Fibronectin-enhanced attachment of gelatin-coated erythrocytes to isolated hepatic Kupffer cells.

The phagocytic process is a combination of a sequence of events which includes a recognition attachment phase and a subsequent internalization phase. The present study was designed to investigate the effect of plasma fibronectin on the attachment and ingestion of gelatinized sheep erythrocytes to isolated rat Kupffer cells in a monolayer assay. Kupffer cells were isolated by sequential collagenase-pronase digestion followed by metrizamide density gradient centrifugation and subsequent adherence to plastic. Classification as Kupffer cells was confirmed by the presence of functional Fc receptors, a positive peroxidase reaction, and phagocytic activity. Purified plasma fibronectin as well as rat serum containing fibronectin promoted attachment of gelatinized fixed sheep erythrocytes to Kupffer cells in a dose-dependent manner, whereas fibronectin-deficient serum did not. Heparin did not enhance the fibronectin-mediated attachment or ingestion of gelatinized sheep erythrocytes at lower particle doses, whereas at higher particle doses heparin augmented the response. These results indicate that fibronectin can enhance the binding and ingestion of foreign gelatin-coated particulates by Kupffer cells.

Synthesis and antibacterial activity of new C-10 quinolonyl-cephem esters.

A series of cephalosporins derived from cephalothin containing an ester-linked quinolonyl substituent at the C-10 position (C-10 quinolonyl-cephem esters) has been prepared and evaluated for in vitro antibacterial activity. The C-10 quinolonyl-cephem esters exhibited a broadened spectrum of activity when compared with cephalothin and the corresponding quinolones, including activity against beta-lactamase-producing bacteria.

Preparation and antimicrobial assessment of 2-thioether-linked quinolonyl-carbapenems.

This reports the synthesis and in vitro antimicrobial properties of a series of 2-thioether-linked quinolonyl-carbapenems. Although the title compounds exhibited broad spectrum activity, the MICs were generally higher than those observed for selected benchmark carbapenems, quinolonyl-penems, and quinolones. Enzyme assays suggested that the title compounds are potent inhibitors of penicillin binding proteins and inefficient inhibitors of bacterial DNA-gyrase. Uptake studies indicated that the new compounds are not substrates for the norA encoded quinolone efflux pump.

Anticomplementary activity of tuberculin: relationship to platelet aggregation and lytic response.

Experiments were performed to examine the interaction of tuberculin with platelets and complement. Hemolytic complement titrations show that tuberculin consumes complement in human, rabbit, and guinea pig serum. Evidence in support of classical pathway activation was provided by observation of C1 consumption and failure to detect significant conversion of alternative pathway factor B to B by immunoelectrophoresis. Platelets in plasma from guinea pigs deficient in the fourth component of complement were not affected by tuberculin. However, studies on platelet aggregation in plasma chelated with ethyleneglycolbis(beta-aminoethyl ether)-N,N-tetraacetic acid indicated that tuberculin may initiate sluggish activation of the alternative pathway. That the reaction between tuberculin and platelets is a lytic one was evidenced by observing the release of the cytoplasmic enzyme lactic dehydrogenase and efflux of rubidium-86. Studies with C6-deficient rabbits indicated that platelet release of exogenously supplied tritiated serotonin is caused by platelet lysis.

Effect of fibronectin fragments on macrophage phagocytosis of gelatinized particles.

Rat plasma fibronectin enhances the binding and ingestion of gelatin-coated, formalin-fixed, or tanned sheep erythrocytes by elicited rat peritoneal macrophages. Fibronectin binding to the gelatinized erythrocytes is required for this enhancement, because macrophages preferentially recognize the surface bound molecule. This enhancement of particle uptake by fibronectin required the presence of a renewable, trypsin-sensitive component(s) on the macrophage surface (fibronectin receptor). When subjected to plasminolysis for 3 hr, fibronectin was degraded into gelatin-binding fragments (170 to 210 kd) and a 25-kd nongelatin binding fragment. The 170 to 210 kd gelatin binding fragments retained uptake-enhancing activity but were less active on a weight and molar basis than intact, dimeric fibronectin. The nongelatin binding 25 kd fragment alone did not enhance uptake. These results indicate that the sites for interaction with both the gelatinized erythrocyte surface and macrophages are retained on 170 to 210 kd fragments. However, the fibronectin dimeric structure is required for maximal expression of opsonic activity.

Fibronectin augments binding of fibrin to macrophages.

Because of the demonstrated ability of fibronectin to mediate particle uptake by macrophages and the demonstrated affinity of plasma fibronectin for fibrin, we investigated the ability of plasma fibronectin to augment macrophage binding of fibrin. Fibronectin significantly increased fibrin binding by elicited peritoneal macrophages and isolated hepatic Kupffer cells. The binding of fibrinogen was not augmented in the presence of fibronectin. The small amount of macrophage-associated fibrin observed in the absence of fibronectin was primarily internalized, whereas the increment in fibrin binding in the presence of fibronectin remained primarily surface bound, as indicated by susceptibility to removal by trypsin. An amino terminal fibrin-binding fragment of plasma fibronectin could similarly support binding of fibrin by peritoneal macrophages. Greater quantities of fibrin were associated with the macrophages in the presence of protease inhibitors, which inhibited elastase activity, but not in the presence of those that inhibited cathepsin activity, suggesting that an elastase-like protease may degrade surface-bound fibrin. Uptake of both fibrin and fibronectin was inhibited by prior treatment of cells with trypsin. Competitive binding studies suggested the presence of a high-affinity fibronectin receptor on peritoneal macrophages. Data from the current study thus support the conclusion that fibronectin augments binding of fibrin to the surface of mononuclear phagocytes.