RESUMO
Helicobacter pylori, a gram-negative spiral bacterium, is the cause of chronic superficial (type B) gastritis and peptic ulcer disease. The urease enzyme of H. pylori was expressed as an inactive recombinant protein in Escherichia coli, purified as particulate structures of 550-600 kDa molecular mass with a diameter of approximately 12 nm. Given orally, 5 micrograms of urease with an appropriate mucosal adjuvant, such as the labile toxin of E. coli, protected 60%-100% of mice against challenge with virulent Helicobacter felis. Protection correlated with the level of secretory IgA antibodies against urease. Oral administration of antigen was as effective or better than intragastric administration. Parenteral injection of antigen or intragastric administration of high-dose antigen without adjuvant elicited serum IgG but no IgA antibodies and did not confer protection. Recombinant urease as an oral vaccine candidate deserves further investigation as an approach to the prevention of Helicobacter-induced chronic gastroduodenal diseases in humans.
Assuntos
Vacinas Bacterianas/imunologia , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/prevenção & controle , Helicobacter pylori/imunologia , Imunoglobulina A Secretora/biossíntese , Urease/imunologia , Vacinas Sintéticas/imunologia , Adjuvantes Imunológicos , Administração Oral , Animais , Formação de Anticorpos , Vacinas Bacterianas/administração & dosagem , Sequência de Bases , Clonagem Molecular , Primers do DNA , Fezes/microbiologia , Mucosa Gástrica/microbiologia , Helicobacter/isolamento & purificação , Helicobacter pylori/enzimologia , Humanos , Imunoglobulina A Secretora/sangue , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Saliva/imunologia , Estômago/microbiologia , Urease/biossíntese , Urease/genética , Vacinas Sintéticas/administração & dosagemRESUMO
The proteolytic activity of bovine splenic cathepsin D was evaluated by using the digestion of tritiated hemoglobin. Acid-denatured human serum was an enhancer of cathepsin D activity. Concentrated serum sources showed decreased activity because of competition of serum albumin with tritiated hemoglobin for cathepsin D. The enhancing activity of serum did not separate with any isolated protein as assessed by Sephadex G-50-80 chromatography or protein electrophoresis. The extraction of serum with ethanol-ether and chloroform-methanol showed that the enhancing activity separated with the phospholipid fraction, which had 2.6 times as much activity as the total lipid fraction. The activity was stable to heat at 56 degrees C for 30 minutes and to freeze-thawing, and was not dependent on metal ions.
Assuntos
Fenômenos Fisiológicos Sanguíneos , Catepsina D/metabolismo , Animais , Bovinos , Relação Dose-Resposta a Droga , Humanos , Baço/enzimologiaRESUMO
Uridine injection in 0.6% saline elevated rabbit temperatures (mean = 0.9 degree C) in the USP XX pyrogen test. Hyperthermia was delayed in onset and peaking 3-4 h post injection, but the injection was negative in the limulus amoebocyte lysate (LAL) assay. Uridine from five lots of different sources exceeded USP XX limits in the rabbit pyrogen test and proved negative in the LAL assay. Because the dose of uridine was high, several procedures were used to determine if an impurity was the cause of temperature elevation. Uridine remained pyrogenic in spite of ultrafiltration (10 000 nominal mol. wt), recrystallization and preparative scale HPLC. Sterile filtration and autoclaving also did not affect the response. Hyperthermia, therefore, appears to be an inherent property of uridine. Uridine was also found to release endogenous pyrogen in-vitro from human mononuclear cells. Uridine has been reported to induce fever in man, thus the USP rabbit pyrogen test predicted for the clinical response.
Assuntos
Temperatura Corporal/efeitos dos fármacos , Pirogênios , Uridina/farmacologia , Animais , Tolerância a Medicamentos , Leucócitos/fisiologia , Coelhos , Fatores de Tempo , UltrafiltraçãoRESUMO
A sensitive in vitro assay for generation of human leukocytic pyrogen has been used to study the pathogenesis of fever accompanying administration of human alpha-interferon. Unlike other potent pyrogens, two recombinant interferon preparations tested over a wide concentration range did not stimulate release of leukocytic pyrogen. This result suggests that interferon may cause fever by a novel mechanism not dependent on leukocytic pyrogen.
Assuntos
Interferon Tipo I/toxicidade , Monócitos/fisiologia , Pirogênios , Animais , Linhagem Celular , Células Cultivadas , Cães , Endotoxinas/toxicidade , Humanos , Interferon Tipo I/genética , Rim , Cinética , Monócitos/efeitos dos fármacos , Coelhos , Staphylococcus epidermidisRESUMO
To attempt to locate functionally important regions of the interferon (IFN) molecule, recombinant human IFN-alpha 2 was subjected to proteolytic digestion. The bacterial proteinase thermolysin produced two major complementary fragments, HuIFN-alpha 2-(1-110) and HuIFN-alpha 2-(111-153). After reduction with 2-mercaptoethanol and separation of the two major fragments on NaDodSO4/polyacrylamide gel electrophoresis, antiviral activity persisted in the larger, Mr 12,000, fragment consisting of the amino-terminal 110 amino acids.
Assuntos
Clonagem Molecular , Interferon Tipo I , Interferon Tipo I/genética , Interferon Tipo I/isolamento & purificação , Fragmentos de Peptídeos/isolamento & purificação , Animais , Ligação Competitiva , Bovinos , Linhagem Celular , DNA Recombinante , Fibroblastos , Humanos , Interferon Tipo I/toxicidade , Interferon-alfa , Interferons/metabolismo , Rim , Cinética , Masculino , Peso Molecular , Fragmentos de Peptídeos/toxicidade , Receptores de Superfície Celular/metabolismo , Receptores de Interferon , Proteínas Recombinantes , Pele , Termolisina , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacosRESUMO
Two active site inhibitors of acid proteinases were tested for their effects on human neutrophil chemotaxis and lysosomal enzyme release. Both bromphenacyl bromide (BPAB) and epoxy-p-nitrophenoxypropane (EPNP) inhibited chemotaxis and chemotaxin-induced enzyme release elicited by pepstatin and formylmethionyl peptides, which share membrane receptors, and also by zymosan-activated serum, the major active component of which (C5a) occupies a different receptor. In contrast to the previously tested acid protease inhibitor diazoacetylnorleucine methyl ester, neither BPAB nor EPNP blocked binding of [3H]fMLP to neutrophils. Thus BPAB and EPNP inhibit chemotaxin-mediated neutrophil functions, but not by interaction with the chemotaxin receptor.
Assuntos
Acetofenonas/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Lisossomos/enzimologia , Nitrofenóis/farmacologia , Inibidores de Proteases/farmacologia , Fosfatase Ácida/metabolismo , Dipeptídeos/farmacologia , Compostos de Epóxi/farmacologia , Humanos , Lisossomos/efeitos dos fármacos , Muramidase/metabolismo , N-Formilmetionina/análogos & derivados , N-Formilmetionina/farmacologia , N-Formilmetionina Leucil-Fenilalanina , Neutrófilos/efeitos dos fármacos , Oligopeptídeos/farmacologia , Pepstatinas/farmacologiaRESUMO
Soluble human serum albumin anti-albumin immune complexes (IC) have been shown to bind to freshly isolated human peripheral blood monocytes and polymorphonuclear leucocytes (PMN) in vitro. Binding sites on both cell types were saturable and specifically inhibited by heat aggregated IgG1 and IgG3 subclasses. PMN contained a greater number of binding sites than monocytes although the affinity was similar for both cell types. The enhanced binding of IC by both cell types observed after incubation at low pH (pH 6.0) was a consequence of increased affinity of the PMN binding site and an increase in the number of sites in monocytes. Binding of IC by both cell types was inhibited by normal human serum. Enhanced IC binding associated with enhanced affinity and number of sites was observed in PMN and monocytes preincubated in suspension with trypsin. However, monocytes exposed to trypsin while adherent to microexudate coated flasks demonstrated a marked increase in affinity without any change in the number of sites.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Monócitos/imunologia , Neutrófilos/imunologia , Animais , Afinidade de Anticorpos , Sítios de Ligação de Anticorpos , Células Cultivadas , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G/imunologia , Masculino , Coelhos , Fatores de Tempo , Tripsina/farmacologiaRESUMO
Treatment of human neutrophils with a reagent (diazoacetylnorleucine methyl ester plus copper ion) which covalently labels the active site of the acid proteases, pepsin and cathepsin D, inhibits neutrophil chemotaxis and enzyme release stimulated by the chemoattractants pepstatin and formylmethiony peptides. In contrast, chemotaxis and enzyme release in response to zymosan activated serum are not affected. Furthermore, diazoacetylnorleucine methy ester plus copper competes with [3H]formylmethionyl leucylphenylalanine for binding to neutrophils. Since pepstatin shares binding sites with formylmethionyl leucylphenylalanine, the present data suggest that diazoacetylnorleucine methyl ester plus copper reacts with the neutrophil receptor for pepstatin and formylmethionyl peptides, and thus may be useful in further characterization of this structure.
Assuntos
Fosfatase Ácida/sangue , Quimiotaxia de Leucócito/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Pepsina A/antagonistas & inibidores , Adulto , Sítios de Ligação , Fatores Quimiotáticos/sangue , Fatores Quimiotáticos/farmacologia , Cobre , Humanos , Técnicas In Vitro , N-Formilmetionina/análogos & derivados , N-Formilmetionina Leucil-Fenilalanina , Neutrófilos/fisiologia , Norleucina/análogos & derivados , Oligopeptídeos , Pepstatinas/farmacologiaRESUMO
Pepstatin, a chemotactic microbial pentapeptide, competes with f-Met-Leu-[3H]Phe for binding to human neutrophils. Furthermore, porcine neutrophils, which neither specifically bind nor respond chemotactically to the synthetic f-methionyl peptides, also fail to respond chemotactically to pepstatin. These results suggest that pepstatin shares a receptor on the neutrophil with f-methionyl peptides, despite their completely different amino acid compositions. The specificity of this cytotaxin receptor may therefore be broader than expected and depend on ligand characteristics distinct from primary structure.
Assuntos
Ligação Competitiva , Fatores Quimiotáticos/metabolismo , Dipeptídeos/metabolismo , Metionina/análogos & derivados , N-Formilmetionina/análogos & derivados , Neutrófilos/metabolismo , Oligopeptídeos/metabolismo , Pepstatinas/metabolismo , Animais , Sítios de Ligação , Quimiotaxia de Leucócito , Humanos , N-Formilmetionina/metabolismo , Neutrófilos/fisiologia , SuínosRESUMO
Human peripheral blood monocytes were maintained in in vitro culture for periods up to 4 months using a non-human serum source. Monocytes were cultured in Dulbecco's modified Eagle's medium buffered with 20 mM HEPES and containing 10% horse serum and 10% foetal calf serum. The metabolic and morphological changes which occur in vitro were investigated using microtitre, Linbro and T 25 cultures. During culture, monocytes increased in size, had increased membrane activity as visualized by SEM, and differentiated into a morphologically heterogeneous population of fusiform and epithelioid shapes. These cell types retained the ability to phagocytose E glut and EA and to rosette with EA and EAC. Larger giant polynucleated cells were also observed during culture; many of these lacked the ability to bind or phagocytose inert or antibody-coated erythrocytes. Increases in lysozyme release and acid phosphatase activity also occurred during culture. Cultured monocytes exhibited characteristic profiles of leucine and uridine uptake with maximal activity observed by 5 days of culture. There was no detectable uptake of thymidine. Detailed analysis of regulatory processes involved in monocyte growth and differentiation could be performed with this in vitro system.
Assuntos
Monócitos/citologia , Células Cultivadas , Meios de Cultura , Humanos , Leucina/metabolismo , Microscopia Eletrônica de Varredura , Microscopia de Contraste de Fase , Monócitos/metabolismo , Monócitos/ultraestrutura , Fagocitose , Formação de Roseta , Fatores de Tempo , Uridina/metabolismoAssuntos
Tecido Linfoide/anatomia & histologia , Animais , Linfócitos B/citologia , Medula Óssea/anatomia & histologia , Bolsa de Fabricius/anatomia & histologia , Comunicação Celular , Diferenciação Celular , Movimento Celular , Sistema Digestório/anatomia & histologia , Humanos , Linfonodos/anatomia & histologia , Linfócitos/citologia , Linfócitos/ultraestrutura , Tecido Linfoide/imunologia , Macrófagos/citologia , Monócitos/citologia , Fagócitos/citologia , Plasmócitos/citologia , Sistema Respiratório/anatomia & histologia , Baço/anatomia & histologia , Linfócitos T/citologia , Timo/anatomia & histologiaRESUMO
Extracts of human neutrophils, lymphocytes and platelets enzymically deformylate N-formyl-L-methionine. Enzyme activity is stimulated by Co2+, inhibited by bivalent-cation chelators and unaffected by inhibitors of serine, thiol and carboxyl proteinases. Leucocyte or platelet N-formylmethionine deformylase may be important in modulation of neutrophil responses to chemoattractant formylmethionyl peptides or similar compounds.
Assuntos
Amidoidrolases/sangue , Plaquetas/enzimologia , Leucócitos/enzimologia , Cobalto/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Técnicas In Vitro , N-FormilmetioninaAssuntos
Anticorpos , Eritrócitos/imunologia , Monócitos/imunologia , Animais , Adesão Celular , DNA/biossíntese , Vidro , Humanos , Fragmentos Fc das Imunoglobulinas , Imunoglobulina G , Látex , Microesferas , Fagocitose , OvinosRESUMO
Human and rat alveolar macrophages produce haemolytic C2 during in vitro culture. We conclude that C2 synthetic ability is maintained during in vivo maturation of human monocytes to macrophages and that production of complement components by mature tissue macrophages may be important for optimal generation of inflammatory responses.
Assuntos
Complemento C2/biossíntese , Macrófagos/imunologia , Alvéolos Pulmonares/imunologia , Animais , Células Cultivadas , Complemento C2/antagonistas & inibidores , Cicloeximida/farmacologia , Temperatura Alta , Humanos , RatosAssuntos
Imunidade Celular , Leucemia Mieloide/imunologia , Quimiotaxia de Leucócito , Citotoxicidade Imunológica , Humanos , Leucemia Mieloide/patologia , Teste de Inibição de Aderência Leucocítica , Leucócitos/imunologia , Masculino , Pessoa de Meia-Idade , Monócitos/ultraestrutura , Receptores de Antígenos de Linfócitos B/análiseRESUMO
Human blood monocytes, but not other Ficoll-Hypaque cells, adhere to plastic surfaces from which confluent BHK cells have been removed. EDTA (3mM) rapidly and completely removes monocytes, providing a simple technique for preparation of human monocytes in suspension. The method should be valuable for studies of monocyte function where monocyte monolayers are not suitable.
Assuntos
Monócitos , Animais , Adesão Celular , Separação Celular , Cricetinae , Ácido Edético/farmacologia , Ficoll , Humanos , CamundongosAssuntos
Leucemia Linfoide/imunologia , Linfócitos/imunologia , Idoso , Citotoxicidade Celular Dependente de Anticorpos , Membrana Celular/imunologia , Eritrócitos/imunologia , Feminino , Humanos , Leucemia Linfoide/genética , Leucemia Linfoide/ultraestrutura , Ativação Linfocitária , Linfócitos/ultraestrutura , Masculino , Mitógenos/farmacologia , Linhagem , Receptores de Antígenos de Linfócitos B , Formação de RosetaRESUMO
The host defense responses to Nocardia asteroides are not known. We have investigated a patient with common variable adult onset hypogammaglobulinemia who developed fatal disseminated nocardiosis. The patient had low levels of serum immunoglobulins; the total lymphocyte count was normal as were the percentages of circulating T and B cells. Transmission electron microscopic studies demonstrated typical N. asteroides within the brain parenchyma and nonspecific inflammatory changes in the central nervous system. The findings suggest that humoral immunity may play a major role in the host defense of patients with this disease.