Maternal gene expression profiling during pregnancy and preeclampsia in human peripheral blood mononuclear cells.

UNLABELLED: Preeclampsia is a major obstetrical complication affecting maternal and fetal health. While it is clear that there is a substantial placental contribution to preeclampsia pathogenesis, the maternal contribution is less well characterized. We therefore performed a genome-wide transcriptome analysis to explore disease-associated changes in maternal gene expression patterns in peripheral blood mononuclear cells (PBMCs). METHODS: Preeclampsia was defined as gestational hypertension, proteinuria and hyperurecimia. Total RNA was isolated from PBMCs obtained from women with uncomplicated pregnancies (n = 5) and women with preeclamptic pregnancies (n = 5). Gene expression analysis was carried out using Agilent oligonucleotide microarrays. Biological pathway analysis was undertaken using Ingenuity Pathway Analysis software. Quantitative real-time PCR (QRTPCR) was performed to validate the gene expression changes of selected genes in normotensive and preeclamptic patients (n = 12 each). RESULTS: We identified a total of 368 genes that were differentially expressed in women with preeclampsia compared to normal controls with false discovery rate (FDR) controlled at 10%. In follow up experiments we further analyzed the expression levels of a number of genes that were identified as altered by the microarray data including survivin (BIRC5), caveolin (CAV1), GATA binding protein-1 (GATA1), signal tranducer and activator of transcription 1 (STAT1), E2F transcription factor-1 (E2F1), fibronectin-1 (FN1), interleukin-4 (IL-4), matrix metalloprotease-9 (MMP-9) and WAP four disulfide domain protein (WFDC-1) by QRTPCR. Additionally we performed immuno blot analysis and zymography to verify some of these candidate genes at the protein level. Computational analysis of gene function identified an anti-proliferative and altered immune function cellular phenotype in severe preeclamptic samples. CONCLUSIONS: We have characterized the genome-wide mRNA expression changes associated with preeclampsia-specific genes in circulating maternal blood cells at the time of delivery. In addition to providing information relating to the biological basis of the preeclampsia phenotype, our data provide a number of potential biomarkers for use in the further characterization of this disease.

Conformational dynamics of a transposition repressor in modulating DNA binding.

The repressor of bacteriophage Mu functions in the establishment and maintenance of lysogeny by binding to Mu operator DNA to shut down transposition. A domain at its N terminus functions in DNA binding, and temperature-sensitive mutations in this domain can be suppressed by truncations at the C terminus. To understand the role of the C-terminal tail in DNA binding, a fluorescent probe was attached to the C terminus to examine its environment and its movement with respect to the DNA binding domain. The emission spectrum of this probe indicated that the C terminus was in a relatively hydrophobic environment, comparable to the environment of the probe attached within the DNA-binding domain. Fluorescence of two tryptophan residues located within the DNA-binding domain was quenched by the probe attached to the C terminus, indicating that the C terminus is in close proximity to this domain. Addition of DNA, even when it did not contain operator DNA, reduced quenching of tryptophan fluorescence, indicating that the tail moves away from the DNA-binding domain as it interacts with DNA. The presence of the tail also produced a trypsin hypersensitive site within the DNA-binding domain; mutant repressors with an altered or truncated C terminus were relatively resistant to cleavage at this site. Interaction of the wild-type repressor with DNA greatly reduced cleavage at the site. A repressor with a temperature-sensitive mutation in the DNA-binding domain was especially sensitive to cleavage by trypsin even in the presence of DNA, and the C-terminal tail failed to move in the presence of DNA at elevated temperatures. These results indicate that the tail sterically inhibits DNA binding and that it moves during establishment of repression. Such conformational changes are likely to be involved in communication between repressor protomers for cooperative DNA binding.

Single-isomer beta-agonists.

A new generation of bronchodilators is being developed for acute asthma management-single-isomer beta-agonists. These drugs consist only of the active bronchodilatory isomer (eutomer); they do not have the inactive and potentially harmful isomer (distomer) that is present in marketed racemic beta-agonists. Clinical studies comparing the effectiveness of (R)-albuterol (levalbuterol) with racemic albuterol established a strong rationale for using single-isomer beta-agonists in place of the racemic mixture: reduced dosages provide equivalent bronchodilatory effects with fewer beta-mediated side effects. Higher dosages achieve superior bronchodilation in episodes of severe asthma and may reduce costs of emergency department treatment.

Dose-response evaluation of levalbuterol versus racemic albuterol in patients with asthma.

Albuterol, in all marketed forms, is sold as a racemate, composed of a 50:50 mixture of (R)- and (S)-isomers. Racemic albuterol and the single isomer version (R)-albuterol (levalbuterol) were compared in a randomized, double-blind, dose-ranging five-way crossover study in patients (n = 20) with mild persistent to moderate persistent asthma. Placebo, racemic albuterol (2.50 mg), or levalbuterol (0.31, 0.63, or 1.25 mg) were delivered as single, nebulized doses to 5 male and 15 female nonsmoking patients with asthma aged 18-50 years. Serial pulmonary function was assessed at 15-min intervals and mean time to onset of activity and duration of improvement of forced expiratory volume in 1 sec (FEV1) were measured. In addition, blood chemistries, electrocardiogram (ECG) readings, and patient subjective assessment of adverse symptoms were recorded. Levalbuterol was found to provide significant bronchodilatory activity and was well tolerated. Levalbuterol 1.25 mg provided the greatest increase and duration in FEV1 improvement, whereas racemic albuterol (2.50 mg) and levalbuterol 0.63 mg provided comparable effects. The lower doses of levalbuterol were associated with a less marked effect on heart rate and potassium than racemic albuterol or high-dose levalbuterol. These data suggest that 0.63 mg levalbuterol provides bronchodilation equivalent to 2.50 mg racemic albuterol with less beta-mediated side effects.

Monitoring of the European corn borer (Lepidoptera: Crambidae) in central Maine.

Pheromone trap types and within-field trap locations were compared for their effectiveness in monitoring the flight activity of European corn borer, Ostrinia nubilalis (Hübner), and its relationship to egg mass density and crop damage in sweet corn in central Maine from 1995 to 1996. The use of both 3:97 Z:E-11-tetradecenyl acetate and 97:3 Z:E-11 tetradecenyl acetate pheromone blends confirmed that European corn borer in central Maine is attracted to both pheromone lure types. European corn borer moths were captured predominantly with the E-lure type than with the Z-lure type in both years. The Scentry Heliothis trap was more effective than the Multi-Pher trap, but similar to the pheromone-baited water pan trap for monitoring European corn borer flights. With the Scentry Heliothis trap, the grassy border and 1st corn rows were the best locations for moth capture during the early flight period, but during the peak flight period, traps located in the middle of the field caught the most moths. Corn damage was recorded before moth captures in some sites and before egg mass counts in others, indicating poor efficacy of traps for early flights. Significant and positive correlations were found between moth captures in the midfield location and egg mass counts, and corn leaf damage, and between egg mass counts and corn leaf damage. However, low coefficients of variation suggest that pheromone trap captures were not good predictors of European corn borer leaf damage in sweet corn.

New millennium bronchodilators for asthma: single-isomer beta agonists.

Racemic beta2 agonists are composed of a 50:50 mixture of R and S isomers. The R isomer exhibits virtually all the bronchodilation, whereas the S isomers are generally considered inert. However, (S)-albuterol was shown to enhance bronchial reactivity to methacholine, eosinophil activation, and histamine-induced influx of fluid, proteins, and neutrophils into the airspaces. Actions such as these may compress the potency and foreshorten the duration of (R)-albuterol. Accordingly, pure (R)-albuterol provides bronchodilation at lower doses than racemate, allowing for fewer beta-adrenergic-mediated side effects. In addition, differential metabolism may allow for the progressive accumulation of (S)-albuterol. This logic is applicable to long-acting beta2 agonists: the therapeutically active (R,R)-formoterol is currently being developed in the United States, and preliminary results suggest rapid improvements in FEV1 with up to 24-hour duration of action. These combined observations with the R isomers of beta2 agonists suggest that potential improvements in therapeutic indices can be achieved with isomerically pure versions of existing racemic drugs.

The asthma-like pharmacology and toxicology of (S)-isomers of beta agonists.

Beta(2 ) agonists were designed to emulate the bronchodilation and mast cell suppression effects of human adrenaline, an endogenous neuromediator. Endogenous adrenaline is produced exclusively as the single enantiomer or isomer, (R)-adrenaline, although all selective beta(2 ) agonists are marketed as racemic drugs, composed of a precise 50:50 mixture of R and S isomers. Isomers are compounds that are nonsuperimposable mirror images. The R isomers of beta agonists, essentially all congeners of (R)-adrenaline, exhibit the observed bronchodilation and clinical benefit of the racemate. The S isomers of the racemic beta agonists are devoid of clinical benefit, are assumed to be benign, and have not been studied until recently. In contradistinction to their assumed benign status, extensive studies with (S)-albuterol have shown that it opposes the bronchodilation effects of (R)-albuterol (levalbuterol), may be proinflammatory, and exhibits the potential to exacerbate airway reactivity to a variety of spasmogens by enhancing contractile responses. Clinically, (S)-albuterol can increase airway reactivity and, because of its slow metabolism, exists in higher and prolonged plasma concentrations than levalbuterol. The sustained presence of (S)-albuterol may help to explain why racemic beta agonists have not demonstrated a significant clinical anti-inflammatory potential. Furthermore, the adverse effects (S)-albuterol may contribute to paradoxic bronchospasm and the occurrence of severe reagenic-like reactions seen with racemic albuterol. These adverse effects of (S)-albuterol are completely avoided with single isomer version of (R)-albuterol (levalbuterol). The removal of (S)-albuterol increased the clinical potency of levalbuterol, such that bronchodilator efficacy is achieved at one-fourth the dose of racemic albuterol, but with marked reduction in side effects. Levalbuterol, a third generation beta agonist, retains the clinical benefit of racemic albuterol without the proinflammatory and potentially detrimental effects of (S)-albuterol.

Effects of S-salbutamol on human isolated bronchus.

A range of stimuli have been used to determine the effect of S-salbutamol on contractile responses of human isolated bronchus. Significant augmentation of contraction was evident during responses to histamine or LTC4 but responses to EFS, methacholine, bradykinin and capsaicin were not influenced and allergic bronchospasm was significantly impaired by prior exposure to S-salbutamol. Since R-salbutamol relaxes human isolated bronchus, the capacity of S-salbutamol to enhance contractile responses to histamine or LTC4 cannot be attributed to activation of beta2-adrenoceptors. It is concluded, therefore, that these effects of S-salbutamol represent distinct pharmacological actions of the distomer. It is also suggested that the capacity of S-salbutamol to augment contraction of airway smooth muscle may contribute to hyperreactivity towards spasmogens when racemic salbutamol is used for symptom relief in asthma and chronic obstructive pulmonary disease (COPD).

A force plate system for measuring low-magnitude reaction forces in small laboratory animals.

We present a force plate system which measures low-magnitude vertical reaction forces generated by small laboratory animals. The force plate mechanical design minimizes radiated transverse waves, acoustic reverberation, and standing waves caused by impacts on the force plate surface. A secondary force plate and PC-based software algorithm minimize floor vibrational artifact. The force plate was used to measure function of rats during two tests: forelimb/hindlimb hopping reaction and surface righting reaction. In control rats, forelimb hopping rate exceeded hindlimb hopping rate during 16 weeks of repeated testing. Subchronic intraperitoneal (i.p.) dosing of 10 mg/kg/day acrylamide produced a selective impairment of hindlimb hopping. In contrast, single doses of haloperidol (1-5 mg/kg, i.p.) slowed the righting reaction and produced a relatively selective impairment of forelimb hopping. The force plate system presents new opportunities for performing quantitative neurological assessments of small laboratory animals when previously such tests had been performed subjectively and qualitatively.

Clinical endoscopic evaluation of the gastroduodenal tolerance to (R)- ketoprofen, (R)- flurbiprofen, racemic ketoprofen, and paracetamol: a randomized, single-blind, placebo-controlled trial.

Ketoprofen, a nonsteroidal anti-inflammatory drug (NSAID) of the 2-arylpropionic acid class, causes gastroduodenal hemorrhages and erosions in 10-15% of patients. The (S)- enantiomer exhibits most of the anti-inflammatory properties, with concomitant gastrointestinal toxicity. The (R)- enantiomer, however, was recently found to have analgesic properties independent of prostaglandin inhibition. Seventy-two healthy male volunteers not receiving NSAIDs, alcohol, or anti-ulcer drugs, were enrolled in a randomized, investigator-blind, placebo-controlled trial to evaluate the gastroduodenal tolerance of (R)- ketoprofen 100 mg b.i.d., (R)- flurbiprofen 100 mg b.i.d., racemic ketoprofen 100 mg b.i.d., and paracetamol 1,000 mg b.i.d. Gastroduodenal endoscopies at baseline and after 2.5 days of dosing were used to detect newly occurring hemorrhages and erosions. Adverse events were also recorded. The incidence of submucosal hemorrhages was 4/16 in the (R)- ketoprofen group, 5/16 in the (R)- flurbiprofen group, 12/16 in the racemic ketoprofen group, 1/16 in the paracetamol group, and 1/8 in the placebo group. The incidence of erosions was 2/16 in the (R)- ketoprofen group, 4/16 in the (R)- flurbiprofen group, 10/16 in the racemic ketoprofen group, 0/16 in the paracetamol group, and 2/8 in the placebo group. The differences in hemorrhages and erosions among treatments were statistically significant (gastric hemorrhages P = 0.0008; duodenal hemorrhages P = 0.00062; gastric erosions P = 0.0004; duodenal erosions P = 0.0062, Kruskal-Wallis test). At 100 mg b.i.d., (R)- ketoprofen caused fewer gastroduodenal hemorrhages and erosions than racemic ketoprofen (P = 0.019, P = 0.0112, P = 0.0097, P = 0.0139 for gastric, duodenal hemorrhages and gastric, duodenal erosions, respectively). The difference between 100 mg b.i.d. (R)- ketoprofen and 100 mg b.i.d. (R)- flurbiprofen was not statistically significant. The dissociation between analgesic and anti-inflammatory properties for (R)- ketoprofen suggests that it may represent a unique analgesic with a favorable safety profile.

Preclinical enantioselective pharmacology of (R)- and (S)- ketorolac.

Many of the nonsteroidal anti-inflammatory drugs (NSAIDs) are marketed as racemic mixtures, composed of (R)- and (S)- enantiomers. Racemic NSAIDs are potent cyclooxygenase (COX) inhibitors only through the action of the (S)- enantiomers, as the (R)- enantiomers do not exhibit COX inhibition. However, the (R)- enantiomer of ketoprofen exhibits potent analgesic activity and minimal ulcerogenic potential. To extend these observations, we examined the (R)- and (S)- enantiomers of RS- ketorolac, (S)- ketorolac exhibited potent COX1 and COX2 enzyme inhibition, whereas (R)- ketorolac was > 100-fold less active on both COX subtypes. Both enantiomers did not affect norepinephrine or serotonin uptake sites, and nitric oxidase or lipoxygenase activities, nor did they demonstrate any affinity for opioid receptors (mu, delta, or kappa). In experimental models, (S)- ketorolac exhibited about 10-fold greater activity than (R)- ketorolac in the murine phenylquinone writhing model. In this model, morphine sulfate was effective at much lower doses, however, and neither (R)- nor (S)- ketorolac showed any morphine-sparing effect. In the rat gait test for analgesia in the foot paw after injection of brewers yeast suspension, neither (R)- nor (S)- ketorolac affected paw volume. However, both provoked changes in gait scores, the (S)- enantiomer being 30-fold more potent than the (R)- enantiomer. A similar reduction was observed with respect to ulcerogenic potential, measured by direct microscopic changes after test conclusion. These findings suggest that (R)- ketorolac may possess analgesic activity that is independent of COX inhibition and may be associated with reduced ulcerogenic potential compared to effects exhibited by (S)- ketorolac.

Sympathomimetic enantiomers and asthma.

Airways of asthma patients can become hyperresponsive to airway spasmogens following regular use of isoprenaline or beta 2-selective sympathomimetics. Hyper-reactivity that results from acute exposure of animals to these drugs is pre-empted by vagal section (a procedure which does not influence spasmolytic efficacy of sympathomimetics), is not diminished by antagonism of beta 2-adrenoceptors and is not associated with loss of responsivity of beta 2-adrenoceptors in the airways. Since activation, modulation, or blockade of beta 2-adrenoceptors does not determine this form of hyperreactivity, the possibility that distomers may induce hyperreactivity must be considered. Ocular and vascular responses to distomers of sympathomimetics have long been recognised and, more recently, comparable observations have been made for the airways. Thus, reactivity of guinea-pig airways to spasmogens was increased following exposure to S-isoprenaline, S-salbutamol, or S-terbutaline and exposure to S-isoprenaline or S-salbutamol can intensify symptoms in asthmatics. Regular exposure to the racemate, especially during or following an allergic reaction, predisposes to expression of hyper-reactivity, which is nullified, acutely, by the eutomer. These observations imply that biological effects of sympathomimetic distomers may contribute to morbidity and mortality in asthma patients.

The pursuit of precision pharmaceuticals: divergent effects of beta2 agonist isomers.

Beta2 agonists are the most commonly used treatment for acute bronchoconstriction. However, during regular use there is a progressive decline of protective efficacy of bronchodilators. This progressive decline has long been considered anomalous because with short-acting beta agonists, there is no corresponding change in bronchodilator efficacy. Airway hyper-responsiveness is itself a feature of asthma and there maybe however, there may be an increase in airway hyper-responsiveness following regular use of beta2 agonist. Airway hyperresponsiveness could diminish the capacity of beta agonists to protect from or result in paradoxical bronchospasm and there effects of racemic salbutamol. There have been reports of increased morbidity and mortality associated with excessive use of beta(2) agonists. As all beta agonists used clinically are racemates composed of 1:1 mixtures of R and S isomers, conducted on the possible involvement of the isomers in hyper-responsiveness. Hyper-responsiveness cannot be attributed to the R isomer, whose capacity to activate beta adrenoceptors will nullify this effect. In contrast, extensive evidence indicated that the S isomer might cause hyper-responsiveness and potential airway inflammation. Further, the S isomer shows a propensity to activate human eosinophils and alter muscarinic M(2) receptor functions. The S isomer, which makes no contribution to therapeutic efficacy and may exacerbate asthma, might therefore be excluded from asthma therapy.

Levalbuterol hydrochloride.

Racemic salbutamol (racemic albuterol) ameliorates symptoms of asthma by activating beta-adrenoceptors on nerve, smooth muscle and inflammatory cells within the airways. Racemic salbutamol comprises equal proportions of 2 isomers: (S)-salbutamol and (R)-salbutamol, with the latter being exclusively responsible for activation of beta-adrenoceptors. Accordingly, within racemic salbutamol it is (R)-salbutamol that efficiently relieves obstruction of asthmatic airways and affords highly effective protection from bronchoconstrictor stimuli, including allergens. During regular use of racemic salbutamol, there is a progressive decline of protective efficacy and a corresponding intensification of airway responsiveness. This decline is largely absent during regular use of (R)-salbutamol. Consequently, bronchodilator responses to sub-maximal doses of (R)-salbutamol exceed responses to the equivalent dose of (R)-salbutamol given as the racemate. For example, in asthmatics with baseline FEVs

Therapeutic advantages of third generation antihistamines.

A third generation of antihistamines is emerging for the treatment of allergic rhinitis and chronic urticaria. First generation antihistamines are among the most widely used drugs in the world, and provide symptomatic relief from allergies and the common cold to millions of patients, mainly in OTC combination preparations. Their full potential is limited by the sedation caused by their effects on histamine receptors in the brain. Second generation antihistamines (terfenadine, astemizole, loratadine and cetirizine), which block peripheral H1 receptors without penetrating the blood-brain barrier, were developed and introduced from 1981 onwards to provide comparable therapeutic benefit without the CNS side-effects. Although largely successful in this goal, terfenadine and astemizole were found to cause potentially serious arrhythmias when plasma concentrations became elevated subsequent to impaired metabolism. It was established that the cardiac toxicity was mainly due to the parent drugs. As active metabolites could account for most of the clinical benefit, the goal for the third generation of antihistamines became to develop therapeutically active metabolites that were devoid of cardiac toxicity. The first of these drugs, fexofenadine (the active metabolite of terfenadine), was approved in July 1996, after an unusually rapid development programme. Its introduction set a new standard of safety that led the FDA to request the withdrawal of terfenadine in 1997 on the grounds that a safer version of an equivalent drug was now available. Norastemizole and descarboethoxy loratadine, the metabolites of astemizole and loratadine, respectively, are also in clinical development. These offer comparable or superior clinical benefits.

Quantification of the hindlimb extensor thrust response in rats.

This report describes a procedure for measuring the extensor thrust response (ETR) and summarizes the results of initial validation experiments using adult Long-Evans rats. The ETR can be quickly elicited and the force measured by pressing against the hindlimb footpads with a small rectangular plate or bar attached to a digital force gauge. Output of the force gauge is analyzed and displayed with commercially available hardware and software. The first experiment compared the acute effects of i.p. injection of chlorpromazine (CPZ; 1, 4, or 7 mg/kg) or amphetamine (AMP; 0.3, 1, or 3 mg/kg) on the ETR and forelimb/hindlimb grip strength (FL/HL-GS) in male and female rats. CPZ decreased both ETR and FL/HL-GS values. Both 1 and 3 mg/kg AMP increased grip strength values but decreased ETR values. A second experiment compared the evolution of changes in ETR, FL/HL-GS, and peripheral neurophysiological measures during 8 weeks of daily oral dosing of 10 mg/kg acrylamide (ACR) monomer. ACR-treated rats exhibited a progressive decrease in ETR beginning after 3 weeks of dosing, whereas a reduction of HL-GS was observed beginning much later, after 7 weeks of dosing. The deficit in ETR progressed in the absence of any changes in spontaneous or evoked electrophysiological abnormalities in neuromuscular function, but was accompanied by a decrease in peripheral nerve conduction velocity. Taken together, the results indicate that the ETR can be used to characterize functional effects in both single dose and repeated dose experiments. The data also indicate that the ETR does not merely duplicate the information provided by FL/HL-GS, and suggest a hypothesis that the ETR may be sensitive to neurotoxicant-induced changes in somatosensory function.

Effect of enantiomeric forms of albuterol on stimulated secretion of granular protein from human eosinophils.

We studied the effect of R-, S- and R,S-albuterol in inhibiting the eosinophil peroxidase (EPO) secretion caused by 10(-10) to 10(-6) M formyl-met-leu-phe + 5 micrograms/ml cytochalasin B (FMLP/CB) in non-allergic and allergic subjects. Total RAST score obtained for allergic subjects was 4.12 +/- 0.21 vs 0.36 +/- 0.17 for non-allergic subjects (P < 0.0001). Stimulated EPO secretion was comparable in allergic [2,051 +/- 567 ng/10(6) eosinophils (eos)] and non-allergic subjects [2,337 +/- 488 ng/10(6) eos (P = NS)]. At all concentrations used, both R- and R,S-enantiomers caused comparable (27-32%) inhibition of FMLP/CB stimulated secretion of EPO in allergic and non-allergic subjects. Pretreatment with S-albuterol caused no augmentation of EPO secretion in either allergic (115 +/- 34.6%) or non-allergic subjects (114 +/- 23.7%) subjects, and there was no significant difference in secretion caused by FMLP/CB alone in either experimental group. Similar results were obtained for subjects stratified according to serum IgE concentration. Our data demonstrate that both R- and R,S-albuterol are equivalently effective in inhibiting stimulated secretion of EPO in both normal and allergic subjects and that there is no paradoxical augmenting effect of S-albuterol in stimulated eosinophil secretion.