Detection of fibronectin expression by human endothelial cells using a enzyme-linked immunosorbent assay (ELISA): enzymatic degradation by activated plasminogen.

An enzyme-linked immunosorbent assay (ELISA) has been developed to measure cellular fibronectin (cFN) in association with human umbilical vein endothelial cells (HUVEC) in culture. The expression of a number of functional domains on the cFN molecule was demonstrated using three specific murine monoclonal antibodies. This system was found to be sensitive, detecting as little as 0.156 microg/ml of cFN, and required only 1.3 x 10(5) cells per well confluent cells per experimental condition. This allowed multiple experiments to be performed on one batch of endothelial cells. cFN was detected on both viable and methanol fixed endothelial cells without significant non-specific antibody binding. The utility of this experimental model was studied by exploring the effect of urokinase activated plasminogen, a potent protease, on the expression of cFN and its functional domains.

Detection of glycosaminoglycans on the surface of human umbilical vein endothelial cells using gold-conjugated poly-L-lysine with silver enhancement.

Endothelial glycosaminoglycans are important in a diverse range of vascular functions. In the course of a biochemical and histological study exploring the role of glycosaminoglycans in inflammation, we have investigated the use of gold-conjugated poly-L-lysine with silver enhancement to establish the nature and physical location of glycosaminoglycans on the surface of cultured human umbilical vein endothelial cells. Cationic gold was effective in locating anionic sites in both cultured endothelial cells and in paraffin-embedded renal tissue. By manipulating pH, and by using enzymes specific for degrading glycosaminoglycans, it was found that, at pH 1.2, staining was directed primarily at glycosaminoglycans. The surface of human umbilical vein endothelial cells was found to be extensively covered in heparan sulphate, the histological appearance of which was dependent upon the fixation procedure employed. Heparan sulphate was also seen to co-distribute with the extracellular matrix protein, fibronectin, when endothelial cultures were simultaneously stained with cationic gold and an antibody to cellular fibronectin.

Reduction of the anticoagulant activity of glycosaminoglycans on the surface of the vascular endothelium by endotoxin and neutrophils: evaluation by an amidolytic assay.

The processes that underlie the coagulopathy observed in severe infection are not fully understood, but seem to be due to an imbalance in the antithrombotic, and prothrombotic properties of the vascular endothelium. Sulphated glycosaminoglycans (GAGs) present on the vessel wall represent an important component of the non-thrombogenic nature of the endothelium. We have modified an amidolytic assay to study the functional ability of GAGs on human umbilical vein endothelial cells (HUVECS), and investigate the effect of E. coli endotoxin and neutrophils on HUVEC surface anticoagulant activity (SAA). Neither endotoxin alone, nor separated neutrophils at lower concentrations (less than 10(6) neutrophils per ml), had major effects on endothelial SAA. When activated neutrophils were incubated with HUVECS pre-stimulated with endotoxin, a significant decrease in SAA was seen using either plasma (mean percentage of control 67.8% +/- sem 7.8; p < 0.02) or purified ATIII (mean percentage of control 69% +/- sem 4.6; p < 0.001). We suggest that alterations in endothelial surface GAGs may occur during sepsis and inflammation, and that this may have important consequences for vascular function. This system will allow the further study of the role of GAGs in the intravascular thrombosis of severe sepsis, and other inflammatory diseases.

Alteration in glycosaminoglycan metabolism and surface charge on human umbilical vein endothelial cells induced by cytokines, endotoxin and neutrophils.

There is increasing evidence that the glycosaminoglycan (GAG) component of the vascular endothelium is important in regulating vascular permeability, thromboresistance and cellular interactions. We have investigated the GAG metabolism of cultured human umbilical vein endothelial cells (HUVEC) in response to a range of inflammatory stimuli. Using both chemical measurement of cellular and supernatant GAGS and 35S labelling to identify newly synthesised GAGS, interleukin 1 (IL1), tumour necrosis factor (TNF) and interferon gamma (IFN gamma) were shown to influence sulphated GAG metabolism significantly. IL1 and TNF caused a marked increase in culture supernatant GAGS and a concomitant reduction in cell-associated GAGS. This was shown histochemically to be associated with a marked reduction and redistribution of endothelial surface anionic sites. The addition of neutrophils to HUVEC pretreated with Escherichia coli endotoxin, IL1 or TNF resulted in a further reduction in both cellular GAGS and surface anionic sites. These results suggest that changes in endothelial cell GAG metabolism during inflammation may contribute to the disturbance of vascular endothelial homeostasis associated with infectious and inflammatory states.

Deficiency of prostacyclin production in meningococcal shock.

A deficiency of prostacyclin (PGI2) production by the vascular endothelium might underline the severe vasoconstriction and intravascular thrombosis that characterise meningococcal shock. The effect on PGI2 synthesis by human umbilical vein endothelial cells (HUVEC) in culture was examined in sera from children with meningococcal shock, healthy adults, and children with other febrile illnesses. In comparison with adult controls, PGI2 synthesis was reduced when HUVEC were incubated with the sera from 10 of 13 patients with meningococcal shock. A similar defect was observed with only four of 20 sera from children with other febrile illnesses. The effect of sera from patients with meningococcal shock on HUVEC was reversible with normal serum, and seems to be due to the absence of a factor necessary for PGI2 production rather than an inhibitor. These findings suggest that a deficiency of PGI2 may have a role in the pathogenesis of meningococcal shock and that exogenous PGI2 may be of therapeutic benefit.

Circulating antigens and antibodies in human and mouse dermatophytosis: use of monoclonal antibody reactive to phosphorylcholine-like epitopes.

The presence of circulating antibodies in the sera of patients infected with either Trichophyton concentricum or Trichophyton rubrum, and in the sera of BALB/c mice chronically infected with Trichophyton quinckeanum, was determined by ELISA. High levels of antibody to dermatophyte cytoplasmic antigens were detected both in infected humans and in mice. Partial inhibition of this reaction was observed by pretreatment of the sera with the hapten phosphorylcholine (PC). Moreover, antibodies were shown to have some reactivity with PC when tested by ELISA against PC conjugated to bovine serum albumin. Significant levels of circulating antigen were detected in patients with T. concentricum and T. rubrum infections, but not in uninfected subjects, by an immunoradiometric assay using a monoclonal antibody, Tq-1, which reacts with the PC-like epitopes of dermatophytes. It is possible that this dermatophyte antigen may play a role in modulating the cell-mediated immune responses, which would appear to be defective in most patients with these chronic forms of dermatophytosis.

Production and immunological characterization of a monoclonal antibody to Trichophyton quinckeanum: interaction with phosphorylcholine-bearing components.

A monoclonal antibody (Tq-1) that interacts with the phosphorylcholine (PC)-bearing antigens of Trichophyton quinckeanum was produced by fusion of myeloma cells (JKAg-8) with spleen cells of BALB/c mice immunized with an alum-precipitated fraction of T. quinckeanum cytoplasmic antigen. It was characterized as an IgM class antibody by immunodiffusion using anti-Ig heavy chain specific reagents, ELISA using immunoglobulin-specific peroxidase-conjugated antibodies, and by gel filtration chromatography; it showed high affinity for Staphylococcus aureus protein-A. Interaction of Tq-1 with PC-like antigens of T. quinckeanum was demonstrated by inhibition studies using ELISA, immunoblotting, immunoprecipitation and immuno electron microscopy techniques. The binding activity of Tq-1 antibody with a range of dermatophyte proteins was completely inhibited by prior incubation with PC hapten. Moreover, dermatophyte antigens reacting with the monoclonal antibody reacted strongly with sera from chronically infected mice. Dermatophyte antigens derived from both young (24 h) and old (20 d) cultures reacted with Tq-1 and this binding was inhibited by PC, suggesting that Tq-1 target antigen PC appears at an early stage during fungal growth and remains throughout its life.

Susceptibility of Trichophyton quinckeanum and Trichophyton rubrum to products of oxidative metabolism.

Two dermatophyte strains, Trichophyton quinckeanum and Trichophyton rubrum, were highly susceptible to in vitro killing by components of the H2O2-peroxidase-halide system. Both strains were, however, resistant to relatively high concentrations of reagent H2O2 or H2O2 enzymatically generated by glucose and glucose oxidase, KI, or lactoperoxidase (LPO) alone. Resistance to hydrogen peroxidase killing was found to be in part due to the presence of endogenous catalase in the fungi; susceptibility was increased by pretreatment of the fungi with a catalase inhibitor. Kinetic studies using small quantities of reagent or enzymatically generated H2O2 and LPO-KI showed that the system was lethal for both fungal strains within 1 min. Furthermore, using the glucose-glucose oxidase-LPO-KI system, it was shown that catalase, superoxide dismutase and histidine scavengers of H2O2, superoxide anion and singlet oxygen, respectively, prevented the killing of fungus, whereas scavengers of hydroxyl radicals such as benzoate and mannitol had no effect. T. quinckeanum was found to contain large quantities of superoxide anion, as judged by the nitroblue-tetrazolium test. Consequently, the xanthine (or hypoxanthine) and xanthine oxidase system in which the main product is superoxide anion had no toxic effect on the fungus. The high sensitivity of dermatophytes to killing by the H2O2-peroxidase-halide system active in polymorphonuclear neutrophils and macrophages may account in part for fungal toxicity in vivo.