Long live the worms: methods for maintaining and assessing the viability of intestinal stages of Parascaris spp. in vitro.

In vitro maintenance of helminth parasites enables a variety of molecular, pharmaceutical and immunological analyses. Currently, the nutritional and environmental in vitro requirements of the equine ascarid parasite, Parascaris spp., have not been determined. Additionally, an objective method for assessing viability of Parascaris spp. intestinal stages does not exist. The purpose of this study was to ascertain the in vitro requirements of intestinal stages of Parascaris spp., and to develop a viability assessment method. A total of 1045 worms were maintained in a total of 212 cultures. Worms obtained from naturally infected foals at necropsy were immediately placed in culture flasks containing 200 mL of culture media. A variety of media types, nutrient supplementation and environmental conditions were examined. A motility-based scoring system was used to assess worm viability. Worms maintained in Roswell Park Memorial Institute-1640 had significantly better viability than any other media (P < 0.0001) and all media types supplemented with any of the nutrients examined (P < 0.0001). The use of a platform rocker also significantly improved viability (P = 0.0305). This is the first study to examine the requirements for maintaining Parascaris spp. intestinal stages in vitro and to evaluate their viability based on movement using an objective scoring system.

Attenuation of ketamine-induced impairment in verbal learning and memory in healthy volunteers by the AMPA receptor potentiator PF-04958242.

There is a need to develop treatments for cognitive impairment associated with schizophrenia (CIAS). The significant role played by N-methyl-d-aspartate receptors (NMDARs) in both the pathophysiology of schizophrenia and in neuronal plasticity suggests that facilitation of NMDAR function might ameliorate CIAS. One strategy to correct NMDAR hypofunction is to stimulate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) as AMPAR and NMDAR functioning are coupled and interdependent. In rats and nonhuman primates (NHP), AMPAR potentiators reduce spatial working memory deficits caused by the nonselective NMDAR antagonist ketamine. The current study assessed whether the AMPAR potentiator PF-04958242 would attenuate ketamine-induced deficits in verbal learning and memory in humans. Healthy male subjects (n=29) participated in two randomized treatment periods of daily placebo or PF-04958242 for 5 days separated by a washout period. On day 5 of each treatment period, subjects underwent a ketamine infusion for 75 min during which the effects of PF-04958242/placebo were assessed on ketamine-induced: (1) impairments in verbal learning and recall measured by the Hopkins Verbal Learning Test; (2) impairments in working memory on a CogState battery; and (3) psychotomimetic effects measured by the Positive and Negative Syndrome Scale and Clinician-Administered Dissociative Symptoms Scale. PF-04958242 significantly reduced ketamine-induced impairments in immediate recall and the 2-Back and spatial working memory tasks (CogState Battery), without significantly attenuating ketamine-induced psychotomimetic effects. There were no pharmacokinetic interactions between PF-04958242 and ketamine. Furthermore, PF-04958242 was well tolerated. 'High-impact' AMPAR potentiators like PF-04958242 may have a role in the treatment of the cognitive symptoms, but not the positive or negative symptoms, associated with schizophrenia. The excellent concordance between the preclinical (rat, NHP) and human studies with PF-04958242, and in silico modeling of AMPAR-NMDAR interactions in the hippocampus, highlights the translational value of this study.

Pathogenic Helicobacter pylori strains translocate DNA and activate TLR9 via the cancer-associated cag type IV secretion system.

Helicobacter pylori (H. pylori) is the strongest identified risk factor for gastric cancer, the third most common cause of cancer-related death worldwide. An H. pylori constituent that augments cancer risk is the strain-specific cag pathogenicity island, which encodes a type IV secretion system (T4SS) that translocates a pro-inflammatory and oncogenic protein, CagA, into epithelial cells. However, the majority of persons colonized with CagA+ H. pylori strains do not develop cancer, suggesting that other microbial effectors also have a role in carcinogenesis. Toll-like receptor 9 (TLR9) is an endosome bound, innate immune receptor that detects and responds to hypo-methylated CpG DNA motifs that are most commonly found in microbial genomes. High-expression tlr9 polymorphisms have been linked to the development of premalignant lesions in the stomach. We now demonstrate that levels of H. pylori-mediated TLR9 activation and expression are directly related to gastric cancer risk in human populations. Mechanistically, we show for the first time that the H. pylori cancer-associated cag T4SS is required for TLR9 activation and that H. pylori DNA is actively translocated by the cag T4SS to engage this host receptor. Activation of TLR9 occurs through a contact-dependent mechanism between pathogen and host, and involves transfer of microbial DNA that is both protected as well as exposed during transport. These results indicate that TLR9 activation via the cag island may modify the risk for malignancy within the context of H. pylori infection and provide an important framework for future studies investigating the microbial-epithelial interface in gastric carcinogenesis.

PDE5 inhibition improves acquisition processes after learning via a central mechanism.

In previous studies, we have shown that phosphodiesterase type 5 inhibitors (PDE5-Is) can improve early consolidation of object memory. These conclusions were based on the timing of drug administration relative to the learning trial (i.e. before or after). However, there are very little pharmacological data available about the pharmacokinetic profile of orally administered PDE5-Is in the rat. Furthermore, there is still debate whether these effects are achieved via central or peripheral mechanisms and if acquisition processes are improved. In the current study, we tested the effects of the PDE5-I vardenafil in a cholinergic-deficit model and compared the effects after intracerebroventricular (ICV) versus oral (PO) administration. We found that PO vardenafil restored a scopolamine-induced memory impairment when dosed within 2 min after the learning trial while ICV vardenafil was able to restore memory when injected within 4 min after learning. Because the test trial was within 10 min after the learning trial, this suggests that these effects on object memory are related to acquisition processes that may still be ongoing in a time window after the learning trial. To further elucidate the extent of this acquisition window, we investigated the pharmacokinetic profile of vardenafil after PO administration where it was detected within 4 min post-dose. Taken together, our data suggest that PDE5 is involved in acquisition processes, which may linger for at least 4-6 min after learning. Further studies are needed to exclude that these effects could also be explained on basis of an effect on early consolidation processes. Additionally, the effectiveness of ICV-administered vardenafil provides further experimental evidence that PDE5-Is improve memory via a central mechanism.

Neurophysiological signals as potential translatable biomarkers for modulation of metabotropic glutamate 5 receptors.

The Group I metabotropic glutamate receptor subtype 5 (mGluR5) is widely distributed in the brain with dense expression in the cerebral cortex, hippocampus, and basal ganglia. These receptors have been implicated in psychiatric and neurological disorders such as schizophrenia, Fragile X syndrome, addiction, anxiety/depression, Parkinson's disease and neuropathic pain. The present study evaluated the effects of the mGluR5 negative allosteric modulators (NAMs) 4-difluoromethoxy-3-(pyridine-2-ylethynyl)phenyl)5H-pyrrolo[3,4-b]pyridine-6(7H)-yl methanone (GRN-529) and methyl (3aR,4S,7aR)-4-hydroxy-4-[(3-methylphenyl)ethynyl]octahydro-1H-indole-1-carboxylate (AFQ056) on polysomnographic (PSG) and quantitative electroencephalographic (qEEG) measures in freely moving rats. Furthermore, the anxiolytic profile of GRN-529 was characterized in anesthetized rats by measuring stimulation-induced hippocampal theta oscillation. The present findings demonstrate that inhibition of mGluR5 via its allosteric site profoundly modulates high-level neuronal network activities as indicated by changes in sleep-wake activity and power distribution of qEEG. Both GRN-529 and AFQ056 reduced the total time spent in rapid-eye movement with AFQ056 producing a significant increase in wakefulness at the highest dose tested. Additionally, qEEG revealed significant compound-induced increases in delta power concomitant with more subtle decreases in theta and alpha band power. Receptor occupancy (RO) studies revealed that GRN-529 and AFQ056 at all doses resulted in over 45% mGluR5 occupancy. Furthermore, GRN-529 dose-dependently decreased elicited hippocampal theta frequency, consistent with previous findings using clinically active anxiolytic compounds. The described changes in neurophysiological signals identified in freely moving rats may be considered suitable translational biomarkers for the clinical evaluation of mGluR5 NAMs.

Rhythmic theta and delta activity of cortical and hippocampal neuronal networks in genetically or pharmacologically induced N-methyl-D-aspartate receptor hypofunction under urethane anesthesia.

N-Methyl-d-aspartate receptor (NMDAR) antagonists mimic several symptoms of schizophrenia in healthy subjects, and are used in preclinical disease models. In the present study, the impact of pharmacologically and genetically induced NMDAR hypofunction was assessed in rats and mice, including the NMDAR hypomorphic (Grin1) mice, with respect to neuronal network oscillations. Field potentials were recorded from the ventro-medial prefrontal cortex (mPFC) and hippocampus (CA1) in rats, as well as spontaneous and elicited hippocampal theta oscillations in response to brainstem stimulation in Grin1 and wild-type (WT) mice under anesthesia. Effects of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor positive allosteric modulator LY451395 were tested in Grin1 mice and in WT mice following an MK-801 challenge. Recordings from the mPFC and CA1 in rats revealed regular delta and theta oscillations, respectively, which were disrupted by MK-801. In WT mice, MK-801 reduced both spontaneous and elicited hippocampal theta power. Age-matched Grin1 mice showed abnormal hippocampal field potentials, resembling activity seen after administration of MK-801 in WT mice, but also epileptiform discharges. Administration of MK-801 achieved high levels of NMDAR occupancy (84-98%) in both rats and mice, which is comparable to the approximately 90-95% reduction of NMDAR expression in the Grin1 mouse. Impaired elicited CA1 theta oscillation in WT mice following MK-801, or Grin1 mice was significantly improved by LY451395. These findings demonstrate similar, although not identical, changes in network activity following reduction in functioning NMDARs induced by acute pharmacological or genetic manipulations, indicating that these novel neurophysiological models could be used in evaluating drug candidates targeting glutamate neurotransmission.

N-dealkylation of an N-cyclopropylamine by horseradish peroxidase. Fate of the cyclopropyl group.

Cyclopropylamines inactivate cytochrome P450 enzymes which catalyze their oxidative N-dealkylation. A key intermediate in both processes is postulated to be a highly reactive aminium cation radical formed by single electron transfer (SET) oxidation of the nitrogen center, but direct evidence for this has remained elusive. To address this deficiency and identify the fate of the cyclopropyl group lost upon N-dealkylation, we have investigated the oxidation of N-cyclopropyl-N-methylaniline (3) by horseradish peroxidase, a well-known SET enzyme. For comparison, similar studies were carried out in parallel with N-isopropyl-N-methylaniline (9) and N,N-dimethylaniline (8). Under standard peroxidatic conditions (HRP, H(2)O(2), air), HRP oxidizes 8 completely to N-methylaniline (4) plus formaldehyde within 15-30 min, whereas 9 is oxidized more slowly (<10% in 60 min) to produce only N-isopropylaniline (10) and formaldehyde (acetone and 4 are not formed). In contrast to results with 9, oxidation of 3 is complete in <60 min and affords 4 (20% yield) plus traces of aniline. By using [1'-(14)C]-3, [1'-(13)C]-3, and [2',3'-(13)C]-3 as substrates, radiochemical and NMR analyses of incubation mixtures revealed that the complete oxidation of 3 by HRP yields 4 (0.2 mol), beta-hydroxypropionic acid (17, 0.2 mol), and N-methylquinolinium (16, 0.8 mol). In buffer purged with pure O(2), the complete oxidation of 3 yields 4 (0.7 mol), 17 (0.7 mol), and 16 (0.3 mol), while under anaerobic conditions, 16 is formed quantitatively from 3. These results indicate that the aminium ion formed by SET oxidation of 3 undergoes cyclopropyl ring fragmentation exclusively to generate a distonic cation radical (14+*) which then partitions between unimolecular cyclization (leading, after further oxidation, to 16) and bimolecular reaction with dissolved oxygen (leading to 4 and 17 in a 1:1 ratio). Neither beta-hydroxypropionaldehyde, acrolein, nor cyclopropanone hydrate are formed as SET metabolites of 3. The synthetic and analytical methods developed in the course of these studies should facilitate the application of cyclopropylamine-containing probes to reactions catalyzed by cytochrome P450 enzymes.

Effect of aerosolized uridine 5'-triphosphate on mucociliary clearance in mild chronic bronchitis.

Previous studies show that uridine 5'-triphosphate (UTP), a P2Y(2) receptor agonist, is effective at acutely enhancing mucociliary clearance in healthy, nonsmoking adults. UTP solution for inhalation is being developed by Inspire Pharmaceuticals under the compound number INS316. In a double-blind, randomized, crossover, placebo-controlled study we tested the single-dose effect of UTP in chronic smokers with mild chronic bronchitis (n = 15) by measuring the clearance of (99m)Tc-Fe(2)O(3) particles (4.0 microm mass median aerodynamic diameter [MMAD]) after inhalation of nebulized placebo (0.9% saline) and two doses of UTP (20 and 100 mg in the nebulizer). On each study day, gamma camera scanning was performed over a 2-h period. After an initial deposition scan, subjects inhaled placebo or UTP during the first 20 min of scanning. Analysis of whole lung clearance showed that the retention-time curves for each day were biphasic and that the earliest break point in the average curves occurred at 50 min. Mean particle clearance rate (Clr in %/min) through 50 min for placebo treatment was Clr = 0.65 +/- 0.27 whereas treatment with UTP showed Clr significantly increased to 0.95 +/- 0.48 and 0.93 +/- 0.44 for the 20-mg and 100-mg dose respectively, p < 0.005 for both as compared with placebo. These data show that mucociliary clearance associated with mild chronic bronchitis is acutely improved with minimal doses of aerosolized UTP, presumably because of its stimulation of ciliary beating and hydration of airway secretions.

A multicenter evaluation of gentamicin therapy in the neonatal intensive care unit.

STUDY OBJECTIVE: To evaluate traditional nomogram (TN) versus individualized pharmacokinetic gentamicin dosing practices in neonatal intensive care units, focusing on achieving target therapeutic concentrations (peak > 8 microg/ml, trough < 2 microg/ml), number of dosing changes, number of concentrations obtained, and evidence of nephrotoxicity. DESIGN: Retrospective chart review. SETTING: Three neonatal intensive care units. PATIENTS: Three hundred nine infants prescribed gentamicin. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: Sixty-seven percent of patients receiving pharmacokinetic dosing had initial peak concentrations of 8 microg/ml or greater compared with 7% of patients receiving TN dosing (p<0.001). Trough concentrations exceeding 2 microg/ml were reported in 23% of patients receiving TN dosing compared with 2% of pharmacokinetic-dosed patients (p<0.001). Forty-two percent and 6%, respectively, required dosage adjustments (p<0.01). The mean number of concentrations obtained per patient was 2.8 and 2.1, respectively (p<0.01). Neither group had evidence of gentamicin-related nephrotoxicity. CONCLUSION: Compared with TN dosing, administering gentamicin loading doses and performing initial pharmacokinetic analysis resulted in rapid attainment of desired concentrations and fewer dosage adjustments, and allowed for a decrease in the number of gentamicin concentrations.

Acute hemodynamic effects of the prostacyclin analog 15AU81 in severe congestive heart failure.

This multicenter, open-label study provides the first assessment of the safety and acute hemodynamic effects of a short-term infusion of 15AU81, a chemically stable analog of prostacyclin, in patients with New York Heart Association class III or IV heart failure. Twelve patients underwent sequential dose escalation by increasing the rate of the infusion at 15-minute intervals until the drug was no longer tolerated. Patients then received a 90-minute infusion at their maximum tolerated dose. The infusion was then discontinued and the subjects were observed during a 90-minute washout segment. Serial hemodynamic measurements were made throughout the dose-ranging, maintenance, and washout segments. A significant decrease in systemic vascular resistance (1,935 +/- 774 vs 1,243 +/- 351 dynes.s.cm-5; p < 0.001) and pulmonary vascular resistance (395 +/- 335 vs 223 +/- 198 dynes.s.cm-5; p = 0.008) occurred from the infusion of vehicle to the maximum tolerated dose. During dose titration, there was a a significant increase in cardiac index (1.9 +/- 0.7 vs 2.6 +/- 0.6 liters/min/m2; p < 0.001) and a tendency for a mild reduction in pulmonary artery wedge pressure (18 +/- 7 vs 17 +/- 6; p = 0.055) for the 8 patients with values on vehicle and maximum tolerated dose. These hemodynamic changes persisted during the maintenance infusion and disappeared rapidly during the washout segment. The most common adverse event to limit dose-ranging was headache, which occurred at a mean maximum tolerated dose of 36 +/- 15 ng/kg/min. Administration of 15AU81 was associated with significant acute hemodynamic improvement in patients with severe heart failure.(ABSTRACT TRUNCATED AT 250 WORDS)

Interexaminer agreement for readings of dip slide tests for salivary mutans streptococci and lactobacilli.

This study reports the interexaminer agreement of three pairs of evaluators of salivary dip slide tests for mutans streptococci and lactobacilli. 717 Cariescreen SM and Bactotest LB dip slides were available for assessment by 2 dentists and 1 dental hygienist. A single calibration session was held prior to the onset of the study. Each dip slide was read once by each examiner independently. Among the three pairs of examiners, Pearson R values ranged from 0.84 to 0.90 for Cariescreen and from 0.78 to 0.87 for Bactotest. Kappa statistic values ranged from 0.56 to 0.61 for Cariescreen and from 0.70 to 0.74 for Bactotest. The range of agreement was from 72.2 to 76.9% for Cariescreen and from 86.4 to 88.3% for Bactotest. The majority of disagreements were of one category in magnitude, though there were a few disagreements of greater magnitude. This study found moderately strong agreement among the three examiners; it suggests that multiple examiners of dip slide tests carefully calibrated initially and periodically to ensure a high level of agreement.

Caries risk assessment in a longitudinal discrimination study.

Our objective was to develop and perfect a model for the assessment of risk of dental caries onset in children. Even though dental caries prevalence in children is continuing to decline, there is still a significant minority for whom it is a problem. In this study, we sought to ascertain whether a set of variables selected in a previous cross-sectional study could be used to differentiate between caries-free six-year-olds who would or would not subsequently present with clinically-detectable caries. A total of 472 caries-free six-year-olds--286 from a fluoridated community and 186 from a fluoride-deficient community--was selected. Clinical examinations for DMFS, dental fluorosis, and plaque were conducted. Stimulated whole saliva was collected for analysis of mutants streptococci, lactobacilli, total viable flora, and fluoride, calcium, and phosphate concentrations. A questionnaire was used for collection of demographic data as well as information on prior fluoride exposure, dietary habits, and oral hygiene practices. By means of linear discriminant analyses, it was possible to predict correctly which children would develop caries within six to 12 months (sensitivity) in 82.8% of cases and which children would not develop caries during that period (specificity) in 82.4% of cases.

Caries risk assessment by a cross-sectional discrimination model.

Although the prevalence of dental caries is continuing to decline, it still affects a majority of the US population and can be a serious problem for those afflicted. The objective of this project was to develop and perfect a model for assessment of risk of dental caries onset in children. In the first study, reported herein, a set of clinical, microbiological, biochemical, and socio-demographic variables was identified that distinguished, with an acceptable level of sensitivity and specificity, between children who had no previous caries experience and children who had high caries levels. A total of 313 children--age 12-15 years, 140 from a fluoridated community and 173 from a fluoride-deficient community--was selected on the basis of previous caries experience, either zero DMFS or high DMFS (> or = 6 in the fluoridated or > or = 8 in the fluoride-deficient community). Clinical exams for DMFS, dental fluorosis, and plaque were conducted. Stimulated whole saliva was collected for analysis of mutans streptococci, lactobacilli, total viable flora, and fluoride concentration. A questionnaire was used for collection of demographic data as well as information on prior fluoride exposure, dietary habits, and oral hygiene practices. By means of discriminant analyses, with use of seven key clinical and laboratory variables, it was possible for zero-DMFS subjects to e classified correctly (specificity) in 77.6% of cases in the fluoridated community and in 86.1% of cases in the fluoride-deficient community. High-caries subjects were classified as such (sensitivity) in 79.3% and 88.1% of cases, respectively.