RESUMEN
Protective properties of immunoglobulin A (IgA) monoclonal antibodies (MAbs) directed against O and H antigens of Salmonella enterica serotype Enteritidis (S. enteritidis) were evaluated in a model of generalized infection after intranasal (i.n.) inoculation of BALB/c mice. Passive i.n. instillation of antibodies 1 h before i.n. challenge did not prevent infection, and mice developed rapid inflammatory response in the lower respiratory tract. The passive systemic immunization was partially protective and a single intravenous (i.v.) injection of both O and H antigen specific IgA antibodies prolonged survival period of the infected animals. Permanent secretion of O:9 specific IgA MAb 177E6 into the respiratory tract in a "backpack" tumor model protected 50% of animals infected i.n. with a high dose of virulent S. enteritidis strain. Thus, secretory IgA (S-IgA) directed against O:9 antigen alone can prevent bacterial invasion in the respiratory epithelium.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígenos Bacterianos/inmunología , Inmunoglobulina A/inmunología , Enfermedades Pulmonares/microbiología , Antígenos O/inmunología , Salmonelosis Animal/inmunología , Salmonella enteritidis/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antibacterianos/farmacología , Anticuerpos Monoclonales/inmunología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Inmunización Pasiva/métodos , Inmunoglobulina A/farmacología , Cinética , Enfermedades Pulmonares/inmunología , Enfermedades Pulmonares/prevención & control , Ratones , Ratones Endogámicos BALB C , Salmonelosis Animal/microbiología , Salmonelosis Animal/prevención & controlRESUMEN
Mouse plasmacytomas are appropriate models to study the biology of human multiple myeloma (MM). Growth of murine interleukin-6 (IL-6)-dependent hybridoma/plasmacytoma lines can be stimulated by bacterial lipopolysaccharides (LPS). However, the molecular mechanisms of this phenomenon are still not elucidated. In this study the in vitro action of bacterial LPS on the mouse IL-6-dependent B9 hybridoma/plasmacytoma cell line and two IL-6-dependent hybridomas was investigated. The involvement of different signal transduction pathways was established using specific kinase inhibitors in proliferation assays and immunoblotting analysis of the kinase activity. Selective mitogen-activated protein kinase (MAPK) kinase inhibitor PD989059 inhibited both IL-6- and LPS-induced B9 cell proliferation. In contrast, in H187 and H188 cells, PD98059 inhibited only LPS-, but not IL-6-stimulated cell growth. The kinetics of MAPK activation in all cell lines showed that phosphorylation of p42 MAPK (encoded as ERK2) but not of p44 MAPK (ERK1), was considerably increased after treatment with LPS. We found that in H187 and H188 hybridomas IL-6 induced proliferation by a different STAT3-dependent mechanism. This study demonstrates the key role of the MAPK pathway in LPS-stimulated growth of mouse IL-6-dependent plasmacytoma cells. These findings suggest the presence of signaling mechanism in MM cells inducible by bacterial mitogens and possibly mediated by Toll-like receptors (TLR)--evolutionarily conserved molecules playing a central role in the microbial recognition and initiation of the cellular innate immune response.
Asunto(s)
Interleucina-6/metabolismo , Lipopolisacáridos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Plasmacitoma/metabolismo , Animales , División Celular/fisiología , Humanos , Ratones , Salmonella/metabolismoRESUMEN
Monoclonal antibody (MAb) 8aC10 against Salmonella O:5 antigen was obtained after immunization of BALB/c mice with live attenuated mutant of Salmonella typhimurium. Antigen specificity of the MAb was characterized by ELISA, immunoblotting, passive hemagglutination (PHA), passive hemolysis and agglutination tests. In ELISA, PHA and immunoblotting the MAb reacted only with lipopolysaccharides (LPS) of Salmonella strains from group O:4 (B), expressing O:5 antigen. The MAb agglutinated in addition Salmonella strains with O:8 antigen from group C(2)-C(3) but did not react with purified LPS. These results demonstrate O:5 specificity of MAb 8aC10. Cross-agglutination with group C(2)-C(3) suggests the presence of similar but not identical epitope in O:8 expressing strains, which is possibly localized onto O-acetyl-abequose and abequose residues bound with a alpha-1-->3 linkage to the basic polysaccharide backbone of Salmonella LPS with O:5 and O:8 antigen respectively.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Antígenos O/inmunología , Salmonella/inmunología , Pruebas de Aglutinación , Animales , Especificidad de Anticuerpos , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Hemaglutinación , Hemólisis , Immunoblotting , Lipopolisacáridos/inmunología , Ratones , Salmonella/clasificación , Salmonella typhimurium/inmunología , SerotipificaciónRESUMEN
Three cross-reactive monoclonal antibodies (MAbs) of IgM and IgG2b isotype were generated in two separate fusions after immunization of BALB/c mice with heat killed Salmonella minnesota R595 of Re chemotype and acid-treated bacteria, coated with Re lipopolysaccharide (LPS) antigen. The specificity of the MAbs was demonstrated as the Re LPS antigen. The activity and cross-reactivity against purified elementary bodies of Chlamydia trachomatis and various S- and R-LPS antigens of other Gram-negative bacteria were characterized in enzyme-linked immunosorbent assay, passive hemolysis assay, immunoblot and immunofluorescence with the available chlamydial strains. The results demonstrated cross-reaction between the Re LPS antigen, the genus-specific chlamydial LPS and the LPS antigens of Escherichia coli O119 and Acinetobacter baumannii, suggesting the presence of identical or similar epitopes in the lipopolysaccharide antigens. The findings are implying the necessity of novel approaches, improving the specificity of serologic assays in the laboratory diagnosis of chlamydial infections.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Chlamydia trachomatis/inmunología , Lipopolisacáridos/inmunología , Salmonella/clasificación , Salmonella/inmunología , Animales , Anticuerpos Antibacterianos/análisis , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Bacterias Gramnegativas/inmunología , Immunoblotting , Ratones , Ratones Endogámicos BALB C , Modelos Animales , Sensibilidad y EspecificidadRESUMEN
A murine monoclonal antibody (MAb) 202D7 of IgG3 isotype recognizes a lipopolysaccharide (LPS) epitope of Chlamydia spp. and cross-reacts with the Re chemotype LPS of Salmonella and Escherichia coli. The antibody exhibits strong complement activating properties and stimulates phagocytosis of Salmonella enterica serovar Minnesota Re mutant by murine macrophages. Salmonella Re mutants are non-invasive for cell monolayers but still can enter and replicate in L-929 murine fibroblast cells. The entry of bacteria within the cells increases five-fold in the presence of MAb 202D7. The antibody mediates attachment and enhances five-fold the infectivity of Chlamydia pneumoniae into L-929 cells, which suggests a possible IgG-mediated mechanism of entry and survival of the pathogen in fibroblast cells.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Chlamydophila pneumoniae/patogenicidad , Inmunoglobulina G/inmunología , Lipopolisacáridos/inmunología , Salmonella enterica/patogenicidad , Animales , Acrecentamiento Dependiente de Anticuerpo , Línea Celular , Chlamydophila pneumoniae/inmunología , Reacciones Cruzadas , Epítopos/inmunología , Fibroblastos/microbiología , Humanos , Macrófagos Peritoneales/microbiología , Ratones , Ratones Endogámicos BALB C , Fagocitosis , Salmonella enterica/genética , Salmonella enterica/inmunologíaRESUMEN
Hybridomas were generated after intragastral immunization of BALB/c mice with live Salmonella suberu and subsequent fusion between isolated spleen lymphoblasts and myeloma cells. Three monoclonal antibodies (MAbs) of immunoglobulin A (IgA) isotype were selected and characterized. All of them were found to recognize the H:g epitope in enzyme-linked immunosorbent assay and immunoblotting but did not react with all H:g-expressing strains in slide agglutination test. All MAbs strongly agglutinated Salmonella enteritidis type strain and a large number of S. enteritidis clinical isolates. They were not bactericidal in the presence of complement. All hybridoma clones produced secretory IgA forms, which were found in the gastrointestinal tract of mice bearing hybridoma as a subcutaneous 'backpack' tumor or after intravenous application of purified MAbs. The IgA MAbs stability demonstrated in different tests together with their antigen specificity and strong agglutination ability make them a useful diagnostic tool for serotyping of Salmonella strains.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Antígenos Bacterianos/inmunología , Flagelina/inmunología , Inmunoglobulina A Secretora/biosíntesis , Salmonella enteritidis/inmunología , Animales , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Hibridomas , Immunoblotting , Inmunoglobulina A Secretora/inmunología , Ratones , Ratones Endogámicos BALB CRESUMEN
The protective potential of immunoglobulin A (IgA) monoclonal antibodies (MAbs) directed against O and H antigens of Salmonella enterica serotype Enteritidis to prevent bacterial adhesion to and invasion of HEp-2 cells was evaluated. Although anti-flagellar IgA MAbs showed strong agglutinating capacities, they did not protect cell monolayers. In contrast, IgA MAbs specific for the O:9 epitope of Salmonella lipopolysaccharide antigen alone prevented S. enterica serotype Enteritidis entry and replication within HEp-2 cells, and the protection was not mediated by direct binding of antibodies to bacterial adhesins or by agglutination of microorganisms.