, we investigated the relationship between disease severity and biomarkers. METHODS: The subjects were 32 children aged 0 to 14 years, 23 of whom had aseptic meningitis and 9 of whom were meningitis-free controls. Aseptic meningitis was retrospectively categorized into two subgroups, namely mumps meningitis (MM) and viral meningitis excluding that caused by mumps (EM). We defined a novel aseptic meningitis severity score (AMSS) from the signs and symptoms of aseptic meningitis and thus evaluated disease severity. We analyzed the profiles of cytokines in the patients' cerebrospinal fluid (CSF). RESULTS: The AMSS in MM was significantly higher than that in EM. IL-4, IL-6, IL-8, IL-10, and G-CSF levels in MM and EM CSF were higher than those in control CSF. IFN-γ levels were higher in MM than in controls (p<0.01). IL-10 and IFN-γ levels in MM were higher than those in EM. CONCLUSIONS: MM was more severe than EM. One likely reason is the higher CSF cytokine levels in MM. IFN-γ may be a potentially strong biomarker of MM severity. Our findings would help further understanding
Assuntos
Líquido Cefalorraquidiano/imunologia , Citocinas , Meningite Asséptica , Adolescente , Biomarcadores/líquido cefalorraquidiano , Criança , Pré-Escolar , Citocinas/líquido cefalorraquidiano , Citocinas/classificação , Feminino , Humanos , Lactente , Masculino , Meningite Asséptica/diagnóstico , Meningite Asséptica/etiologia , Meningite Asséptica/fisiopatologia , Prognóstico , Projetos de Pesquisa , Índice de Gravidade de Doença , Estatística como AssuntoRESUMO
Sphingobacterium spiritivorum has five unusual sphingophospholipids (SPLs). Our previous study determined the complete chemical structures of these SPLs. The compositions of the long-chain bases/fatty acids in the ceramide portion, isoheptadecasphingosine/isopentadecanoate or isoheptadecasphingosine/2-hydroxy isopentadecanoate, are characteristic. The immune response against bacterial lipid components is considered to play important roles in microbial infections. It is reported that several bacterial sphingolipids composed of ceramide are recognized by CD1-restricted T and NKT cells and that a non-peptide antigen is recognized by γδ T cells. In this study, we demonstrated that these bacterial SPLs activated murine bone marrow macrophages (BMMs) via Toll-like receptor (TLR) 4 but not TLR2, although they slightly activated CD1d-restricted NKT and γδT cells. Interestingly, this TLR 4-recognition pathway of bacterial SPLs involves the fatty acid composition of ceramide in addition to the sugar moiety. A non-hydroxy fatty acid composed of ceramide was necessary to activate murine BMMs. The bacterial survival was significantly higher in TLR4-KO mice than in TLR2-KO and wild-type mice. The results indicate that activation of the TLR4-dependent pathway of BMMs by SPLs induced an innate immune response and contributed to bacterial clearance.
Assuntos
Carga Bacteriana/imunologia , Ácidos Graxos/metabolismo , Macrófagos/microbiologia , Sphingobacterium/fisiologia , Esfingolipídeos/metabolismo , Receptor 4 Toll-Like/fisiologia , Animais , Medula Óssea/imunologia , Medula Óssea/metabolismo , Proliferação de Células , Feminino , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/metabolismo , Infecções por Bactérias Gram-Negativas/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos T gama-delta/fisiologia , Transdução de Sinais , Baço/imunologia , Baço/metabolismo , Baço/microbiologia , Receptor 2 Toll-Like/fisiologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
We previously demonstrated the colonization of Mycobacterium avium complex in bathrooms by the conventional culture method. In the present study, we aimed to directly detect M. avium organisms in the environment using loop-mediated isothermal amplification (LAMP), and to demonstrate the efficacy of LAMP by comparing the results with those obtained by culture. Our data showed that LAMP analysis has detection limits of 100 fg DNA/reaction for M. avium. Using an FTA(®) elute card, DNA templates were extracted from environmental samples from bathrooms in the residences of 29 patients with pulmonary M. avium disease. Of the 162 environmental samples examined, 143 (88%) showed identical results by both methods; 20 (12%) and 123 (76%) samples were positive and negative, respectively, for M. avium. Of the remaining 19 samples (12%), seven (5%) and 12 (7%) samples were positive by the LAMP and culture methods, respectively. All samples that contained over 20 colony forming units/primary isolation plate, as measured by the culture method, were also positive by the LAMP method. Our data demonstrate that the combination of the FTA elute card and LAMP can facilitate prompt detection of M. avium in the environment.
Assuntos
Monitoramento Ambiental/métodos , Complexo Mycobacterium avium/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Quartos de Pacientes , Microbiologia da Água , Humanos , Japão , Complexo Mycobacterium avium/genética , Complexo Mycobacterium avium/crescimento & desenvolvimento , Infecção por Mycobacterium avium-intracellulare/microbiologia , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Sensibilidade e EspecificidadeRESUMO
Tuberculosis remains one of the most deadly infectious diseases worldwide and is a leading public health problem. Although isoniazid (INH) is a key drug for the treatment of tuberculosis, tolerance to INH necessitates prolonged treatment, which is a concern for effective tuberculosis chemotherapy. INH is a prodrug that is activated by the mycobacterial enzyme, KatG. Here, we show that mycobacterial DNA-binding protein 1 (MDP1), which is a histone-like protein conserved in mycobacteria, negatively regulates katG transcription and leads to phenotypic tolerance to INH in mycobacteria. Mycobacterium smegmatis deficient for MDP1 exhibited increased expression of KatG and showed enhanced INH activation compared with the wild-type strain. Expression of MDP1 was increased in the stationary phase and conferred growth phase-dependent tolerance to INH in M. smegmatis. Regulation of KatG expression is conserved between M. smegmatis and Mycobacterium tuberculosis complex. Artificial reduction of MDP1 in Mycobacterium bovis BCG was shown to lead to increased KatG expression and susceptibility to INH. These data suggest a mechanism by which phenotypic tolerance to INH is acquired in mycobacteria.
Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Isoniazida/farmacologia , Mycobacterium/fisiologia , Pró-Fármacos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catalase/genética , Catalase/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Farmacorresistência Bacteriana/fisiologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/fisiologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologiaRESUMO
Bacillus Calmette-Guérin (BCG) Tokyo 172 is a predominant World Health Organization Reference Reagent for the BCG vaccine. Recently, the BCG Tokyo 172 substrain was reported to consist of two subpopulations with different colony morphologies, smooth and rough. Smooth colonies had a characteristic 22-bp deletion in Rv3405c of the region of difference (RD) 16 (type I), and rough colonies were complete in this region (type II). We hypothesized that the morphological difference is related to lipid phenotype and affects their antigenicity. We determined the lipid compositions and biosynthesis of types I and II. Scanning electron microscopy showed that type I was 1.5 times longer than type II. Phenolic glycolipid (PGL) and phthiocerol dimycocerosate (PDIM) were found only in type I. Although it has been reported that the RD16 is involved in the expression of PGL, type II did not possess PGL/PDIM. We examined the ppsA-E gene responsible for PGL/PDIM biosynthesis and found that the existence of PGL/PDIM in types I and II is caused by a ppsA gene mutation not regulated by the RD16. PGL suppressed the host recognition of total lipids via Toll-like receptor 2, and this suggests that PGL is antigenic and involved in host responses, acting as a cell wall component. This is the first report to show the difference between lipid phenotypes of types I and II. It is important to clarify the heterogeneity of BCG vaccine substrains to discuss and evaluate the quality, safety, and efficacy of the BCG vaccine.
Assuntos
Vacina BCG/metabolismo , Mycobacterium bovis/genética , Animais , Carboidratos/química , Cromatografia em Camada Fina/métodos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Ácidos Graxos/metabolismo , Genótipo , Glicolipídeos/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Mycobacterium bovis/imunologia , Ácidos Micólicos/metabolismo , Fenótipo , Piruvato Sintase/genéticaRESUMO
During the 2009-2010 season, a significant numerical increase of genotype GII.2 norovirus (NoV)-associated outbreaks was observed in Osaka City, Japan. The most common genotype in that season was GII.2 (44.6%), followed by GII.4 (39.2%). Mostly, GII.2 strains were associated with outbreaks in children and with person-to-person contact. The National Infectious Disease Surveillance Center reported that GII.2 NoV infections were widespread in Japan in that season. Comparative phylogenetic analysis of RNA-dependent RNA polymerase (RdRp) and capsid sequences revealed that this GII.2 epidemic resulted from two genetic strains. The first, GII.2p2 strains, had an identical genotype in the RdRp and capsid genes. GII.2p2 strains in the 2009-2010 season were a different genetic cluster from the strains of spring 2004, the previous epidemic of GII.2 NoV, but showed no unique amino acid change. The second, GII.2 chimera virus (GII.2p16), had GII.16 RdRp and GII.2 capsid genotypes, suggesting prior recombination at the junction of ORF1 and ORF2. GII.2p16 strains had four significant amino acid changes in the P2 subdomain, suggesting antigenic changes. Before the 2009-2010 season, GII.2 chimera viruses had been observed only sporadically. This spreading of GII.2p16 strains in the 2009-2010 season might be the first epidemic of GII.2 chimera virus. This study revealed that the NoV epidemic in the 2009-2010 season differed considerably from the prior season, when GII.4 was predominant. Furthermore, GII.2 strains persisted in human populations by drastic recombination and gradual accumulation of mutations, indicating a prevalent pattern of non-GII.4 genotypes with genetic evolution.
Assuntos
Infecções por Caliciviridae/epidemiologia , Surtos de Doenças , Norovirus/classificação , Estações do Ano , Sequência de Aminoácidos , Infecções por Caliciviridae/diagnóstico , Infecções por Caliciviridae/transmissão , Proteínas do Capsídeo/genética , Genótipo , Humanos , Japão/epidemiologia , Dados de Sequência Molecular , Norovirus/genética , Filogenia , Prevalência , Alinhamento de SequênciaRESUMO
The Mycobacterium avium-M. intracellulare complex (MAIC) is divided into 28 serotypes by a species-specific glycopeptidolipid (GPL). Previously, we clarified the structures of serotype 7 GPL and two methyltransferase genes (orfA and orfB) in serotype 12 GPL. This study elucidated the chemical structure, biosynthesis gene, and host innate immune response of serotype 13 GPL. The oligosaccharide (OSE) structure of serotype 13 GPL was determined to be 4-2'-hydroxypropanoyl-amido-4,6-dideoxy-ß-hexose-(1 â 3)-4-O-methyl-α-L-rhamnose-(1 â 3)-α-L-rhamnose-(1 â 3)-α-L-rhamnose-(1 â 2)-α-L-6-deoxy-talose by using chromatography, mass spectrometry, and nuclear magnetic resonance (NMR) analyses. The structure of the serotype 13 GPL was different from those of serotype 7 and 12 GPLs only in O-methylations. We found a relationship between the structure and biosynthesis gene cluster. M. intracellulare serotypes 12 and 13 have a 1.95-kb orfA-orfB gene responsible for 3-O-methylation at the terminal hexose, orfB, and 4-O-methylation at the rhamnose next to the terminal hexose, orfA. The serotype 13 orfB had a nonfunctional one-base missense mutation that modifies serotype 12 GPL to serotype 13 GPL. Moreover, the native serotype 13 GPL was multiacetylated and recognized via Toll-like receptor 2. The findings presented here imply that serotypes 7, 12, and 13 are phylogenetically related and confirm that acetylation of the GPL is necessary for host recognition. This study will promote better understanding of the structure-function relationships of GPLs and may open a new avenue for the prevention of MAIC infections.
Assuntos
Glicolipídeos/química , Glicolipídeos/metabolismo , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Especificidade de Hospedeiro , Complexo Mycobacterium avium/química , Complexo Mycobacterium avium/fisiologia , Infecção por Mycobacterium avium-intracellulare/microbiologia , Acetilação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Carboidratos , Linhagem Celular , Glicolipídeos/genética , Glicopeptídeos/genética , Humanos , Dados de Sequência Molecular , Família Multigênica , Complexo Mycobacterium avium/classificação , Complexo Mycobacterium avium/genética , Infecção por Mycobacterium avium-intracellulare/metabolismo , Especificidade da Espécie , Receptor 2 Toll-Like/metabolismoRESUMO
Measles virus (MV) is the causative agent for acute measles and subacute sclerosing panencephalitis (SSPE). Although numerous mutations have been found in the MV genome of SSPE strains, the mutations responsible for the neurovirulence have not been determined. We previously reported that the SSPE Osaka-2 strain but not the wild-type strains of MV induced acute encephalopathy when they were inoculated intracerebrally into 3-week-old hamsters. The recombinant MV system was adapted for the current study to identify the gene(s) responsible for neurovirulence in our hamster model. Recombinant viruses that contained envelope-associated genes from the Osaka-2 strain were generated on the IC323 wild-type MV background. The recombinant virus containing the M gene alone did not induce neurological disease, whereas the H gene partially contributed to neurovirulence. In sharp contrast, the recombinant virus containing the F gene alone induced lethal encephalopathy. This phenotype was related to the ability of the F protein to induce syncytium formation in Vero cells. Further study indicated that a single T461I substitution in the F protein was sufficient to transform the nonneuropathogenic wild-type MV into a lethal virus for hamsters.
Assuntos
Vírus do Sarampo/genética , Panencefalite Esclerosante Subaguda/virologia , Proteínas Virais de Fusão/genética , Animais , Chlorocebus aethiops , Cricetinae , Vírus do Sarampo/patogenicidade , Especificidade da Espécie , Células Vero , Proteínas Virais de Fusão/fisiologia , Virulência/genéticaRESUMO
In spite of the importance of hyaluronan in host protection against infectious organisms in the alveolar spaces, its role in mycobacterial infection is unknown. In a previous study, we found that mycobacteria interact with hyaluronan on lung epithelial cells. Here, we have analyzed the role of hyaluronan after mycobacterial infection was established and found that pathogenic mycobacteria can grow by utilizing hyaluronan as a carbon source. Both mouse and human possess 3 kinds of hyaluronan synthases (HAS), designated HAS1, HAS2, and HAS3. Utilizing individual HAS-transfected cells, we show that HAS1 and HAS3 but not HAS2 support growth of mycobacteria. We found that the major hyaluronan synthase expressed in the lung is HAS1, and that its expression was increased after infection with Mycobacterium tuberculosis. Histochemical analysis demonstrated that hyaluronan profoundly accumulated in the granulomatous legion of the lungs in M. tuberculosis-infected mice and rhesus monkeys that died from tuberculosis. We detected hyaluronidase activity in the lysate of mycobacteria and showed that it was critical for hyaluronan-dependent extracellular growth. Finally, we showed that L-Ascorbic acid 6-hexadecanoate, a hyaluronidase inhibitor, suppressed growth of mycobacteria in vivo. Taken together, our data show that pathogenic mycobacteria exploit an intrinsic host-protective molecule, hyaluronan, to grow in the respiratory tract and demonstrate the potential usefulness of hyaluronidase inhibitors against mycobacterial diseases.
Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Ácido Hialurônico/metabolismo , Mycobacterium tuberculosis/fisiologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Contagem de Colônia Microbiana , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Glicosaminoglicanos/farmacologia , Histocitoquímica , Humanos , Hialuronan Sintases , Ácido Hialurônico/farmacologia , Pulmão/química , Pulmão/metabolismo , Pulmão/microbiologia , Macaca mulatta , Masculino , Camundongos , Mycobacterium bovis/fisiologia , Mycobacterium tuberculosis/metabolismo , RatosRESUMO
The Schwarz FF-8 (FF-8) and AIK-C measles virus vaccine strains are currently used for vaccination in Japan. Here, the complete genome nucleotide sequence of the FF-8 strain has been determined and its genome sequence found to be remarkably similar to that of the AIK-C strain. These two strains are differentiated only by two nucleotide differences in the phosphoprotein gene. Since the FF-8 strain does not possess the amino acid substitutions in the phospho- and fusion proteins which are responsible for the temperature-sensitivity and small syncytium formation phenotypes of the AIK-C strain, respectively, other unidentified common mechanisms likely attenuate both the FF-8 and AIK-C strains.
Assuntos
Genoma Viral , Vacina contra Sarampo/genética , Vírus do Sarampo/genética , Fosfoproteínas/genética , Polimorfismo Genético , Proteínas Virais/genética , Substituição de Aminoácidos , Humanos , Japão , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Mutação Puntual , Análise de Sequência de DNA , Vacinas AtenuadasRESUMO
Attenuated live vaccines of measles virus (MV) have been developed from clinical isolates by serial propagation in heterologous cells, mainly chicken embryonic cells. The safety and effectiveness of these vaccines have been well established. However, the molecular mechanism of their attenuation remains a subject of investigation. The CAM-70 MV vaccine strain was developed from the Tanabe strain by serial propagation in chicken embryonic cells. In the present study, we assessed the contribution of each gene in the CAM-70 strain to efficient growth in chicken embryonic fibroblasts (CEF). We used a cloned MV IC323 based on the wild-type IC-B strain and generated a series of IC323s that possess one or more of the CAM-70 genes. Then, we examined the infection of CEF and CEF expressing human signaling lymphocyte activation molecule with the recombinant MVs. Our results demonstrated that MV needs to adapt to CEF at both the entry and postentry steps and that the CAM-70 matrix protein gene plays an important role in adaptation to CEF at the early stage of the virus replication cycle. The CAM-70 large protein gene was responsible for the efficient transcription and replication in CEF, and the CAM-70 hemagglutinin and fusion protein genes were responsible for efficient entry. Investigations focusing on these genes might elucidate unknown molecular mechanisms underlying the attenuation of MV.
Assuntos
Hemaglutininas Virais/genética , Vacina contra Sarampo/genética , Vírus do Sarampo/genética , Animais , Western Blotting , Linhagem Celular , Embrião de Galinha/virologia , Clonagem Molecular , DNA Recombinante/genética , Fibroblastos/virologia , Citometria de Fluxo , Genes Virais/fisiologia , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/genética , Transdução Genética , Proteínas da Matriz Viral/genética , Proteínas Virais/genética , Internalização do Vírus , Replicação Viral/genéticaRESUMO
In seasons from 1996-1997 through 2008-2009, noroviruses (NoVs) were detected in 505 outbreaks (71%) of nonbacterial gastroenteritis in Osaka City, Japan using molecular diagnosis with reverse transcription (RT)-PCR or real-time RT-PCR. The occurrences of NoV-associated outbreaks were related with the cold season during November-March (85.3%), and occasionally small epidemics of NoVs occurring during April-June were observed. Oyster-associated outbreaks were dominant transmission modes (25-61.1%) before the 2003-2004 season, and decreased (5-20.5%) from the 2003-2004 season, although outbreaks attributable to food-borne transmission (except for oysters) and person-to-person contact increased from the 2003-2004 season. The NoV strains were characterized into genotypes based on sequence analysis of partial capsid regions. Genotyping analyses identified at least 30 genotypes (12 in genogroup I [GI] and 18 in genogroup II [GII]) of NoV. The most common genotype was GII.4 (44.6%), followed in order by GII.3, GII.6, GII.2, and GII.5. The number of GII.4 NoVs increased greatly from the 2003-2004 season, eventually comprising a large share among the NoV- associated outbreaks (97.4%) of the 2006-2007 season. Occasional increased prevalence of genotypes other than GII.4 was observed during this study period. This study showed the appearance, spread, and disappearance of various genotypes and the change of NoV epidemic in a limited geographic region. Continuous NoV molecular surveillance is important for understanding NoV infections and for improving measures for their control and prevention.
Assuntos
Infecções por Caliciviridae/epidemiologia , Surtos de Doenças , Gastroenterite/epidemiologia , Epidemiologia Molecular , Norovirus/genética , Estações do Ano , Infecções por Caliciviridae/virologia , Gastroenterite/virologia , Genótipo , Humanos , Japão/epidemiologia , Dados de Sequência Molecular , Norovirus/classificação , Norovirus/isolamento & purificação , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
Monocyte-derived dendritic cells (mDCs) recognize viral RNA extrinsically by Toll-like receptor (TLR) 3 on the membrane and intrinsically retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5) in the cytoplasm to induce type I IFNs and mDC maturation. When mDCs were treated with live or UV-irradiated respiratory syncytial virus (RSV), early ( approximately 4 h) induction of IFN-beta usually occurs in other virus infections was barely observed. Live RSV subsequently replicated to activate the cytoplasmic IFN-inducing pathway leading to robust type I IFN induction. We found that RSV initial attachment to cells blocked polyI:C-mediated IFN-beta induction, and this early IFN-beta-modulating event was abrogated by antibodies against envelope proteins of RSV, demonstrating the presence of a IFN-regulatory mode by early RSV attachment to host cells. By IFN-stimulated response element (ISRE) reporter analysis in HEK293 cells, polyI:C- or LPS-mediated ISRE activation was dose dependently inhibited by live and inactive RSV to a similar extent. Of the RSV envelope proteins, simultaneously expressed or exogenously added RSV G or soluble G (sG) proteins inhibited TLR3/4-mediated ISRE activation in HEK293 cells. sG proteins expressed in cells did not affect the RIG-I/MDA5 pathway but inhibited the TLR adaptor TRIF/TICAM-1 pathway for ISRE activation. Finally, extrinsically added sG protein suppressed the production of IFN-beta in mDCs. Although the molecular mechanism of this extrinsic functional mode of the RSV G glycoprotein (G protein) remains undetermined, G proteins may neutralize the fusion glycoprotein function that promotes IFN-mediated mDC modulation via TLR4 and may cause insufficient raising cell-mediated immunity against RSV.
Assuntos
Interferon beta/metabolismo , Vírus Sincicial Respiratório Humano/patogenicidade , Receptor 3 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/antagonistas & inibidores , Proteínas do Envelope Viral/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Células Dendríticas/imunologia , Regulação da Expressão Gênica , Humanos , Interferon beta/genética , Vírus Sincicial Respiratório Humano/metabolismo , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteínas do Envelope Viral/imunologiaRESUMO
Medical treatment of pulmonary Mycobacterium avium complex (MAC) disease does not always provide curative effects and is frequently hampered by recurrence. This suggests the presence of a reservoir for MAC in the environment surrounding patients. We previously reported the recovery of MAC isolates from the residential bathrooms of outpatients. In the present study, to ascertain the colonizing sites and the possibility of an MAC reservoir in the bathrooms of patients, we tested the recovery and the genetic diversity of MAC isolates from 6 sites of specimens, including 2 additional sampling sites, inside the showerhead and the bathtub inlet, in the residential bathrooms of patients with pulmonary MAC disease. MAC isolates were recovered from 15 out of the 29 bathrooms (52%), including specimens from 14 bathtub inlets and 3 showerheads. Nearly half of these bathrooms (7/15) contained MAC strains that were identical or similar to their respective clinical isolates Additionally, in 5 out of 15 bathrooms, polyclonal colonization was revealed by pulsed-field gel electrophoresis. The results imply that colonization of MAC organisms in the bathrooms of MAC patients occurs predominantly in the bathtub inlets, and there is thus a risk of infection and/or reinfection for patients via use of the bathtub and other sites in the bathroom.
Assuntos
Banhos , Contaminação de Equipamentos , Hospitais , Complexo Mycobacterium avium/isolamento & purificação , Quartos de Pacientes , Biofilmes , Distribuição de Qui-Quadrado , Eletroforese em Gel de Campo Pulsado , Zeladoria Hospitalar , Humanos , Japão , Complexo Mycobacterium avium/genética , Complexo Mycobacterium avium/crescimento & desenvolvimento , Infecção por Mycobacterium avium-intracellulare/microbiologia , Engenharia Sanitária , Estatísticas não Paramétricas , Microbiologia da ÁguaRESUMO
Mycobacterium avium-Mycobacterium intracellulare complex (MAC) is the most common isolate of nontuberculous mycobacteria and causes pulmonary and extrapulmonary diseases. MAC species can be grouped into 31 serotypes by the epitopic oligosaccharide structure of the species-specific glycopeptidolipid (GPL) antigen. The GPL consists of a serotype-common fatty acyl peptide core with 3,4-di-O-methyl-rhamnose at the terminal alaninol and a 6-deoxy-talose at the allo-threonine and serotype-specific oligosaccharides extending from the 6-deoxy-talose. Although the complete structures of 15 serotype-specific GPLs have been defined, the serotype 16-specific GPL structure has not yet been elucidated. In this study, the chemical structure of the serotype 16 GPL derived from M. intracellulare was determined by using chromatography, mass spectrometry, and nuclear magnetic resonance analyses. The result indicates that the terminal carbohydrate epitope of the oligosaccharide is a novel N-acyl-dideoxy-hexose. By the combined linkage analysis, the oligosaccharide structure of serotype 16 GPL was determined to be 3-2'-methyl-3'-hydroxy-4'-methoxy-pentanoyl-amido-3,6-dideoxy-beta-hexose-(1-->3)-4-O-methyl-alpha-L-rhamnose-(1-->3)-alpha-L-rhamnose-(1-->3)-alpha-L-rhamnose-(1-->2)-6-deoxy-alpha-L-talose. Next, the 22.9-kb serotype 16-specific gene cluster involved in the glycosylation of oligosaccharide was isolated and sequenced. The cluster contained 17 open reading frames (ORFs). Based on the similarity of the deduced amino acid sequences, it was assumed that the ORF functions include encoding three glycosyltransferases, an acyltransferase, an aminotransferase, and a methyltransferase. An M. avium serotype 1 strain was transformed with cosmid clone no. 253 containing gtfB-drrC of M. intracellulare serotype 16, and the transformant produced serotype 16 GPL. Together, the ORFs of this serotype 16-specific gene cluster are responsible for the biosynthesis of serotype 16 GPL.
Assuntos
Desoxiaçúcares/química , Genes Bacterianos , Glicolipídeos/análise , Glicopeptídeos/química , Complexo Mycobacterium avium/química , Complexo Mycobacterium avium/genética , Sequência de Carboidratos , Glicolipídeos/química , Glicopeptídeos/genética , Redes e Vias Metabólicas/genética , Família Multigênica , Complexo Mycobacterium avium/classificação , Complexo Mycobacterium avium/metabolismoRESUMO
Between March and May 2004, a GII.2 genotype norovirus strain caused an epidemic of acute gastroenteritis in Osaka, Japan. Phylogenetic analysis showed that this strain was distinct from all other GII.2 strains detected in Osaka City between April 1996 and March 2005.
Assuntos
Infecções por Caliciviridae/epidemiologia , Gastroenterite/epidemiologia , Norovirus/classificação , Norovirus/isolamento & purificação , Adulto , Infecções por Caliciviridae/virologia , Criança , Surtos de Doenças , Gastroenterite/virologia , Genótipo , Humanos , Japão/epidemiologia , Epidemiologia Molecular , Dados de Sequência Molecular , Norovirus/genética , Filogenia , RNA Viral/genética , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
Measles virus (MV) is the causative agent of subacute sclerosing panencephalitis (SSPE) and viruses isolated from brains of the patients contain numerous mutations. We have previously demonstrated that the hemagglutinin (H) protein of MV SSPE strains can interact with the signaling lymphocyte activation molecule (SLAM) and an unidentified molecule on Vero cells, but not with CD46, as a receptor. The mechanism by which MV SSPE strains can induce cell-cell fusion in SLAM-negative Vero cells is not understood. We report here on the effect of mutations in the fusion (F) proteins of three MV SSPE strains on syncytium formation. The F proteins of the three SSPE strains were functional and co-expression with H protein from the MV wild-type or SSPE strains in this study induced formation of large syncytia in Vero cells as well as in cell lines expressing SLAM or CD46. Expression of chimeric F proteins of SSPE strains showed that amino acid substitutions in the F protein extracellular as well as cytoplasmic domain contributed to enhanced cell-cell fusion in Vero cells. These findings suggest a common molecular mechanism and a key role of the F protein for syncytium formation in cells expressing an unidentified third receptor for MV.
Assuntos
Células Gigantes/virologia , Vírus do Sarampo/genética , Vírus do Sarampo/patogenicidade , Panencefalite Esclerosante Subaguda/virologia , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Substituição de Aminoácidos/genética , Animais , Chlorocebus aethiops , Humanos , Vírus do Sarampo/isolamento & purificação , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Células VeroRESUMO
Macrophages (Mø) and dendritic cells (DC) are thought to be targets of measles virus (MeV) at the early stage of infection. We compared the growth of Edmonston-derived vaccine strains and fresh clinical isolates of MeV in monocytes, monocyte-derived granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced Mø (GM-Mø) and in monocyte-derived DC (Mo-DC). Neither vaccine strains nor fresh isolates thrived in monocytes and GM-Mø and no differences were evident among them. On the other hand, infectious virus production was robust in Mo-DC infected with fresh isolates, but below the limits of detection in those infected with vaccine strains. Although the vaccine strains infected Mo-DC and replicated comparably with the fresh isolates, they accumulated far less matrix (M) protein. This was attributed to a difference in the stability of M protein produced in Mo-DC between the strains. Impaired production of infectious viruses in DC may be one cause of vaccine strain attenuation.
Assuntos
Células Dendríticas/virologia , Sarampo/virologia , Monócitos/virologia , Morbillivirus/crescimento & desenvolvimento , Animais , Células Cultivadas , Criança , Pré-Escolar , Células Dendríticas/metabolismo , Humanos , Monócitos/metabolismo , Proteínas da Matriz Viral/metabolismoRESUMO
Measles virus (MV) is the causative agent of subacute sclerosing panencephalitis (SSPE). We previously reported that the F gene of the SSPE Osaka-2 strain is the major determinant of MV neurovirulence. Because the sites and extents of mutations differ among SSPE strains, it is necessary to determine the mutations responsible for the SSPE-specific phenotypes of individual viral strain. In this study, recombinant viruses containing the envelope-associated genes from the SSPE Osaka-1 strain were generated in the IC323 wild-type MV background. Hamsters inoculated with MV containing the H gene of the Osaka-1 strain displayed hyperactivity and seizures, but usually recovered and survived. Hamsters inoculated with MV containing the F gene of the Osaka-1 strain displayed severe neurologic signs and died. Amino acid substitutions in the heptad repeat A and C regions of the F protein, including a methionine-to-valine substitution at amino acid 94, play major roles in neurovirulence.
Assuntos
Substituição de Aminoácidos/genética , Vírus do Sarampo/genética , Vírus do Sarampo/patogenicidade , Panencefalite Esclerosante Subaguda/virologia , Proteínas Virais de Fusão/genética , Animais , Encéfalo/virologia , Células CHO , Callithrix , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Cricetulus , Humanos , Vírus do Sarampo/isolamento & purificação , Estrutura Terciária de Proteína , Células VeroRESUMO
Cytochrome P450(BSbeta) is a peroxygenase that catalyzes the alpha- or beta-hydroxylation of myristic acid by utilizing H(2)O(2). The wild-type enzyme not only hydroxylated myristic acid, but oxidized 3,5,3',5'-tetramethylbenzidine (TMB), a peroxidase substrate, in a myristic acid-dependent reaction. Study of inhibition of hydroxylation of myristic acid by TMB indicates these two substrates compete for the same highly reactive intermediate during the course of their respective reactions. When deuterated myristic acid was used as a substrate to decrease hydroxylation activity, the rate of TMB oxidation increased. This increased rate of TMB oxidation was greatly enhanced when the R242K mutant enzyme bound with deuterated myristic acid was used. These results suggest that there are critical structural elements at the distal active site which determine whether this enzyme acts as a peroxygenase or a peroxidase.