RESUMEN
A monoclonal antibody (5A2) recognizing a segment near the C-terminus of the fibrin(ogen) A alpha-chain (A alpha #529-539) was found to inhibit alpha-chain crosslinking catalyzed by coagulation factor XIIIa and by tissue-transglutaminase. The rapid gamma-chain cross-linking by factor XIIIa was not affected by the antibody. Results obtained from direct binding and competitive immunoassay established that the antigenic determinant recognized by 5A2 was included within the CNBr fragment referred to as CNBr X (A alpha #518-584), and that it survived trypsin digestion but was destroyed by treatment with Staph V-8 protease or chymotrypsin. Reverse-phase (C-18) high performance liquid chromatography (HPLC) was employed to obtain a CNBr X tryptic fingerprint, which was subsequently characterized by compositional and NH2-terminal analysis. Assay of the HPLC column effluent revealed a single peak of 5A2 immunoreactivity that coincided with elution of the eleven-residue tryptic peptide, A alpha #529-539. When this isolated peptide and its parent CNBr fragment were employed as solution phase competitors in the 5A2 immunoassay, the relative cross-reactivities (18.3%, peptide: fragment) indicated that a significant proportion of the 5A2 epitope was preserved within the small peptide. This is a region that is released from fibrinogen early in its degradation by plasmin. Thus, the antibody can be used as a probe for intact fibrin(ogen) and C-terminal (A) alpha-chain fragments, in addition to assessing roles of the A alpha-chain C-terminus in cross-linking.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Mapeo Epitopo , Fibrinógeno/inmunología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Transglutaminasas/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Bromuro de Cianógeno , Sustancias Macromoleculares , Ratones , Ratones Endogámicos BALB C , Mapeo Peptídico/métodos , Trombosis/sangreRESUMEN
Endotoxemia induced by gram-negative bacteria leads to endotoxic shock pathogenetically stemming from the integral component of the bacterial wall--lipid A. The study made to define the ability of lipid A monoclonal antibodies to correct hemodynamic disturbances due to endotoxemia in dog experiments showed the efficacy of the antibodies administration. ReLPS isolated from Salmonella Minnesota was used as an antigen. Administration of the complex monoclonal antibodies-endotoxin caused no hemodynamic impairment.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Infecciones por Bacterias Gramnegativas/terapia , Inmunoglobulina M/uso terapéutico , Lípido A/inmunología , Choque Séptico/terapia , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Perros , Evaluación Preclínica de Medicamentos , Infecciones por Bacterias Gramnegativas/complicaciones , Infecciones por Bacterias Gramnegativas/fisiopatología , Hemodinámica/efectos de los fármacos , Inmunización , Inmunoglobulina M/aislamiento & purificación , Lipopolisacáridos/inmunología , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos BALB C , Salmonella/inmunología , Choque Séptico/etiología , Choque Séptico/fisiopatologíaRESUMEN
Apolipoprotein B (apo B), fibrinogen/fibrin, blood platelets, factor VIII-related antigen of the blood coagulation system, and smooth muscle cells (SMC) were identified in the intima of normal and atherosclerotic human aorta and large arteries by the indirect immunofluorescence technique. Fibrinogen/fibrin was revealed by a monoclonal antibody (monAb) against the C-terminal region of human fibrinogen A alpha-chain. Fibronectin was visualized by monAb to the cellular form and against an epitope shared by different fibronectin subunit variants. In normal intima, fatty streaks, small amounts of fibrinogen/fibrin together with large amounts of apo B were observed. Fibronectin detected by two types of monAb was not found in extracellular matrix (ECM), whereas cellular fibronectin encircled SMC. According to the data obtained, fibrinogen/fibrin accumulates in plaques as a result of intramural thrombus incorporation, blood insudation, intramural haemorrhage, and in or around cells, apparently macrophages.
Asunto(s)
Apolipoproteínas B/metabolismo , Arterias/metabolismo , Arteriosclerosis/metabolismo , Fibrina/metabolismo , Fibrinógeno/metabolismo , Fibronectinas/metabolismo , Anciano , Anticuerpos Monoclonales , Aorta/metabolismo , Plaquetas/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Persona de Mediana Edad , Factor de von Willebrand/metabolismoAsunto(s)
Anticuerpos Monoclonales/uso terapéutico , Enfermedades Cardiovasculares/prevención & control , Lípido A/inmunología , Choque Séptico/prevención & control , Animales , Presión Sanguínea/efectos de los fármacos , Enfermedades Cardiovasculares/etiología , Perros , Ensayo de Inmunoadsorción Enzimática , Hemodinámica/efectos de los fármacos , Inmunoglobulina M/inmunología , Radioisótopos de Yodo , Ratones , Ratones Endogámicos BALB C , Salmonella/inmunología , Choque Séptico/complicaciones , Choque Séptico/fisiopatologíaRESUMEN
Flow cytometry was used for the isolation of hybrid cells immediately after fusion. Precursor cells were stained by two lipophilic fluorescent probes: perylenoyl-labeled triglyceride (perylenoyl-TG, green fluorescence, 520 nm) and rhomdaminyl-labeled triglyceride (rhodaminyl-TG, red fluorescence, greater than 580 nm). Since the maximum emission of perylenoyl-TG coincides with the maximum absorbance of rhodaminyl-TG, the two fluorescent dyes form an effective donor-acceptor pair. Cells stained by perylenoyl-TG (0.25-1 microgram/ml) at the excitation wavelength of 457 nm displayed high intensity of fluorescence in the green region (520 nm), and low intensity of fluorescence in the red region (greater than 580 nm). Using the same conditions, cells that were stained by rhodaminyl-TG displayed a low intensity of fluorescence in both regions. When cells were simultaneously labeled by perylenoyl-TG and rhodaminyl-TG (used in a concentration ratio of 1:10, respectively) essentially total energy transfer was observed, and the cells exhibited a high intensity of red fluorescence. After the fusion of cells which had been separated stained by perylenoyl-TG and rhodaminyl-TG, the hybrid cells containing the two fluorescent probes had a high intensity of red fluorescence. Resonance exitation energy transfer between the two fluorescent dyes permits effective sorting of hybrid cells by flow cytofluorometry.
Asunto(s)
Separación Celular/métodos , Citometría de Flujo/métodos , Células Híbridas , Fusión Celular , ADN/análisis , Transferencia de Energía , Colorantes Fluorescentes , Humanos , Técnicas In Vitro , Lípidos , SolubilidadRESUMEN
The cells of the arterial wall are heterogeneous. To study the functions and peculiarities of various subpopulations of arterial cells the authors have generated an IgM mouse monoclonal antibody, designated 3-Lena, which interacts with human aortic cells (demonstrated by RIA and flow cytofluorometry). The cellular antigens interacting with monoclonal antibody 3-Lena are gangliosides GM3, GD3, GD1a, and GT1b. Presumably, the epitope is represented by the structure -NeuAc alpha 2----3Gal beta 1, a component of the oligosaccharide moiety of the ganglioside molecule. In addition to aortic cells, those dissociated from other large human vessels as well as cells of the myometrium and lung parenchyma interact with 3-Lena. Cells dissociated from the spleen and renal cortex exhibit a substantially weaker interaction, while liver and myocardial cells do not react. Compared with human aortic cells, aortic cells of other mammalian species stain less effectively (dog, swine) or do not stain at all (bovine, ram). Flow cytofluorometric analysis demonstrates that practically all human aorta medial cells interact with antibody 3-Lena, whereas a certain portion of cells in the intimal population do not. In the outer intimal sublayer adjoining the media, the cells reacting with monoclonal antibody 3-Lena make up the bulk of the cell population, and the inner sublayers are characterized by a prevalence of nonreactive cells. After separation of reactive and nonreactive human aortic intimal cells by a cytofluorometer-cell sorter, 3-Lena+ cells were found to have an elongated bipolar shape, whereas most 3-Lena- cells have multiple processes and variable elongated, stellate, or irregular shapes.(ABSTRACT TRUNCATED AT 250 WORDS)