Effects of N-acyl-2-hydroxymethyl aziridines on in vitro proliferative responses of human lymphocytes stimulated by mitogens.

Aziridines have been shown to possess marked immunotropic activity. The aim of this work was to study the in vitro effects of different concentrations of three novel aziridines, 2-hydroxy-methyl-1-(N-phtaloylglycyl) aziridine (aziridine 1), 2-hydroxy-methyl-1-(N-phtaloylalanyl) aziridine (aziridine 2) and 2-hydroxy-methyl-1-(N-phtaloylphenylalanyl) aziridine (aziridine 3), on the proliferative responses of human lymphocytes stimulated by mitogens (concanavalin A (Con A) and lipopolysaccharide (LPS)), and interleukin-2 (IL-2), interleukin-6 (IL-6) secretion. The results showed that aziridines 1 and 3 significantly stimulated the resting and Con A or LPS lymphocyte proliferation at concentrations between 1 micromol/l and 1 mmol/l, in a dose-dependent manner, the action of aziridine 3 being the highest. They also increased IL-2 and IL-6 secretion. However, aziridine 2 had no effect on the resting lymphocyte proliferation in the absence of mitogens, at any concentration used, reduced Con A-stimulated T lymphocyte proliferation and LPS- stimulated B lymphocyte proliferation in a dose dependent manner and diminished IL-2 and IL-6 production. None of the three aziridines affected cell viability. In conclusion, the three aziridines used in this study displayed immunomodulatory properties. Aziridines 1 and 3 are potentially immunostimulant while aziridine 2 is immunosuppressive and could be used to provide nonspecific cell-mediated immune responses.

[Fish proteins moderate triacylglycerols, activities of hepatic triacylglycerol lipase and tissue lipoprotein lipases in hypertensive and diabetic rats]. / Les protéines de poisson modulent le métabolisme des triglycerides ainsi que les activités de la triglycéride lipase hépatique et des lipoprotéines lipases tissulaires chez le rat spontanément hypertendu (SHR) et le SHR diabétique.

Fish proteins effect compared with casein was determined on triacylglycerols (TG) metabolism and activities of hepatic triacylglycerol lipase (HTGL) and tissue lipoprotein lipases (LPL), in SHR and diabetic SHR. Two groups of rats (n=24) were fed, 2 months, diet containing 20% casein (CAS) or 20% fish proteins (FP). One month before sacrifice, diabetes was induced in one group of rats by a single intraperitoneal injection of streptozotocin (STZ) (60 mg/kg of body weight). FP vs. CAS showed a significant decrease of TG concentrations in plasma (-41%) and liver (-66%), in SHR-STZ. VLDL-LDL mass, which represented the amount of apolipoproteins, TG, phospholipids (PL), unesterified cholesterol (UC), and cholesteryl esters (CE), decreased by 21 and 16% with FP vs. CAS, in SHR and SHR-STZ, respectively, and was concomitant with its low TG. Indeed, TG values was 1.35- and 1.82-fold lower in SHR and SHR-STZ, respectively. In HDL2, a TG diminution of 13% was noted, in SHR with FP vs. CAS. In SHR-STZ with FP, TG and PL were enhanced by 11% and 27%, respectively compared to SHR. HTGL activity decreased by 22% in SHR fed FP compared to those fed CAS. In SHR-STZ with CAS vsSHR, this activity was decreased by 21%. LPL activity in heart was enhanced by 19% in SHR-STZ vs. SHR with FP, when that of muscle was diminished 1.5-fold in SHR with FP vs. CAS and 1.8-fold in SHR-STZ vs. SHR with CAS. Adipose LPL activity was 1.36-fold higher in SHR with FP than CAS. In conclusion, it appears that fish proteins have a hypotriglyceridemic effect, which the mechanism can differ in SHR or SHR-STZ. It may be of interest to propose these fish proteins as lipid metabolism regulator in diseases with hypertriglyceridemia.

[Gestational or essential hypertension in pregnant women limits the capacity to stock triglycerides by the placenta despite raised lipoprotein-lipase activity]. / L'hypertension gravidique ou essentielle chez la femme en gestation limite la capacité de stockage des triglycérides du placenta malgré une activité lipoprotéine lipase élevée.

Placenta lipoprotein lipase (LPL) activity as well as serum VLDL and placenta lipids composition were determined in pregnant hypertensive women at term. 46 patients aged from 29 +/- 2 years with gravidic hypertension (HTA-G) and 38 patients with essential hypertension (HTA-E) aged 30 +/- 1 years were compared with 20 normotensive women aged 27 +/- 1 years. Serum triacylglycerols (TG) concentrations were 1.3-fold higher in the both hypertensive patients compared with controls. However, serum phospholipids (PL) and total cholesterol (TC) values were similar in the three groups. VLDL mass and their apolipoproteins, unesterified cholesterol (UC) and cholesteryl esters (CE) contents were significantly increased in hypertensive women compared with controls. In HTA-G and HTA-E patients, respectively. TG-VLDL concentrations were increased by +43% and +36% compared with those of controls (P < 0.01). In placenta, the values were lower 2.2- and 1.9-fold for TG, 2.8 and 2.5-fold for PL and two- and threefold for TC, in HTA-G and HTA-E patients than in controls. Placenta LPL activity was 2.7-fold higher in HTA-G and HTA-E patients compared with that of controls. In conclusion, although placenta LPL activity is higher it is not permit a decrease of serum TG-VLDL on the one hand, and an increase of placenta ability in TG storage on the other hand.

Implication of acyl chain of diacylglycerols in activation of different isoforms of protein kinase C.

We synthesized diacylglycerols (DAGs) containing omega-6 or omega-3 polyunsaturated fatty acids [i.e., 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG), 1-stearoyl-2-docosahexaenoyl-sn-glycerol (SDG), and 1-stearoyl-2-eicosapentaenoyl-sn-glycerol (SEG)] and assessed their efficiency on activation of conventional (alpha, beta I, gamma) and novel (epsilon, delta) protein kinase C (PKC). SAG exerted significantly higher stimulatory effects than SDG and SEG on activation of PKC alpha and PKC delta. Activation of PKC beta I by SEG and SDG was higher than that by SAG. Activation of PKC gamma did not differ significantly among DAG molecular species. Addition of SAG to assays containing SEG and SDG exerted additive effects on activation of alpha and epsilon, but not on beta I and gamma, isoforms of PKC. SDG- and SEG-induced activation of PKC delta was significantly curtailed by the addition of SAG. Three DAG species significantly curtailed the PMA-induced activation of beta Iota, gamma, and delta, but not of alpha and epsilon, isoforms of PKC. Our study demonstrates for the first time that in vitro activation of different PKC isoenzymes vary in response to different DAG species, and one can envisage that this differential regulation may be responsible for their in vivo effects on target organs.

A freeze-fracture and thin section study of the interaction of blood platelets with native type I collagen fibrils.

The membranes of non-activated and activated human blood platelets have been studied with freeze-fracture and conventional electron microscopy. Aggregated platelets activated by ADP or by native collagen fibrils did not show any intramembrane particle clustering. No intramembrane modification is detectable at the contact regions with collagen fibrils. However, a local densification of the cortical cytoplasm is noted in thin sections where platelets make contact with collagen fibrils. These results suggest that either the membrane receptor for collagen is not visualized by freeze-fracturing or that, as suggested previously, the binding site on collagen may not have a specificity and affinity as high as expected from a conventional receptor-ligand interaction.

Effects of nefopam on visual tracking.

The effects of 2 doses of nefopam, d-amphetamine, pentazocine, and placebo were studied in healthy male sleep-deprived volunteers to determine whether the drugs improved or impaired coordination and whether they induced subjective effects. A critical tracking task was used to study hand-eye coordination. D-amphetamine, 10 mg orally, significantly improved tracking performance and made subjects feel better able to perform tasks but more anxious. It also made them feel more alert, steady, sociable, and strong. Pentazocine, 45 mg intramuscularly, caused deterioration in tracking performance and was followed by reports of depression, gloominess, dreaminess, nausea, and injection site pain. There was no significant change in tracking performance or subjective effects after both doses of nefopam and placebo.

Hepatic storage and transport of n-3 and n-6 polyunsaturated fatty acids by very-low-density lipoproteins in growing rats fed low- or adequate-protein diets with sunflower, soybean, coconut, and salmon oils.

Protein and essential fatty acid (EFA) deficiencies may both occur in chronic malnutrition and have common symptoms. To determine the interactions between dietary protein intake and EFA availability, rats were fed purified diets containing 20% or 2% casein and 5% as one of four fats (sunflower, soybean, coconut, or salmon oil) that differed particularly in their n-6 and n-3 polyunsaturated fatty acids (PUFAs). Protein malnutrition enhanced hepatic triacylglycerol and cholesterol concentrations while decreasing hepatic protein and phospholipid contents and mass and components of very-low-density lipoprotein (VLDL). The ratio of PUFAs to saturated fatty acids (SFAs) was consistently depressed by protein malnutrition in liver and VLDL triacylglycerol and phospholipid. Total n-6 and n-3 fatty acids were diminished by protein malnutrition, except with salmon oil, with which a decrease in 20:5n-3 was compensated for by an increase in 22:6n-3. The ratio of 20:4n-6 to 18:2n-6 was enhanced in liver phospholipid and VLDL triacylglycerol, and modified little in liver triacylglycerol. Generally, the ratio of 20:3n-9 to 20:4n-6, an index for EFA deficiency, was raised with protein malnutrition in liver triacylglycerol and phospholipid and in VLDL triacylglycerol. The extent of changes in each fatty acid proportion varied according to the oil fed. Overall, VLDL-apolipoprotein concentrations were, in general, strongly reduced with protein malnutrition. In conclusion, protein malnutrition may accelerate marginal EFA deficiency and decrease long-chain PUFA bioavailability and thus increase EFA requirement.

Drugs effect on platelet survival time: comparison of two pyrimido-pyrimidine derivatives in patients with aortic or mitral replacement.

A prospective randomized trial of the effects of 2 antiplatelet aggregating drugs, dipyridamole (375 mg/d), a related substance RA 233 (1500 mg/d) and placebo, concomitantly with oral anticoagulants, was carried out in patients with prior valvular replacement. The study was aimed to determine effect on platelet survival time (PST) of these 2 agents. The trial sample consisted of 40 males and 15 females aged 40-70 years (average 53 years). 32 received Björk-Shiley valve in aortic position, 23 underwent mitral valve replacement: 3 with Cooley-Cutter, 11 with Lillehei-Kaster 500 and 9 with Starr-Edwards 6120 prostheses; 28 patients had aortic stenosis, 21 aortic insufficiency. All the PST measured after 3 months of treatment were within normal ranges and not different between placebo, dipyridamole or RA 233 treated subjects: averages in days were, respectively, 7.49, 7.11 and 6.88. The present study did not support the claim that modern valve prosthesis could lead to a shortened PST.

In vivo release of a heparin-like factor in dogs during profound hypothermia.

Experimentally induced hypothermia (20 degrees C) for 60 minutes in dogs provokes a significant decrease in the platelet count, which reverses during subsequent rewarming, and the constant release of a heparin-like factor, which reacts as a specific inhibitor of factor Xa. This phenomenon is also rapidly reversible, and heparin values are not significantly different from control levels after 90 minutes of rewarming. The mean maximal concentration of heparin-like material is 0.54 U/ml, or about double control levels. Its half-life is approximately 90 minutes. The level of circulating antithrombin III was not modified during hypothermia and rewarming.

Immunization by bovine thrombin used with fibrin glue during cardiovascular operations. Development of thrombin and factor V inhibitors.

Brief case histories of three patients aged 58, 38, and 44 years are reported. All underwent cardiovascular operations. Subsequently hemostasis test abnormalities developed between the seventh and eighth postoperative days after exposure to bovine thrombin used with fibrin glue. These were characterized by an increased activated partial thromboplastin time (64 to 147 seconds), prothrombin time (19 to 24 seconds), bovine thrombin time (> 120 seconds) and a markedly reduced factor V level (< 10% in two patients and 16% in the third patient). A patient plasma dilution of 1 in 200 with a normal plasma pool was necessary to correct bovine thrombin time. No fast-acting or progressive inhibitor against factor V could be detected by coagulation tests, and fresh frozen plasma perfusion had no effect. Plasmapheresis was performed preventatively to avoid bleeding, and factor V levels stabilized at around 50% after two to four exchanges. Immunologic studies showed that the inhibitors were directed not only against bovine factors but also against human ones. Therefore factor V decrease could have been the result of rapid clearance from the circulation of complexes formed with a nonneutralizing inhibitor that is not detected by clotting tests. These antibodies were purified by standard methods and immunoaffinity. Fast immunization could be explained by a prior sensitization to bovine thrombin exposure during previous operations. It is suggested that bovine thrombin used with fibrin glue contains small amounts of factor V and may be responsible for these abnormalities. This is in agreement with previous literature reports. However, these described neutralizing factor V inhibitors, which were easily detected.

Effect of iodinated contrast media on blood clotting.

Recently, blood clot formation in catheters used for the injection of nonionic contrast media (CM) during angiography has been reported as being due to activation of hemostasis in the catheter. However, CM exhibit inhibitory properties regarding coagulation and platelet functions. The effect on blood clotting of iohexol, iopamidol, ioxaglate, diatrizoate, and ioxitalamate at a ratio of 10% v/v with nonanticoagulated human whole blood was evaluated using the kinetics of fibrinopeptide A (FpA) generation. Blood aliquots were taken every 2 minutes until blood clot occurred. Two groups of contrast media were identified: (1) iohexol and iopamidol, which increased the clotting time, and (2) ioxaglate, diatrizoate, and ioxitalamate, for which all clotting times were over 30 minutes and no FpA generation occurred.

In vitro interactions of gadolinium DOTA meglumine and gadolinium DTPA meglumine on hemostasis.

The interactions of two gadolinium complexes (Gd-DOTA meglumine and Gd-DTPA meglumine) with hemostatic function have been analyzed using: (1) coagulation reactions (extrinsic and intrinsic pathways and fibrinoformation) and (2) platelet function investigations (aggregation, release of Ca++ and ATP after stimulation with collagen 2.5 micrograms/mL). The data obtained with Gd-DTPA meglumine (Mgl) exhibited a significant increase of the intrinsic coagulation pathway and a delay in fibrinoformation, although there is no alteration of the effect of thrombin on fibrinogen (fibrinopeptid A determinations). Platelet aggregation and release are moderately modified. In contrast, Gd-DOTA Mgl exerts no effect on the coagulation system and only minor effects on platelet functions. It is suggested that at least one mechanism involves the complexation of ionized calcium because Gd-DTPA Mgl and Gd-DOTA Mgl complex, respectively, 45% and 23% of ionized calcium in plasma. However, other mechanisms such as an alteration of fibrin polymerization are not unlikely.

In vitro biocompatibility evaluation of a heparinizable material (PUPA), based on polyurethane and poly(amido-amine) components.

The biocompatibility of a new heparinizable material based on polyurethane and poly(amido-amine) (PUPA) was evaluated both in the heparinized and non-heparinized forms. The quantity of heparin present on the material was measured using radiolabelled heparin and biological tests. Heparin release in plasma from heparinized PUPA was investigated using in vitro methods. The behaviour of PUPA towards cellular and plasmatic blood components was studied. The influence of sterilization on the cytocompatibility response of both heparinized and non-heparinized PUPA was investigated; gamma-rays were found to be a suitable method of sterilization as no toxic response was noticed.

Fetal macrosomia related to maternal poorly controlled type 1 diabetes strongly impairs serum lipoprotein concentrations and composition.

AIMS: To determine the effects of fetal macrosomia related to maternal type 1 diabetes on the lipid transport system. METHODS: Serum lipoprotein concentrations and composition and lecithin:cholesterol acyltransferase (LCAT) activity were investigated in macrosomic newborns (mean birth weight, 4650 g; SEM, 90) and their mothers with poorly controlled type 1 diabetes, in appropriate for gestational age newborns (mean birth weight, 3616 g; SEM, 68) and their mothers with well controlled type 1 diabetes, and macrosomic (mean birth weight, 4555 g; SEM, 86) or appropriate for gestational age (mean birth weight, 3290 g; SEM, 45) newborns and their healthy mothers. RESULTS: In mothers with well controlled type 1 diabetes, serum lipids, apolipoproteins, and lipoproteins were comparable with those of healthy mothers. Similarly, in their infants, these parameters did not differ from those of appropriate for gestational age newborns. Serum triglyceride, very low density lipoprotein (VLDL), apolipoprotein B100 (apo B100), and high density lipoprotein (HDL) triglyceride concentrations were higher, whereas serum apo A-I and HDL3 concentrations were lower in mothers with diabetes and poor glycaemic control than in healthy mothers. Their macrosomic newborns had higher concentrations in all serum lipids and lipoproteins, with high apo A-I and apo B100 values compared with appropriate for gestational age newborns. In macrosomic infants of healthy mothers, there were no significant differences in lipoprotein profiles compared with those of appropriate for gestational age infants. LCAT activity was similar in both groups of mothers and newborns. CONCLUSION: Poorly controlled maternal type 1 diabetes and fetal macrosomia were associated with lipoprotein abnormalities. Macrosomic lipoprotein profiles related to poor metabolic control of type 1 diabetes appear to have implications for later metabolic diseases.

In vitro study of the inhibition of coagulation induced by different radiocontrast molecules.

The anticoagulant activity of seven intravascular radiocontrast molecules (RCM) was evaluated in different in vitro systems using citrated human plasma. Each RCM was tested in a concentration range of 5 to 50 mM. The thrombin time and the reptilase time showed a dose-dependent lengthening of fibrinoformation, the recording of fibrinoformation exhibited a significant delay of fibrin monomer generation and polymerization although the amplitude of the fibrinoformation was not decreased. The interfering effect with fibrin clot formation impairs also global coagulation tests and monospecific coagulation tests using fibrinoformation as the final step of the assay, but a possible interaction between RCM and some specific coagulation factors cannot be excluded. RCM potentiated the anti-thrombin action of heparin but the inhibition or delay of fibrinoformation is not related to an antithrombinic effect of contrast media. The thrombin amidolytic activity is not modified by RCM but the generation of FpA is delayed and decreased. The ultrastructure of the fibrin clot is not altered at the end of the polymerization.

Immunolocalization of factor V in adult and fetal rat liver.

Immunolocalization of the factor V was performed in adult and fetal rat liver by means of an indirect peroxidase labelling. This could be done owing to the production in our laboratory of a mono-specific antiserum anti rat factor V. In all the cases (perfused and non-perfused adult liver and fetal one) the observation of the sections has revealed an intense circular or granular labelling into all the hepatocytes whatever was their localization in the hepatic lobule. Hepatic endothelial cells seemed to be negative for factor V and this aspect of our results was discussed.

Biosynthesis of factor V by normal adult rat hepatocytes.

The synthesis of coagulation factor V was investigated in isolated rat hepatocytes maintained in long-term primary culture. Two culture conditions were compared. A clotting assay and an immunoprecipitation experiment with rabbit anti rat factor V IgG were used to demonstrate not only the presence of factor V in the cells but also active secretion into the culture medium. Both the inhibition of the clotting reaction in presence of the antibody and absence of thrombin in culture media confirmed the specificity of the clotting assays. Electron microscopic examination located factor V in the endoplasmic reticulum and Golgi apparatus of hepatocytes in common with other liver specific plasma proteins. Examination of liver tissue sections confirmed the production of factor V in hepatocytes but not in hepatic endothelial cells although it did not exclude a transit pathway of factor V through these cells. Addition of Russell viper venom factor V activating enzyme to the culture medium had no effect on the factor V activity. In contrast, treatment of cell extracts did increase the coagulant activity. This suggests that hepatocytes contained principally an unactivated form or procofactor, whereas factor V present into the culture medium was mainly in an activated form. These data provide evidence for synthesis and secretion of an hepatocytic factor V.

Contrast agents used for excretory urography impair platelet function in human patients.

The platelet function was investigated in 65 patients submitted to diagnostic excretion urography (injection of 60 ml contrast medium). Blood sampling was performed before (T0) 90 seconds after (T1) and 30 minutes after (T2) injection of the radiocontrast molecule (RCM). Five RCM of different osmolality ionicity and nature of the lateral chain have been tested. Platelet aggregation, platelet release of ATP, osmolality, total calcemia and ionized calcium level were determined on each plasma sample as well as RCM concentration at T1 and T2. Decrease (20 - 40%) of platelet aggregation occurred at T1, whichever platelet aggregating agent (ADP, collagen, Ristocetin or thrombin) were used (significant after Iopamidol 300 and Na Meg Ioxaglate). Platelet release of ATP induced by collagen was also decreased or delayed. These changes were rapidly reversible and a tendency to improvement was observed at T2. Among the five RCM tested, one (Na Diatrizoate) might be a proaggregating agent. No changes of osmolality occurred in the plasma and no correlation could be established between RCM concentration and platelet inhibition. A pathogenic hypothesis is suggested by the significant fall of total and ionized calcium level after RCM injection.

Thromboresistance of bulk heparinized catheters in human.

Bulk heparinized catheters (1 mm internal diameter) containing 10% heparin ionically bound, were tested in four human volunteers. Catheters containing 0% and 10% heparin were compared in each individual using ultrasound microflow velocimetry, permeability test, sequential determinations of activated partial thromboplastin time, heparin levels and generation of Fibrinopeptide A, beta thromboglobulin and Platelet factor 4. Although the release of heparin expressed by its anti-IIa activity is of similar range in the four individuals the release of anti-Xa activity is variable and generally of greater magnitude, suggesting a privileged migration of low molecular weight components of heparin. These antiproteasic activities of heparin are sufficient to inhibit fibrin formation and blood coagulation despite their relative inability to prevent platelet activation.

Effect of ioxaglate--an ionic low osmolar contrast medium--on fibrin polymerization in vitro.

Ioxaglate, an iodinated contrast agent, decreases the rates of fibrin clot formation induced by thrombin or reptilase. This effect is not related to an increase in the ionic strength of the medium since a specific control of equivalent composition does not induce such variation. The concentration of ioxaglate which led to a 50% decrease of the control clot turbidity induced by thrombin was 17.5 +/- 2 mM. Macroscopically, clots formed with ioxaglate were larger and less turbid than the isotonic control. An increase in fibrin fibre diameters and a decrease in their densities were observed. During the fibrin polymerization process, all the fibrinogen was converted into fibrin, as for both the control and ioxaglate quantitative analysis of clots and supernatants showed (1) an identical quantity of FpA in clot supernatants, (2) the same quantities of protein incorporated into clots, and (3) no trace of fibrin monomers in the clot supernatants. Furthermore, dissolution in urea of clots formed in the presence of ioxaglate occurred more rapidly than in the control. Total incorporation of fibrinogen into clots, associated with a decrease in clot turbidity, indicated the existence of a qualitative abnormality in the construction of the three-dimensional fibrin structure. Using differential scanning calorimetry, it was observed that the two domains (D and E) of fibrinogen were modified by ioxaglate, showing the absence of specificity in the interaction between ioxaglate and a particular domain.