Unveiling the mysteries of clopidogrel metabolism and efficacy.

Clopidogrel is an important antiplatelet agent, but a considerable variability in the biological effect of the drug has been observed. Additionally, patients with insufficient platelet reactivity inhibition following a loading dose (LD) of clopidogrel have a poor outcome. The mechanisms of variability are dependent on genetic polymorphisms of enzymes involved in clopidogrel metabolism. Paraoxonase 1 has been identified as the main determinant of the biological and clinical efficacy of clopidogrel. This finding could enable the use of pharmacogenomics to tailor antiplatelet agents.

Total and fetal cell-free DNA analysis in maternal blood as markers of placental insufficiency in intrauterine growth restriction.

OBJECTIVE: To compare total and fetal DNA levels in the maternal plasma in three groups: pregnancies with intrauterine growth restriction (IUGR) due to placental insufficiency (PI) and other causes, and in control pregnancies. METHODS: Total as well as fetal DNA was quantified in 78 maternal plasma samples. In 19 pregnancies, the fetus presented IUGR due to PI (group A), and in 31 pregnancies due to other causes (group B). The control group comprised 28 patients (group C). DNA quantification was done using real-time quantitative PCR with a standardized pool of plasmid calibrators. DNA concentrations of the three groups were compared using non-parametric tests (Kruskal-Wallis or Mann-Whitney tests). RESULTS: The three groups did not statistically differ regarding maternal age (mean +/- SD: 30.5 +/- 5.4 years), gestational age (30 +/- 5.3 weeks) or the proportion of male fetuses (48.2%). Plasma total DNA was significantly higher in group A compared to groups B and C (p = 0.001 for both). An increase in fetal DNA was only observed in group A for patients beyond 28 weeks of gestation. CONCLUSIONS: The plasma total DNA level is higher in patients with IUGR due to PI. These results suggest the presence of maternal endothelial damage independently of preeclampsia.

Characterization of ovarian function in granulocyte-macrophage colony-stimulating factor-deficient mice.

During the estrous cycle and early pregnancy, lymphohemopoietic cytokines and chemokines contribute to the regulation of ovarian function by orchestrating the recruitment and activation of leukocytes associated with the ovulatory follicle and corpus luteum. The purpose of this study was to investigate the physiological role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the ovary, utilizing mice genetically deficient in GM-CSF. Our results show that the mean duration of the estrous cycle in GM-CSF-deficient (GM-/-) mice was extended by 1.5 days (mean +/- SE, 4.9 +/- 0.3 vs. 6.5 +/- 0.5 days for GM+/+ and GM-/- mice, respectively). Similar ovulation rates were observed in immature superovulated mice (31.8 +/- 7.7 vs. 28.9 +/- 6.4 oocytes per mouse) and adult naturally cycling mice (10.4 +/- 0.8 vs. 10.3 +/- 0.8 oocytes per mouse). Furthermore, comparable numbers of oocytes were released from GM+/+ and GM-/- ovaries in an in vitro perfusion model. However, ovaries in pregnant GM-/- mice were found to comprise fewer cells and synthesize less progesterone (141.6 +/- 10.3 vs. 116.5 +/- 6 nM plasma), although the duration of pseudopregnancy was unaltered by GM-CSF deficiency (11.0 +/- 0.2 vs. 11.0 +/- 0.5 days). Immunohistochemical staining of leukocytes in the ovary during the periovulatory period indicated that the size and composition of ovarian leukocyte populations were unaltered in the absence of GM-CSF. However, an effect of GM-CSF deficiency on the activation phenotype of ovarian leukocytes was indicated by a 57% increase in mean secretion of nitric oxide in in vitro-perfused GM-/- ovaries, and diminished major histocompability complex (MHC) class II (Ia) expression in ovarian macrophages and/or dendritic cells (30.5 +/- 7. 2% vs. 9.1 +/- 1.8% positive stain in GM+/+ and GM-/- ovaries, respectively). Furthermore, ovarian macrophages and neutrophils were diminished in number after parturition, with significantly decreased CD11b+ (Mac-1) staining in the stromal region of postpartum GM-/- ovaries (6.7 +/- 0.6 vs. 3.6 +/- 0.7% positive stain). In summary, GM-CSF does not appear to be essential for ovarian function but may play a role in fine-tuning the activation status and adhesive properties of ovarian myeloid leukocytes. Aberrant activation of these cells appears to compromise the luteinization process and the steroidogenic capacity of the corpus luteum during early pregnancy in GM-CSF-deficient mice.

Colony stimulating factor-1 concentrations in blood and follicular fluid during the human menstrual cycle and ovarian stimulation: possible role in the ovulatory process.

To evaluate a possible role for colony stimulating factor-1 (CSF-1) in human ovarian function, the peripheral blood CSF-1 concentration throughout the human menstrual cycle and during ovarian stimulation was monitored. Blood was sampled across the menstrual cycle (n = 10) and at specific times during ovarian stimulation. In addition, the CSF-1 concentrations in follicular fluid (FF) during the follicular phase and during the luteinizing hormone (LH) surge of natural cycles, as well as 35-37 h after human chorionic gonadotrophin (HCG) during ovarian stimulation, were determined. There was no significant variation in CSF-1 concentrations during the natural menstrual cycle (median 470, range 212-1364 pg/ml). CSF-1 concentrations in FF (n = 11) were about four-fold higher (P < 0. 0001) than those in plasma of the same patients. CSF-1 concentrations in these FF showed some stage dependent variability, with significantly higher values during the ovulatory phase (median of 2017 pg/ml, range 1131-2236 pg/ml), compared to mid-follicular phase (median 961 pg/ml, range 830-1340 pg/ml; P = 0.02). During ovarian stimulation (n = 20), the plasma concentrations were similar to a time prior to stimulation up to and including 35-37 h after HCG. On day 9 after HCG, the values (median 644, range 357-1352 pg/ml) were significantly higher compared to pre-stimulation (median 422, range 253-1598 pg/ml; P < 0.05) and 35-37 h after HCG (median 458, range 250-658 pg/ml; P < 0.01). FF concentrations (n = 27) of CSF-1 at oocyte retrieval (median 3116, range 1824-5883 pg/ml) were about seven-fold higher than blood concentrations (median 472, range 250-1055 pg/ml; P < 0.0001). These results suggest that the intra-ovarian CSF-1, possibly induced by LH/HCG, plays an important role during ovulation and luteinization.

Inhibition of nitric oxide: effects on interleukin-1 beta-enhanced ovulation rate, steroid hormones, and ovarian leukocyte distribution at ovulation in the rat.

The ovulatory process resembles an inflammatory reaction with an infiltration of leukocytes, production of inflammatory mediators such as cytokines, and a general edema and hyperemia. Nitric oxide (NO), a potent vasodilator and the main mediator of macrophage tumoricidal and bacteriocidal activities, is known to participate in inflammatory reactions and has been shown to mediate the interleukin-1 beta (IL-1 beta)-directed tissue-remodeling events within the ovary. The regulation by NO of ovulation rate, leukocyte distribution, and steroid release in the rat ovary was investigated through use of a combination of in vivo and in vitro models of ovulation and a competitive inhibitor, N-omega-nitro-L-arginine methyl ester (L-NAME), of the NO synthase (NOS) enzyme. Subcutaneous L-NAME (1.5 x 10(-4) mol/kg) administration significantly reduced the in vivo ovulation rate of eCG/hCG-primed rats (L-NAME-treated: 10.6 +/- 1.8 [mean +/- SEM] oocytes per ovary [O/O], 11.0 +/- 1.2 rupture sites per ovary [RS/O]; saline-treated: 18.0 +/- 1.8 O/O, 19.4 +/- 1.1 RS/O; p < 0.01) at 20 h post-hCG. These results were reflected in vitro, where addition of L-NAME (3.5 x 10(-5) mol/L) to LH (0.1 microgram/ml)-perfused ovaries decreased ovulation rate from 8.2 +/- 1.6 to 2.7 +/- 1 ovulations per ovary (p < 0.05) and simultaneously decreased nitrite accumulation at the completion of perfusions from 16.5 +/- 1.9 to 4.1 +/- 0.5 nmol/ml (p < 0.001). The addition of L-NAME to LH+IL-1 beta (4 ng/ml)-perfused ovaries decreased ovulation rate from 15.2 +/- 2.4 to 0.8 +/- 0.8 ovulations per ovary (p < 0.001) and simultaneously decreased nitrite accumulation at 22 h from 22.8 +/- 2.2 to 1.9 +/- 0.6 nmol/ml (p < 0.001). Studies analyzing and manipulating perfusion flow rate indicated that the L-NAME effects on ovulation rate are primarily due to a reduction in flow rate resulting from inhibition of NO, which may be a consequence of the known vasoconstrictor effects of NOS inhibitors. The observed reduction of in vivo ovulation rate by NO inhibition at 20 h post-hCG was associated with a significant reduction in thecal MCA149+ neutrophils at 12 h post-hCG, the expected time of ovulation (L-NAME-treated: 98.4 +/- 9.2 cells per thecal area; saline-treated: 211.5 +/- 11.5 cells per thecal area; p < 0.001), while ED1+ monocytes/macrophages underwent similar but nonsignificant changes. Plasma (20 h post-hCG) and perfusate progesterone were not different with L-NAME treatment, while perfusate estradiol levels were markedly reduced upon addition of L-NAME, suggesting a role for NO in ovulation but not in the process of luteinization. In summary, deprivation of NO by use of the competitive inhibitor, L-NAME, led to fewer ovulations, reduced accumulation of nitrite, a decreased neutrophil count in the theca of preovulatory follicles, and reduced estradiol secretion, while progesterone release remained unaffected. The NO pathway may therefore play an important role in the regulation of ovulation and the mediation of IL-1 beta's pro-ovulatory effects. There are likely to be primarily vascular effects, but also a nonvascular component, to the NO regulation of ovulation, with both components indirectly affecting ovulatory leukocyte distribution and steroid secretion.

Reduction of ovulation rate in the rat by administration of a neutrophil-depleting monoclonal antibody.

To investigate the effect of systemic neutrophil depletion on ovulation rate, rats were synchronised with eCG and hCG, and concurrently were administered neutrophil-specific, cytotoxic RP-3 monoclonal antibody (mAb), or an irrelevant, class-matched mAb. Neutrophils in the peripheral blood and in the thecal-luteal area of corpora lutea were detected by immunohistochemical analysis with the neutrophil-specific mAb MCA149 and were found to be reduced in number by 70% and 38% respectively following RP-3 treatment compared to the control group. Ovulation rate, as assessed by counting the number of oocytes in the ampullary region of the oviduct 20 h after hCG administration, was found to be reduced by 27% in the neutrophil-depleted rats. This result provides further evidence that neutrophilic granulocytes play an active role in ovulation in the rat.

Effects of tumour necrosis factor alpha (TNF alpha) on ovulation in the rat ovary.

The effects of tumour necrosis factor alpha (TNF alpha) on ovulation rate, ovarian secretion of steroids, and on tissue concentrations of the ovulatory mediators plasminogen activator (PA) and prostaglandins were examined in rat ovaries perfused in vitro for 20 h. Unstimulated control ovaries did not ovulate whereas luteinizing hormone (LH; 100 ng mL-1) induced ovulations in all ovaries (2.6 +/- 0.7). TNF alpha (40 ng mL-1) induced ovulations in 3 of 5 ovaries (0.8 +/- 0.4). When TNF alpha was added concomitantly with LH, the LH-induced ovulation rate was increased four-fold (10.6 +/- 1.3). This effect was not seen when TNF alpha was added 5 h after LH (3.2 +/- 0.6). More progesterone was released when TNF alpha was combined with LH by comparison with LH alone. Tissue concentrations of prostaglandin F2 alpha (PGF2 alpha) and PGE were increased by the addition of TNF alpha when compared with the control group, but did not exceed those observed in the LH group, when TNF alpha was combined with LH. PA activity in the tissue was unaffected by TNF alpha and the LH-induced increase in PA activity was inhibited when TNF alpha was combined with LH. The results demonstrate that the proinflammatory cytokine TNF alpha promotes ovulations in the rat ovary.

Interleukin-1ß inhibits luteinizing hormone-induced plasminogen activator activity in rat preovulatory folliclesin vitro.

The effects of interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNFα) on ovulation-associated plasminogen activator (PA) activity were investigated using preovulatory follicles excised 48h after equine chorionic gonadotrophin (16IU)-priming of immature rats. Follicles were incubated for 6 and 14h with a single dose of LH (1 µg/ml) only, or various cytokine doses in the presence or absence of LH. PA activity in follicular homogenates was determined by a radioactively labelled fibrin-coated plate method and secreted levels of the ovulatory mediators progesterone (P) and prostaglandin E (PGE) were measured by radioimmunoassay. LH induced timedependent rises in PA (2.5-fold over control at 6h and fourfold over control at 14h), while IL-1ß and TNFα alone had no effect over either time period. LH and cytokine coincubations over 14h revealed that IL-1ß dosedependently inhibited the LH-induced increase in PA activity, up to 85%. The effects of TNFα on LH-induced PA activity were not significant. Both IL-1ß and TNFα increased P and PGE secretion time- and dose-dependently. In summary, IL-1ß dose-dependently inhibits the LH-induced increase in PA activity in rat preovulatory folliclesin vitro while, as with TNFα, increasing P and PGE synthesis. This study, shows that the earlier reported pro-ovulatory action of IL-1ß is not likely to be mediated by activation of the PA-system and suggests that IL-1ß may mediate a regulatory loop controlling the extent and distribution of LH-induced PA activity in rat preovulatory follicles.