PO-23 - Expression of heparanase in cancer as biomarker of malignancies: overexpression in an aggressive, poor survival gastric cancer "gastric signet ring cell carcinoma" compared with that of other gastric cancers.

INTRODUCTION: Gastric signet ring cell carcinoma (GSRC) is a distinct entity among of other gastric cancer. With unknown etiopathology, their incidence is increasing and it presents a low sensitivity to chemotherapy. AIM: Here, we studied the expression of the heparanase (HPA) in cancer tissues from GSRC patients and several cancer cell lines. HPA is an endo-ß-D-glucuronidase, capable of cleaving the lateral chains of heparan sulfate on cell surfaces and the extracellular matrix at acidic pH. Apart from its well-characterized enzyme activity, HPA also has independent enzymatic functions, such as up-regulation of vascular endothelial growth factor (VEGF) -A and VEGF-C and activation of intracellular signaling pathways involved in, survival, migration and proliferation of tumor cells. It can also induce an hypercoagulability by a non-enzymatic manner. MATERIALS AND METHODS: HPA was tested in several cancer cell lines from ovaries (OVCAR-3, SKOV-3), breast (MDAMB231, MCF7) colon (LS-174), lung (A549), uterus (HELA) and gastric (adenocarcinoma (AGS) and GSRC (KATO-III) using several techniques such as RT-PCR, Q-PCR, immunocytochemistry, flow cytometry and degradation of Fondaparinux at pH 5, evaluated by its anti Xa activity evaluated by Factor Xa amidolytic activity. The amount of HPA mRNA in the biopsy simples of gastric adenocarcinoma (n=10) and GSRC (n=11) in tumors and their environment were analyzed. RESULTS: HPA gene is expressed in all cancer cell lines, but its level varies depending on the tumor cell line. In biopsies of gastric cancer, the HPA gene is more expressed in the tumor regions (p=0.0002) and tumor environment (p=0.015) in GSRC than in gastric adenocarcinoma. B) The activity of HPA, evaluated by degradation of Fondaparinux at pH 5, 1) in the supernatants of 10(6) cancer cells: the residual activity of Fondaparinux after 2 hours incubation at 37°C with OVCAR-3 supernatant was of 70% of control value, and of 80% with KATO-III cell supernatants. 2) in patient's plasmas, this technique cannot be used because the site of degradation of fondaparinux by heparanase is masked by AT present in plasma. CONCLUSIONS: Heparanase was found in in many cancer cell lines and its level depends on origin of tumor cells and on its aggressivity. Taking into account the pro-metastatic functions, proangiogenic and procoagulant activity of HPA and its overexpression in gastric signet ring cell carcinoma of poor prognosis and its cell line, HPA can be considered as a biomarker of malignancy and as a therapeutic target in GSRC patients.

[A chromogenic method for rapid identification of Staphylococcus aureus]. / Méthode chromogénique d'identification rapide de Staphylococcus aureus.

Staphylococcus aureus is responsible for septicaemia and serious nosocomial infections. A rapid and specific identification of this species is of great importance in clinical microbiology. Current methods for S. aureus identification require a 18 to 24 h-incubation. We describe a two hour-identification method based on the detection of the staphylocoagulase, using human prothrombin and a chromogenic substrate. 242 staphylococcal strains (160 S. aureus, 82 coagulase-negative staphylococci (CNS)) were collected from 4 French hospitals. They have been identified by the following methods: (i) clotting of citrated rabbit plasma, which is considered as reference method; (ii) biochemical tests (Rapidec Staph and Api Staph or ID 32 Staph); (iii) and agglutination test (Pastorex Staph or Pastorex Staph-plus). A strain of S. intermedius was provided by the Collection of the Pasteur Institute (Paris). An adapted culture medium is inoculated with staphylococci and adjusted to 2 Mac Farland unities. This medium is then mixed to an equal volume with a human prothrombin solution and the chromogenic substrate. After 1 to 2 hours incubation at 37 degrees C, the strength of the yellow colour of the mixture is observed to the naked eye, or measured at 405 nm with a spectrophotometer. Fifteen chromogenic tripeptides having a thrombin-like affinity and paranitroanilin as leaving group were compared. With the substrate which has the higher hydrolysis velocity and enzymatic affinity (SQ149), all S. aureus strains gave a positive result: 94.7% of the methicillin-susceptible S. aureus were detected after 1 hour incubation, but only 52.3% of the methicillin-resistant S. aureus. 98.4% of the methicillin-resistant S. aureus were detected after 2 hours. No false positive result was observed for the 82 CNS strains. The chromogenic method shows good within-run and day-to-day precision tests. It doesn't need any complementary test. The sensitivity and the specificity are 99.4% and 100% respectively.

A new gel tube method for the direct detection, identification and susceptibility testing of bacteria in clinical samples.

We recently developed a simple new method which is designed to separate and concentrate bacteria from a sample by centrifugation in a gel system. Bacterial enzyme activity is then detected inside the gel without further manipulation using a colorimetric or fluorogenic substrate. The method provides a rapid, direct means of detecting bacteria in clinical samples, dispensing with the 24-h period normally required to isolate colonies on agar. Various applications of the method are described below, e.g. screening of negative urine samples, identification of Escherichia coli in urine samples, identification of Staphylococcus aureus in blood culture broths and detection of oxacillin-resistant S. aureus in blood culture broths. The advantages of the gel system and other applications are discussed.

Validation protocol of analytical hemostasis systems: measurement of anti-Xa activity of low-molecular-weight heparins.

A standard validation protocol adapted to the chromogenic assay of anti-Xa activity of low-molecular-weight heparins was used in a multicenter study to assess its suitability for comparing and evaluating analytical hemostasis systems. The protocol included: familiarization with the system (repeatability); assessment of limits of linearity, detection limits, and cross-contamination; and validation (reproducibility and accuracy of measurements of treated patients' plasmas). We calibrated the systems with the same range of lyophilized plasmas daily and evaluated repeatability and reproducibility by using a single batch of lyophilized plasmas at three anti-Xa activities. The two automated systems tested [SB 300 (Gilford) and ACL (IL)] and the two semiautomated systems [ST 888 (D. Stago) and Chromotimer (Behring)] gave similar mean values. Dispersion of results was lower with the automated systems than with the semiautomated ones, especially at low anti-Xa activities, a tendency that also was observed for reproducibility. Because each analytical system gave linear results for activities as great as 1000 IU/L, suitable sample dilution is advisable for higher anti-Xa activities. Accuracy was greater in the automated systems. We conclude that this protocol is feasible and is applicable to validation of other analytical hemostasis instruments, in particular the latest generation of fully automated instruments.

Functional assay of protein S in 70 patients with congenital and acquired disorders.

A functional assay for the selective measurement of the active form of protein S in plasma, based on the prolongation of an APTT, was previously developed. This assay is sensitive, reproducible and specific, not affected by other clotting factors including FVIII. This method was applied to the measurement of protein S activity in congenital and acquired disorders. Results of protein S activity were compared to those of total and free antigen measured by ELISA. In 30 controls, there was an excellent correlation between protein S activity and free antigen. In patients with inflammatory disease, protein S activity and free antigen were normal, despite high levels of both C4b-binding protein and total protein S antigen. In dicoumarol-treated patients, protein S activity was lower than free antigen due to the presence of acarboxylated forms. Surprisingly, in liver cirrhosis, free antigen was only slightly decreased whereas protein S activity was significantly reduced. In 23 patients with congenital deficiency, protein S activity was consistently decreased, from less than 5% to 60% and showed good correlation with the free antigen. This functional assay allows the rapid diagnosis of congenital or acquired deficiency of protein S.

Determination of plasminogen activator inhibitor (PAI) activity of human plasma after dilution in a PAI-depleted plasma.

A simple and discriminating assay for the determination of the fast-acting plasminogen activator inhibitor (PAI) activity in human plasma is described. The method is based on the inhibition of purified tissue plasminogen activator (tPA) by plasma diluted with a PAI-depleted plasma and the subsequent measurement of residual tPA in the presence of CNBr-fibrinogen fragments, purified plasminogen and a plasmin sensitive chromogenic substrate CBS 10.65. This assay does not require any acidification step, and allows PAI determination directly on plasma. Since dilutions are made in PAI-depleted plasma, all the serine-protease inhibitors, except PAI, are kept constant in their effect on the assay. Thus, any detectable degree of inhibition can only be ascribed to PAI. Under these conditions, parallel titration curves of tPA are obtained in plasma and the values of PAI are reproducible when measured at different dilutions. The PAI levels of 31 normal volunteers ranged from 0.3 to 8.7 IU/ml (mean: 3.5 IU/ml). After venous occlusion, variations of PAI were associated with the release of tPA. A marked increase of PAI levels was observed in the post-operative period and in pregnancy. In this case both PAI-1 and PAI-2 related activities were measured. Due to its simplicity, the assay can be easily used for the screening of patients with thrombotic diseases.

[Rapid determination of protein C activity]. / Dosage rapide de l'activité de la protéine C.

The diagnosis of Protein C (PC) congenital deficiency is of first importance because it leads, more frequently than Antithrombin III deficiency, to serious thromboembolic accidents in young patients, even when PC levels are slightly decreased (40 p. cent up to 60 p. cent). In order to measure PC activity, the authors developed a new method using the activator from Agkistrodon C. Contortrix snake venom and the synthetic chromogenic substrate CBS 65-25. Results obtained on plasma from normal individuals, congenital and acquired deficiencies, are comparable on the one hand, to those found with an ELISA method, and on the other hand with the clotting method, except for patients under anticoagulant therapy. This new rapid and sensitive method can be performed manually and is easily adapted on instruments used in clinical chemistry laboratories. This method is potentially available for routine use.

Measurement of tPA and tPA-PAI-1 complexes by ELISA, using monoclonal antibodies: clinical relevance.

Two ELISA methods using monoclonal antibodies, are described for the measurement of tPA:Ag and tPA-PAI-1 complexes. On normal population tPA:Ag was found with a mean value of 5 ng/ml. Furthermore, despite that tPA activity was very low, only 50% (mean value) was measured as stable complexes with PAI-1. Important increase of tPA:Ag was observed in various pathologies (cardiac infarction, septicemia, respiratory distress syndrome). In liver disease, tPA:Ag reached high levels up to 100 ng/ml. Impaired liver clearance can potentiate the increased concentration which results from endothelial release. In all patients with elevated tPA:Ag level, 70 to 100% of tPA was complexed to PAI-1. Excess release of PAI-1 accompanys the increased release of tPA as it is proved by presence of high residual PAI-1 activity. Addition of exogeneous tPA to these pathological plasmas induced a high increase in tPA-PAI-1 complexes. Venous stasis in normal population resulted in a parallel increase of tPA:Ag and tPA-PAI-1 complexes. Although about a two fold increase was obtained for both parameters, post venous stasis plasma presented a much higher fibrinolytic activity while PAI-1 activity was moderately elevated. tPA:Ag and tPA-PAI-1 complexes have diagnosis and prognosis value in various pathologies as indicators of stimulated release of fibrinolysis activator and inhibitor.

Release pattern of the vascular plasminogen activator and its inhibitor in human postvenous occlusion plasma as assessed by a spectrophotometric solid-phase fibrin-tPA activity assay.

Vascular or tissue-type plasminogen activator (plasma t-PA) is the circulating physiological fibrinolytic enzyme of endothelial cell origin which function is regulated by fibrin and a specific inhibitor (PAI). To study the pattern of release of t-PA and the behavior of t-PA-PAI complexes in plasma we determined t-PA activity in 44 healthy subjects before and after 10 min of forearm venous occlusion using a new spectrophotometric solid-phase fibrin-tPA activity assay. The assay is based on 1) the high affinity binding of t-PA to fibrin, and 2) the detection of fibrin-bound t-PA by measuring the release of pNA from a chromogenic substrate in the presence of plasminogen. Values at rest were rather undetectable in plasma (0.05 +/- 0.03 IU/ml, in 23 out of 44 samples) but were positively detected in all the euglobulins: 0.88 +/- 0.68 IU/ml. After venous occlusion the majority of plasmas (36 out of 44) showed a slight increase in t-PA activity (0.65 +/- 0.63 IU/ml) as compared to the important level observed in all the euglobulins (9.78 +/- 9.58 IU/ml). So, the ratio plasma/euglobulin t-PA activity was very low (0.06) and remained identical in both pre- and postocclusion samples. However, when diluted plasmas were tested the inhibitory effect disappeared and t-PA activity increased indicating that although t-PA circulates in a neutralized state it can be available for fibrinolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Low molecular weight heparin fractions as an alternative therapy in heparin-induced thrombocytopenia.

Eight patients with a delayed-onset heparin-induced thrombocytopenia, with thrombotic complications requiring immediate anticoagulation in 7 of them, were given low molecular weight heparin (LMWH) fractions as alternative therapy. This treatment led to normalization of platelet count within 3-5 days in 6 patients with clinical recovery in 5. In 2 patients, thrombocytopenia persisted despite LMWH therapy. In vitro platelet aggregation tests performed in all patients gave evidence of a relationship between the presence (or absence) of a LMWH-dependent platelet-aggregating factor in the patients' plasma and the persistence (or correction) of the thrombocytopenia with LMWH therapy. Although positive in vitro tests may not necessarily be associated with thrombocytopenia, in vitro testing may prove to be a useful guide before giving LMWH fractions as an alternative therapy in patients with heparin-induced thrombocytopenia requiring immediate anticoagulation.

Treatment of acute pulmonary embolism by a low molecular weight heparin fraction. A preliminary study.

We evaluated the antithrombotic efficacy of the low molecular weight heparin (LMWH) fraction PK 10169 in nine consecutive patients with acute pulmonary embolism documented by pulmonary angioscan and angiography. Therapy with PK 10169 was initiated by an i.v. bolus of 0.5 mg/kg, followed by a continuous intravenous infusion during the first 10 days; the drug was then given subcutaneously twice daily during the following 15 days. The dosage of PK 10169 was adjusted by daily measurements of anti-Xa and anti-IIa activities using amidolytic methods. For a dosage ranging from 1.4 to 4.1 mg/kg per day during the i.v. period and from 0.7 to 3.5 mg/kg per day during the s.c. period, the anti-Xa activity ranged from 4 to 8.7 PK U/ml and from 4.5 to 7.2 PK U/ml respectively. Clinical improvement was observed in all the patients and was consistent with progressive reperfusion evaluated by successive angioscans. No recurrence of pulmonary embolism occurred. No deleterious hemorrhagic side-effects were observed, even in two patients at high risk of bleeding. In this pilot study, the LMWH fraction PK 10169 proved to be an effective anticoagulant therapy during the first three weeks after pulmonary embolism in man.

Heparin inactivation during blood storage: its prevention by blood collection in citric acid, theophylline, adenosine, dipyridamole-C.T.A.D. mixture.

An attempt was made to prevent heparin inactivation during the time that lapses between blood collection and laboratory assay. The blood collection in a combination of citric acid, theophylline, adenosine, dipyridamole - C.T.A.D. mixture - was found to reduce greatly the heparin loss during blood centrifugation and storage whatever the temperature for these two steps (centrifugation at 4 degrees C, 12 degrees C or 25 degrees C; storage at 4 degrees C or 20-25 degrees C). With this mixture the influence of centrifugation temperature upon the heparin loss appeared negligible. The heparin loss during the initial 5 hours storage at 20 degrees-25 degrees C was found to be 4.4 +/- 4% for a heparin concentration of 0.45 +/- 0.05IU/ml. The combination of the platelet active drugs with citric acid in C.T.A.D. mixture did not interfere either with the amidolytic heparin assay (anti IIa) or clotting tests such as activated partial thromboplastin time and calcium thrombin time. Thus the C.T.A.D. mixture appears to be useful in routine use because it is effective, cheap, stable, does not interfere with the tests currently used for monitoring heparin therapy and thus helps overcome the main cause of error in heparin assays.