RESUMO
Bordetella bronchiseptica PagP (PagPBB) is a lipid A palmitoyl transferase that is required for resistance to antibody-dependent complement-mediated killing in a murine model of infection. B. parapertussis contains a putative pagP homolog (encoding B. parapertussis PagP [PagPBPa]), but its role in the biosynthesis of lipid A, the membrane anchor of lipopolysaccharide (LPS), has not been investigated. Mass spectrometry analysis revealed that wild-type B. parapertussis lipid A consists of a heterogeneous mixture of lipid A structures, with penta- and hexa-acylated structures containing one and two palmitates, respectively. Through mutational analysis, we demonstrate that PagPBPa is required for the modification of lipid A with palmitate. While PagPBB transfers a single palmitate to the lipid A C-3' position, PagPBPa transfers palmitates to the lipid A C-2 and C-3' positions. The addition of two palmitate acyl chains is unique to B. parapertussis. Mutation of pagPBPa resulted in a mutant strain with increased sensitivity to antimicrobial peptide killing and decreased endotoxicity, as evidenced by reduced proinflammatory responses via Toll-like receptor 4 (TLR4) to the hypoacylated LPS. Therefore, PagP-mediated modification of lipid A regulates outer membrane function and may be a means to modify interactions between the bacterium and its human host during infection.
Assuntos
Aciltransferases/metabolismo , Bordetella parapertussis/enzimologia , Lipídeo A/metabolismo , Palmitatos/metabolismo , Aciltransferases/genética , Bordetella parapertussis/química , Bordetella parapertussis/genética , Análise Mutacional de DNA , Lipídeo A/química , Espectrometria de MassasRESUMO
Clostridium sordellii, an anaerobic pathogen, has recently been associated with rapidly fatal infections following medically induced abortions and injecting drug use. Patients with C. sordellii infection display few signs of inflammation such as fever, or redness and pain at the site of infection. We hypothesized that this could be due to reduced recognition of the organism by Toll-like receptors (TLRs) of the innate immune system. An ELAM-NF-kappaB luciferase reporter system in TLR-transfected HEK cells was used to measure TLR-dependent recognition of washed, heat-killed C. sordellii and other pathogenic clostridial species. Results demonstrated that all clostridia were well recognized by TLR2 alone and that responses were greatest when TLR2 was co-expressed with TLR6. Further, isolated human monocytes produced the pro-inflammatory cytokine TNFalpha and the immunoregulator IL-10 in response to C. sordellii. In addition, C. sordellii-stimulated monocytes produced 30% less TNFalpha following treatment with an anti-TLR2 blocking antibody. These data demonstrate that innate immune recognition of, and response to, cell-associated components of C. sordellii and other clostridial pathogens are mediated by TLR2 in combination with TLR6. We conclude that the characteristic absence of inflammatory signs and symptoms in C. sordellii infection is not related to inadequate immune detection of the organism, but rather is attributable to a species-specific immune system dysfunction that remains to be elucidated.
Assuntos
Infecções por Clostridium/imunologia , Infecções por Clostridium/microbiologia , Clostridium sordellii/imunologia , Receptores Toll-Like/imunologia , Bioensaio , Linhagem Celular , Infecções por Clostridium/patologia , Clostridium sordellii/isolamento & purificação , Citocinas/metabolismo , Genes Reporter , Humanos , Imunidade Inata , Luciferases/genética , Luciferases/metabolismo , Monócitos/imunologia , Monócitos/microbiologiaRESUMO
Toll-like receptor (TLR) 2 and TLR4 play important roles in the early, innate immune response to microbial challenge. TLR2 is preferentially involved in the inflammatory response to lipoteichoic acid, lipopeptides, and glycans from a variety of microbes, whereas TLR4 is essential for a complete response to LPSs. We report here that TLR2 transduces the response to phenol-soluble modulin, a factor secreted by Staphylococcus epidermidis. The TLR2-mediated response to this modulin was enhanced by TLR6 but inhibited by TLR1, indicating a functional interaction between these receptors. We also demonstrate that a response to phenol-soluble modulin mediated by TLR2 and TLR6 was more refractory to inhibition by TLR1 than one mediated by TLR2 alone.
Assuntos
Proteínas de Bactérias/fisiologia , Toxinas Bacterianas/metabolismo , Proteínas de Drosophila , Glicoproteínas de Membrana/fisiologia , Receptores de Superfície Celular/fisiologia , Staphylococcus epidermidis/imunologia , Animais , Proteínas de Bactérias/antagonistas & inibidores , Toxinas Bacterianas/antagonistas & inibidores , Linhagem Celular , Clonagem Molecular , Espaço Extracelular/imunologia , Humanos , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Dados de Sequência Molecular , Fenóis , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Solubilidade , Receptor 1 Toll-Like , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptor 6 Toll-Like , Receptores Toll-Like , TransfecçãoRESUMO
Toll-like receptors (TLRs) have been shown to participate in the recognition of pathogens by the innate immune system, but it is not clear how a restricted family of receptors has the capacity to recognize the wide spectrum of TLR stimuli known to exist. We report here that two members of the TLR family, TLR2 and TLR6, together coordinate macrophage activation by Gram-positive bacteria and the yeast cell-wall particle, zymosan. TLR6 and TLR2 both are recruited to the macrophage phagosome, where they recognize peptidoglycan, a Gram-positive pathogen component. By contrast, TLR2 recognizes another component, bacterial lipopeptide, without TLR6. The requirement for TLR cooperation is supported by the finding that TLR2 needs a partner to activate tumor necrosis factor-alpha production in macrophages. Dimerization of the cytoplasmic domain of TLR2 does not induce tumor necrosis factor-alpha production in macrophages, whereas similar dimerization of the TLR4 cytoplasmic domain does. We show that the cytoplasmic domain of TLR2 can form functional pairs with TLR6 or TLR1, and this interaction leads to cytokine induction. Thus, the cytoplasmic tails of TLRs are not functionally equivalent, with certain TLRs requiring assembly into heteromeric complexes, whereas others are active as homomeric complexes. Finally, we show that TLR6, TLR2, and TLR1 are recruited to macrophage phagosomes that contain IgG-coated erythrocytes that do not display microbial components. The data suggest that TLRs sample the contents of the phagosome independent of the nature of the contents, and can establish a combinatorial repertoire to discriminate among the large number of pathogen-associated molecular patterns found in nature.
Assuntos
Proteínas de Drosophila , Sistema Imunitário/imunologia , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Sequência de Bases , Células CHO , Cricetinae , Primers do DNA , Fungos/imunologia , Fungos/patogenicidade , Bactérias Gram-Positivas/imunologia , Bactérias Gram-Positivas/patogenicidade , Camundongos , Dados de Sequência Molecular , Receptor 1 Toll-Like , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptor 6 Toll-Like , Receptores Toll-LikeRESUMO
Macrophages orchestrate innate immunity by phagocytosing pathogens and coordinating inflammatory responses. Effective defence requires the host to discriminate between different pathogens. The specificity of innate immune recognition in Drosophila is mediated by the Toll family of receptors; Toll mediates anti-fungal responses, whereas 18-wheeler mediates anti-bacterial defence. A large number of Toll homologues have been identified in mammals, and Toll-like receptor 4 is critical in responses to Gram-negative bacteria. Here we show that Toll-like receptor 2 is recruited specifically to macrophage phagosomes containing yeast, and that a point mutation in the receptor abrogates inflammatory responses to yeast and Gram-positive bacteria, but not to Gram-negative bacteria. Thus, during the phagocytosis of pathogens, two classes of innate immune receptors cooperate to mediate host defence: phagocytic receptors, such as the mannose receptor, signal particle internalization, and the Toll-like receptors sample the contents of the vacuole and trigger an inflammatory response appropriate to defence against the specific organism.
Assuntos
Proteínas de Drosophila , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Fagossomos/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antígenos de Diferenciação/metabolismo , Células CHO , Linhagem Celular , Cricetinae , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C3H , Dados de Sequência Molecular , Fator 88 de Diferenciação Mieloide , Fagocitose , Mutação Puntual , Receptores de Superfície Celular/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salmonella/metabolismo , Transdução de Sinais , Staphylococcus aureus/metabolismo , Receptor 2 Toll-Like , Receptores Toll-Like , Fator de Necrose Tumoral alfa/biossíntese , Leveduras/metabolismo , Zimosan/metabolismoRESUMO
Fourteen MS patients took pentoxifylline at varying doses for up to 24 months. In vitro production of tumor necrosis factor alpha was reduced in patients taking 2,400 to 3,200 mg/day of pentoxifylline for 12 weeks or more. Twelve of the 14 patients experienced worsening of the disease during the study according to clinical, MRI, or visual evoked potential criteria. These results provide no hint of efficacy for pentoxifylline as a treatment for MS in progression phase.
Assuntos
Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/fisiopatologia , Pentoxifilina/uso terapêutico , Adulto , Encéfalo/patologia , Progressão da Doença , Relação Dose-Resposta a Droga , Potenciais Evocados Visuais/efeitos dos fármacos , Potenciais Evocados Visuais/fisiologia , Humanos , Linfócitos/imunologia , Imageamento por Ressonância Magnética , Esclerose Múltipla/imunologia , Fatores de Tempo , Falha de Tratamento , Fator de Necrose Tumoral alfa/biossínteseRESUMO
An efficient method for the isolation of human immunodeficiency virus type 1 (HIV-1) nucleic acids from dry cervical swabs was developed. HIV-1 gag and env were detected in 96% (25 of 26) and 81% (21 of 26), respectively, of the samples tested by PCR from HIV-1-seropositive women in a Kenyan cohort study. Eighty-eight percent of the swabs (22 of 25) were positive for gag RNA, and 85% (17 of 20) were positive for env RNA. Fewer than 1,000 copies of HIV-1 gag RNA were detected in four swabs in which a competitive quantitative PCR assay was used. The method described here may be useful for both qualitative and quantitative analyses of HIV RNA in mucosal secretions as well as amplification and cloning of full-length viral genes for functional studies.
Assuntos
Colo do Útero/virologia , Infecções por HIV/virologia , HIV-1/isolamento & purificação , RNA Viral/isolamento & purificação , Estudos de Coortes , Feminino , Genes env , Genes gag , HIV-1/genética , Humanos , Quênia , Reação em Cadeia da Polimerase/métodos , RNA Viral/genéticaRESUMO
Reports of the success of treatment using endosseous implants vary, and claims about the usefulness of some implant systems are supported by little hard data. The authors of this report followed the progress of patients who had been implanted with the ITI Bonefit implant system over a period of 5 years. They present the results of their study and give recommendations on how general dental practitioners (GDPs) should be trained to use such implants.
Assuntos
Implantação Dentária Endóssea , Implantes Dentários , Arcada Parcialmente Edêntula/reabilitação , Adolescente , Adulto , Idoso , Implantação Dentária Endóssea/métodos , Feminino , Odontologia Geral/educação , Humanos , Arcada Parcialmente Edêntula/cirurgia , Masculino , Pessoa de Meia-Idade , Seleção de Pacientes , Estudos Retrospectivos , Resultado do Tratamento , Reino UnidoRESUMO
In this report, we describe a recombinant provirus generated during in vitro passage that contains a short region of adenosine-to-guanosine hypermutation. The hypermutated region is restricted to complementary sequences present in the recombinant provirus. We propose that a duplex was formed in the recombinant RNA prior to reverse transcription. This duplex was a substrate for double-stranded RNA adenosine deaminase, an activity found in all cells examined that deaminates A in double-stranded RNA, converting it to inosine, which is further converted to a guanosine by reverse transcription. It appears that cis viral sequences facilitated the A-->G transitions.
Assuntos
Adenosina Desaminase/metabolismo , Vírus do Sarcoma Aviário/genética , Mutação , RNA Viral/metabolismo , Sequências Repetitivas de Ácido Nucleico , Adenosina , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Genes env , Genes pol , Guanosina , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Provírus/genética , Codorniz , RNA/biossíntese , RNA/metabolismo , RNA Antissenso , RNA Viral/biossíntese , Proteínas de Ligação a RNA , Especificidade por SubstratoRESUMO
Using a recently described system to generate recombinants between avian leukosis viruses (ALV) and cellular neo mRNA (A.M. Hajjar and M.L. Linial, J. Virol. 67:3845-3853, 1993), we isolated a recombinant containing 7S L sequences. Analysis of this recombinant revealed that it most likely arose during reverse transcription of three copackaged RNAs: 7S L RNA, neo RNA, and ALV genomic RNA. Reverse transcription appears to have initiated on the 7S L RNA. A model for the generation of this recombinant is described.
Assuntos
Vírus da Leucose Aviária/genética , RNA Viral/genética , RNA/genética , Recombinação Genética , Transcrição Gênica , Animais , Sequência de Bases , Humanos , Modelos Genéticos , Dados de Sequência Molecular , CodornizRESUMO
A current model for the generation of transforming retroviruses proposes that read-through RNAs, containing both viral and cellular sequences, are copackaged with viral genomic RNA. It is, however, possible that a cellular mRNA is occasionally encapsidated into a retroviral particle, even though viral packaging sequences are absent. We have generated recombinant proviruses following copackaging of an avian leukosis viral genomic RNA and a neo-containing RNA completely devoid of retroviral sequences. In these studies, we used the packaging cell line SE21Q1b, which has the unique ability to randomly package cellular mRNA into retroviral particles. We describe 10 recombinants obtained following copackaging of nonhomologous RNAs. Our data show that recombination is not occurring at the DNA level in the parental SE21Q1b cells but is occurring at the RNA level, during reverse transcription. These data further suggest that reverse transcriptase can preferentially jump between templates at short stretches of homology in otherwise unrelated RNAs. We conclude that retroviral sequences are not required for packaged mRNA to be reverse transcribed and to be included in integrated proviruses.
Assuntos
Vírus da Leucose Aviária/genética , RNA Viral/genética , Recombinação Genética , Animais , Sequência de Bases , Coturnix , Técnicas In Vitro , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , DNA Polimerase Dirigida por RNA/genética , TransfecçãoRESUMO
RNA packaging signals (psi) from the 5' ends of murine and avian retroviral genomes have previously been shown to direct encapsidation of heterologous mRNA into the retroviral virion. The avian 5' packaging region has now been further characterized, and we have defined a 270-nucleotide sequence, A psi, which is sufficient to direct packaging of heterologous RNA. Identification of the A psi sequence suggests that several retroviral cis-acting sequences contained in psi+ (the primer binding site, the putative dimer linkage sequence, and the splice donor site) are dispensable for specific RNA encapsidation. Subgenomic env mRNA is not efficiently encapsidated into particles, even though the A psi sequence is present in this RNA. In contrast, spliced heterologous psi-containing RNA is packaged into virions as efficiently as unspliced species; thus splicing per se is not responsible for the failure of env mRNA to be encapsidated. We also found that an avian retroviral mutant deleted for both nucleocapsid Cys-His boxes retains the capacity to encapsidate RNA containing psi sequences, although this RNA is unstable and is thus difficult to detect in mature particles. Electron microscopy reveals that virions produced by this mutant lack a condensed core, which may allow the RNA to be accessible to nucleases.
Assuntos
Vírus da Leucose Aviária/crescimento & desenvolvimento , Capsídeo/metabolismo , Genes gag/genética , RNA Viral/genética , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Vírus da Leucose Aviária/genética , Sequência de Bases , Linhagem Celular , Cisteína , Histidina , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Codorniz , Splicing de RNA/genética , RNA Viral/metabolismo , Vírion/genética , Vírion/crescimento & desenvolvimentoRESUMO
Sialodacryoadenitis virus (SDAV) was detected in athymic rats subcutaneously implanted with human tumor cell lines. Clinical signs included sneezing, dyspnea, weight loss and death. Necropsy revealed both upper and lower respiratory tract disease from which Staphylococcus aureus, Pasteurella pneumotropica and Pseudomonas aeruginosa were recovered. Histopathological changes consisted of suppurative rhinitis and bronchopneumonia. Lesions characteristic of SDAV infection were found in lacrimal and salivary glands, and viral antigens were detected in the salivary glands and respiratory tract by immunohistochemistry. Submaxillary salivary gland. Harderian gland and lung homogenates from affected athymic rats were inoculated intranasally into euthymic rats as a rat antibody production test. All euthymic rats seroconverted to SDAV. Seroconversion to SDAV was demonstrated in consecutive pairs of sentinel euthymic rats housed for 6 months with infected athymic rats. Inoculation of supernatants of the original tumor cell lines into euthymic rats did not result in seroconversion. The source of the virus was not determined. In this study, spontaneously acquired SDAV infection persisted for at least 6 months in athymic rats.
Assuntos
Animais de Laboratório , Infecções por Coronaviridae/veterinária , Surtos de Doenças/veterinária , Ratos Nus , Animais , Antígenos Virais/análise , Infecções Bacterianas/epidemiologia , Infecções Bacterianas/microbiologia , Infecções Bacterianas/patologia , Infecções Bacterianas/veterinária , Doença Crônica , Conjuntivite Viral/epidemiologia , Conjuntivite Viral/microbiologia , Conjuntivite Viral/patologia , Conjuntivite Viral/veterinária , Infecções por Coronaviridae/epidemiologia , Infecções por Coronaviridae/microbiologia , Infecções por Coronaviridae/patologia , Feminino , Masculino , RatosRESUMO
A cytopathogenic virus with size and structural characteristics of a Herpesviridae was isolated from a cheetah with severe ulcerative dermatitis. Restriction endonuclease analysis and cross-hybridization studies revealed that the isolate was related to feline herpesvirus type 1 (FHV-1). Antigenic comparison studies using anti-FHV-1 serum demonstrated the presence of common antigens in the FHV-1 and the isolate from the cheetah.
Assuntos
Acinonyx/microbiologia , Animais de Zoológico/microbiologia , Carnívoros/microbiologia , DNA Viral/análise , Herpesviridae/classificação , Úlcera Cutânea/veterinária , Animais , Eletroforese em Gel de Poliacrilamida , Herpesviridae/análise , Herpesviridae/isolamento & purificação , Herpesviridae/ultraestrutura , Técnicas Imunológicas , Hibridização de Ácido Nucleico , Polimorfismo de Fragmento de Restrição , Homologia de Sequência do Ácido Nucleico , Úlcera Cutânea/microbiologia , Proteínas Virais/análiseRESUMO
The purpose of this randomized study was to compare the effects of two methods of hemostasis--photocoagulation using YAG Neodyme laser and liquid monopolar electrocoagulation--on acute experimental bleeding ulcers created in the dog stomach with an ulcer-maker. One hundred and fifty-three lesions were made and randomized into 3 groups; 51 lesions were treated with photocoagulation and complete hemostasis was achieved in all cases. Hemostasis was obtained in 80 p. 100 of 51 ulcers treated with liquid electrocoagulation. Control untreated ulcers remained hemorrhagic after 45 min of observation. The mean external muscle injury on day 7 was 55 p. 100 after photocoagulation and 65 p. 100 after liquid electrocoagulation. On day 14, mean external injury was 60 p. 100 after photocoagulation and 75 p. 100 after liquid electrocoagulation (non-significant difference). On day 7, the mean re-epithelization index, expressed as the percentage of the original ulcer diameter, ranged from 8 to 10 p. 100 in each trial group. On day 14, reepithelization covered 78 p. 100 of control ulcers and 72 p. 100 of photocoagulated ulcers (NS). This percentage falls to 47 p. 100 in ulcers treated with liquid electrocoagulation (p less than 0.01 when compared with ulcers treated with photocoagulation). Photocoagulation seemed to be more efficient in ensuring hemostasis and external muscle injury was correlated with the energy delivered. External muscle injury could not be controlled by liquid electrocoagulation. However the difference in the percentages of mean external muscle injury between the two methods was not significant. Therefore, in man, the risk of perforation is certainly slight and not very different whatever the method of hemostasis considered.