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.

Activation of pro-urokinase and plasminogen on human sarcoma cells: a proteolytic system with surface-bound reactants.

Human HT-1080 fibrosarcoma cells produce urokinase-type plasminogen activator (u-PA) and type 1 plasminogen activator inhibitor (PAI-1). We found that after incubation of monolayer cultures with purified native human plasminogen in serum-containing medium, bound plasmin activity could be eluted from the cells with tranexamic acid, an analogue of lysine. The bound plasmin was the result of plasminogen activation on the cell surface; plasmin activity was not taken up onto cells after deliberate addition of plasmin to the serum-containing medium. The cell surface plasmin formation was inhibited by an anticatalytic monoclonal antibody to u-PA, indicating that this enzyme was responsible for the activation. Preincubation of the cells with diisopropyl fluorophosphate-inhibited u-PA led to a decrease in surface-bound plasmin, indicating that a large part, if not all, of the cell surface plasminogen activation was catalyzed by surface-bound u-PA. In the absence of plasminogen, most of the cell surface u-PA was present in its single-chain proenzyme form, while addition of plasminogen led to formation of cell-bound two-chain u-PA. The latter reaction was catalyzed by cell-bound plasmin. Cell-bound u-PA was accessible to inhibition by endogenous PAI-1 and by added PAI-2, while the cell-bound plasmin was inaccessible to serum inhibitors, but accessible to added aprotinin and an anticatalytic monoclonal antibody. A model for cell surface plasminogen activation is proposed in which plasminogen binding to cells from serum medium is followed by plasminogen activation by trace amounts of bound active u-PA, to form bound plasmin, which in turn serves to produce more active u-PA from bound pro-u-PA. This exponential process is subject to regulation by endogenous PAI-1 and limited to the pericellular space.

Porcine serum cobalophilin and transcobalamin. Identification, isolation and properties including electrofocusing patterns.

Pooled porcine serum was found to contain cobalophilin (also called transcobalamin I) and transcobalamin (also called transcobalamin II). The two proteins were harvested by batchwise absorption with vitamin B-12 covalently coupled to Sepharose, and then separated from each other either by gel filtration or using an immunoadsorbent. Both proteins were finally isolated as single proteins using a second vitamin B-12-Sepharose chromatography step. Cobalophilin and transcobalamin complexed with vitamin B-12 had molecular weights by gel filtration of 135 000 and 38 000 and by the formula of Svedberg 104 000 and 44 000, Stokes radii 4.97 nm and 2.65 nm, and sedimentation coefficients 5.39 S and 3.75 S, respectively. Electrofocusing resolved the cobalophilin complex into three main isoproteins isoelectric at pH 3.23, 3.42 and 3.69, and transcobalamin into only the main component isoelectric at a value as low as pH 3.47. Neither protein was capable of binding to the ileal intrinsic factor receptor.

Identification and characterization of intrinsic factor and cobalophilin from pig ileal and pyloric mucosa.

Pig ileal mucosa was found to bind about 240 ng vitamin B12/g and to contain two vitamin B12-binding proteins. One was highly active in the Schilling test, behaved immunologically as intrinsic factor and was responsible for about half of the total vitamin B12-binding capacity. The other binder was identified as cobalophilin (R-protein). Immunochemical purification of these proteins from pig ileum and pylorus was performed and the molecular characteristics (sedimentation and diffusion coefficients, Stokes radii, frictional ratios and molecular weights) of their vitamin B12 complexes were estimated. Isoelectric focusing revealed differences between the ileal and pyloric intrinsic factors but not between the cobalophilins. The mean isoelectric points of the pyloric and ileal intrinsic factors were pH 5979 and 5.30, respectively, whereas the corresponding figures for the cobalophilins were 4.13 and 4.10.

Isolation of vitamin B-12-binding proteins by combined immuno and affinity chromatography. Comparative studies on the isolated and unisolated proteins.

Intrinsic factor or cobalophilin were removed by incubating human gastric juice and pig pyloric extract with purified anti-intrinsic factor and anti-cobalophilin immunoglobulin-G, respectively, covalently coupled to Sepharose. Cobalophilin (transcobalamin I) was also removed from pig serum either by using anti-cobalophilin immunoglobulin-G Sepharose or by Sephadex G-200 chromatography. The one remaining semipurified vitamin B-12-binding protein (intrinsic factor, cobalophilin or transcobalamin II) was then isolated by vitamin B-12-Sepharose affinity chromatography. Intrinsic factors, cobalophilins and transcobalamin II isolated by this two-step procedure were compared by double isotope techniques with the corresponding protein not subjected to affinity chromatography and found to be identical in reaction to antiserum, gel filtration and electrofocusing. The avidity of the isolated and unisolated intrinsic factors for the ileal intrinsic factor receptor was also the same.

Antibody status to HHV-6 in children with leukaemia.

Forty children with acute lymphoblastic (33) or myeloid leukaemia (seven) were studied for IgG and IgM antibodies and IgG avidity against human herpesvirus 6 (HHV-6) at the time of diagnosis, and compared with age-, sex- and season-matched children with various neurological diseases of suspected viral origin. Of the children with leukaemia, 97.5% had IgG antibodies and 40% IgM antibodies to HHV-6 compared with 92.3% and 7.7% of reference subjects (P = 0.005). A seronegative child with leukaemia seroconverted 3 weeks after the diagnosis. The avidity of IgG antibodies (based on the resistance to urea treatment) was high in all children with leukaemia. One reference child had HHV-6-specific IgG antibodies with low avidity, which together with his positive IgM indicated an acute infection. The presence of specific IgM antibodies in 40% of children with leukaemia and the high avidity of IgG suggest a reactivation or an inaproppriate primary response to HHV-6 infection. The results support the conclusion of the role of the HHV-6 infection at the onset of childhood leukaemia.

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.

Human glioma U-251 cells contain type 1 plasminogen activator inhibitor in a rapidly releasable form.

Because recent information suggests that the localized deposition of protease inhibitors is one mechanism by which cells regulate pericellular proteolysis during tissue invasion, the distribution of type 1 plasminogen activator inhibitor (PA1-1) associated with the invasive human glioma cell line U-251 was investigated. Direct and reverse fibrin zymography indicated the presence of urokinase-like plasminogen activator (u-PA) and PAI-1 in U-251 conditioned media and cell lysates. PA1-1 antigen was detected immunologically in cytoplasmic granules present within cellular processes of U-251 cells and these organelles could be isolated on Percoll density gradients in a high density band. In contrast, u-PA activity and another secreted protein, amyloid beta-protein precursor, were only present in the low density region of the gradients. Functional analysis of PAI-1 in the granules contained within the high density fractions revealed the presence of active PAI-1. Incubation of U-251 cells with the secretagogue, 8-bromoadenosine 3':5'-cyclic monophosphate, resulted in a 3-fold increase in the release of PAI-1 in the media conditioned by these cells. These data suggest that the human glioma cell line U-251 contains PAI-1 in a rapidly releasable form, which may provide another mechanism by which these tumors could regulate proteolytic activity in a localized manner.

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.

Immobilization of viral and mycoplasma antigens and of immunoglobulins on polystyrene surface for immunoassays.

The immobilization of purified influenza virus, rubella virus, crude nuclear cytomegalovirus antigen and of mycoplasma on polystyrene tubes was studied using radio-iodinated preparations. The antigen activities on tube surfaces were determined using sequentially specific human antibodies and alkaline phosphatase-conjugated anti-human IgG in an enzyme-immunoassay (EIA) reaction. In addition, immobilization of radio-iodinated human IgG, IgM, and IgA, serving as model proteins, was studied using the respective anti-immunoglobulin conjugates in EIA directly. Pretreatment of the surface with albumin and glutaraldehyde inhibited the adsorption and antigenicity of IgG. Increase of temperature and thus of speed of adsorption did not affect the fraction of antigen eluted during the test procedure. Only with IgG and IgA was it necessary to saturate the polystyrene surface in order to achieve maximal reactivity in EIA. With other antigens, maximal reactivity in EIA was obtained with amounts of protein much lower than the maximal amount that could be adsorbed per tube. IgM was found to have an exceptionally high affinity to polystyrene.

Rapid solid-phase enzyme immunoassay for antibodies to viruses and other microbes: effects of polyethylene glycol.

A rapid and sensitive solid-phase enzyme immunoassay (EIA) was developed for determination of serum antibodies, including addition of 4% (w/v) polyethylene glycol (PEG) to the diluent buffers. This modification accelerated the antigen-antibody reactions and made it possible to complete the assay within 2 h and to incubate at room temperature only. The enhancing effect of the polymer was particularly prominent in the second solid-phase immune reaction, the interaction between the antigen-bound antibody and the enzyme-labeled anti-immunoglobulin. The PEG-EIA procedure was successfully applied in the assay of antibodies to rubella, influenza A, herpes simplex and cytomegalo viruses, to Mycoplasma pneumoniae and Toxoplasma gondii.

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.

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.

Procollagen-III in serum, plasminogen activation and fibronectin in bronchoalveolar lavage fluid during and following irradiation of human lung.

In the search for predictors of late radiation-induced lung injury we studied procollagen type III peptide concentration (P-III-P) in serum as well as fibronectin and plasminogen activation in bronchoalveolar lavage (BAL) fluid during and following irradiation of human lung. The patients received either high-dose hemithorax irradiation for pleural mesothelioma (11 patients) or high-dose irradiation with individually shaped fields for non-small cell lung cancer (12 patients). The severity of radiation fibrosis was assessed clinically from CT scans 6 months and 12 months after treatment. Four scores were used: severe, moderate, mild, or normal. Radiological lung injury varied from "severe" (9 patients) to near absence of injury-"normal" (6 patients). Serum levels of P-III-P, when measured weekly during the 5-week period of radiotherapy or at several time-points after treatment, did not show consistent changes, nor did the levels correlate with the score for radiation fibrosis as assessed by CT scanning. Changes in fibronectin levels or in markers of plasminogen activation in BAL fluid did not correlate with the development of late lung injury. The levels of BAL fluid plasmin and plasminogen activator as assessed zymographically, but not the free net enzyme values, showed a tendency to be elevated in patients with severe radiation-induced lung injury, suggesting a possible role for inhibitors of the plasminogen activation cascade in the process of radiation-induced lung injury.

Cerebrospinal fluid IgG bands and virus-specific IgG, IgM, and IgA antibodies in herpes simplex virus encephalitis.

To characterize the immune response of the central nervous system in herpes simplex virus (HSV) encephalitis, cerebrospinal fluid (CSF) specimens of 7 biopsy proven and 7 presumptive herpes simplex virus (HSV) encephalitis patients were studied, using sodium dodecyl sulfate polyacrylamide gel electrophoresis for the presence of CSF IgG bands, and solid-phase enzyme immunoassays for HSV-specific antibodies. IgG bands were detected in all CSF specimens of the patients, as early as day 6 and up to day 1088. A novel, unidentified, 120 000 dalton polypeptide was found in the CSF of most of the patients, in a total of 25/50 specimens, but not in the controls. This polypeptide was evident by day 6, its intensity fluctuated and it was present in specimens collected as late as day 855. HSV-specific antibodies, of either IgG, IgM, or IgA class, were not detected in the CSF during the first week of illness. IgG antibodies appeared later in all patients and persisted to the end of the follow-up of 3 years. The fact that CSF IgG bands were present in some patients before the appearance of HSV antibodies, and also persisted longer, suggests that the IgG response is not restricted to HSV-specific antigenic determinants.

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.

Fibronectin concentration in cerebrospinal fluid reflects early central nervous system involvement in children with acute lymphoblastic leukemia.

We measured concentrations of fibronectin (FN) in the cerebrospinal fluid (CSF) in long-term follow-up patients with acute lymphoblastic leukemia (ALL). In 11 patients with neuroleukemia the CSF-FN level was elevated already at the time of diagnosis of ALL, 3.8 +/- 0.6 mg/l, increased during therapy to 4.7 +/- 0.5 mg/l, and at the time of concurrent blast cell finding it was 5.5 +/- 1.0 mg/l. In 11 patients with no subsequent CNS leukemia, the mean CSF-FN level was 2.4 +/- 0.6 mg/l at the time of diagnosis of ALL and 2.8 +/- 0.6 mg/l during therapy, and increased to 3.2 +/- 0.8 mg/l. The neuroleukemia rate was 43% in patients with initial CSF-FN levels greater than 2 mg/l, compared with 5% in patients with CSF-FN levels less than or equal to 2 mg/l (p less than 0.005) in a group of 45 long-term follow-up patients with ALL. Regression analysis on the 21 clinical or laboratory parameters studied showed that the only variable independently associated with CSF-FN was the total protein concentration in the CSF; this, however, explained only 14% of the observed variation in the CSF-FN concentration and did not show any correlation with CNS involvement. We conclude that the CSF-FN test at diagnosis of ALL showed significant differences between groups of patients with and without CNS leukemia, and may prove to be a new early marker for neuroleukemia.

Rubella antibody determination from heparinised finger-tip blood by single radial haemolysis and enzyme immunoassay.

Rubella antibodies were determined by single radial haemolysis (SRH) and a micromodification of enzyme immunoassay (EIA) of samples of heparinised finger-tip blood or plasma collected into transportable vials, and the results were compared with antibody titres obtained from conventional samples of venous serum. The antibody titres of finger-tip specimens gave a high correlation with those of venous serum. For SRH only 5 microliters of heat-inactivated finger-tip plasma was needed, and for EIA only a single dilution, 1:100, in duplicate, of heparinised finger-tip plasma or while blood was sufficient. The minimal inconvenience in sample collection makes the finger-tip test particularly suitable for large-scale immunity screening when assessing the need for, or efficacy of, rubella vaccination.

C3c-binding in inflammatory rheumatic diseases.

Sera from 73 patients, 24 with rheumatoid arthritis (RA), 10 with Sjögren's syndrome (SS, 4 with and six without RA), 17 with Reiter's syndrome (RS), 10 with systemic lupus erythematosus (SLE), 9 with Yersinia arthritis (YA), and 3 with mixed connective tissue disease (MCTD) were examined by enzyme-immunoassay (EIA) for the presence of C3c-binding IgG, IgM, and IgA activity (C3cBIgG, C3cBIgM, C3cBIgA). This activity probably consists of immunoconglutinins and immunoglobulin aggregates. Significantly elevated C3cBIgG was found in the sera of patients with RA (p less than 0.01), SS, and SLE (p less than 0.02). C3cBIgM was elevated in SS and SLE (p less than 0.02). C3cBIgA was increased in RA (p less than 0.005), in SS and YA (p less than 0.02). Correlations of C3c-binding with erythrocyte sedimentation rate, hemoglobin, C-reactive protein, IgG-binding onto platelets, serum IgG, IgM, and IgA levels, and with rheumatoid factors of IgM and IgA classes were computed. C3cBIgG, C3cBIgM, and C3cBIgA correlated significantly with each other, but usually not with other laboratory variables. The exceptions were positive correlations of C3cBIgM with IgM-rheumatoid factors in RA (p less than 0.05), and with serum IgM levels in the whole series of patients (p less than 0.05). The use of C3c represents a new principle to detect abnormalities in the sera of patients with inflammatory rheumatic diseases. However, the pathogenic significance of elevated C3c-binding remains unknown.

Plasmin and fibronectin degradation in chronic secretory otitis media.

Mucoid effusions from 39 children with secretory otitis media, altogether 42 specimens, were analyzed for proteolytic activity using radial caseinolysis procedures, for fibronectin using a solid-phase enzyme immunoassay, and for fibronectin fragmentation using immunoblotting. All samples contained proteolytic activity, tentatively identified as plasmin on the basis of comigration with purified human plasmin in zymographic analysis. In 19 specimens the plasmin level exceeded 1 microgram/mg of protein; the highest value recorded was 18.7 micrograms/mg. Low levels of net plasminogen activator activity were found in 12 specimens and identified as urokinase according to comigration with the urokinase standard in zymography. Fibronectin was detected in all but one of the 42 specimens; in seven specimens the levels exceeded those in normal plasma, calculated per milligram of total protein. Extensive fragmentation of fibronectin was found in 19 specimens, correlating with high plasmin levels. The results are indicative of an ongoing proteolytic process in secretory otitis media and suggest that plasmin-caused degradation of the fibronectin-containing basement membrane and subsequent formation of granulation tissue may be involved in the development of adhesive middle ears.