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Biomedical subjects

E M Salonen

Publications and source records attributed to E M Salonen.

At least 55 records · Page 3Linked to original sources
PubMed1988

Rapid appearance of plasmin in tear fluid after ocular allergen exposure.

Specific allergens were applied topically to the conjunctivae of 18 allergic patients. In cases with positive allergic reaction, proteolytic activity, identified as plasmin using zymographic analysis and a monoclonal anticatalytic antibody to plasmin, was found to appear within 3-5 min in the tear fluid, reaching concentrations of 2.2-28.6 micrograms/ml. No plasmin was detected, either in the tear fluid of 30 unchallenged non-atopic control persons, or atopic patients provoked with a test material that had given no reaction in a skin prick test. These findings suggest that generation of plasmin is a consequence of the conjunctival allergic reaction.

Administration, Topical↗
PubMed1987

Microplate immunocapture assay for plasminogen activators and their specific inhibitors.

We report a convenient sensitive enzyme activity assay for urokinase and tissue-type plasminogen activators, based on a solid-phase microtitre plate method using readily available polyclonal antibodies. The sensitivities for urokinase (active and proenzyme) and tissue activator were better than 1 ng/ml. The specificity was very high, with no significant contribution of urokinase in tissue activator assays or vice versa. This method is particularly useful for the assay of urokinase proenzyme in samples containing inhibitors. We describe how this assay may also be used to measure specific inhibitors of plasminogen activators, making use of their rapid formation of stable complexes with solid-phase activator. Inhibitors may be assayed in samples containing proenzymes.

Binding, Competitive↗
PubMed1987

Fibronectin-binding 36 kDa protein in human fibroblasts.

A 36 kDa fibronectin-binding protein was identified from electrophoretically separated proteins of the deoxycholate-soluble fraction of cultured fibroblasts by blotting with fibronectin and using poly- or monoclonal antibodies and immunoperoxidase staining to detect the bound fibronectin. The 36 kDa protein was purified by preparative electrophoresis and used to raise specific antibodies. Solid-phase 36 kDa protein bound plasma and fibroblast fibronectins equally well. The 36 kDa protein is an amphipathic protein with pI 5.9. It is monomeric with a tendency to dimerize and appears to be distinct from the cell surface fibronectin receptors which interact with the Arg-Gly-Asp recognition site in the fibronectin molecule.

Cells, Cultured↗
PubMed1987

Distinct localizations of urokinase-type plasminogen activator and its type 1 inhibitor under cultured human fibroblasts and sarcoma cells.

We studied the immunocytochemical localization of urokinase-type plasminogen activator (u-PA) and the type 1 plasminogen activator inhibitor (PAI-1) in human fibroblasts and sarcoma cells, using both polyclonal and monoclonal antibodies. The u-PA was found to be located at discrete cell-substratum contact sites, and also at areas of cell-cell contacts, whereas PAI-1 was distributed as a homogeneous carpet excluding strialike areas on the substrate under the cells. To confirm the extracellular localization of u-PA and PAI-1, we stained the cells live at 0 degree C before fixation. A double-labeling experiment showed different distribution of u-PA and PAI-1 under the cells, and especially their peripheral parts. The staining pattern of u-PA and PAI-1 resisted treatment with 0.2% saponin followed by mechanical removal of cells, a method previously reported to isolate focal contact membranes of fibroblasts. We further demonstrated the deposition of u-PA to the contact areas of cells obtained by saponin treatment by zymography, and that of PAI-1 by metabolic labeling, reverse zymography, immunoblotting, and immunoprecipitation. Fibronectin was also present in the preparations. The deposition of both PAI-1 and fibronectin by the sarcoma cells was enhanced, after treating the cells with 10(-6) M dexamethasone. The confinement of u-PA to discrete contact sites and the more uniform distribution of PAI-1 on the cell substratum may explain how cells producing large amounts of enzyme inhibitors can produce PA-mediated focal proteolysis.

Cell Line↗
PubMed1987

Plasmin in tear fluid of patients with corneal ulcers: basis for new therapy.

In a patient with chronic corneal ulcer, resistant to conventional therapy, analysis of tear fluid revealed a high plasmin activity which could be inhibited by aprotinin, an inhibitor of serine proteinases. Therapy with topical aprotinin resulted in rapid epithelialization. After this initial patient, within a period of four months tear fluid specimens of altogether 48 patients with corneal lesions were analyzed, and 32 were found to be positive for proteolytic activity. Of these 18 were treated with topical aprotinin which rapidly promoted corneal epithelial healing. Six of these patients had been treated with conventional therapy for 3-10 weeks but proved to be completely therapy-resistant. Our observations on three successfully treated patients with chemical burns of the cornea indicated appearance of plasmin in tear fluid after a few days correlating with cessation of epithelialization. In all patients, in which tear fluid plasmin activity was followed, the activity disappeared during aprotinin therapy correlating with corneal re-epithelialization. In some patients with low proteolytic activity aprotinin was combined with fibronectin with a beneficial therapeutic effect. No proteolytic activity was found in the tear fluid of control individuals. These preliminary data indicate that in patients with treatment-resistant corneal lesions inhibition of proteolytic activity can assist in epithelial healing. Such an inhibition is likely to be a prerequisite for the proteinase-sensitive cell adhesion proteins such as fibronectin to promote epithelialization.

Administration, Topical↗
PubMed1986

Single-serum radial hemolysis to detect recent rubella virus infection.

In the radial hemolysis test for rubella virus-specific antibodies, sera obtained shortly after primary rubella produce a characteristically altered hemolysis zone, termed soft hemolysis. The occurrence of soft hemolysis was analyzed statistically in 6,723 sera. The specificity of soft hemolysis for infection with rubella virus was 99.0%. Its sensitivity in primary infections was 97.3%, and the predictive value for rubella serodiagnosis (increase in IgG level or detectable IgM) was 91.2%. The temporal association of soft hemolysis with rubella was determined with sera obtained from 129 persons during late convalescence (70-310 days after onset of symptoms). Soft hemolysis disappeared by 190 days after the onset of symptoms and the presence of ordinary hemolysis ruled out primary rubella in the preceding 70-100 days. Soft hemolysis was caused by IgG antibody to rubella virus, and thus represents a new serodiagnostic principle. This technique, together with an Igm assay, allows rapid diagnosis of recent rubella from a single serum sample.

Antibodies, Viral↗
PubMed1985

Plasminogen and tissue-type plasminogen activator bind to immobilized fibronectin.

Fibronectin immobilized onto polystyrene surface was found to bind plasminogen and tissue-type plasminogen activator (t-PA) but only slightly the urokinase type as determined using mono- and polyclonal antibodies against the activators. Of the defined fibronectin fragments tested, the Mr 120,000-140,000 fragment was found to bind both plasminogen and t-PA. Proteolytically modified plasminogen (Lys-plasminogen) bound considerably better than the native form (Glu-plasminogen). Experiments with 125I-plasminogen yielded Kd = 9.1 X 10(-8) M for the binding to immobilized fibronectin. The partially or completely inactive single-chain form of t-PA (pro-t-PA) bound considerably better than the activated two-chain form. Lysine at greater than 3 mM inhibited the binding of plasminogen. The interaction was independent of calcium ions. CaCl2 (greater than 0.5 mM) and NaCl (greater than 0.2 M) inhibited the binding of pro-t-PA and of t-PA. Fibronectin-bound t-PA retained its ability to activate plasminogen. The observed interactions may operate in directional proteolysis localizing plasminogen and plasminogen activator to degrade fibronectin-containing extracellular matrix including fibrin clots.

Epitopes↗
PubMed1985

Fibronectin in formation and degradation of the pericellular matrix.

Fibronectin is a large multifunctional glycoprotein present both in soluble form in plasma and other body fluids and in an insoluble form in interstitial connective tissues and in association with most basement membranes. The protein seems to provide a link between circulating body fluids, cell surfaces and the extracellular matrix. At sites of injury, fibronectin, covalently cross-linked to the fibrin clot, may provide a growth substratum for the invading cells and appears through its multiple interactions with other matrix components and cell surfaces to be involved in the organization of the matrix that will replace the clot. Fibronectin deposition may serve as a useful early marker for connective tissue formation in pathological processes and its fragmentation may serve as an indicator of pathological conditions involving proteolytic events.

Animals↗
PubMed1985

Kinetics of specific IgA, IgD, IgE, IgG, and IgM antibody responses in rubella.

Rubella-specific IgD and IgE antibodies were determined with a solid-phase enzyme immunoassay using enzyme-labeled heavy-chain specific anti-immuno-globulins, and the antibody responses in rubella infection were compared to IgM, IgA, and IgG antibodies. IgD and IgE antibodies increased rapidly after the onset of infection, remained at a high level for at least 2 months, and declined slightly by 6 months. In comparison, the IgM antibodies decreased more rapidly, whereas the IgG antibodies persisted longer at a steady level. By 6 months the mean levels of the different antibodies had declined from their maximal mean levels as follows: IgM, 52%; IgA, 42%; IgE, 35%; IgD, 29%; and IgG, 8%. Thus IgD and IgE antibodies, in spite of their known short half lives, persisted longer than IgM and IgA antibodies, which limits their diagnostic value. The IgA antibody responses were found too variable to substitute for IgM antibody determination in diagnosis of a recent rubella virus infection from a single serum specimen. Comparison of maternal and cord blood sera indicated that, in addition to IgG antibodies, rubella-specific IgD antibodies were found to cross the placenta.

Adult↗
PubMed1984

A rapid and highly sensitive solid-phase enzyme immunoassay specific for human fibronectin using a characterized monoclonal antibody.

A solid-phase enzyme immunoassay (EIA) was developed for the quantitation of human fibronectin in body fluids and cell culture media. In the assay a human fibronectin-specific murine monoclonal IgG1 (f-33) was used as capture antibody and polyclonal rabbit anti-fibronectin as detector antibody. The antibody showed no reactivity to purified monkey, dog, rabbit, horse, sheep, mouse, bovine or chicken fibronectins. The determinant of the monoclonal antibody was mapped to the cell-binding region of the fibronectin molecule. This localization was based on the use of purified fragments of fibronectin, immunoblotting, EIA and inhibition of fibroblast adhesion and spreading by the antibody. The detection limit of the fibronectin assay was 2 ng/ml. The assay was used for the quantitation of fibronectin in human plasma, urine and cerebrospinal fluid specimens and culture media of human cells.

Animals↗
PubMed1984

Tissue type plasminogen activator, but not urokinase, exerts transformation-enhancing activity.

Previous studies have established that plasma cryoprecipitates of tumor patients, culture media of transformed cells and defined proteolytic fragments of fibronectin enhance the morphological cell transformation ( TEF activity) in cultures of chicken embryo fibroblasts infected with temperature-sensitive mutants of Rous sarcoma virus. We now report that purified human tissue type plasminogen activator (t-PA), but not urokinase (u-PA), has a similar TEF activity, at doses as low as 2 ng/ml (30 pM). Specific antibodies effectively neutralized the activity. No significant contamination (less than or equal to 1%) between the preparations of t-PA and fibronectin (FN) or its fragments ( FNdp ) was detected. The results suggest that t-PA may have a direct role in the process of morphological cell transformation in vitro.

Cell Transformation, Neoplastic↗
PubMed1984

Binding of fibronectin by the acute phase reactant C-reactive protein.

Following tissue injury, the concentration of C-reactive protein (CRP) is known to increase in plasma rapidly, while that of fibronectin often decreases. We now report that CRP immobilized onto polystyrene surfaces binds soluble plasma fibronectin (Kd = 1.5 X 10(-8) M). The binding of fibronectin by CRP was relatively sensitive to ionic conditions, being maximal at physiological NaCl concentrations. A decrease of pH from neutral to 5-6 greatly enhanced the binding of fibronectin by CRP. Ca2+ ions at greater than 1 mM inhibited binding. No binding was observed between fibronectin and CRP in soluble phase. CRP was found also to bind fibrinogen, which competed with fibronectin for CRP-binding sites. This was shown to explain why fibronectin was effectively bound from serum but not from plasma by immobilized CRP. The amount of CRP immobilized was critical in binding fibronectin; a too dense molecular layer of CRP inhibited the binding, as did the postsaturation of free surfaces with albumin, which itself was not bound by CRP. Soluble fibronectin agglutinated CRP-coated latex particles. Most or all of the CRP-binding activity in the fibronectin molecule was localized to the 120-140-kilodalton fragment, which also contains cell-binding and heparin-binding domains of fibronectin. The results provide a link between acute phase response and tissue repair.

C-Reactive Protein↗
PubMed1984

Integrity of the pericellular fibronectin matrix of fibroblasts is independent of sulfated glycosaminoglycans.

The pericellular matrix fibers of cultured human fibroblasts contain fibronectin, other glycoproteins, and heparan and chondroitin sulfate proteoglycans. In the present study, cell-free pericellular matrices were isolated from metabolically labeled fibroblast cultures. The isolated matrices were digested with heparinase from Flavobacterium heparinum, and then analyzed for sulfated glycosaminoglycans (GAGs). Nitrous acid degradation was used to distinguish the N-sulfated GAGs (heparan sulfate) from chondroitin sulfate. Fibronectin and the other major matrix polypeptides were studied using gel electrophoresis, enzyme immunoassay and immunofluorescence. Upon heparinase digestion, greater than 95% of sulfated GAGs were degraded in the matrix without detectable release of fibronectin or other matrix polypeptides or alteration of the fibrillar matrix structure. We conclude that in fibroblast cultures the integrity of the fibrillar matrix is independent of sulfated GAGs. Together with earlier observations, this suggests that filamentous polymerization of fibronectin forms the backbone of early connective tissue matrix.

Cells, Cultured↗
PubMed1984

Retrovirus RD114 p30 is bound by the extracellular matrix proteins, fibronectin and laminin.

Purified retrovirus RD114 p30 was found to bind to the extracellular matrix proteins, fibronectin and laminin. The purified matrix proteins were immobilized onto polystyrene and the binding of p30 was quantitated using radioiodinated proteins and enzyme immunoassay (EIA). The dissociation constants were for the p30-fibronectin binding Kd = 5.3 X 10(-8) M and for the p30-laminin Kd = 7.3 X 10(-8) M. The molecular ratio in the binding from 4000 ng/ml of p30 was 1.9 mol per mol fibronectin and 2.5 mol per mol laminin. The interaction between fibronectin and RD114 retrovirus was also detected using a modified immunoblotting procedure.

Fibronectins↗
PubMed1983

Monoclonal antibody against the N-terminal end of human plasma fibronectin.

Purified human plasma fibronectin was digested with cathepsin G and the degradation products were tested for reactivity towards a monoclonal antibody. In an immunoblotting assay, after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the digestion products, the 85 000-Mr and 72 000-Mr gelatin- and heparin-binding fragments as well as the N-terminal 30 000-Mr heparin-binding fragment reacted with the antibody, whereas the 64 000-Mr gelatin- and heparin-binding fragment did not. In enzyme immunoassay the antibody reacted with intact fibronectin and the 30 000-Mr fragment but not with a 40 000-Mr gelatin-binding fragment. The alignment of the binding domains in these fragments and in the intact molecule [Vartio (1982) Eur. J. Biochem. 123, 223-233] localizes the antigenic determinant to the 21 000 Da N-terminal Staphylococcus aureus-binding region of fibronectin.

Antibodies, Monoclonal↗
PubMed1982

A rapid and sensitive solid-phase enzyme immunoassay for C-reactive protein.

C-reactive protein (CRP) is an acute-phase reactant the concentration of which increases significantly following tissue injury or inflammation. I report here the development of a rapid and sensitive solid-phase enzyme immunoassay (EIA) for determination of serum CRP. The inclusion of 4% (w/v) polyethylene glycol (PEG) in the diluent buffers made it possible to quantitate CRP within 4 h at room temperature. The minimum detectable amount was about 500 pg CRP/ml. Excellent correlation in determination of serum CRP was found between solid-phase EIA and nephelometric assay, radial immunodiffusion and the qualitative latex agglutination tests.

Animals↗