Comparison of the efficacy of differing starter doses of recombinant follicle stimulating hormone (rFSH) for IVF and intracytoplasmic sperm injection cycles when the treatment is administered both subcutaneously and intramuscularly.

The procyanidin-rich French maritime pine bark extract Pycnogenol (PBE) has been investigated for its effect in protecting human skin against solar UV-simulated light-induced erythema. Twenty-one volunteers were given an oral supplementation of Pycnogenol: 1.10 mg/kg body weight (b. wt.)/d for the first 4 weeks and 1.66 mg/kg b. wt./d for the next 4 weeks. The minimal erythema dose (MED) was measured twice before supplementation (baseline MED), once after the first 4 weeks of supplementation, and a last time at the end of the study. The UVR dose necessary to achieve 1 MED was significantly increased during PBE supplementation. Since the activation of the pro-inflammatory and redox-regulated transcription factor NF-kappaB is thought to play a major role in UVR-induced erythema, the effect of PBE was also investigated in the human keratinocyte cell line HaCaT. PBE, added to the cell culture medium, inhibited UVR-induced NF-kappaB-dependent gene expression in a concentration-dependent manner. However, NF-kappaB-DNA-binding activity was not prevented, suggesting that PBE affects the transactivation capacity of NF-kappaB. These data indicate that oral supplementation of PBE reduces erythema in the skin. Inhibition of NF-kappaB-dependent gene expression by PBE possibly contributes to the observed increase in MED.

Immunosuppression may have an important impact on early graft coronary endothelial injury. We investigated functional and morphologic coronary alterations, myocardial expression, and cardiac release of possible mediators of allograft vasculopathy within 6 months after cardiac transplantation with respect to different immunosuppressive regimens. Epicardial and microvascular endothelium-dependent and endothelium-independent vasomotor function and epicardial intimal thickening were measured in 8 transplant recipients treated with cyclosporin A (CyA), azathioprine, and prednisone (group 1), 9 transplant recipients treated with tacrolimus (TKL), azathioprine, and prednisone (group 2), and 14 patients treated with TKL, mycophenolate mofetil (MMF), and prednisone (group 3). The gene expressions of inducible and endothelial nitric oxide synthase (iNOS and eNOS), endothelin-1, prostacyclinsynthase, and thromboxansynthase were analyzed in endomyocardial biopsy specimens using semiquantitative reverse transcription polymerase chain reaction. Transcardiac cytokine release, endothelin-1, and nitrate-release were determined from plasma samples. Epicardial endothelial dysfunction (vasoconstriction to acetylcholine > 10%) and microvascular smooth muscle cell dysfunction (flow velocity increase to adenosine and nifedipine < 2.0) were enhanced in heart transplant recipients immunosuppressed with TKL, azathioprine, and prednisone. The prevalence of epicardial dysfunction was 78% in group 2 versus 44% and 46% in group 1 and 3 (p < 0.05), respectively. The prevalence of microvascular dysfunction was 56% in group 2 versus 13% and 7% in group 1 and 3 (p < 0.02), respectively. Coronary vasomotor dysfunction was associated with increased myocardial iNOS expression (p < 0.05), decreased eNOS expression (p < 0.05), and enhanced cardiac immunoreactive interleukin-6 (p < 0.01). Coronary intimal thickening was not different between the groups. The combination of TKL and MMF appears to be superior to TKL and azathioprine (and comparable to CyA and azathioprine) concerning preservation of early coronary vasomotor function, eNOS expression, iNOS suppression as well as cardiac interleukin-6 release. This may have an important impact on subsequent development of transplant coronary atherosclerosis.

Triiodothyronine (T3) increases mitochondrial respiration and promotes the uncoupling between oxygen consumption and ATP synthesis. T3 effect is mediated partly through transcriptional control of genes encoding mitochondrial proteins. We determined the effect of T3 on mRNA levels of uncoupling proteins (UCP) and proteins involved in the biogenesis of the respiratory chain in human skeletal muscle and on UCP2 mRNA expression in adipose tissue. Ten young, healthy males received 75 to 100 5g of T3 per day for 14 days. The increase in plasma-free T3 levels was associated with an increase of resting metabolic rate and a decrease of respiratory quotient. In skeletal muscle, treatment with T3 induced a twofold increase of both UCP2 and UCP3 mRNA levels (p c oxidase subunits 2 and 4, nuclear respiratory factor 1, mitochondrial transcription factor A, and the co-activator PGC1 did not change during the treatment. In adipose tissue, UCP2 mRNA levels increased threefold. The direct effect of T3 on skeletal muscle an d adipose tissue UCP2 and UCP3 mRNA expression was demonstrated in vitro in human primary cultures. Our data show that T3 induces UCP2 and UCP3 mRNA expression in humans. In skeletal muscle, UCP regulation by T3 is not associated with the transcriptional regulation of respiratory chain proteins.

The clinical features of congestive heart failure (CHF) result from a complex interaction between reduced ventricular function, neurohormonal activation, and impaired endothelial function. Although endothelial dysfunction has been well documented, the mechanisms that contribute to this abnormality remain unknown. Recent studies, however, indicate a potential therapeutic role for supplemental L-arginine, suggesting the presence of an underlying disorder of L-arginine metabolism.

Platelet alpha(2A)-adrenoceptors (alpha(2A)AR) and imidazoline binding sites (subtype I(1)) have been proposed as peripheral markers of brain stem receptors that mediate sympathetic outflow and are reported to be elevated in major depression.

The aims of these experiments were to determine the effect of a therapeutic regimen of dexamethasone on cytochrome P4503A4 (CYP3A4) activity in healthy volunteers; and the concentration-effect relationship between dexamethasone and CYP3A4 activity in primary human hepatocyte cultures.

P-Glycoprotein is an efflux pump in many epithelial cells with excretory function. It has been demonstrated that rifampin (INN, rifampicin) induces P-glycoprotein, particularly in the gut wall. We therefore hypothesized that rifampin affects pharmacokinetics of the P-glycoprotein substrate talinolol, a beta1-blocker without appreciable metabolic disposition but intense intestinal secretion in human beings.

Several reports indicate that vascular endothelial growth factor (VEGF) expression is increased in endometrial glands and stroma during the menstrual phase in the human endometrium. Here we report that VEGF receptor type 2 (KDR), normally expressed only in the vascular endothelium, was dramatically up-regulated in the stromal cells of the superficial endometrial zones during the premenstrual phase in both human and macaque endometrium. This increase was detectable by Northern analysis, in situ hybridization, and immunocytochemistry and was cell specific, zone specific, cycle phase specific, and VEGF receptor type specific. That is, it only occurred during the premenstrual/menstrual phase, did not occur in glandular epithelium, endothelium, or stromal cells of the deepest endometrial zones, and was not observed for VEGF receptor type 1. The upregulation of stromal KDR was induced by progesterone (P) withdrawal in both women and macaques, and adding back P 24 h after P withdrawal in macaques blocked stromal, but not vascular, endothelial KDR expression. Promatrix metalloproteinase-1 (MMP-1) was coordinately up-regulated in the same stromal cell population by P withdrawal. Because of reports that VEGF can enhance MMP expression, we hypothesize that VEGF-KDR interactions may influence MMP expression in the superficial zones of the primate endometrium during the premenstrual phase, and that these interactions play a role in the induction of menstruation.

To investigate whether the expression of uncoupling proteins (UCP2 and UCP3) was affected by a very low calorie diet (VLCD) and growth hormone (GH) treatment for 4 weeks.

Mutations in the HSD11B2 gene encoding the kidney (11-HSD2) isozyme of 11beta-hydroxysteroid dehydrogenase cause apparent mineralocorticoid excess, a form of familial hypertension. Because the hypertension associated with AME is of the salt-sensitive type, it seemed possible that decreases in 11-HSD2 activity might be associated with salt sensitivity. To examine this, Italians with mild hypertension underwent a protocol consisting of a rapid intravenous saline infusion and subsequent furosemide diuresis. To determine whether there were genetic associations between HSD11B2 and salt sensitivity, 198 Italians were genotyped for a CA repeat polymorphism (11 alleles) in the first intron. Increased differences in mean arterial pressure between the sodium loaded and depleted states were correlated with shorter CA repeat length (R=0.214, P=0. 0025). The effect behaved as a recessive trait. This suggested that decreased HSD11B2 expression was associated with shorter CA repeat length. Furthermore, activity of renal 11-HSD2 as measured by an increase in the ratio of urinary-free cortisol/urinary-free cortisone was lower in 33 salt-sensitive subjects (urinary-free cortisol/urinary-free cortisone 0.89+/-0.04 [mean+/-SE]) compared with 34 salt-resistant subjects (0.71+/-0.04, P<0.001). However, when minigenes containing either 14 or 23 CA repeats were transfected into rabbit or human kidney cortical collecting duct cells, the construct with 14 repeats was instead expressed at levels 50% higher than those of the construct with 23 repeats, as determined by reverse transcription-polymerase chain reaction. We conclude that polymorphisms in HSD11B2 and decreased 11-HSD2 activity are associated with sensitivity to sodium loading, but a functional explanation for these associations remains to be elucidated.

The effect of intravenous administration of lipid emulsions enriched with omega-3 (n3) and omega-6 (n6) fatty acids on equine monocyte phospholipid fatty acid composition and the synthesis of inflammatory mediators in vitro was evaluated. In a randomized crossover design, horses were infused intravenously with 20% lipid emulsions containing n3 or n6 fatty acids. Monocytes were isolated from the horses before and 0 h, 8 h, 24 h, and 7 days after lipid infusion. Monocyte fatty acid analysis demonstrated incorporation of the parenteral n3 and n6 fatty acids in monocyte phospholipids immediately after infusion, with changes in the fatty acid composition persisting for up to 7 days after infusion. In vitro production of the inflammatory mediators thromboxane B2/thromboxane B3 (TXB(2/3)) and tumor necrosis factor-alpha (TNFalpha) by peripheral blood monocytes was diminished by n3 lipid infusion and was unchanged or increased by n6 lipid infusion. The results of this study demonstrate that short-term infusions of n3 and n6 fatty acid-enriched lipid emulsions alter the fatty acid composition of equine monocyte phospholipids and modify the inflammatory response of these cells in vitro. These results also support further investigation into the use of parenteral n3 fatty acids as part of the supportive therapy of patients with multiple organ dysfunction (MODS) or systemic inflammatory response syndrome (SIRS).

Recent preclinical studies have shown that lithium (Li) robustly increases the levels of the major neuroprotective protein, bcl-2, in rat brain and in cells of human neuronal origin. These effects are accompanied by striking neuroprotective effects in vitro and in the rodent central nervous system in vivo. We have undertaken the present study to determine if lithium exerts neurotrophic/ neuroprotective effects in the human brain in vivo.

Animal and post mortem studies indicate that neuroleptic therapy may induce D2-dopamine receptor upregulation in the basal ganglia. To address this phenomenon in a clinical study, we investigated the D2-dopamine receptor binding in 15 DSM-III-R schizophrenics in the drug-naive state and three days after completion of a standardized neuroleptic therapy (benperidol 12-16 mg/d for 25 days) using single photon emission computed tomography (SPECT). SPECT scans were obtained 2 h after intravenous injection of 185 MBq 123I-IBZM. For analysis, basal ganglia to frontal cortex (BG/FC) ratios were calculated and the patient sample was subgrouped into patients with a favourable versus a poor treatment response. Neuroleptic treatment led to decreased BG/FC ratios in patients with a favourable response, but increased ratios in the poor responders (df = 1, F = 4.1, p = 0.06). Changes of BG/FC ratios were significantly correlated with extrapyramidal side effects, but not with neurological soft signs (NSS). Our findings indicate that neuroleptic therapy induces D2-dopamine receptor upregulation in a subgroup of patients characterized by poor treatment response and pronounced extrapyramidal side effects.

Thymidine phosphorylase (TP) is an essential enzyme for the biochemical activation of 5-fluorouracil (5-FU). Interferon upregulates TP in vivo, although the dose and schedule of interferon for optimal biomodulation of 5-FU is not known. In this study, TP activity was measured in peripheral blood lymphocytes (PBLs) from patients with advanced carcinoma receiving treatment with 5-FU and folinic acid. Cohorts of patients were treated with interferon alpha (IFNalpha), immediately prior to 5-FU/folinic acid, at doses of 3 MIU m(-2), 9 MIU m(-2) and 18 MIUm(-2). IFNalpha was administered on day 0 cycle two, day-1 and day 0 cycle three and day-2, day-1 and day 0 cycle four. A fourth cohort was treated with IFNalpha 9 MIU m(-2) three times per week from cycle 2 onwards. Twenty-one patients were entered into the study with 19 evaluable for response. Six patients (32%) had stable disease and 13 (68%) progressive disease. There were no grade-IV toxicities. TP activity was detected in PBLs from all patients with wide interpatient variability in constitutive TP activity prior to chemotherapy, and in response to IFNalpha. 5-FU/folinic acid alone did not induce TP activity but a single dose of IFNalpha led to upregulation of TP within 2 h of administration with a further increase by 24 h (signed rank test, P = 0.006). TP activity remained elevated for at least 13 days (signed rank test, P= 0.02). There were no significant differences in TP activity between schedules or with additional doses of IFNalpha. A single dose of IFNalpha as low as 3 MIU m(-2) can cause sustained elevation of PBL TP activity in vivo indicating that biochemical markers are important pharmacodynamic endpoints for developing optimal schedules of IFNalpha for biomodulation of 5-FU.

The in vivo effects of beta-adrenergic receptor (betaAR) agonists given chronically by metered-dose inhaler (MDI) on the molecular components of the beta-adrenoceptor system expressed by human respiratory cells are poorly understood. This study examined the effects of inhaled albuterol (180 microg four times daily for 7 days) on betaAR function of airway epithelial cells (AECs) and alveolar macrophages (AMs) freshly isolated from 10 normal subjects. Responses were related to beta2AR genotype in codons 16 and 27, regions which affect chronic responses to beta2-agonists. In AEC, betaAR density and adenosine cyclic 3',5'-phosphate (cAMP) production in response to isoproterenol (ISO) were significantly lower in the albuterol versus placebo treatment arm (p < 0.01 for both). Moreover, in AEC, albuterol treatment increased betaAR-kinase (betaARK) protein immunoreactivity. In contrast, in AM, albuterol tended to decrease betaAR density and cAMP production but changes did not achieve statistical significance (p > 0.20 for both) and had no effect on betaARK immunoreactivity. Changes in betaAR density occurred in all subjects but tended to be greater in subjects with the glycine 16 genotype. In cultured cells exposed to equal concentrations of beta-agonist in vitro, the magnitude of betaAR down-regulation (p < 0.05) and cAMP densensitization (p < 0.05) was greater in AEC than AM. These results indicate that albuterol taken by inhalation in a therapeutically relevant dose for 1 week produces betaAR down-regulation, densensitizes the cAMP response of airway epithelial cells to a beta2-adrenergic agonist, and increases betaARK immunoreactivity. Greater densensitization of AEC than AM in response to chronic albuterol inhalation likely reflects cell type-specific responses.

The phosphorylation of glucose to glucose-6-phosphate (G-6-P) is the first committed step in glucose uptake in skeletal muscle. This reaction is catalyzed by hexokinase (HK). Two HK isoforms, HKI and HKII, are expressed in human skeletal muscle, but only HKII is regulated by insulin. The present study was undertaken to determine the time course for the regulation of HK activity and expression by physiological plasma insulin concentrations in human skeletal muscle in vivo. A hyperinsulinemic-euglycemic glucose clamp and percutaneous muscle biopsy were performed in separate groups of healthy subjects after 60, 120, 180, and 360 minutes of euglycemic hyperinsulinemia. Muscle biopsies were subfractionated into soluble and particulate fractions to determine HKI and HKII activities. RNA was extracted from a separate portion of the muscle biopsy, and HKI and HKII mRNA content was determined using an RNase protection assay. Glycogen synthase (GS) activity and fractional velocity were also determined. HKII mRNA was increased 2-fold by 120 minutes and remained high versus the basal value for up to 360 minutes. HKI mRNA was unchanged throughout the study. HKII activity increased after 360 minutes of insulin infusion, and this increase was limited to the soluble fraction. In contrast, insulin induced a 1.5- to 2-fold increase in GS fractional velocity that was sustained for 360 minutes. The time course of the ability of hyperinsulinemia to increase HKII mRNA indicates that insulin is likely a physiological regulator of HKII expression in human skeletal muscle in vivo.

Imiquimod (IQ) has been successfully used in treatment of genital warts. In clinical settings, patients responded well but wart reduction rates varied. Our aim was to find a correlation between clinical responses and pretreatment (constitutive) levels of genes that might be involved in the molecular action of IQ. Since IQ is a cytokine inducer, we analyzed levels of expression of genes of the JAK/STAT signaling pathway and their inhibitors as well as interferon response factors (IRFs) in pretreatment biopsy specimens from complete responders (99 to 100% wart reduction rate) versus incomplete responders (75 to 92% wart reduction rate) by reverse transcription-PCR. We found that mRNA levels of signal transducer and activator of transcription 1 (STAT1) and IRF1 were higher in complete responders than in incomplete responders. Incomplete responders expressed larger amounts of STAT3, IRF2, and protein inhibitor of activated STAT1 (PIAS1) mRNAs compared to complete responders before IQ treatment. We hypothesize that high-level expression of STAT1 and IRF1 is advantageous for a better IQ response. The observed differences in constitutive mRNA levels of these genes may be the consequence of alterations in cellular differentiation and/or variable expression of endogenous interferons. Previous in vitro studies showed that keratinocyte differentiation coordinates the balance between positive and negative signals along the JAK/STAT pathway by regulating the IRF1:IRF2 and STAT1:PIAS1 ratios and thus affecting induction of IQ-inducible genes. Specifically, differentiation supports constitutive expression of STAT1 and IRF1 mRNAs but not expression of IRF2 and PIAS1. Our data are in good agreement with studies that showed the importance of STAT1 in cytokine induction and activation of interferon-responsive genes by IQ.

Insulin regulates expression and production of leptin in rodents but whether this is also true in humans remains unclear. To test the effects of acute hyperinsulinemia on expression of leptin mRNA in humans, percutaneous needle biopsies of abdominal subcutaneous adipose tissue were performed at baseline and immediately following a 200-min two-step hyperinsulinemic-euglycemic glucose clamp in 16 Pima Indians (8M/8F). Leptin mRNA was quantified by reverse transcription, PCR amplification and expressed relative to actin mRNA. Leptin mRNA levels were higher in women than men (25.6 +/- 1.7 v 16.9 +/- 2.1 relative units, P = 0.003) at baseline. Baseline levels were directly related to percentage body fat (r = 0.54, P = 0. 03) and fasting plasma glucose concentrations (r = 0.57, P = 0.02) and were negatively correlated to glucose disposal at physiologic insulin concentrations (750 +/- 40 pmol/L) during the clamp (r = -0. 51, P = 0.04). Acute hyperinsulinemia (final insulin concentration 11560 +/- 950 pmol/L) increased leptin mRNA levels in 13 of 16 individuals an average of 13% (21.3 +/- 1.7 to 24.2 +/- 1.2 relative units, P = 0.01). Changes in leptin mRNA were directly related to glucose disposal rates during physiologic hyperinsulinemia (r = 0.54, P < 0.04). These results suggest that the expression of leptin mRNA is regulated by insulin in humans, as it is in rodents.

The effect and clinical significance of tolcapone withdrawal on erythrocyte catechol-O-methyltransferase (COMT) activity, levodopa pharmacokinetics, and levodopa requirements were investigated in 59 patients with fluctuating parkinsonism who were randomized to receive placebo or tolcapone 100 or 200 mg three times daily for 6 weeks. Tolcapone withdrawal caused a transient elevation in COMT activity by 64% in patients receiving 100 mg three times daily and by 128% in those receiving 200 mg three times daily at approximately 1-2 weeks after discontinuation of drug. Thereafter, COMT activity was declining but did not reach baseline values by the 12-week study endpoint. However, this had no effect on plasma levodopa and 3-O-methyldopa (3-OMD) concentrations or on levodopa requirements. During treatment, tolcapone increased "on" time and decreased "off" time; after discontinuation of study medication and levodopa dose adjustment, on and off times were similar to baseline. Withdrawal was generally well tolerated; no patients withdrew from the trial during the posttreatment period, and no serious adverse events were observed. In conclusion, the transient increase in erythrocyte COMT activity observed after discontinuation oftolcapone is not associated with changes in peripheral levodopa metabolism and therefore has no significant clinical consequences in terms of levodopa requirements, clinical symptoms, or adverse events.