RESUMO
Immunization of Balb/c mice with conjugates of oligosaccharide haptens and crystalline bacterial surface-layer proteins (S-layers) primed the mice for a strong, hapten-specific, delayed-type hypersensitivity (DTH) response. Conjugates of haptens with bovine serum albumin produced only weak DTH responses but, when mixed with aluminium hydroxide, elicited DTH responses comparable to those against S-layer conjugates. Surface-layer conjugates also elicited strong anti-hapten DTH responses when administered by an oral/nasal route. Apparently, the natural assembly of S-layer proteins into large, two-dimensional arrays endows them with intrinsic adjuvant properties.
Assuntos
Antígenos/imunologia , Proteínas de Bactérias/imunologia , Imunização/métodos , Oligossacarídeos/imunologia , Linfócitos T/imunologia , Adjuvantes Imunológicos , Administração Oral , Hidróxido de Alumínio/imunologia , Animais , Antígenos/administração & dosagem , Proteínas de Bactérias/isolamento & purificação , Sequência de Carboidratos , Parede Celular/química , Parede Celular/imunologia , Cristalização , Ciclofosfamida/farmacologia , Portadores de Fármacos , Feminino , Haptenos/administração & dosagem , Haptenos/imunologia , Hipersensibilidade Tardia/imunologia , Imunidade Celular/efeitos dos fármacos , Imunoterapia Adotiva , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C/imunologia , Dados de Sequência Molecular , Oligossacarídeos/administração & dosagem , Soroalbumina Bovina/imunologiaRESUMO
Mycobacterial strains from the Mycobacterium avium complex were compared with each other and with Mycobacterium phlei isolates by restriction endonuclease digestion of chromosomal DNA with SspI and analysis by pulsed-field gel electrophoresis. Characteristic profiles were observed for known typed strains, and five groups were identified. Primary bovine isolates identified as Mycobacterium paratuberculosis by classical methods were shown to fall into both the M. paratuberculosis- and M. avium-like groups. M. paratuberculosis 18 was in the latter category. Two Mycobacterium intracellulare strains of different Schaefer serotypes had different digestion profiles. In addition, this system was exploited for the preparation of DNA probes by the isolation, digestion, and subcloning of DNA fragments separated by pulsed-field gel electrophoresis. Probe JC12 hybridized only to M. avium complex strains, but not to M. phlei, showing characteristic hybridization profiles for each of the groups previously identified by pulsed-field gel electrophoresis. The approach taken in the study lends itself to the comparative analysis of members of the M. avium complex and to the isolation and characterization of DNA probes with specificity for these mycobacteria.
Assuntos
Sondas de DNA/isolamento & purificação , DNA Bacteriano/genética , Complexo Mycobacterium avium/genética , Mycobacterium avium subsp. paratuberculosis/genética , DNA Bacteriano/isolamento & purificação , Desoxirribonucleases de Sítio Específico do Tipo II , Eletroforese em Gel de Campo Pulsado , Complexo Mycobacterium avium/isolamento & purificação , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Polimorfismo de Fragmento de RestriçãoAssuntos
Anticorpos Heterófilos/isolamento & purificação , Sítios de Ligação de Anticorpos , Imunoglobulina G/análise , Imunoglobulina M/análise , Transplante Heterólogo/imunologia , Animais , Sequência de Carboidratos , Carboidratos/imunologia , Haptenos , Humanos , Técnicas de Imunoadsorção , Rim/imunologia , Modelos Biológicos , Dados de Sequência Molecular , Miocárdio/imunologia , Perfusão , Soroalbumina Bovina , SuínosRESUMO
Hybridoma cell growth and monoclonal antibody production in dialyzed continuous suspension culture were investigated using a 1.5-L Celligen bioreactor. Medium supplemented with 1.5% fetal bovine serum was fed directly into the reactor at a dilution rate of 0.45 d(-1). Dailysis tubing with a molecular weight cut-off (MWCO) of 1000 was coiled inside the bioreactor. Fresh medium containing no serum or serum substitutes passed through the dialysis tubing at flow rates of 2 to 5 L/d. The objective was to remove low molecular weight inhibitors, such as lactic acid and ammonia, by diffusion through the tubing, while continuously replenishing essential nutrients by the same mechanism. Due to the low MWCO of the dialysis tubing high molecular weight components such as growth factors and antibody were not removed by the dialyzing stream. In the batch start-up phase, the monoclonal antibody (MAb) titer was almost 3 times that achieved in typical batch cultures (i.e., 170 to 180 mg/L). During dialyzed continuous operation, a substantial increase (up to 40%) in cell density, monoclonal antibody (MAb) titer, and reactor MAb productivity was observed, as compared with a conventional continuous suspension culture. The cell viability and the specific MAb productivity remained practically constant at different dialysis rates. This finding suggests that the steady state growth and death rate in continuous suspension hybridoma cultures are not direct functions of the nutrient or inhibitor concentrations.
RESUMO
The serological specificities of twelve hybridomas were compared as to their chemical reactivity as determined using direct binding to synthetic carbohydrate structures. All anti-Lea cross-react with type-1-precursor structures and three different variants of anti-Lea could be defined by their binding to type-3-precursor chains, sialylated compounds and the monosaccharide D-galactose. Three major reactivity patterns were also identified among anti-Leb reagents. Anti-LebL cross-react with Lea and do not significantly bind to H-related structures. Anti-LebH,L had both anti-LebL-like activity (cross-reaction with Lea) and anti-LebH-like activity (cross-reaction with H). Finally, anti-LebH cross-reacts strongly with H compounds and do not bind to Lea. The binding pattern of anti-LebL suggests that these antibodies have lower affinity for ALeb and BLeb pentasaccharides than anti-LebH. All these specificities are not absolute, but rather are expressed as members of a quantitative progressive varying series, suggesting the existence of a whole range of antibody specificities gradually changing from Lea----Lea,b----LebL----LebH,L----LebH. The results suggest that anti-LebL will always cross-react with Lea and that anti-LebH will always cross-react with H related structures. However, under certain well-defined conditions these cross-reactions may not be apparent and antibodies might behave as specific anti-Lea or anti-Leb in certain tests.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos do Grupo Sanguíneo de Lewis/imunologia , Oligossacarídeos/imunologia , Especificidade de Anticorpos/imunologia , Sequência de Carboidratos , Análise por Conglomerados , Reações Cruzadas/imunologia , Ensaio de Imunoadsorção Enzimática , Testes de Hemaglutinação , Humanos , Antígenos do Grupo Sanguíneo de Lewis/genética , Dados de Sequência MolecularRESUMO
An enzyme-linked immunosorbent assay (ELISA) has been developed for the Lewis blood-group associated alpha-(1----4)-fucosyltransferase activity. Microtiter plates coated with the bovine serum albumin conjugate of a synthetic beta-D-Galp-(1----3)-beta-D-GlcpNAc disaccharide are incubated with a fucosyltransferase preparation in the presence of guanosine 5'-diphosphofucose. The resulting immobilized Lewis-a active trisaccharide beta-D-Galp-(1----3)-[alpha-L-Fucp-(1----4)]-beta-D-GlcpNAc is then detected and quantitated using a monoclonal anti-Lewis-a antibody. Product formation detected in this manner is linear with time, proportional to enzyme activity, and reproducibly quantitated in the 50-400 fmol range.
Assuntos
Fucosiltransferases/sangue , Hexosiltransferases/sangue , Antígenos do Grupo Sanguíneo de Lewis , Sequência de Carboidratos , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Dados de Sequência MolecularRESUMO
A fucosyltransferase from mung bean seedling was found to transfer L-fucose from GDP-fucose to the Type 1 disaccharide beta-D-Galp-(1----3)-beta-D-GlcpNAc-OR [R = (CH2)8COOMe]. The product, which was detected by an anti-Lea antibody in a novel ELISA assay, was isolated and shown to be the human Lea blood-group determinant beta-D-Galp-(1----3)-[alpha-L-Fucp-(1----4)]-beta-D-GlcpNAc-OR by 1H-n.m.r. spectroscopy. This enzyme activity is distinct from that of the human Lewis-fucosyl-transferase since alpha-L-Fucp-(1----2)-beta-D-Galp-(1----4)-beta-D-Glcp-OR is a very poor substrate, while the Type 2 disaccharide beta-D-Galp-(1----4)-beta-D-GlcpNAc-OR is not an acceptor. In common with the Lewis fucosyltransferase, the H-Type 1 trisaccharide alpha-L-Fucp-(1----2)-beta-D-Galp-(1----3)-beta-D-GlcpNAc-OR was an excellent substrate for the enzyme. This new enzyme activity was further characterized with respect to pH, nucleotide, Mn2+ dependence, and acceptor specificity against a panel of synthetic oligosaccharides.
Assuntos
Fabaceae/enzimologia , Fucosiltransferases/metabolismo , Hexosiltransferases/metabolismo , Antígenos do Grupo Sanguíneo de Lewis , Plantas Medicinais , Sequência de Carboidratos , Ensaio de Imunoadsorção Enzimática , Humanos , Concentração de Íons de Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Especificidade por SubstratoAssuntos
Sistema ABO de Grupos Sanguíneos/imunologia , Anticorpos Monoclonais , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Ensaio de Imunoadsorção Enzimática , Animais , Sítios de Ligação de Anticorpos , Configuração de Carboidratos , Humanos , Indicadores e Reagentes , Camundongos , Relação Estrutura-AtividadeAssuntos
Sistema ABO de Grupos Sanguíneos/imunologia , Isoanticorpos/imunologia , Oligossacarídeos/imunologia , Ligação Competitiva , Humanos , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Técnicas de Imunoadsorção , Isoanticorpos/isolamento & purificação , Oligossacarídeos/síntese químicaRESUMO
Alpha 1-protease inhibitor (alpha 1Pi), an acute-phase reactant, is the major inhibitor of neutral proteases causing lung tissue injury, such as elastase. While examining the acute-phase reaction to the nematode parasite Nippostrongylus brasiliensis, we noticed that the alveolar macrophage was closely associated with alpha 1Pi when the larvae were present in the lung. Histologic examination revealed marked edema and hemorrhage with numerous alveolar macrophages that stain intensely for intracellular alpha 1Pi. Isolation of these cells by bronchoalveolar lavage showed the macrophage to be activated. Cultured alveolar macrophages from normal and infected animals synthesized and secreted alpha 1Pi, as revealed by [35S]-methionine incorporation, but the amounts were insignificant compared with that synthesized by hepatocytes. There was, however, no apparent difference in alpha 1Pi synthetic activity between normal and activated macrophages. The presence of demonstrable intracellular alpha 1Pi in the parasite-activated alveolar macrophage likely represents endocytosis as host protease- and/or parasite protease-antiprotease complexes. Although alpha 1Pi is synthesized primarily by hepatocytes, synthesis by alveolar macrophages may provide immediate local protection in the microenvironment of the lung during an acute inflammatory response.
Assuntos
Proteínas Sanguíneas/biossíntese , Macrófagos/metabolismo , Alvéolos Pulmonares/metabolismo , Animais , Proteínas Sanguíneas/análise , Feminino , Imunoquímica , Fígado/citologia , Pneumopatias/metabolismo , Macrófagos/análise , Camundongos , Camundongos Endogâmicos CBA , Alvéolos Pulmonares/análise , Ratos , Ratos Endogâmicos , Estrongiloidíase/metabolismo , alfa 1-AntitripsinaRESUMO
Systemic inflammatory reactions are a prominent feature of many parasitic infections and the cellular and humoral components of the acute phase reaction may have an impact on the host-parasite relationship. We examined serum changes of four acute phase reactants: alpha 1-proteinase inhibition (alpha 1Pi); complement C3; serum amyloid A protein (SAA); and serum amyloid P component (SAP), in mice undergoing a primary infection with Nippostrongylus brasiliensis. SAA and SAP showed changes within the first 2 days of infection indicating the presence of an acute phase response associated with inflammation in the lung. Alpha 1Pi and C3 serum levels were not altered. However, all four acute phase reactants were synthesized in greater amounts by primary cultures of hepatocytes taken from infected animals at this time. Subsequently, as parasite-mediated inflammatory changes occur in the gut, both serum and hepatocyte cultures demonstrate an acute inflammatory response in all four reactants. It is proposed that the early reaction between parasites and macrophage/monocyte lead to the release of a mediator of inflammation which initiates the hepatocyte response. In this infection, at least one of the APR is shown to localize to the site of inflammation influencing the host-parasite relationship.