Synthesis and secretion of apolipoproteins by pig intestinal mucosa in organ culture. Lack of inhibition of apolipoprotein A-I secretion by colchicine.

Pig duodeno-jejunal mucosa was maintained in organ culture for up to 24 h in Eagle's minimum essential medium containing 10% foal serum. Viability was controlled by determination of alkaline phosphatase and sucrase activity in the tissue. [14C]Leucine incorporation into proteins decreased 3-fold between 2 and 24 h. Newly synthesized secreted proteins were analyzed by SDS-polyacrylamide gel electrophoresis of the whole culture medium. Apolipoprotein A-I specifically measured by immunoelectrophoresis represented 10-20% of newly secreted proteins. Only 10% of apolipoprotein A-I secreted was recovered with the lipoprotein fraction (d less than 1.21). Recombination of the medium with porcine lipoproteins or DMPC vesicles prior to ultracentrifugation allowed, respectively, the recovery of 40 and 80% of apolipoprotein A-I secreted. The lipoprotein fractions also contained some apolipoproteins B and C and, after DMPC recombination, an apolipoprotein of Mr 45 000, most likely apolipoprotein A-IV, representing about 3.5% of newly secreted proteins. The d greater than 1.21 fractions all contained a high Mr protein, identified as IgA, and an unidentified protein of Mr approximately 45 000. The addition of colchicine (125 microM) to the culture medium did not significantly modify either tissue enzyme activities or [14C]leucine incorporation. It reduced total secretion by about 40% between 2 and 8 h of incubation, without interfering with apolipoprotein A-I secretion, which then represented up to 35% of secretion products. This raises the question of the mode of secretion of apolipoprotein A-I, which may be related to the high proportion of its which is secreted free.

Determination of apolipoprotein A-I synthesis in intestinal explants from fetal and neonatal rats.

The ability of rat intestine and liver to synthesize the main constitutive apoproteins of HDL (apolipoproteins (apo) A-I, A-IV and E) was studied by incorporation of [3H]leucine in vitro at different stages of perinatal life. In both organs, apoprotein synthesis was barely detectable at day 18 of gestation; it was initiated 2 days before the end of gestation. Apo A-I synthesis leveled off at birth in the intestine but kept increasing in the liver during suckling. Intestinal apo A-IV and hepatic apo E synthesis became stable 5 days after birth. Hormonal determination of apo A-I synthesis was examined at different ages in jejunum cultured for 48 h in vitro in the presence of effectors. The addition of dexamethasone to the culture medium was without effect on intestine explanted either at day 18 of gestation or at different postnatal ages (0, 2 and 5 days), but induced the specific stimulation of apo A-I synthesis at day 20 of gestation. At this stage, triiodothyronine alone was ineffective, whereas it enhanced the dexamethasone-induced stimulation. Apo A-I synthesis remained unaffected by insulin alone or combined with the glucocorticoid. Administration of cortisone acetate to pregnant rats from day 14 of gestation onwards resulted in a stimulation of apo A-I synthesis only when it was prolonged after the 20th day of gestation. No effect of dietary substrates could be obtained in vitro. It is concluded that glucocorticoids specifically potentiate prenatal apo A-I synthesis in the rat intestine but that their action is limited to the days immediately preceding birth. They cannot induce early maturation nor stimulate existing synthesis.

Oleic acid-rich fats increase the capacity of postprandial serum to promote cholesterol efflux from Fu5AH cells.

Cell cholesterol efflux to serum is stimulated after an oral fat load. The impact of meal fatty acid composition was explored by measure of serum promoted cholesterol efflux from Fu5AH cells after ingestion of 4 different fats: sunflower (Sf), oleic-sunflower (Ol), a mixed oil (Mx), and beef tallow (Bt). High density lipoprotein (HDL)2 and HDL3 were isolated and analyzed. Cholesterol efflux increased regularly after Ol (P<0.05 at 4 h and P<0.02 at 8 h), and 8 h after Mx (P<0.02) or Bt (P<0.05), but not after Sf. Percent HDL3 phospholipids increased after Ol (P<0.05 at 6 h and P<0.01 at 8 H) and 8 h after Mx (P<0.01). After Ol, variations in efflux and percent phospholipids in HDL3 (but not HDL2) were positively correlated (r=0.929; P=0.007 at 6 h). Using HDL3, efflux increased 6 h after Ol (P<0.05) but not after Sf, and efflux was correlated with HDL3 phospholipid concentration in medium (r=0.913; P=0.011). Thus postprandial increase in cholesterol efflux in influenced by ingested fats in relation to increased phospholipid availability on HDL3. The protective effect of monounsaturated fatty acids against atherogenesis might be partly mediated by an enhanced ability of postprandial serum to accept cell cholesterol.

Binding of HDL to basolateral membranes of the renal cortex. Evidence for two components in the HDL-membrane association.

The binding of porcine 125I-HDL to purified basolateral membrane fractions isolated from pig kidney cortex displays two categories of sites, one with high affinity ((Kd = (3.0 +/- 0.7) x 10(-9) M) and low capacity (Bmax = 52 +/- 32 ng/mg proteins) another with low affinity (Kd = (5.3 +/- 0.7) x 10(-8) M) but a higher capacity (Bmax = 795 +/- 115 ng/mg proteins). Binding was competitively inhibited to the same extent by unlabeled HDL from swine, human or rat, demonstrating an absence of species specificity. Porcine LDL partially competed for binding even in the presence of 30 mM EDTA which prevents apo B/E specific binding. Membrane proteins solubilized with CHAPS were analyzed by electrophoresis followed by ligand blotting using porcine 125I-HDL and 125I-apoAI-HDL to show that HDL bound to two proteins of respective molecular masses 120 +/- 2 and 95 +/- 9 kDa. 125I-apoAI associated mostly with the 95 kDa protein. A 100-fold excess of unlabeled HDL greatly decreased binding to the 95 kDa protein but less to the 120 kDa protein. We conclude that part of HDL binding occurs through the lipid moiety, while another is the result of a specific interaction between apoAI and a membrane protein of 95 kDa.

[Cell therapy product (Dermagen) for treatment of hard-to-heal skin wounds]. / Produit de thérapie cellulaire Dermagen pour le traitement de plaies difficiles.

Epidermal sheets were developed for the treatment of hard-to-heal skin wounds. Products currently marketed in France, e.g. Epibase, are very efficient but not totally satisfactory in cases of very deep dermal injury which often occurs in burn victims. A function dermis or "dermis equivalent" association a three-dimensional matrix (dermal substrate) and allogenic dermal fibroblasts able to secrete numerous cytokines, growth factors, and protease inhibitors, was developed in our biotechnology laboratory using good manufacturing standards (ISO9000). This cell therapy product which is histologically and physiologically close to normal dermis is obtained after culturing fibroblasts obtained from a microbiologically and virologically validated cell bank for 21 days in a patented collagenous sponge. Our laboratories successfully developed a freezing process allowing preservation of the product at -80 C. This allows us to perform high-performance quality control of the batches. The product is easy to use and instructions can be described in a few lines. When clinicians order one or more Dermagen sheets, depending on the wound area to treat, Dermagen is thawed and culturel at 37 C for 48 to 72 hours. Metabolic activity is recovered and the product is then packaged in a specific blister with a semi-solid medium to ensure good viability during transportation. In some indications, Dermagen acts like a true transplant and in others like a biological dressing secreting numerous mediators such as growth factors or matrix proteins.

Serum lipids and apolipoproteins in the rat refed after starving: influence of the molecular form of nitrogen (protein, peptides, or free amino acids).

Efficient treatment of deep denutrition should promote the restoration of normal intestinal villous structure and the return to a positive nitrogen balance. To determine whether the plasma measurement of lipoproteins could serve as sensitive indexes of villous architecture and/or nitrogen balance, these parameters were compared in rats starved for three days and refed three types of diets containing either whey proteins (WP), whey protein hydrolysate (WPH), or amino acids, known to differ in their capacity to promote restoration of normal villous architecture. Starvation lowered the concentration of triglycerides and phospholipids but not cholesterol. Apolipoprotein AI and AIV concentrations were also significantly lowered (30% and 40%, respectively), but ApoE was significantly increased by 40%. Upon refeeding with all three diets, plasma lipids progressively returned to control values except for triglycerides, which were significantly elevated by the protein and peptide diets. Apoprotein AI continued to decrease for 24 hours on the peptide and amino acid diets. Control levels were restored in all groups after 48 hours. ApoAIV increased progressively in parallel with the restoration of the intestinal mucosa; after 48 hours of refeeding, plasma concentrations of apo AIV were significantly correlated with jejunal villous height and protein content (P less than .01). ApoE was depressed below control levels in the WP and WPH groups at 24 and 48 hours and restored only after 96 hours. Because ApoE was affected, both in the fed state and during refeeding by the form of dietary nitrogen, it may prove to reflect nitrogen balance and/or insulin: glucagon balance.(ABSTRACT TRUNCATED AT 250 WORDS)

[Anisoylated plasminogen streptokinase complex during the acute phase of myocardial infarction. Results of a multicenter double-blind study versus heparin]. / Le complexe streptokinase plasminogène acylé à la phase aiguë de l'infarctus du myocarde. Résultats d'une étude multicentrique en double aveugle versus héparine.

Two hundred and thirty-one patients admitted to hospital within 5 hours of the onset of symptoms of a primary myocardial infarction were randomised into 2 groups: one received thrombolytic therapy [anisoylated plasminogen streptokinase activator complex (APSAC): 30 IU in 5 minutes] and the other was given conventional heparin therapy (5,000 IU). Heparin was given to both groups 4 hours later (500 IU/kg/day); the APSAC (N = 119) was identical with respect to age, location of infarct, Killip classification, delay before randomisation (188 +/- 62 minutes). Coronary angiography and ventriculography were performed after 3.4 +/- 1.2 days, and angioscintigraphy and myocardial scintigraphy after 19 +/- 2.5 days to determine the size of the infarct and the quality of left ventricular function. Coronary patency was much higher in the APSAC group (77%) than the heparin group (37%) (p less than 0.001). The angiographic ejection fraction was significantly greater in the thrombolytic group than in the heparin group (53 +/- 13% vs 47 +/- 12%, p less than 0.002), the difference being statistically significant in the anterior and inferior infarct subgroups. At the third week, the difference remained significant in the anterior infarct subgroup: a 31 per cent reduction in necrosed myocardial mass was observed in the APSAC group (33% in anterior infarcts: p less than 0.05 and 16% in inferior infarcts: NS). The limitation of infarct size explained the smaller reduction in left ventricular systolic function (r = 0.73; p less than 0.01). The hospital and one year mortality was comparable in the two groups which was not surprising given the small number of patients.(ABSTRACT TRUNCATED AT 250 WORDS)

[Combination of slow-release diltiazem and a beta-blocker in arterial hypertension. 2 cases of cardiogenic shock with severe bradycardia]. / Association de diltiazem à libération prolongée et d'un bêta-bloquant dans l'hypertension artérielle. Deux cas de choc cardiogénique avec bradycardie extrême.

Patient 1 received carteolol and captopril for hypertension. Three days after a slow-release diltiazem preparation (300 mg) had been introduced, he developed cardiogenic shock and sinus bradycardia (heart rate: 30/mn) with acidosis and severe hyperkaliemia. He was successfully treated by temporary pacing and dobutamine. Patient 2 had received sotalol and captopril for several years. Twelve hours after slow release diltiazem had been added, he was found in cardiogenic shock and extreme bradycardia with wide QRS, acidosis and hyperkaliemia. He died one hour later despite intensive emergency treatment. Concomitant use of beta-blockers and calcium channel blockers has been reported in patients suffering of severe coronary heart disease. However, several adverse reactions similar to our cases have been described. Slow-release diltiazem should be avoided in hypertensive patients taking beta-blockers.

[Autologous epidermal sheets production for skin cellular therapy]. / Production de feuillets épidermiques autologues pour la thérapie cellulaire cutanée.

Cell therapy is becoming a very interesting solution to replace degenerated or damaged tissues. In January 1998, Genevrier Laboratories inaugurated a new department especially designed for the production of cultured cells as therapeutic agents. Meeting clinician therapeutic needs by providing autologous keratinocytes and chondrocytes in the near future, represents the primary aim of the Biotechnology department. Concrete cell-based products are already being used for the treatment of burns and cutaneous chronic wounds such as the EPIBASE graft, which corresponds to an epidermis sheet composed of cultured autologous keratinocytes.

[Extrinsic allergic alveolitis and hematologic diseases (lymphoproliferative syndromes)]. / Alvéolite allergique extrinsèque et affections hématologiques (syndromes lympho-prolifératifs).

The authors report 2 cases of hypersensitivity pneumopathy associated with haematological abnormalities: benign lymphoproliferative syndrome in one case, and malignant lymphadenopathy with fatal outcome in the other case. Such cases, now observed with increasing frequency, can be regarded either as chance associations of 2 types of disease, or as the results of immune disorders with repercussions on the blood.

[Isotopes and myocardial ischemia]. / Isotopes et ischémie myocardique.

Single photon emission computed tomography (SPECT) is superior to conventional radionuclide scintigraphy. The most widely used marker is thallium-201, despite the fact that it is not exclusively flow-dependent. Thallium-201 SPECT can be performed in patients at rest, during exercise followed by a redistribution study, under dipyridamole or nitrate infusion, or combined with spasm provocation test. When performed at rest, it is helpful to diagnose and, above all, quantify a myocardial infarction. Under nitrate infusion, it delineates the viable muscle remaining around the infarct. During exercise or under dipyridamole infusion, it is more effective than any other non-invasive method in detecting myocardial ischaemia (sensitivity 95%, specificity 90%). Following surgical revascularization, a normal exercise SPECT indicates a functional aorto-coronary bypass in 80% of the cases. It is a good method to detect post-angioplasty restenosis (sensitivity 82%, specificity 86%, negative predictive value 93%). Like all paraclinical examinations, SPECT has its limitations: left bundle branch block, reverse redistribution, non-ischaemic (i.e. dilated or hypertrophic) cardiopathies.

[General physiopathology of chronic left ventricular insufficiency]. / Physiopathologie générale de l'insuffisance ventriculaire gauche chronique.

The failing heart is unable to provide some organs, notably the brain and the myocardium, with the amount of blood flow they require. To this myocardial inadequacy and resulting "circulatory insufficient" the body reacts by setting in action compensatory mechanisms which are "intracardiac" first (Starling's heterometric regulation, ventricular hypertrophy), then neurohormonal, with the activation of vasoconstrictor systems (noradrenergic system, renin-angiotensin-aldosterone system, arginine-vasopressin system) counterbalanced by the activation of vasodilator systems (vasodilator prostaglandins, atrial natriuretic factor and kinins). However, the vasoconstrictor systems outweigh the vasodilator systems. They create an excessive arterial and venous vasoconstriction, together with water-and-salt retention, which leads to an increase of left ventricular work during both systole and diastole and to a gradual worsening of the heart failure. The present-day treatment of heart failure aims at reducing the water-and-salt retention and at restoring the balance between the vasoconstrictor and vasodilator systems.

Lipoproteins of the newborn rat. Reciprocal development of low density lipoproteins and apoprotein E-rich high density lipoproteins.

Plasma lipids increase sharply with the onset of suckling in the neonatal rat. Much of the variation has been attributed to the high fat content of milk. Apoproteins AI, E and AIV were found in low concentrations in the fetus. They increased during suckling. Apoprotein E and apoprotein AIV did not exceed adult values whereas apoprotein AI concentration in the late suckling period was twice that of the adult. On the contrast, fetal apoprotein B was nearly 2.5-fold above adult concentration and was under the form of LDL, the main lipoprotein class in the late fetal period. Apoprotein B concentration decreased progressively as LDL was replaced by an apoprotein E-rich HDL. The latter class constituted an important transitory cholesterol carrier during the shift from the neonatal lipoprotein pattern dominated by LDL to the typical adult pattern in which HDL are predominant. Lack of active cholesterol ester transfer protein is believed to be one of the reasons for low LDL concentration in adult rats. However, in vitro incubation of radioactively-labelled HDL cholesteryl esters with rat plasma demonstrated that the juveniles' lipoprotein depleted plasma induced as little transfer of the label from HDL to lower density lipoproteins as that of the adult. Thus a transient cholesteryl ester transfer activity could not have contributed to the composition of the LDL pool in the fetus and the early suckling rat. It is more likely that LDL are secreted directly by the liver. Each apolipoprotein exhibited a characteristic developmental pattern different from that of adult rats fed hyperlipidic diets. It therefore appears that each apoprotein is controlled independently by a combination of programmed ontogenic development and nutritional factors leading to the progressive establishment of the adult lipoprotein profile.

Response of the exocrine pancreas to quantitative and qualitative variations in dietary lipids.

In the rat, pancreatic amylase and, to a lesser extent, lipase adapt quantitatively to the amount of their respective substrates in the diet by an increase in specific activity and total contents (range of variation, fivefold for amylase and twofold for lipase). Colipase responded to protein intake (r = 0.85, P less than 0.01) and not to lipids provided protein intake was below 3.5 g or above 6.0 g. With this latter amount of protein, a maximal level was obtained, even with 2% lipid in the diet. Between 3.5 and 6.0 g, lipid intake was found to modulate colipase in parallel with lipase. When different types of fat were compared, the degree of saturation was found to have no impact on lipase, colipase, and amylase. Diets containing medium-chain triglycerides (C8-C10) did not maximally increase specific activity and total content of lipase and colipase, whereas they did not repress amylase as much longer chain triglycerides did. With coconut oil (45% C12), lipase was maximally stimulated but amylase was not maximally repressed, showing that the regulation of the hydrolases may be partly reciprocal and partly independent.

Fatty acid composition of an oral load affects chylomicron size in human subjects.

HDL-phospholipids are determinants in reverse cholesterol transport. They are mostly derived from triacylglycerol (TG)-rich lipoproteins. Chylomicron size is important, therefore, because it is related to the ratio surface phospholipids: core TG and, thus, determines the availability of postprandial phospholipids for transfer to HDL. Eleven healthy young women each ingested four different fat loads supplemented with retinyl palmitate and containing 60 g sunflower oil (SO), oleic-sunflower oil (OSO), mixed oil (MO; (g/kg) linoleic acid 480, oleic acid 380, linolenic acid 13) or beef tallow (BT). At the peak of TG absorption for all loads (4 h) chylomicron diameters, determined by agarose-gel filtration, were larger after SO compared with OSO (P < 0.05) and BT (P = 0.06) and after MO compared with BT (P < 0.05). At 6 h chylomicron size was larger after the vegetable oils compared with BT (P < 0.05 in each case). After each fat load chylomicron size decreased at 6 and 8 h compared with that at 4 h (P < 0.05) except for OSO. Retinyl ester and TG concentrations were lower in chylomicrons after BT than after the other fats but not in the chylomicron-free serum (containing chylomicron remnants), suggesting absorption in the form of very small particles. Compared with the fasting value, the concentration of the Svedberg unit of flotation 20-400 fraction, which contains VLDL and chylomicron remnants, was lower 8 h after MO, the only fat to contain significant amounts of linolenic acid. We conclude that chylomicron size is dependent on the fatty acid composition of ingested fats and the time-course of digestion, being larger for polyunsaturated fatty acid-rich fats and in the early phase of digestion. On the basis of retinyl ester concentration there were no differences between fats in chylomicron-remnant clearance.

Contributions of studies on uncoupling proteins to research on metabolic diseases.

The coupling of O2 consumption to ADP phosphorylation in mitochondria is partial. This is particularly obvious in brown adipocyte mitochondria which use a regulated uncoupling mechanism generating heat production from substrate oxidation, and catalysing thermogenesis in rodents or infants in response to cold, and arousing hibernators. In the case of brown adipose tissue, the uncoupling mechanism is related to a specific protein in the inner mitochondrial membrane referred to as UCP1. Although the biological importance of UCP1 in human adults is not demonstrated, genetic analysis of various human cohorts suggested a participation of UCP1 to control of fat content and body weight. Very recently, the cloning of UCP2 and UCP3, two homologues of UCP1, has renewed the field of research on the importance of respiration control in metabolic processes and metabolic diseases. UCP2 is widely expressed in organs, whereas UCP3 is mainly present in muscles. These proteins may explain why the coupling of respiration to ADP phosphorylation is less than perfect. Their biological importance should be studied. They also represent new putative targets for drugs against metabolic diseases such as obesity.