Metabolism of plasminogen in healthy subjects: effect of tranexamic acid.

The metabolism of human plasminogen labeled with radioactive iodine was studied in 12 healthy men. The labeled plasminogen had a high specific activity and the same elution on Sephadex G-100 as the plasminogen activity in plasma. Immunoelectrophoresis revealed a single precipitin line. Polyacrylamide gel electrophoresis revealed six main bands, all with plasminogen properties and radioactivity. The purified plasminogen behaved as a homogeneous protein in the turnover experiments. The plasma radioactivity data were adequately approximated by a sum of two exponential terms. The metabolism of plasminogen was therefore represented by a two-compartment mammillary model. Results in the 12 normal subjects were as follows: plasma plasminogen concentration 20.8+/-1.9 mg/100 ml; intravascular plasminogen pool 0.66+/-0.14 g; intravascular fraction 0.59+/-0.06; fractional catabolic rate 0.55+/-0.09 of the plasma pool per day; half-life of the plasma radioactivity 2.21+/-0.29 days. Circulating large-molecular-weight degradation products of labeled plasminogen could not be detected by Sephadex G-100 gel filtration. The plasminogen turnover rate was normal in a patient with Behçet's syndrome and low circulating plasminogen activator activity. This finding supports the concept that under normal conditions the primary pathway of plasminogen catabolism is not via the formation of plasmin. The in vivo effect of tranexamic acid, a potent inhibitor of plasminogen activation, on the turnover of labeled plasminogen was studied in five normal subjects. When 1 g was administered perorally t.i.d. to three of them, one showed an increased plasminogen turnover. A 2 g dose administered t.i.d. to the other two caused markedly increased catabolism in both. This increase may be attributable to a direct reversible effect of tranexamic acid on the plasminogen molecule.

Transfer factor therapy in multiple sclerosis: a three-year prospective double-blind clinical trial.

One hundred five patients with MS were divided into three groups matched for age, sex, and disability, and treated with either placebo, transfer factor prepared from leukocytes of random donors, or transfer factor from leukocytes of family members living with the patients. There were no differences in the three treatment groups for changes in disability, activities of daily living, or evoked potentials. Eighteen months of transfer factor therapy had no effect on gamma-interferon production or natural killer cell activities.

Action of brinase on human fibrinogen and plasminogen.

Brinase added to human plasma in vitro caused a decrease in fibrinogen concentration, positive paracoagulation tests and formation of a friable clot in sequence. Agarose gel filtration of these samples revealed the presence of fibrinogen derivatives both larger and smaller than the parent molecule. Infusion of the enzyme in vivo resulted in a decreased fibrinogen level, a prolonged thrombin time and an increase in fibrinogen related antigen (FRA) in serum. The elution pattern of FRA in the plasma samples obtained after infusion of Brinase was similar to that of the in vitro samples. The plasma pool of fibrinogen was partially consumed by infusion of Brinase, but the turnover of plasminogen remained unaffected. Purified plasminogen was partially degraded by addition of the enzyme but this was accompanied by a generation of proteolytic activity. These findings confirm that Brinase induces a proteolytic degradation of fibrinogen in plasma without activation of the plasminogen-plasmin system. Exposure of polymerization site(s) in the fibrinogen molecule is probably responsible for the reported clot promoting effect of the enzyme.

A pilot study of the prevalence of hepatitis C virus antibodies and hepatitis C virus RNA in southern Cameroon.

Information is lacking on the prevalence of hepatitis C virus (HCV) infection in most African countries. An algorithm based on a combination of enzyme immunoassays (EIAs) with different formats (a commercial test, an HCV antibody [Ab] III test, and an HCV core Ab EIA) was used to estimate the prevalence of HCV infection in different population groups from southern Cameroon. An overall high prevalence was observed, with a significant increasing trend for both sexes with respect to age. A high proportion (67.4%) of HCV-positive sera were viremic as demonstrated by the reverse transcription-polymerase chain reaction. We conclude that the prevalence of HCV is high in southern Cameroon and increases linearly with age.

Interferon production and natural killer (NK) activity in leukocyte cultures from multiple sclerosis patients.

Peripheral blood leukocyte (PBL) cultures from only 37% of MS patients produced detectable HuIFN-gamma in response to ConA as opposed to 85% of the cultures derived from normal blood donors. However, the yields in patient-derived cultures that were responsive, were not lower than those in cultures from controls. Production of HuIFN-alpha after stimulation with Sendai virus was not aberrant in cells taken from MS patients. The difference in HuIFN-gamma response rate between MS and normal donor-derived cells was more pronounced when DR2+ carriers were compared amongst each other than when DR2-k carriers were compared. Among the MS patients, the failure of PBLs to produce HuIFN-gamma in response to ConA was not correlated with age, sex, disease duration and type of disease. However, positive correlations were found with current disability indices and past disease progression rates. Unstimulated NK-activities of MS patient-derived PBLs were not different from those of normal donor-derived cells. the degree of augmentation of the activity by stimulation with ConA and interferon-alpha was also normal. Within the MS patients group, but not in the control group, there was a trend for DR2+ carriers to have lower spontaneous and stimulated NK-activities than DR2- individuals.

A simplified method for hand-made oblique graphical reconstructions.

The present paper deals with a convenient, manual method for making oblique graphical reconstructions from serial sections. The method is based on a projection on the plane of drawing of a three-dimensional coordinate system, which is submitted to two successive and predetermined spatial rotations. Restriction to two rotations simplifies the mathematical approach to a coordinate transformation from a three-dimensional into a two-dimensional system. The resulting formulae, which are quite easily applied, lead to a quick visualization procedure.

Streptococcal preparation OK-432-induced interferon in human leukocytes: purification and characterization.

Interferon production was induced in leukocyte suspensions from human buffy coats after stimulation with the streptococcal preparation OK-432. At day 2-3 the induced interferon reached a maximal level of 0.9 units/1,000 cells. By a combination of batch adsorption/elution on silicic acid, batch adsorption to DEAE-Sephacel, affinity chromatography on concanavalin A-Sepharose and on poly(U)-Sepharose, this interferon could be purified to a specific activity of 10(7.5) units/mg protein. The antiviral activity was characterized as being solely due to gamma-type interferon by a variety of physicochemical, biochemical and serological criteria. Its molecular weight as determined by gel filtration amounted to 53,000 daltons, and its activity was completely neutralized by highly specific antisera to human gamma-type interferon (45K). The OK-432-induced interferon, as the crude supernatant of stimulated leukocytes, and at several stages of its purification, was found to stimulate the natural killer cell activity of fresh human lymphocytes.

Association of hepatitis C virus carrier state with the occurrence of hepatitis C virus core antibodies.

An enzyme immunoassay (EIA) was developed for the determination of antibodies against the "putative" core protein of hepatitis C virus (HCV). Antigens used were recombinant fragments (amino acids 6-77 or 6-143) of the HCV core protein, produced in Escherichia coli with truncated hepatitis B core (HBc) as fusion protein. Evaluation of 385 sera positive for HCV antibodies by first generation EIA, revealed 98 (25.4%) with HCV core antibodies. HCV-RNA, determined by the polymerase chain reaction (PCR), was exclusively found in the sera positive for HCV core antibodies (89 PCR positives). In random screening of 3,708 sera, 3 sera with HCV core antibodies were found PCR positive. Only 2 of these sera were positive in the first generation EIA. It is concluded that HCV core antibody determination is a reliable test for identifying HCV carriers among blood donors.

A microcomputer-based graphical reconstruction technique for serial sectioned objects, with hidden line removal.

The presented software package fulfills the need for processing serial sections with a microcomputer configuration, enabling three-dimensional reconstruction with hidden line removal. The language used is an interpreted BASIC-dialect (HPL). The input is performed via an interactive program. The object can be rotated in space. The Hidden Line algorithm does not depend upon a raster technique. Points of intersection of successive contours are calculated and inserted, thus providing drawings of high resolution and quality. The handling time can be said to be short, especially when considering the capacities of the microcomputer configuration used.

Confirmation of hepatitis C virus positive blood donors by immunoblotting and polymerase chain reaction.

In a series of 385 sera obtained from volunteer blood donors positive for the first-generation hepatitis C virus assay (Ortho), the viral genome was detected by polymerase chain reaction (PCR) in 89 sera (23%). Most PCR-positive sera were found positive with the c100-3 neutralisation assay (Abbott) and by two second-generation enzyme immunoassays (Abbott, Ortho). However overall specificity of these assays was rather low. By immunoblotting (Innogenetics and Chiron/Ortho) the specificity could be considerably improved and the best correlation with carrier state was obtained when analysing the results for lane-specific reaction: all 89 viral carriers and only 9 other donors had antibodies against structural 'core' epitopes. From the present data we can conclude that in screening a volunteer blood donor population the confirmation of antibodies against 'core' epitopes by immunoblotting is strongly associated with viral carriage.

Hepatitis C virus confirmation in blood donor screening.

A combination of different enzyme immunoassays (EIAs) was used for the serological confirmation of sera that were positive in a hepatitis C virus (HCV) second-generation screening EIA. Different reaction patterns were related with the probability of the HCV-carrier state as determined by polymerase chain reaction (PCR). Five hundred and eight sera of volunteer blood donors were send for confirmation and at first reexamined with both Abbott and Ortho second-generation screening EIA. A group of 195 sera, positive in both assays, was further evaluated by the Abbott Supplemental Assay, the Monolisa anti-HCV and an EIA with only the amino terminal part of the nucleocapsid protein as antigen. In addition PCR on the 5'-noncoding region of the viral genome was performed. We observed that 75 of the 78 PCR-positive sera were found in a group of 89 sera that were strongly positive in the four EIAs used. Moreover all but 1 PCR-positive sera were reactive against the nucleocapsid protein of the virus. Hence we concluded that a genuine antibody response to the nucleocapsid protein is highly suggestive for the HCV-carrier state.

Evaluation of third-generation screening and confirmatory assays for HCV antibodies.

A third-generation (gen.) screening and immunoblot assay (Ortho EIA-3.0; Chiron RIBA-3 prototype), using antigens derived from the capsid and different nonstructural regions (NS3, NS4 and NS5) of the hepatitis C virus viral genome, were evaluated in comparison with the corresponding second-gen. assays (Ortho EIA-2.0; revised Ortho EIA-2.5; Chiron RIBA-2). In 203 depository sera of blood donors, positive in EIA-2.0, specificity of the screening assays was improved as shown by an increase in positive predictive value for viral carrier state from 0.23 (EIA-2.0) to 0.37 (EIA-2.5) and 0.52 (EIA-3.0). Comparing the confirmation patterns on RIBA-2 and RIBA-3, this amelioration was mainly due to the specific elimination of false-positive c22-3 and c100-3 reactions. Antibody response to the newly added NS5 antigen was not as prevalent as to the other antigens and had only a minor influence in sample allocation. In contrast, screening of 1,560 volunteer blood donors and 47 hemodialysis patients revealed 3 additional positive sera, only reacting with the NS5 antigen. However none of these isolated NS5 reactions could be confirmed on synthetic peptides [INNO-LIA: NS5(p)] and none was PCR positive. A documented seroconversion, detected earlier with EIA-3.0, was related to a better immunological response to the NS3 antigen and not to the additional NS5. From this pilot study third-gen. assays appeared extremely useful in the reevaluation of HCV-seropositive depository sera. However the additional value of the NS5 antigen in blood donor screening is still hypothetical and remains to be established in larger screening studies.

Localization and reactivity of an immunodominant domain in the NS3 region of hepatitis C virus.

Analysis of the amino acid sequences of the nonstructural region 3 (NS3) of the hepatitis C virus type 1 revealed four points with a high average hydrophilicity (Ah). Two of these potential antigenic sites were expressed in E. coli as short fragments. The first fragment of 91 residues (NS3f3: residues 1359-1449) harbors the hexapeptide K-K-K-C-D-E with an Ah of 2.33; the second fragment is 73 residues long (NS3f4: residues 1460-1532) and encompasses the heptapeptide R-S-N-R-R-G-R with an Ah of 1.79. Both fragments were expressed with truncated hepatitis B core (tHBc) as a carrier protein. The fusion proteins were purified from the bacterial lysates by affinity chromatography on immobilized monoclonal antibodies against HBc, and evaluated as antigens in an enzyme immunoassay for the detection of HCV antibodies. In a specificity control panel, reactivity with NS3f3 was only found in proven HCV carriers, while reactivity with NS3f4 was weak in HCV carriers but accounted for some of the nonspecific serological reactions. In a group of 48 genotyped HCV-infected volunteer blood donors, antibodies against NS3f3 were detected in 90% (27/30) of HCV-type 1 infections and in all HCV-type 4 infections (5/5).(ABSTRACT TRUNCATED AT 250 WORDS)

An EIA method on single donor solubilized HLA antigens for the identification of anti-HLA antibodies.

An enzyme immunoassay (EIA) method based on solubilized human leucocyte antigens (HLA) derived from single donor platelets is described. The EIA results on these solubilized single donor HLA antigens (SDszHLA) correlated well with the complement dependent cytotoxicity (CDC) results on the lymphocytes of the same donors and also with the panel reactivity (PRA) in CDC. A concordancy rate of 78% was found for individual HLA specificities. The EIA+/CDC- ('false positive') discrepancies were more pronounced than EIA-/CDC+ ('false negative') discrepancies and varied for the different donors. To confirm discrepancies, our method was compared with a commercial PRA-STAT EIA method (based on secreted soluble HLA antigens). The same discrepancies between CDC and PRA-STAT EIA were found and are probably due to the higher and different sensitivity (e.g. non complement fixing antibodies) of EIA methods. A SDszHLA EIA method allows the identification of HLA specificities of HLA-antisera. The possibility of using individual and selected donors for the production of SDszHLA allows the directed search for well defined HLA specificities in order to confirm anti-HLA specificities found in other anti-HLA screening methods. An individualized HLA panel can be established with the support of blood banks that have HLA typed blood and platelet donors.

Amino-acid sequence of activation cleavage site in plasminogen: homology with "pro" part of prothrombin.

A 38-residue fragment is isolated from carboxymethylated plasminogen. Residues 29-38 have the same sequence as the amino-terminal end of the light chain of plasmin. The sequence 1-28 is therefore the sequence of the carboxyl-terminal end of the heavy chain and contains the specific sequence at which urokinase (EC 3.4.99.26) and other plasminogen-activating serine proteases split. Two of the five carboxymethyl-cysteine residues in the isolated fragment are situated close to the cleavage site and the fragment is not itself a substrate for plasminogen-activators. Residues 1-11 show extensive sequence homology with residues 137-147 and 242-252 in prothrombin, which are located in corresponding regions of the two internally homologous 83-residue structures in the non-thrombin part of the molecule, indicating that such structures may be a common feature of the non-protease part of the larger serine protease zymogens.

Activation of natural cytotoxicity of human peripheral blood mononuclear cells by interferon: a kinetic study and comparison of different interferon types.

Overnight incubation of human peripheral blood mononuclear (PBMN) cells with leucocyte interferon (leucocyte IFN) resulted in a 2-5-fold increase in natural cytotoxicity (NC) against the erythroleukaemic cell line K562. Fibroblast IFN, purified to homogeneity by zinc-chelate chromatography, stimulated NC to the same extent, while partially purified immune IFN was about twice as active. Upon gel filtration of immune IFN, NC stimulating and antiviral activity co-eluted. Treatment of PBMN cells with ammonium chloride buffer (AmCl) abrogated NC nearly completely. Incubation of AmCl-treated cells with leucocyte IFN resulted in a partial regeneration of NC. Kinetic studies revealed that this regeneration required only short exposure to IFN followed by a longer incubation period. The data are interpreted as indicating that in the process of NC activation IFN mainly acts as a trigger for precursor cells to mature into cells with NC.