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Biomedical subjects

D F Mosher

Publications and source records attributed to D F Mosher.

213 records · Page 12Linked to original sources
PubMed1977

Studies of the coagulation and complement systems during experimental Rocky Mountain spotted fever in rhesus monkeys.

We studied the coagulation and complement systems during Rocky Mountain spotted fever in Macaca mulatta experimentally infected with Rickettsia rickettsii. Ninety-one percent of monkeys infected intravenously with a high dose (10(6) plaque-forming units [pfu]) and 56% of monkeys infected with low doses (10(-1)-10(2) pfu) of R. rickettsii died after two to four days of illness. With the onset of fever and rickettsemia, animals developed hyperfibrinogenemia, mild thrombocytopenia, prolonged prothrombin and activated thromboplastin times, and increased serum fibrin/fibrinogen degradation products (FDP). Rickettsemia, thrombocytopenia, and FDP were greater in fatally ill monkeys than in survivors. Hemolytic titers of the second and third components of complement were not depressed except in a single surviving monkey that developed peripheral gangrenous ecchymoses at a time when both rickettsemia and agglutinating antibody were present. Thus, although activation and consumption of complement may occur during Rocky Mountain spotted fever, the hemostatic disturbances in fulminant infections seem to be a direct effect of the infectious vasculitis.

Animals↗
PubMed1977

Disseminated intravascular coagulopathy during experimental pneumococcal sepsis: studies in normal and asplenic rhesus monkeys.

Disseminated intravascular coagulation (DIC) was induced in both normal and asplenic rhesus monkeys by intravenous challenge with Streptococcus pneumoniae. Our observations in the infected monkeys have led us to conclude that (1) pneumococcal capsular polysaccharide (PCP), immune complexes and complement may not have primary roles in the initiation of DIC; (2) intact pneumococci may be catalysts for the development of DIC; (3) the initial event in DIC may be activation of Hageman factor; and (4) evidence of activation of Hageman factor-dependent systems is present regardless of severity of infection.

Animals↗
PubMed1977

Synthesis and secretion of alpha-2-macroglobulin by cultured adherent lung cells. Comparison with cell strains derived from other tissues.

We studied the synthesis and secretion of alpha-2-macroglobulin by cultures of human adherent cells. Much more alpha-2-macroglobulin (measured by radioimmunoassay) accumulated in media of established strains of adherent cells derived from embryonic lung than in media of established strains derived from adult skin or rheumatoid synovium. Alpha-2-macroglobulin accumulated in media of primary cultures of adherent cells from a variety of embryonic tissues. However, the amount of alpha-2-macroglobulin accumulating in media of subsequent passage of these cells declined for all strains except those derived from lung. Immunodiffusion and double-antibody immunoprecipitation studies of cell extracts and media after incubation of cells with l-[(35)S]methionine supported the radioimmunoassay finding that adherent cells from lung synthesized and secreted more alpha-2-macroglobulin than adherent cells from skin. Intracellular alpha-2-macroglobulin could not be detected by radio-immunoassay or visualized by immunofluorescent microscopy, suggesting that synthesized alpha-2-macroglobulin is rapidly secreted. Plasminogen-rich fibrin clots were lysed in culture media of adherent cells from embryonic lung and, to a lesser extent, heart. Adherent cells from other tissues, which produced less alpha-2-macroglobulin, did not lyse fibrin clots. However, all cultures of adherent cells contained pericellular fibronectin, a large, external, transformation-sensitive glycoprotein known to be cleaved by plasmin. We speculate that production of alpha-2-macroglobulin may be a means for protease-secreting normal cells to preserve cell surface integrity and that alpha-2-macroglobulin synthesized locally in lung may protect lung tissues from a variety of proteases.

Cell Adhesion↗
PubMed1976

Action of fibrin-stabilizing factor on cold-insoluble globulin and alpha2-macroglobulin in clotting plasma.

Experiments were performed to investigate whether proteins other than fibrin are substrates for activated fibrin-stabilizing factor (FSF, blood coagulation Factor XIII, plasma transglutaminase) in clotting whole plasma. Three fluorescently labeled polypeptides were identified in serum prepared by clotting normal, but not FSF-deficient, plasma in the presence of the fluorescent amine, N-(5-aminopentyl)-5-dimethyl-aminonaphthalene-1-sulfonamide (dansylcadaverine). The major labeled polypeptide had a Mr (estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate) of 1.6 times 10(5) and was found in the protein fraction precipitated by 33 to 50% saturated ammonium sulfate. The second had a Mr of 2.0 times 10(5), was found in the protein fraction insoluble in 33% saturated ammonium sulfate, and was precipitated by gamma-globulin directed against cold-insoluble globulin. The third had a Mr of 1.1 times 10(5) and was precipitated by 33 to 50% saturated ammonium sulfate. All three polypeptides were found in the first protein peak when labeled serum was chromatographed on Sephadex G-200. The immunoprecipitin arc containing alpha2-macroglobulin was fluorescent when labeled serum was analyzed by immunoelectrophoresis. These results indicate that alpha2-macroglubulin, cold-insoluble globulin, and an unidentified third protein with a subunit of Mr = 1.1 times 10(5) are transamidated by FSF in clotting plasma. The concentration of cold-insoluble globulin was decreased in serum formed at 37 degrees from normal, but not from FSF-deficient, plasma. The depletion of cold-insoluble globulin in normal serum was partially blocked by clotting in the presence of dansylcadaverine and completely blocked by clotting in the absence of calcium ions. Sera formed at 2 degrees from both normal and FSF-deficient plasma contained less cold-insoluble globulin than plasma. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of clots formed at 2 degrees demonstrated cross-linking of cold-insoluble globulin to fibrin in the normal, but not the FSF-deficient, sample. The serum concentration of alpha2-macroglobulin was the same as the plasma concentration irrespective of the conditions of clotting. Thus, the experiments suggest that FSF catalyzes the cross-linking of cold-insoluble globulin (but not alpha2-macroglobulin) to fibrin in clotting plasma.

Binding Sites↗
PubMed1976

Synthesis and secretion of alpha2-macroglobulin by cultured human fibroblasts.

The following observations indicate that cultured human WI-38 fibroblasts synthesize and secrete alpha2-macroglobulin into serum-free medium: (a) after incubation of cultures with [35S]L-methionine, a labeled protein appeared in the medium which was precipitated by antiserum directed against alpha2-macroglobulin; (b) after incubation of cultures with [35S]L-methionine, a major band of radioactivity detected by polyacrylamide gel electrophoresis of the proteins in medium co-migrated with alpha2-macroglobulin; and (c) the amount of alpha2-macroblobulin in the medium, estimated both functionally and immunologically, increased with time in normal but not not puromycin-treated cultures.

Cells, Cultured↗
PubMed1976

Cross-linking of a major fibroblast surface-associated glycoprotein (fibronectin) catalyzed by blood coagulation factor XIII.

The surface proteins of cultured human skin fibroblasts were iodinated and then exposed to one or more of the following blood coagulation proteins: thrombin, fibrinogen, and factor XIII (plasma protransglutaminase). Radiolabeled polypeptides were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. After exposure to physiological concentrations of activated factor XIII (XIIIa), the band of radioactivity corresponding to the major labeled surface protein (fibronectin, molecular weight = 2.2 x 10(5) daltons) was cross-linked to a very high molecular weight complex. The cross-linking reaction was inhibited by fibrin (which is known to bind the catalytic subunit of XIIIa). Cross-linking of labeled cell surface fibronectin to fibrin could not be demonstrated. The fibrillar pattern of surface fibronectin appeared unaffected by cross-linking when studied by immunofluorescence. Cross-linking of cell surface fibronectin by XIIIa requires highly specific enzyme-substrate and protein-protein interactions, and may be an important physiological reaction.

Cell Line↗
PubMed1976

Functional and morphologic changes during experimental Rocky Mountain spotted fever in guinea pigs.

Experimental Rocky Mountain spotted fever was studied in guinea pigs following intraperitoneal inoculation of 10(7) Rickettsia rickettsii. After a 2-day incubation period, animals developed fever, progressive emaciation, and scrotal swelling with necrosis. Vasculitis, with increased small vessel permeability for colloidal carbon, was evident in cremaster muscles as early as 1 day after inoculation. Inflammatory changes in vessels became progressively more severe as numbers of circulating rickettsiae increased. Thrombosis and vascular occlusion were first evident on day 4. Mild thrombocytopenia developed, coinciding with the development of vasculitis, and preceding the appearance of either fibrin-split products in blood or thrombi in vessels. Rickettsiae were first detected in blood on day 2; peak rickettsemia occurred on days 5 to 8. Rickettsiae were demonstrated in inflamed vessels on day 5 and later, but not at earlier stages. Serum lysozyme concentration was moderately elevated and hemolytic complement was moderately depressed throughout the illness. Agglutinating antibody was present in low titers on days 3 to 10. Antibody titers increased on days 12 to 16 after the rickettsiae were cleared from blood. These studies indicate that vasculitis seen early in the course of Rocky Mountain spotted fever is the result of rickettsial infection, but is not dependent on the presence of rickettsiae in endothelial cells or other blood vessel components.

Agglutination Tests↗
PubMed1975

Cross-linking of cold-insoluble globulin by fibrin-stabilizing factor.

Cold-insoluble globulin (CI globulin) was purified from human plasma and identified on the basis of its sedimentation coefficient, electrophoretic mobility, and concentration in normal plasma. CI globulin was distinguished from antihemophilic factor (AHF) by amino acid analysis, position of elution from 4% agarose, and electrophoretic migration in polyacrylamide gels in the presence of sodium dodecyl sulfate without prior reduction. CI globulin and AHF could not be distinguished by polyacrylamide gel electrophoresis in sodium dodecyl sulfate after reduction and probably have very similar subunit molecular weights. CI globulin apparently consists of two polypeptide chains, each of molecular weight 2.0 x 10(5), held together by disulfide bonds. CI globulin was a substrate for activated fibrin-stabilizing factor (FSF, blood coagulation factor XIII). FSF catalyzed the incorporation of a fluorescent primary amine, N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulfonamide, into CI globulin and also catalyzed the cross-linking of CI globulin into multimers, as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate after reduction. In the presence of fibrin, cross-linking of CI globulin by FSF occurred without the formation of CI globulin multimers. Instead, polypeptides with apparent molecular weights of 2.6 x 10(5) and 3.0 x 10(5) were seen. The formation of these polypeptides coincided with the loss of the alpha chain of fibrin and CI globulin. The polypeptides were not seen when fibrin alone was cross-linked. The formation of the polypeptides was greater in fine clots than in coarse clots, and greater in clots incubated at 0 degrees than in clots incubated at 37 degrees. In clots made from purified fibrinogen, CI globulin, and FSF, the concentration of CI globulin in the clot liquor was greater if either FSF or calcium ion was omitted and cross-linking did not take place. These observations suggest that CI globulin is enzymically cross-linked to one of the chains of fibrin, most likely the alpha chain, and is thus covalently incorporated into the fibrin clot. CI globulin is very similar to a protein in the plasma membrane of fibroblasts. The cross-linking of CI globulin to itself and to fibrin may typify reactions also involving the fibroblast membrane protein.

Amino Acids↗
PubMedDate not supplied

Acute surface-induced thrombosis in the canine ex vivo model: importance of protein composition of the initial monolayer and platelet activation.

The initial events occurring at the blood-polymer interface were examined using a canine ex vivo arteriovenous shunt model. Thrombogenic (fibrinogen) and nonthrombogenic (albumin) proteins were preadsorbed on poly(vinyl chloride), polyethylene, and silicone rubber shunt surfaces, and the blood responses were analyzed using the platelet deposition profile as an indicator of surface thrombogenicity. The distributions of preadsorbed protein molecules on the various polymer surfaces were studied using an immunogold bead (colloidal gold particles coated with antibodies) staining technique and shown to be homogeneous. A sequential protein adsorption technique was developed to probe the nature of competitive protein adsorption and to observe the effect of surface protein concentration on thrombogenicity. The thrombogenicity of a surface was determined by the composition of the initial protein layer rather than the total concentration of protein on the polymer surfaces. The composition of this layer determines the extent of platelet activation and the adhesive strength between platelets and the polymer surface.

Adsorption↗
PubMedDate not supplied

Clinical use of fibronectin. (Potential applications).

There is considerable experimental evidence that the RES is an important host defense system in critical illness. The role of fibronectin in the control of RES phagocytic function and the potential for fibronectin therapy to ameliorate or reverse multisystem organ failure in critically ill patients are less well documented. This documentation may be difficult to accrue in the clinical setting because of the multifactorial problems of individual critically ill patients and the heterogeneity of underlying diseases in such patients. More work needs to be done in animal models in which these variables can be controlled. Large scale production of pasteurized biologically active fibronectin should be possible if the cryoprecipitate studies prove the clinical utility of fibronectin concentrates. Fibronectin will certainly not be a panacea for critically ill patients, but the addition of any useful substance to the now meager armamentarium would be most welcome.

Adolescent↗
PubMedDate not supplied

Stability of red cell antigens and plasma coagulation factors stored in new formulation plastic blood containers.

The stability of red blood cell antigens and plasma coagulation factors stored in new plastic blood packs containing either citrate-phosphate-dextrose (CPD) or citrate-phosphate-dextrose-adenine (CPDA-2) was studied. The containers were made of either polyolefin (PL 732TM) plastic without a plasticizer, or polyvinyl chloride with a non-diethylhexyl phthalate (non-DEHP) plasticizer (PL 1240). All studies were done in parallel using standard polyvinyl chloride (PL 146) bags containing DEPH plasticizer as controls. Red blood cell antigen scoring was performed for the A, B, c, D, K, Fya, Lea, Jka, M, and P antigens using cells obtained from both the blood container and tubing segments. Units of fresh frozen plasma and cryoprecipitate were prepared, and age and donor-matched sets of each of these blood components were stored in both test and control plastic containers. The red blood cell antigens showed no decrease in reactivity over 21 (CPD) or 35 (CPDA-2) days of storage in either test plastic compared with controls. For fresh frozen plasma (factors V, VIII, IX) and cryoprecipitate (factor VIII) the factor activity found for the test plastics ranged from 94 to 115 percent of that recorded for the age and donor-matched control plastic. The concentration of fibronectin found in cryoprecipitate stored in the test plastics averaged 90 to 99 percent of the activity found in PL 146 plastic. We conclude that the red blood cell antigens and coagulation factors tested remain stable in the plastic-anticoagulant combinations studied.

Anticoagulants↗
PubMedDate not supplied

Fibronectin in liquid and frozen stored blood components.

To quantitate the concentration of fibronectin in blood components to determine if the fibronectin present in liquid and frozen stored blood products deteriorates in vitro, we measured the concentration of this opsonic protein at the beginning and at the end of storage. In addition, electroblotting studies were performed to ascertain if aggregation or degradation of fibronectin occurred during component preparation or storage. Results showed that fibronectin concentration was stable in whole blood, single donor plasma, platelet concentrates, fresh-frozen plasma, and cryoprecipitate. By electroblotting, aggregated or degraded fibronectin was found in some components, especially cryoprecipitate. Most of the fibronectin, however, appeared to be unaffected by component preparation or storage.

Blood Preservation↗