Effect of chronic treatment with a purified flavonoid fraction on inflammatory granuloma in the rat. Study of prostaglandin E2 and F2 alpha and thromboxane B2 release and histological changes.

S-5682 (Daflon-500 mg), a purified flavonoid fraction, consisting of 90% diosmin (a flavone derivative) and 10% hesperidin (a flavanone derivative), was administered to rats by intubation in the daily dose of 100 mg/d. 15 days after the start of treatment, polyurethane sponges were implanted in the subcutaneous connective tissue in the dorsolumbar region under rapid ether anaesthesia. Similar fragments of sponge were implanted in a group of control animals who received the vehicle (saccharose syrup) only, also by the oral route. The rats were sacrificed in fractions of 7 animals drawn from each of the two groups (control and treated) after 4, 8, 16 and 30 days (only 5 animals from each group on day 30) after implantation of the polyurethane sponges. The granulomas formed were removed, weighed and their prostaglandin (PG)E2, PGF2 alpha and thromboxane (Tx)B2 contents were determined. In addition a full cell count (polymorphs, lymphocytes, macrophages, plasmocytes and giant cells) was performed and the animals were histologically examined. The results show that treatment of the animals with S-5682 had the following effects: 1. A significant fall in the mean weight of the granulomas formed after 4 and 8 days was observed, reflecting inhibition of oedema formation during the early phase of the inflammatory reaction. 2. The synthesis of PGE2 (78.5% inhibition on day 4) and PGF2 alpha (45.2% on day 16) was inhibited. 3. There was very early inhibition of TxB2 synthesis (59.5% inhibition on day 4). 4. A later reduction in cell migration towards the inflammatory focus occurred which was statistically significant on day 16 (49.6% reduction in the total number of migrant cells). 5. Multiple histological aspects of the acute inflammatory reaction (diapedesis of polymorphs, lymphocytes, histiocytes and macrophages) and features of the chronic inflammatory reaction (newly formed microvascularisation of the granuloma tissue, perivascular oedema, presence of collagen fibres) were improved.

Follicular steroids in relation to oocyte development and human ovarian stimulation protocols.

Oocytes of pre-ovulatory follicles were collected by laparoscopy for in-vitro fertilization in 118 women. Patients were treated either during the natural cycle, or after induction of ovulation with clomiphene citrate, or with human menopausal gonadotrophin (HMG), or clomiphene citrate combined with HMG. The oestrogens (oestrone + oestradiol) and total aromatizable androgens (androstenedione, testosterone and dehydroepiandrosterone and its sulphate) were assayed in follicular fluids using an enzymatic method. The levels of progesterone, 17 alpha OH-progesterone, thromboxane, and prostaglandins (PGE2 and PGF2 alpha) were determined by radioimmunoassay. The best follicular fluid indicator of oocyte fertilization in vitro was the A/E ratio, which was less than 1 when the oocyte was fertilized in vitro and led to a pregnancy. The lowest value for the A/E ratio was obtained with spontaneous ovulation protocols. Regardless of oocyte development, the progesterone level was always greater than 2000 ng/ml, and the PGE2/PGF2 alpha ratio was greater than 1 in all stimulated cycles. Our investigations show that a combination of clomiphene citrate and HMG provides the best stimulation, on the basis of follicular fluid analysis and the outcome of fertilized oocytes.

[Pregnancies by fertilization in vitro. Biochemistry of the follicular fluid]. / Grossesses par fécondation in vitro. Biochimie du liquide folliculaire.

Fifty four follicular fluids containing 41 oocytes were sampled from 6 patients in which at least one pregnancy was obtained after IVF. Their biochemical composition has been correlated with the follicle maturity and oocyte quality. Three components have been assayed: steroids, prostanoids, proteolytic enzymes. Determinations of estrogens and total aromatisable androgens were carried out by the enzymatic technic of Nicolas and al.; progesterone and 17 OH progesterone by RIA. Development of in vitro cultured and fertilized oocytes appears to be improved when progesterone and estrogens concentrations in the follicular fluid are above 2 000 and 600 ng/ml respectively, with an A/E ratio under 1. On the other hand, prostanoids (T X B2, PGE2, PGF2 alpha determined by RIA) and proteolytic enzymes : (collagenolytic and kallikrein type activities) seemed related to ovulatory activity rather than to oocyte quality.

[Principle and practice of in vitro fertilization. Role in the treatment of female sterility]. / Principe et réalisation de la fécondation in vitro. Place dans le traitement de la stérilité féminine.

In vitro fertilization (IVF) and embryo transfer (ET) appear to constitute a revolution in the reproductive sciences rather than merely a new technique in the treatment of sterility. Principle of IVF: IVF accomplishes in vitro the process than normally occurs in the oviduct between the ovulation of oocyte II and embryo implantation in the endometrium. This 4 day period (under normal conditions in the woman) involves 4 steps: recovery, fertilization, segmentation and transport. Performance of IVF: Recovery of the oocytes: The oocytes are recovered under celioscopic or echographic observation when they have completed cytoplasmic maturation and their first meiosis. A precise monitoring of ovulation (spontaneous or induced) should be performed using estrogen and LH assays. IVF provides an opportunity for evaluating the methods of ovulation induction and monitoring, as a function of the maturation of the oocytes recovered. Fertilization: When the oocyte has achieved maturing after several hours of incubation, fertilization is obtained 15 h contact with washed and capacitated spermatozoa (100 000/ml). This step is highly dependent on gametocyte quality: oocyte maturity and fecundity of spermatozoa, which can be estimated from the percentage of survival in the insemination medium. Segmentation occurs in culture at pH 7.28 in the presence of 5 per cent CO2 at 37 degrees C (pronucleus 15th, 2 blastomeres 26 h, 4-8 blastomeres 52 h). Embryo transfer is carried out when an embryo is present at 52 h. Only 1/10 of the embryo transfers result in successful implantation, which depends on the quality of the embryo; the quality can only be indirect criteria.(ABSTRACT TRUNCATED AT 250 WORDS)

[New derivatives of benzopyran-4-one with analgesic, anti-inflammatory and antiplatelet clumping activity]. / Nouveaux dérivés de la benzopyrannone-4, à activités analgésique, anti-inflammatoire et antiagrégante plaquettaire.

A series of 35 new derivatives of 3-benzylidene benzopyran-4-one carboxylic acid was synthesized; their antiinflammatory and analgesic activities were investigated and compared with those of acetylsalicylic acid, phenylbutazone, ketoprofene and glafenine. Among these compounds, four were found to be more potent than the references compounds in tests of antiinflammatory and analgesic activity. Seven compounds were tested on rat blood platelet aggregation induced by ADP. From these tests, HD-039, HD-041, HD-044 and HD-049 appear to be promising antiinflammatory, analgesic and anticlotting agents, although this original series shows little structural analogy with classical non-steroidal antiinflammatory agents.

[Failures at in vitro fertilization: electron microscopic studies of unfertilized oocytes and undivided ova]. / Les échecs de la fécondation in vitro: étude au microscope électronique des ovocytes non fécondés et des oeufs non divisés.

We have examined by electron microscopy the non fertilized oocytes and the non divided eggs after assay of in vitro fertilization. We have observed three blocking points; 1) absence of fusion of the gametes, either because of oocyte immaturity or over-maturity, either because of spermatozoa phagocytosis by follicle cells transformed in phagocytic cells, in presence of intra or extra-cellular bacteria; 2) fusion of the gametes but absence of spermatic nucleus decondensation; 3) fusion of the gametes and pronuclei formation but absence of amphimixy. Two causes are evident: failure of oocyte maturation in vivo or in vitro, and bacterial contamination problems after female or male infra-clinical infections.

[In vitro effects of preovulatory human follicular liquid on migration of human spermatozoa]. / Effets in vitro du liquide folliculaire humain préovulatoire sur la migration des spermatozoïdes humains.

In vitro measurements of spermatozoa migration in different media showed a 75% increase in the number of spermatozoa migrating into preovulatory human follicular fluid versus Menezo's fluid. However after 4 h storage in either medium, total and progressive motility of the spermatozoa was lower in follicular fluid than in Menezo's fluid. A positive correlation was evident between the spermatozoa migration in follicular fluid and the concentration or motility of spermatozoa in fresh sperm.

[Effect of the hormonal environment on the human oocyte and its later development in vitro]. / Influence de l'environnement hormonal de l'ovocyte humain sur son évolution ultérieure in vitro.

The biosynthesis of progesterone by the granulosa cells that come from follicles from which the oocyte has been fertilized and has divided in vitro is far superior to that from the cells coming from follicles in which the oocyte has failed to develop. This result is in accordance with the raised levels of progesterone and the influence of androgens on lower levels of oestrogens in follicular fluid that evolved favourably. In the same follicles the ratio of concentrations of prostaglandins PGE2 to PGF2 alpha is twice as high as in the liquid from other follicles.

[Collection of mature ovocytes during cycles with spontaneous ovulation for the purpose of in vitro fertilization]. / Recueil d'ovocytes mûrs au cours de cycles à ovulation spontanée en vue de fécondation in vitro.

In vitro fertilization was attempted during a spontaneous cycle in 17 out of 97 patients. Preovulatory urinary estrogen and LH maximas were assayed in order to time celioscopy 24 hours after the onset of the triggering urinary LH maxima. In 8 patients, comparison with plasmatic LH shows that the timing of spontaneous ovulation can be precisely predicted from urine assays. However, when the triggering LH release is weak, LH must be above or equal to 25 IU/g of creatinine with a creatininuria above or equal to 0.4 g/l in two urine samples at a three-hour interval.

[In vitro development of human ovocytes collected during spontaneous ovulation. Effect of the biochemical composition of the follicular fluids]. / Evolution in vitro d'ovocytes humains recueillis lors d'ovulations spontanées. Influence de la composition biochimique des liquides folliculaires.

Development of the in vitro cultured and fertilized ovocyte appears to be improved when progesterone and estrogen concentrations in the follicular fluid are above 2 000 and 1 000 ng/ml respectively, with a A/E ratio under 1 and a PGE/PGF ratio above 0.8. Such criteria are only infrequently fulfilled: once in seven cases. They were found in one of the two patients who had a pregnancy after in vitro fertilization during induced ovulation. Division of ovocytes from spontaneous ovulation was recorded in several instances in spite of a less propitious composition of the follicular fluid.

Functional assessment of alveolar macrophages: comparison of cells from asthmatics and normal subjects.

Alveolar macrophages (AM) were obtained by bronchoalveolar lavage (BAL) from seven healthy nonallergic, nonasthmatic donors, 15 patients with allergic bronchial asthma, and six patients with aspirin-sensitive asthma. AM were purified by adherence over 2 hr and cultured for an additional 24 hr. Functional assessment of viable cells was carried out for zymosan phagocytosis and for prostaglandin (PG) E2-PGF2 alpha and thromboxane (Tx) B2 release by resting and zymosan-stimulated AM. The eosinophil count in BAL fluid from allergic asthmatics was higher than that from control subjects (3.9% +/- 1.6% vs 0.4% +/- 0.3%, p less than 0.05) and still greater in BAL from patients with aspirin-sensitive asthma (21.7% +/- 9.0%, p less than 0.01). After the 24 hr of incubation, the AM viability was inversely correlated to the percentage of eosinophils in BAL fluid (r = -0.54, n = 21, p less than 0.02). Zymosan phagocytosis was significantly lower by viable cells from both allergic asthmatics and aspirin-sensitive patients as compared with cells from normal donors (p less than 0.05). Zymosan phagocytosis induced a twofold to threefold increase in the release of PGE2, PGF2 alpha, and TxB2 from AM of normal subjects (p less than 0.01) but only a onefold to twofold increase from AM of allergic asthmatic patients. The stimulated AM from aspirin-sensitive patients released smaller quantities of each product than AM from normal subjects or allergic asthmatic patients (p less than 0.05). We conclude that the viability and functional activity of AM are impaired in asthmatic patients and that these deficits correlate with the percent eosinophilia in the BAL; it is therefore suggested that they may be due to an interaction between eosinophils and AM in the bronchoalveolar lumen.

[Mononucleated phagocytes from rheumatoid synovial fluid. Synthesis of prostanoids and first pharmacological applications]. / Phagocytes mononucléés du liquide synovial rhumatoïde. Synthèse de prostanoïdes et premières applications pharmacologiques.

An original and easily reproducible technique for isolating and culturating mononucleated phagocytes from rheumatoid synovial fluid was used by the authors to study the synthesis of PGE2, PGF alpha 2 and TxB2 by macrophages taken from 16 patients with rheumatoid arthritis. The functional importance of these macrophages was evaluated by comparing their ratios of prostaglandin synthesis. PGE2 and TxB2 are released in large quantities and are increased after stimulation with zymosan. Two distinct cell populations having different biochemical characteristics were identified from these macrophages based on their capacity to synthesize prostaglandins. Prostaglandin synthesis was correlated with the clinical progression of rheumatoid arthritic disease as evaluated by Lee's index and the time required to loosen up morning joint stiffness in these patients. No correlation was found between prostaglandin synthesis and the quantity of synovial fluid in the affected joint. The first results of a pharmacological study evaluating the activity of non steroid anti-inflammatory drugs using this experimental model are presented here. The addition of indomethacin to culture medium produces dose-dependent inhibition of prostaglandin synthesis.

Mononuclear phagocytes, prostanoids and rheumatoid arthritis.

We describe a new, simple procedure for obtaining and studying mononuclear phagocytes from inflammatory synovial fluid. The viability and functional characteristics of these macrophages have been carefully checked. This technique has been used to evaluate the macrophagic synthesis of prostaglandins in 16 patients with evolutive rheumatoid arthritis. Significant amounts of prostaglandins, mainly PGE2 and TxB2, are released and increased after zymosan administration. The three tested prostaglandins (PGE2, PGF2 alpha and TxB2) responded in a similar manner. The in vitro PG biosynthesis and the number of harvested adherent cells are negatively correlated with the synovial fluid volume but positively correlated with the disease activity. In fact, we found an heterogeneity among the analyzed macrophages; these can be divided into two populations with marked differences in their PG biosynthesis. The one with the highest synthesis corresponded to the more severe disease.

[Alveolar macrophages in asthmatic patients: study of their phagocytic function]. / Macrophages alvéolaires de l'asthmatique: étude de leur fonction phagocytaire.

Alveolar macrophages (AM) form the first line of defense against aggression in the lungs. The goal of the present study was to evaluate AM phagocytic function in two types of asthmatic patients: allergic asthma (14 patients) and asthma following ingestion of aspirin (6 patients). Macrophages obtained from bronchiolo-alveolar washings were isolated as a result of their adherence to glass and cultured for 24 hours. The results show that AM viability and phagocytosis of zymosan are reduced in the asthmatic patient. Decreased viability in patients sensitive to aspirin may be the result of alveolar eosinophilia, a quasiconstant finding in asthmatics. Although impaired phagocytosis of zymosan may be explained by the presence of surface IgE on AM of the allergic asthma patient, this is not the case for the aspirin sensitive patient.

[Technic for isolation and culture of macrophages from human synovial fluid]. / Technique d'isolement et de culture des macrophages du liquide synovial humain.

Macrophages from synovial fluid were obtained by joint puncture of patients with rheumatoid arthritis or non-rheumatoid exudative arthropathies. This fluid was subsequently centrifuged and the resulting cell pellet was recovered and distributed for in vitro culture (Falcon Petri dishes: 60 x 15 or 35 x 10). Cells were washed after 4 hours in culture to eliminate non-macrophagic cells. A second cell wash was effected 24 hours later to eliminate non-adherent cells. The culture medium employed was fetal calf serum MMP 119 (Gibco). The macrophagic nature of the cultured cells was ascertained by non specific esterase determinations (Burstone method) and estimation of the phagocytic index (using zymosan). This technique permits the conservation of viable monocytes in culture thus facilitating investigation of their metabolic activity (for example: prostaglandins, prostanoids or fatty acids) and the effects of anti-inflammatory agents.

PGE2, PGF2 alpha, and TXB2 biosynthesis by human rheumatoid synovia.

Prostaglandins (PG) are currently suspected to be involved in human rheumatoid arthritis (RA). We have studied the PG [PGE2, PGF2 alpha, and Thromboxane B2 (TXB2)] biosynthesis capacity of normal (from amputations) and pathologic (osteoarthritis (OA) and RA) synovia. The measurement is done on the whole homogenate and microsomal fraction after 40 min incubation, without exogenous arachidonic acid (AA) or with a saturating concentration (25 micrograms/ml) of this compound, using a radioimmunoassay method. There is a considerable increase in PGE2 and PGF2 alpha production by the rheumatoid synovia and more PGE2 and PGF2 alpha are formed. This production is more marked for homogenates than microsomal fraction. The OA group is not homogeneous and does not differ significantly either from normals or from RA. The addition of AA considerably increases the biosynthesis of PG in both normal and pathologic tissue. For TXB2, the first results (four synovia) show no or a small biosynthesis in RA as well as in normals. Nevertheless, the levels before incubation seem higher in RA than in normal tissue.

[Biosynthesis of PGE2 and PGF2 alpha in normal and pathological human synovial fluid]. / Biosynthèse de PGE2 et PGF2 alpha dans les synoviales humaines normales et pathologiques.

The biosynthesis capacity of PGE2 and PGF2 alpha in normal and pathological (rheumatoid arthritis and osteo-arthrosis) synovialis was studied using the radio-immunological method, simultaneously on homogenized tissues and the microsome fraction, with or without addition of exogenous arachidonic acid. Among the results obtained, the most significant concern rheumatoid polyarthritis: a net increase in PGF2 alpha and especially in PGE2, more pronounced in the homogenized emulsion than in the microsomial fraction. Exogenous arachidonic acid increases the biosynthesis capacity of the PG. The prospective interest of this model is underlined: the possible introduction in the incubation medium of various drugs, making it possible to study the various factors taking part in the biosynthesis of PG (phospholipase A2 and prostaglandin-synthetase).