Evidence of complementarity between targeted and non-targeted analysis based on liquid and gas-phase chromatography coupled to mass spectrometry for screening halogenated persistent organic pollutants in environmental matrices.

This study explored the complementarity between targeted (TS) and non-targeted screening (NTS) based on liquid and gas-phase chromatography coupled to (high-resolution) mass spectrometry (LC-/GC-(HR)MS) for the comprehensive characterization of organohalogen fingerprints within a set of Lake Ontario lake trout samples. The concentrations of 86 legacy, emerging and novel halogenated compounds (HCs), were determined through 4 TS approaches involving no less than 6 hyphenated systems. In parallel, an innovative NTS strategy, involving both LC and GC-Q-Orbitrap, was implemented to specifically highlight halogenated signals. Non-targeted HRMS data were processed under the HaloSeeker software based on Cl and Br isotopic ratio and mass defect to extend the screening to unsuspected and unknown HCs. A total of 195 halogenated mass spectral features were characterized in the Lake Ontario lake trout, including well known HCs (PCBs, PBDEs, PBBs, DDT and their degradation products), emerging HCs (novel brominated flame retardants, short-, medium- and long-chain chlorinated paraffins) or suggested molecular formula (mainly polychlorinated ones). Among the 122 HCs highlighted by TS, only 21 were identified by NTS. These results fueled a discussion on the potential and limitations of both approaches, and the current position of NTS within environmental and health monitoring programs.

Do farming conditions influence brominated flame retardant levels in pig and poultry products?

Brominated flame retardants (BFR) are primarily used as flame retardant additives in insulating materials. These lipophilic compounds can bioaccumulate in animal tissues, leading to human exposure via food ingestion. Although their concentration in food is not yet regulated, several of these products are recognised as persistent organic pollutants; they are thought to act as endocrine disruptors. The present study aimed to characterise the occurrence of two families of BFRs (hexabromocyclododecane (HBCDD) and polybrominated diphenyl ethers (PBDE)) in hen eggs and broiler or pig meat in relation to their rearing environments. Epidemiological studies were carried out on 60 hen egg farms (34 without an open-air range, 26 free-range), 57 broiler farms (27 without an open-air range, 30 free-range) and 42 pig farms without an open-air range in France from 2013 to 2015. For each farm, composite samples from either 12 eggs, five broiler pectoral muscles or three pig tenderloins were obtained. Eight PBDE congeners and three HBCDD stereoisomers were quantified in product fat using gas chromatography-high-resolution mass spectrometry, or high-performance liquid chromatography-tandem mass spectrometry, respectively. The frequencies of PBDE detection were 28% for eggs (median concentration 0.278 ng/g fat), 72% for broiler muscle (0.392 ng/g fat) and 49% for pig muscle (0.403 ng/g fat). At least one HBCDD stereoisomer was detected in 17% of eggs (0.526 ng/g fat), 46% of broiler muscle (0.799 ng/g fat) and 36% of pig muscle (0.616 ng/g fat). Results were similar in concentration to those obtained in French surveillance surveys from 2012 to 2016. Nevertheless, the contamination of free-range eggs and broilers was found to be more frequent than that of conventional ones, suggesting that access to an open-air range could be an additional source of exposure to BFRs for animals. However, the concentration of BFRs in all products remained generally very low. No direct relationship could be established between the occurrence of BFRs in eggs and meat and the characteristics of farm buildings (age, building materials). The potential presence of BFRs in insulating materials is not likely to constitute a significant source of animal exposure as long as the animals do not have direct access to these materials.

Subcutaneous fondaparinux versus intravenous unfractionated heparin in the initial treatment of pulmonary embolism.

BACKGROUND: The standard initial treatment of hemodynamically stable patients with pulmonary embolism is intravenous unfractionated heparin, requiring laboratory monitoring and hospitalization. METHODS: We conducted a randomized, open-label trial involving 2213 patients with acute symptomatic pulmonary embolism to compare the efficacy and safety of the synthetic antithrombotic agent fondaparinux with those of unfractionated heparin and to document noninferiority in terms of efficacy. Patients received either fondaparinux (5.0, 7.5, or 10.0 mg in patients weighing less than 50, 50 to 100, or more than 100 kg, respectively) subcutaneously once daily or a continuous intravenous infusion of unfractionated heparin (ratio of the activated partial-thromboplastin time to a control value, 1.5 to 2.5), both given for at least five days and until the use of vitamin K antagonists resulted in an international normalized ratio above 2.0. The primary efficacy outcome was the three-month incidence of the composite end point of symptomatic, recurrent pulmonary embolism (nonfatal or fatal) and new or recurrent deep-vein thrombosis. RESULTS: Forty-two of the 1103 patients randomly assigned to receive fondaparinux (3.8 percent) had recurrent thromboembolic events, as compared with 56 of the 1110 patients randomly assigned to receive unfractionated heparin (5.0 percent), for an absolute difference of -1.2 percent in favor of fondaparinux (95 percent confidence interval, -3.0 to 0.5). Major bleeding occurred in 1.3 percent of the patients treated with fondaparinux and 1.1 percent of those treated with unfractionated heparin. Mortality rates at three months were similar in the two groups. Of the patients in the fondaparinux group, 14.5 percent received the drug in part on an outpatient basis. CONCLUSIONS: Once-daily, subcutaneous administration of fondaparinux without monitoring is at least as effective and is as safe as adjusted-dose, intravenous administration of unfractionated heparin in the initial treatment of hemodynamically stable patients with pulmonary embolism.

Gamma-interferon inhibits extracellular matrix synthesis and remodeling in collagen lattice cultures of normal and scleroderma skin fibroblasts.

Three-dimensional collagen lattice cultures of fibroblasts mimic the in vivo situation better than monolayer cultures. Here, skin fibroblasts from scleroderma patients and healthy controls were cultivated in collagen lattices, and the effects of recombinant human gamma-interferon (IFN-gamma) on these cultures investigated. IFN-gamma inhibited collagen lattice retraction in a dose-dependent way at concentrations ranging from 10 to 10,000 U/ml. This effect was independent of any alteration to the cell proliferation within the lattices. The inhibition was of the same order of magnitude in normal and pathological fibroblasts. The synthesis of collagen and non-collagen proteins, particularly fibronectin, was increased in scleroderma cultures. It was inhibited in both normal and scleroderma fibroblasts by IFN-gamma, with a maximal effect at the concentration 1000 U/ml, but the inhibition of protein synthesis was far more intense in scleroderma than in normal cells. In situ hybridization, Northern blot and dot blot analyses showed that mRNA coding for pro alpha 1(I) collagen was decreased in IFN-gamma-treated cells, indicating an effect at the pretranslational level. IFN-gamma also inhibited glycosaminoglycan synthesis, but in scleroderma cells only. This study shows that IFN-gamma regulates cell behavior in three-dimensional collagen matrices: (i) it decreases protein and specifically glycosaminoglycan synthesis in scleroderma fibroblasts, (ii) it modulates the interactions between cells and matrix that lead to the retraction of the lattice. Whereas collagen synthesis is largely decreased in lattice cultures like in vivo, it remains increased in the case of scleroderma compared to normal fibroblasts and may be down-regulated by IFN-gamma. Similar conclusions may be drawn for fibronectin and glycosaminoglycans. The inhibitory effect of IFN-gamma on the retraction capacity of fibroblasts and on their ability to synthesize increased amounts of extracellular matrix macromolecules may be of potential interest for therapeutic use of IFN-gamma in scleroderma patients.

The lupus anticoagulant and its role in thrombosis.

The lupus anticoagulant is usually found in the plasma of patients with systemic lupus erythematosus. Lupus anticoagulants are antibodies to phospholipids and probably to phosphodiester-linked phosphate groups. A high frequency of thrombotic events in patients with lupus anticoagulant has been reported. Nevertheless the pathogenesis of thrombosis in these patients remains unknown. Endothelium which plays a key role in the antithrombogenic-thrombogenic balance could be a target for the lupus anticoagulant and alterations of some endothelial-cell functions could be responsible for the thrombotic events. The effects of the lupus anticoagulant on the phospholipids of the protein C-thrombomodulin complex may be important although evidence of such a reaction in vivo is awaited.

Effect of lupus anticoagulant on antithrombogenic properties of endothelial cells--inhibition of thrombomodulin-dependent protein C activation.

We have investigated the effect of purified immunoglobulin G (IgG) on endothelial cell functions in 16 patients with lupus anticoagulant, 9 of whom had systemic lupus erythematosus (SLE). Spontaneous or thrombin-stimulated secretion of prostacyclin (PGI2) by cultured human endothelial cells from umbilical cord vein (HUVEC) was not inhibited by the patient's IgG. Nor was spontaneous release of tissue plasminogen activator (t-PA) or of its inhibitor (PAI) modified in the presence of patient's IgG. The rate of activation of purified protein C (PC) by HUVEC in the presence of thrombin was significantly lowered by patient's IgG or Fab' fragment (inhibition of 43%). Neutralization of this effect was obtained by incubation of a greater quantity of phospholipids (phosphatidylcholine, phosphatidylserine) with the patient's IgG. Activation of PC was also performed using purified rabbit thrombomodulin (TM) and a similar inhibition of the patient IgG was observed (inhibition of 48%) but the activation of Gla-domainless PC was not modified.

A comparative study of the anticoagulant and anti-thrombotic effects of unfractionated heparin and a low molecular weight heparin (Fraxiparine) in an experimental model of human venous thrombosis.

We have compared the anticoagulant and the antithrombotic effects of unfractionated heparin (Calciparine) and low molecular weight heparin (Fraxiparine) in an experimental human venous thrombosis model. One single subcutaneous injection of Calciparine or Fraxiparine was administered to healthy male volunteers at one month interval in a randomised and cross-over design. Ten subjects received doses used in man for preventing venous thrombosis (5,000 IU and 3,075 IU, respectively), and seven other subjects received curative doses (12,500 IU and 6,150 IU, respectively). Thrombus formation was measured 3 h and 8 h after drug administration. Non-anticoagulated human blood was drawn for 5 min directly from an antecubital vein over confluent cultured endothelial cells positioned in a parallel-plate perfusion chamber. The cells were previously stimulated for 4 h with lipopolysaccharides (10 micrograms/ml) and interleukin 1 beta (50 U/ml), resulting in optimal expression of biological active tissue factor. The wall shear rate at the cell surface was 50 s-1 and mimicked venous blood flow conditions. Immunologically quantified fibrin deposition on the stimulated cells was reduced only by curative doses of Calciparine and Fraxiparine at 3 h (3.4 +/- 0.8 versus 1.0 +/- 0.2 micrograms/cm/ and 2.6 +/- 0.8 versus 1.0 +/- 0.1 micrograms/cm2, respectively, p < or = 0.05). The influence of Calciparine and Fraxiparine on the formation of thrombin and fibrin was determined by measuring the plasma levels of thrombin-antithrombin III complexes and fibrinopeptide A (FPA) in blood samples collected distally to the perfusion chamber. The generation of these markers was significantly inhibited (50-83%) by both prophylactic and curative doses of Calciparine and Fraxiparine (p < or = 0.05). However, Fraxiparine still significantly inhibited the thrombin and fibrin generation at 8 h (p < or = 0.05), whereas Calciparine did not. The antithrombotic effects of both heparins were correlated with their plasma activities as measured by the antifactor Xa or the antithrombin assays. Thus, it appears in this model that Calciparine and Fraxiparine produce comparable antithrombotic effects at clinically comparable doses. However Fraxiparine has a longer-lasting anticoagulant activity than Calciparine. These results are in good agreement with clinical observations in man, and thus in favour of our model of human venous thrombogenesis for further studies of antithrombotic molecules.

Pharmacokinetics and tolerance of the natural pentasaccharide (SR90107/Org31540) with high affinity to antithrombin III in man.

This paper reports the results of the first administration of the synthetic natural pentasaccharide with high affinity to antithrombin III (NP) in man. This study was mainly focused upon the pharmacokinetic properties and general tolerance of the compound. Subcutaneous injections of doses < 1.43 mg (1000 anti Xa IU) did not generate measurable anti-Xa activities. After subcutaneous injection of increasing doses from 1.43 to 22.9 mg (1000 to 16,000 anti-Xa IU) to young healthy volunteers, it was found that the maximal concentration (Cmax) and the area under curve (AUC) were linearly correlated to the dose, that the total plasma clearances (CI) were constant and almost 3 times lower than those of the current low molecular weight heparins. Cmax were reached between 1 h and 3 h after the injection and the half-lives (t 1/2) were remarkably constant (13.1 h to 13.9 h). During the first 24 h following the injection, around 50% of the total administered dose was recovered in the urine in an active form, indicating that kidney plays a major roles in the elimination of NP. Consistent with these results, when NP was administered to healthy elderly volunteers having a lower creatinine clearance, the half-life of the compound was longer and the clearance lower. At doses exceeding 22.9, Cmax, and AUC were slightly lower than expected, the percentage of the dose recovered in the urine and the total apparent plasma clearance increased, suggesting that the excess of NP unbound to antithrombin III was excreted faster. NP was also administered at various dosages once or twice a day for 7 days to 20 elderly volunteers. Due to the long half-life of the compound the "steady state" was obtained 2 to 3 days after the first injection at which the mean Cmax was increased 1.5 to 2 times. The general tolerance of the compound was excellent. No relevant prolongations of the prothrombin time, of the activated partial thromboplastin time or of the bleeding time were observed. A re-bleeding phenomenon of the bleeding time incision, probably related to friability of the haemostatic plug, occurred in 3 subjects treated with the highest dose regimens: single injection of 26.6 mg (20,000 anti-Xa IU) (young volunteers) and repeated injections of 11.4 mg (8,000 anti-Xa IU) once a day for 7 days (elderly volunteers). At these times, plasma NP concentrations were between 2.9 and 3.6 micrograms.ml-1 (2 and 2.5 anti-Xa IU.ml-1).

Efficacy of a synthetic pentasaccharide, a pure factor Xa inhibitor, as an antithrombotic agent--a pilot study in the setting of coronary angioplasty.

AIM OF THE STUDY: To assess the antithrombotic properties of SR90107/ORG31540. a sulfated pentasaccharide, which enhances specifically antithrombin III mediated inactivation of factor-Xa, in a clinical setting known to promote arterial thrombosis, i.e. coronary angioplasty. METHODS AND RESULTS: Percutaneous transluminal coronary angioplasty (PTCA) was carried out with conventional balloons with a single 5 min intravenous infusion of 12 mg pentasaccharide, and 500 mg intravenous aspirin. Heparin was not allowed before, during PTCA, and within 24 h after PTCA. The primary end point was the rate of abrupt vessel closure during and within 24 h after the procedure. The sample size was set at 60 evaluable patients, in order to be able to conclude with a good level of confidence (>95%) that the abrupt vessel closure rate was less than 10%, if less than 3 abrupt vessel closures were observed. Seventy-one patients were included in the study, of whom 10 needed elective stenting, and were not considered as evaluable for efficacy. Two out of the 61 remaining evaluable patients experienced acute vessel closure during the study period [3.28%, 95% confidence interval (0.4%; 11.4%)]. No major bleeding occurred. The drug plasma concentrations reached 1.91+/-0.39 mg/], 10 min after pentasaccharide injection, and decreased on average to 1. 18+/-0.27 mg/l at 2 h, and to 0.36+/-0.11 mg/l at 23 h after administration of pentasaccharide. Activated clotting time (ACT) and activated partial thromboplastin (aPTT) time remained within normal range. Thrombin-antithrombin complex levels fell from 22+/-17.1 to 4.5+/-3.4 microg/ml, prothrombin fragment 1+2 levels decreased from 2.15+/-1.01 to 1.73+/-0.87, and activated factor VII levels decreased from 43.4+/-16.8 mU/ml to 18.9+/-7.3 mU/ml respectively from baseline to 2 h following injection of the tested drug. CONCLUSIONS: Administration of pentasaccharide led to the inhibition of thrombin generation without modification of aPTT and ACT. The rate of abrupt vessel closure was within range of rates reported in historical series. Thus we conclude that the anti-thrombotic activity of pentasaccharide, as shown in this pilot trial in the setting of coronary angioplasty, deserves further investigation.

Effect of clopidogrel on naproxen-induced gastrointestinal blood loss in healthy volunteers.

The effect of clopidogrel, a potent inhibitor of platelet aggregation, on naproxen-induced faecal blood loss was investigated in 30 healthy volunteers in a randomized, double-blind, placebo-controlled, two parallel treatment groups study. All subjects first received naproxen 250 mg b.i.d. during 7 days, after which they were randomly allocated to additionally receive either clopidogrel 75 mg once daily (n = 15) or matching placebo (n = 15) for 11 days. Faecal blood loss was measured by the 51Cr-labelled erythrocyte method during the last four days of each of the four study periods, i.e. baseline, treatment with naproxen alone, combined treatment and wash-out. Mean daily faecal blood loss was below 0.5 ml/day during the baseline period in both treatment groups and increased during treatment with naproxen alone to (mean +/- SD) 1.14 +/- 0.58 ml/day in the naproxen + placebo group and to 1.93 +/- 1.51 ml/day in the naproxen + clopidogrel group. Addition of clopidogrel to naproxen treatment was associated with an increase of the mean daily blood loss to 6.83 +/- 9.32 ml/day, which was statistically significantly higher than 1.75 +/- 1.40 ml/day observed during treatment with naproxen + placebo. During the wash-out period, mean daily blood loss decreased to 0.98 +/- 0.51 ml/day in the naproxen + placebo group and to 1.07 +/- 0.46 ml/day in the naproxen + clopidogrel group. Based on these results, it can be concluded that clopidogrel increases naproxen-induced gastrointestinal blood loss in healthy volunteers. Caution should therefore be called for when these drugs are coadministered.

Dietary exposure to perfluoroalkyl acids of specific French adult sub-populations: high seafood consumers, high freshwater fish consumers and pregnant women.

Perfluoroalkyl acids (PFAAs) are globally found in various media, including food and especially fishery products. In the present study, the dietary exposure to 15 perfluoroalkyl acids was assessed for 3 French adult populations, namely high seafood consumers, high freshwater fish consumers, and pregnant women. Purified food extracts were analysed by LC-MS/MS and PFBA, PFPA, PFHxA, PFHpA, PFOA, PFNA, PFDA, PFUnA, PFTrDA, PFTeDA, PFBS, PFHxS, PFHpS, PFOS and PFDS were monitored and quantified according to the isotope dilution principle. Under lower bound (LB) hypothesis (i.e. contamination values

Long-term activity of clopidogrel: a three-month appraisal in healthy volunteers.

The pharmacological effects of clopidogrel, administered once daily at a dose of 75 mg for 12 weeks, were monitored in a group of 35 healthy male subjects. The maximum intensity of platelet aggregation induced by 5 microM of ADP and the velocity of aggregation were determined before treatment, and at regular intervals during and after treatment. The long-term effect of clopidogrel on ADP-induced platelet aggregation was analyzed by comparing maximum aggregation intensities at baseline, at steady state (average for days 8, 10, and 12), and at week 12. The percent inhibition in maximum intensity from baseline was calculated for each time point. A paired, one-tailed Student's t-test was used to test for a change of less than 10% in the maximum intensity of platelet aggregation from steady state to week 12. Bleeding time was measured before treatment, on four occasions during treatment, and at follow-up. A sustained inhibition of platelet aggregation was observed from week 1 through the remainder of the 12-week treatment period, with return to baseline within 2 weeks after the end of treatment. Mean percent inhibition was 43+/-11.6% (+/-SD) at steady state and 39+/-17% at week 12. The difference in mean maximum intensity of aggregation between steady state and week 12, 3.28% (95% CI: [-1.46, 8.01]), was significantly less than the specified limit of 10% (p <0.001). No statistically significant difference between these two time points was observed for the velocity of aggregation. The bleeding time prolongation factor during treatment remained stable at 2.1. These results indicate that the activity of clopidogrel on the inhibition of platelet aggregation was maintained with long-term treatment.

[Circulating lupus-type anticoagulant, a risk factor for thrombosis by inhibition of protein C activation]. / L'anticoagulant circulant de type lupus, facteur de risque thrombotique par inhibition de l'activation de la protéine C.

The lupus anticoagulant is a risk factor of thrombosis. The non thrombogenic endothelial surface could be a target for the lupus anticoagulant. We have investigated the effect of purified immunoglobulins G of five patients with LA on the thrombomodulin activity of cultured human endothelial cells from umbilical cord vein. The rate of activation of purified protein C (PC) (30 nM) by the endothelial cells in the presence of thrombin (0.1 U/dish) has been measured by hydrolysis of substrate S 2366. Activated PC has been 7.37 +/- 0.78 pmoles X ml-1 X h-1 in the presence of buffer and 7.2 +/- 0.78 pmoles X ml-1 X h-1 in the presence of control IgG (2 mg/dish). Heat aggregated IgG did not induce any significant change. Patient's IgG lowered significantly the rate of PC activation (4.86 +/- 1.04 pmoles X ml-1 X h-1, p less than 0.001). Fab fragment from two of these patient's IgG displayed the same inhibition. Moreover neutralization of this effect was obtained by addition of phospholipids (70% phosphatidylcholine, 30% phosphatidylserine) in excess to patient's IgG. Activation of PC has been also performed using purified rabbit thrombomodulin and a similar inhibition by patient's IgG was found. These results seem to indicate that antibodies present in the IgG fractions containing LA could be directed against phospholipids associated to thrombomodulin activity. Reduction of PC activation if present in the patients with LA could play a role in the occurrence of thrombosis.

Inhibition of human endothelial cell proliferation by heparin and steroids.

Previous works have reported the controversial effects of heparin and steroids on angiogenesis. In this study, we investigated the effect of these compounds on human endothelial cell (EC) growth in vitro. An antiproliferative heparin activity was found in low human serum concentrations (2%). When EC were exposed to heparin (10(-6) M), their proliferation index was reduced in the presence of endothelial cell growth factor added 6 hours or more later. These results suggest that there is an intracellular effect of heparin which reduces 3H-methylthymidine uptake. Hydrocortisone acetate and tetrahydroS induced inhibition of EC growth in a dose-dependent manner. Steroids inhibited proliferation of EC in culture medium in the presence or the absence of growth factor and in different human serum concentrations. These results suggest a possible synergistic antiangiogenic action of heparin plus steroids.

Native vs. damaged milk fat globules: membrane properties affect the viscoelasticity of milk gels.

The storage modulus G' of rennet and acid milk gels filled with milk fat globules was measured as a function of the fat globule surface composition (native milk fat globule membrane, caseins and whey proteins, or a mixture of the three due to mechanical treatments) and surface area (i.e., the fat globule size). By different technological procedures, it was possible to obtain fat globules of constant surface composition but various sizes, and vice-versa, which had never been done. For both rennet and acid gels, a critical fraction of the fat globule surface covered by caseins and whey proteins was identified (approximately 40%), beyond which G' increased. Below this threshold, the gel viscoelasticity was unaffected by mechanical treatments. When the diameter of native milk fat globules decreased, the G' of rennet gels increased slightly, whereas that of acid gels decreased sharply. For both types of gels, G' increased when the diameter of partially disrupted fat globules decreased. For recombined globules completely covered with caseins and few whey proteins, G' increased with fat globule surface area for rennet gels whereas it decreased for acid gels. With the help of confocal microscopy and in the light of general structural differences between rennet and acid gels, a mechanism is proposed for the effect of fat globule damage and diameter on G', depending on the interactions the globules can undergo with the casein network.

Single-dose pharmacodynamics of clopidogrel.

The inhibition of platelet aggregation by clopidogrel, a novel platelet ADP-receptor antagonist, was evaluated in healthy male volunteers in two single-dose studies. In one study, 10 subjects received, in increasing order, single doses of 100, 200, 400 and 600 mg of clopidogrel or placebo in five study periods, according to a randomized, doubleblind, protocol design. In the second study, 12 subjects received a single 400 mg dose of clopidogrel as capsules and as tablets, according to an open-label, randomized, crossover design. The interval between the two administrations was seven days. Platelet aggregation induced by ADP (2, 5 and 10 microM) and by collagen (0.5 and 1 microg/mL; rising-dose study only) was assessed from blood samples collected over a period of 24 hours to 72 hours postdose. The inhibition of platelet aggregation was expressed as the mean percent change from baseline in maximum platelet aggregation. The effect of clopidogrel on bleeding time was also assessed. Clopidogrel induced a statistically significant inhibition of ADP-induced platelet aggregation at all doses. With 5 microM of ADP, the inhibition was dose-related up to a dose of 400 mg, with no further increase at a dose of 600 mg. At 2 hours, mean inhibition ranged from 12+/-6% (100 mg) to 42 +/-6% (400 mg), and at 24 hours, it ranged from 17+/-7% (100 mg) to 43+/-9% (400 mg). After 400 mg, the inhibition of platelet aggregation remained stable from 2 hours up to 72 hours, with mean percentages of inhibition ranging from 49 to 39%. Clopidogrel only showed a slight-to-moderate inhibitory effect on collagen-induced platelet aggregation. A mean bleeding time prolongation of 1.7 was observed 5 hours after the 400 mg and 600 mg doses; it was statistically significant only following the higher dose. Individual bleeding time prolongations ranged from 1 to 2.85. Clopidogrel was well tolerated at all doses. The results of these studies were part of the rational for the choice of the loading dose.

Prolonged heparin administration during clopidogrel treatment in healthy subjects.

The potential pharmacological interaction between clopidogrel and heparin was assessed in a randomized, placebo-controlled study carried out in 12 healthy male subjects. Clopidogrel 75 mg once daily or placebo was given in a randomized fashion for 12 days during two periods separated by a 21-day washout period. Sodium heparin was administered as a prolonged intravenous infusion at a starting dose of 300 IU/kg/24 hours then adjusted so as to maintain the activated partial thromboplastin time (APTT) ratio for subject versus control within the therapeutic range of 1.7 to 2.3 for 4 days from day 9 through day 13 of each period. For each period, the following main parameters were measured: total heparin consumption; APTT on days 1 and 8 to 12 before drug intake and on days 13, 14 and 26; bleeding time and platelet aggregation induced by 5 microM ADP on days 1, 8 to 10 and 12 before drug intake, and on days 14 and 26; APTT measured 3 times on day 9, i.e., before the start of heparin infusion, then 3 and 6 hours later. During day 9 to 13 period, the subject/control APTT ratio remained within the specified 1.7 to 2.3 range with no statistically significant difference between the clopidogrel and placebo treatments. The mean (+/-s.e.m.) volumes of heparin infused in each group were 81384+/-2793 IU and 79867+/-2788 IU in the clopidogrel and placebo groups, respectively. The 90% confidence interval of the mean heparin volume difference fell within the+/-10% interval of the placebo mean centered on zero. Bleeding time in the placebo group remained practically unchanged throughout the study. In the clopidogrel group, bleeding time prolongation factor was significantly increased from baseline to day 9, with clopidogrel alone then remained stable at 1.2 following co-administration of heparin until the end of treatment; hence, it was not modified by the coadministration of heparin. ADP (5 microM)-induced platelet aggregation was inhibited by 27+/-12% after 7 days of clopidogrel administration alone, and no significant further change was observed when heparin was coadministered. There were no adverse events that could be related to clopidogrel. In conclusion, under the study conditions, no interactions between clopidogrel and heparin were observed.

Interleukin 2 production by peripheral blood lymphocytes in allograft recipients during acute rejection episodes.

In this study we investigate the relationship between the Interleukin 2 (IL-2) yield produced by kidney allograft recipient's peripheral blood lymphocytes (PBL) under lectin stimulation and the occurrence of acute rejection episodes. PBL were harvested prospectively before grafting, after grafting in steady-state period, and at the onset of acute rejection episodes. In addition, we tested retrospectively the ability of PBL of recipients engrafted for more than 1 yr to produce IL-2. IL-2 levels were assessed on the IL-2-dependent CTL-L2 murine cell line. Our data show: 1) before grafting, hemodialysed patients (N = 14) produced normal IL-2 yield compared with healthy donors (N = 21); 2) the IL-2 secretion of PBL of recipients with good graft function (N = 18) is decreased markedly during roughly the first 12 months following transplantation (P less than 0.01); 3) when acute rejection crisis occurred during this time period (N = 24), a sharp and highly significant increment (P less than 0.01) in lectin-induced IL-2 production of recipient's PBL was seen. After 1 yr, the capacity to secrete IL-2 upon lectin stimulation tends to be restored. Finally, our data correlate rejection and high PBL-IL-2 secretion clearly at a time when recipients with well-functioning grafts have markedly impaired IL-2 secretion.

Increased interleukin II production during kidney allograft rejection.

We have studied the lectin-induced production of Interleukin II (IL-2) by peripheral blood leucocytes (PBL) obtained from recipients of kidney grafts. Pre-graft values (haemodialysed patients) are similar to those of healthy individuals. After grafting and during the first year the IL-2 levels produced by PBL from recipients with well functioning grafts are very low (p less than 0.001). Years after grafting the IL-2 yields tend towards restoration. At the onset of an acute rejection the IL-2 production rises significantly. These data show that an increase in IL-2 production may play a role in the rejection process.