In vitro developmental competence of bovine demi-embryos generated by blastomere separation and blastocyst bisection.

The efficiency of bovine in vitro embryo production can be significantly improved by splitting embryos at different stages. However, the blastocyst quality of in vitro-produced demi-embryos remains unexplored. The objective of this research was to compare embryo developmental rates and quality of bovine demi-embryos produced by two different strategies: (a) embryo bisection (BSEC) and (b) 2-cell blastomere separation (BSEP). To determine demi-embryos quality, we evaluated total blastocyst cell number and proportion of SOX2+ cells. Additionally, the expression of SOX2, NANOG, OCT4, CDX2, IFNT, BAX and BCL genes and let-7a and miRNA-30c Micro RNAs was analysed. BSEP resulted in improved blastocyst development, higher ICM cells and a significantly higher expression of IFNΤ than demi-embryos produced by BSEC. Let-7a, which is associated with low pregnancy establishment was detected in BSEC, while miRNA-30c expression was observed in all treatments. In conclusion, BSEP of 2-cell embryos is more efficient to improve in vitro bovine embryo development and to produce good quality demi-embryos based on ICM cell number and the expression pattern of the genes explored compared to BSEC.

Are there potential problems with generic substitution of antiepileptic drugs? A review of issues.

In response to increasing cost pressures, healthcare systems are encouraging the use of generic medicines. This review explores potential problems with generic substitution of antiepileptic drugs (AEDs). A broad search strategy identified approximately 70 relevant articles. Potential problems with generic substitution included: The limited evidence (mainly case reports with some pharmacokinetic studies) appears to support these concerns for older AEDs. As a result, restrictions on use of specific generic AEDs are in place in some countries and recommended by some lay epilepsy organisations. As more AEDs lose patent protection, it is important to examine the question of whether generic substitution may pose problems for patients with epilepsy, and whether there should be safeguards to ensure that both physician and patient are informed when generic substitution occurs.

Low-dose topiramate in adults with treatment-resistant partial-onset seizures.

OBJECTIVES: Based on dose predictions from animal and human volunteer studies, most patients enrolled in initial randomized controlled trials of topiramate as adjunctive therapy in adults with partial-onset seizures were randomized to >or= 600 mg/day topiramate. Subsequent experience suggests that dosage needs were overestimated. This double-blind, placebo-controlled study evaluated 200 mg/day topiramate in adults with treatment-resistant partial-onset seizures receiving a concurrent enzyme-inducing antiepileptic agent (carbamazepine). MATERIALS AND METHODS: After a 4-week baseline, 263 adults receiving carbamazepine who had at least three partial-onset seizures during the baseline period were randomized to placebo or one of two topiramate 200 mg/day treatment arms: topiramate escalated weekly 25 mg/day(8-week escalation) or 50 mg/day(4-week escalation). Therapy was then maintained for the remainder of the 12-week double-blind study. RESULTS: Median percent reduction in seizure frequency from baseline to study end was 44% with topiramate and 20% with placebo (P or=10% incidence in topiramate-treated patients) were somnolence, fatigue, paresthesia, nervousness and anorexia; 8% of topiramate-treated patients and 2% of placebo-treated patients discontinued because of adverse events. As a result of the low incidence of adverse events, differences between titration rates in terms of tolerability were not detected. CONCLUSION: Topiramate 200 mg/day is an appropriate target dose as adjunctive therapy in adults with treatment-resistant partial-onset seizures, even when receiving an enzyme-inducing agent; 100 mg/day also appears to be effective. A significant therapeutic effect may be seen in the second week of treatment with a dose of 100 mg/day.

Remacemide hydrochloride as an add-on therapy in epilepsy: a randomized, placebo-controlled trial of three dose levels (300, 600 and 800 mg/day) in a B.I.D. regimen.

Remacemide hydrochloride is a low-affinity, non-competitive NMDA receptor channel blocker under investigation for the treatment of epilepsy. This double-blind, placebo-controlled, multicentre study assessed the safety and efficacy of adjunctive remacemide hydrochloride or placebo, in adult patients with refractory epilepsy who were already taking up to three antiepileptic drugs (including an enzyme-inducer). Patients (n= 262) were randomized to one of three doses of remacemide hydrochloride (300, 600 or 800 mg/day) or placebo, in a B.I.D. regimen, for up to 14 weeks. Plasma concentrations of carbamazepine (CBZ) and phenytoin (PHT) were controlled throughout. Patients recorded their seizures on a diary card. There was an increase in the percentage of responders (defined as a reduction in seizure frequency from baseline > or = 50 %), from 15 % (9/60) with placebo, to 30 % (18/60) in the 800 mg/day group. A pairwise comparison between remacemide hydrochloride 800 mg/day and placebo was statistically significant (P = 0.049). Most reported adverse events (mainly CNS and gastrointestinal) were mild or moderate in severity and dose-dependent. Adjunctive remacemide hydrochloride treatment was associated with a higher, dose-related responder rate compared with placebo. The difference reached significance at the highest dose tested (800 mg/day). Remacemide hydrochloride was well tolerated.

Phosphatidylinositol 3-kinase and Ras/mitogen-activated protein kinase signaling pathways are required for the regulation of 5-aminolevulinate synthase gene expression by insulin.

Insulin regulates the expression of several hepatic genes. Although the general definition of insulin signaling has progressed dramatically, the elucidation of the complete signaling pathway from insulin receptor to transcription factors involved in the regulation of a specific gene remains to be established. In fact, recent works suggest that multiple divergent insulin signaling pathways regulate the expression of distinct genes. 5-Aminolevulinate synthase (ALAS) is a mitochondrial matrix enzyme that catalyzes the first and rate-limiting step of heme biosynthesis. It has been reported that insulin caused the rapid inhibition of housekeeping ALAS transcription, but the mechanism involved in this repression has not been explored. The present study investigates the role of phosphatidylinositol 3-kinase (PI3-kinase) and mitogen-activated protein kinase pathways in insulin signaling relevant to ALAS inhibition. To explore this, we combined the transient overexpression of regulatory proteins involved in these pathways and the use of small cell permeant inhibitors in rat hepatocytes and HepG2 cells. Wortmannin and LY294002, PI3-kinase inhibitors, as well as lovastatin and PD152440, Ras farnesylation inhibitors, and MEK inhibitor PD98059 abolished the insulin repression of ALAS transcription. The inhibitor of mTOR/p70(S6K) rapamycin had no effect whatsoever upon hormone action. The overexpression of vectors encoding constitutively active Ras, MEK, or p90(RSK) mimicked the inhibitory action of insulin. Conversely, negative mutants of PKB, Ras, or MEK impaired insulin inhibition of ALAS promoter activity. Furthermore, inhibition of one of the pathways blocks the inhibitory effect produced by the activation of the other. Our findings suggest that factors involved in two signaling pathways that are often considered to be functionally separate during insulin action, the Ras/ERK/p90(RSK) pathway and the PI3K/PKB pathway, are jointly required for insulin-mediated inhibition of ALAS gene expression in rat hepatocytes and human hepatoma cells.

Screening for and characterization of phospholipase A1 hypersecretory mutants of Tetrahymena thermophila.

We have described a procedure for the isolation of mutants of Tetrahymena thermophila with hypersecretion of phospholipase A1 (PLA1). Using random chemical mutagenesis, uniparental cytogamy, genetic crossing and a new, fast and effective screening procedure, four PLA1-hypersecretory mutants were isolated. The screening procedure is based on the formation of a halo appearing around cylindrical holes in a lecithin-containing agar plate filled with cell-free supernatants. About 3,940 clones were tested with this procedure in primary screening for hypersecretory features, of which 60 putative hypersecretory mutants were isolated, subcloned and tested in a secondary screening. Of these, four selected mutants showed 1.8-2.2 more PLA1 activity in the cell-free supernatants compared to the wild-type strain CU 438.1. Hypersecretion was only observable for PLA1; no increased activity for two other lysosomal enzymes could be detected. These hypersecretory mutants of T. thermophila can be very useful for increasing the yield of PLA1 in fermentation processes. This is particularly relevant because, in contrast to other phospholipases, PLA1 is not available on the commercial market for fine chemicals and little is known about the role of PLA1 in cell signaling and metabolism.

Long-term open multicentre, add-on trial of vigabatrin in adult resistant partial epilepsy. The Canadian Vigabatrin Study Group.

Vigabatrin (VGB) has been shown in a number of clinical trials with varying designs to be effective and well-tolerated as both add-on therapy and monotherapy in epilepsy with partial seizures with or without secondary generalization as well as in infantile spasms. The present study is an open, long-term (1 year) extension of a randomized double-blind placebo-controlled multicentre Canadian trial of VGB in resistant partial adult epilepsy. The present study was designed to examine the safety and long-term efficacy of VGB. Completers of the preceding double-blind study had their dose of VGB titrated to 4 g/day over 3 weeks. Patients were evaluated every 2-4 weeks and at week 14 were allowed to continue only if they achieved a 50% seizure reduction compared with pre-VGB baseline. In addition to neurological and physical examinations, safety was assessed by a cognitive psychosocial test battery, visual and somatosensory evoked potentials and MRI scans. Ninety-seven of 100 eligible patients entered the study, 53 of whom completed the 52 weeks. Fifty-eight percent of the patients had a greater than 50% seizure reduction in seizures vs. pre-VGB baseline. Seizure reductions of 56% and 45%, respectively, were seen in the VGB and placebo groups from the preceding study. Fifty-four percent of patients were judged by the investigators to have experienced at least a moderate therapeutic effect. Discontinuations were 29% for lack of efficacy and 12% for adverse effects. There was a mean weight gain of 3.7 +/- 0.2 kg by end of study. Neurologica/psychiatric side effects were the most common reason for withdrawal including three behavioral reactions attributed to the drug which required temporary hospitalization. There were no abnormalities on laboratory or special tests and there was a tendency for improvement on most tests of cognitive function and mood. Vigabatrin, as an add-on agent, is well-tolerated and can be of long-term benefit in a substantial proportion of patients with intractable partial epilepsy.

Vigabatrin as add-on therapy for adult complex partial seizures: a double-blind, placebo-controlled multicentre study. The Canadian Vigabatrin Study Group.

Vigabatrin (VGB) is a novel antiepileptic drug effective as adjunctive therapy in patients with partial seizures. In this study, the efficacy and tolerability of VGB as adjunctive therapy were evaluated in patients with refractory epilepsy. Adult patients with a definite diagnosis of complex partial seizures and/or partial seizures secondarily generalized were recruited from 10 Canadian centres. Patients were randomized to receive either active medication or placebo in a double- blind fashion and entered a 36-week titration and maintenance phase with regularly scheduled visits. Both efficacy parameters and safety assessments were monitored. Clinical laboratory, evoked potential studies, MRI, and neuropsychological tests were also performed. Forty-eight percent of VGB-treated patients vs. 26 percent of placebo-treated patients had a 50 percent or greater reduction in the frequency of complex partial seizures and partial seizures secondarily generalized. Vigabatrin was well tolerated by the majority of patients. Minor neurological side effects were observed in a number of patients in both treatment groups. No serious systemic toxicity was observed. No changes in evoked potential studies or MRI findings were noted. Vigabatrin was found to be an effective and well-tolerated antiepileptic drug when used as adjunctive therapy in patients with difficult to control complex partial seizures and for partial seizures secondarily generalized. Vigabatrin is a selective irreversible inhibitor of the GABA- degradating enzyme GABA transaminase and has shown efficacy in a number of clinical trials in patients with difficult to control partial seizures. Vigabatrin has been found most effective against complex partial and secondarily generalized tonic-clonic seizures in both adults and children. Vigabatrin has also been shown to reduce infantile spasms secondary to various aetiologies and is most effective in spasms associated with tuberous sclerosis. The aim of this study was to further extend the clinical experience with VGB as adjunctive therapy in the treatment of adult patients with difficult to control complex partial seizures and/or partial seizures secondarily generalized. In addition to the assessments of efficacy and tolerability to VGB, neuropsychological evaluations were also carried out.

Generic substitution for brand name antiepileptic drugs: a survey.

BACKGROUND/OBJECTIVE: There are presently 26 different generic preparations for five brand name antiepileptic drugs (AEDs) on the Canadian market with others likely to be released in the near future. The purpose of this review is to examine the basis for the controversy surrounding generic substitution for brand name antiepileptic drugs, to present the results of a survey of neurologists' and patients' attitudes toward generic substitution and to increase neurologists' awareness of the issues. METHODS: The current federal and provincial regulations pertaining to generic drug approval and substitution are reviewed. Published anecdotal and survey reports of the effectiveness and tolerability of generic substitution for AEDs are reviewed. A pilot questionnaire survey of 83 patients from four adult epilepsy clinics and 46 neurologists from across Canada was undertaken to determine attitudes toward generic substitution. RESULTS AND CONCLUSIONS: Several authors have suggested that some AEDs, particularly those with a narrow therapeutic index, may pose problems with generic substitution. Although generic AEDs are lower in price, possible increased side effects and morbidity and the need for closer monitoring could partially offset the cost savings. The results of our survey highlight significant unawareness of the process of generic substitution among both patients and neurologists and reveal a general level of discomfort among neurologists to prescribe generic AEDs. Further data should be obtained about the potential consequences of generic substitution in epilepsy patients.

Symptomatic and asymptomatic visual loss in patients taking vigabatrin.

PURPOSE: To investigate the clinical, perimetric, and electrophysiologic findings in patients with visual field loss on long-term treatment with the antiepileptic medication vigabatrin. DESIGN: Consecutive observational case series. PARTICIPANTS: Forty-one consecutive subjects taking vigabatrin referred for screening ophthalmologic assessment were studied. Twelve subjects with evidence of peripheral visual field constriction are presented. METHODS: Twelve subjects with evidence of peripheral visual field constriction on 60-4 perimetry underwent central 30-2 and blue-on-yellow (B/Y) perimetry, as well as electroretinography (ERG), electro-oculography (EOG), and visual-evoked potential (VEP) testing. MAIN OUTCOME MEASURES: Visual acuity; fundus abnormalities; visual field loss; and ERG, EOG, or VEP abnormalities were the main outcome measures. RESULTS: Eight of the 12 subjects with constricted visual fields were asymptomatic. The central 30-2 perimetry demonstrated bilateral visual field constriction in 9 of 12 patients and the B/Y perimetry in 8 of 9 patients tested. Of the ten patients tested electrophysiologically, four had abnormal ERGs, five had abnormal EOGs, and three had delayed VEPs. CONCLUSIONS: The incidence of visual field constriction in patients taking vigabatrin may be higher, and asymptomatic visual field loss more common, than reported previously. The authors postulate a possible Muller cell dysfunction in the peripheral retina. Patients taking vigabatrin should have regular peripheral visual field examinations.

Hormonal contraception and epilepsy.

Attempts to normalize lifestyle and optimize quality of life in women with epilepsy should include the option of a reliable method of birth control, including oral contraceptives (OCs). Despite the well-known effects of estrogen on lowering seizure threshold, it has never been shown that estrogen-containing OCs worsen seizures in epileptic patients. In theory, the presence of progesterone (which is known to inhibit seizures experimentally) in OCs could counterbalance the seizure-promoting effects of estrogen. However, there is evidence that some OCs may fail when combined with antiepileptic drugs (AEDs), possibly because of the latters' inducing effects on endogenous estradiol and progesterone. Physicians have a duty to offer appropriate and accurate counseling to epileptic patients concerning optimal choice in the use of OCs for those considering this method of contraception. Recommendations include possible use of a noninducing AED, or (for patients taking inducing AEDs) use of an OC containing > or = 50 microg estrogen. Patients should be warned that midcycle bleeding indicates possible OC failure and that the absence of such bleeding is not an indication of OC effectiveness. Additional contraceptive measures are also advised.

Lamotrigine-associated rash: risk/benefit considerations in adults and children.

PURPOSE: Lamotrigine (LTG) is an antiepileptic drug (AED) recently released in several countries. It is effective for a variety of seizure types in adults and children both as an add-on agent and in monotherapy, and is generally well tolerated. This report reviews the apparent risk factors for rash associated with LTG to determine whether and how the risk of serious rash can be minimized in practice. METHODS: The panel of experts reviewed all published and unpublished data related to the incidence and risk factors for serious rash with LTG. RESULTS: An allergic skin reaction occurs in approximately 10% of patients, usually in the first 8 weeks. Rashes leading to hospitalization, including Stevens-Johnson syndrome and hypersensitivity syndrome, occurred in approximately one of 300 adults and one of 100 children in clinical trials and appeared to be increased with overrapid titration when starting therapy and with concurrent valproate (VPA). CONCLUSIONS: Recommendations are made for both minimizing the likelihood of serious rash and for management of rash in patients taking LTG. Risk of serious rash may possibly be lessened by strict adherence to manufacturer's dosing guidelines, particularly in patients who are at higher risk: those on concurrent VPA and in the pediatric population.

A method for the preparation of Tetrahymena thermophila phospholipase A1 suitable for large-scale production.

A rapid and economical method for the purification of phospholipase A1 (PLA1) from the extracellular medium of the ciliate Tetrahymena thermophila is presented. Essentially, the procedure, here designated as purification by selective interaction (PSI), entails the incubation of media containing PLA1 with liposomes made of soy bean phospholipids. The PLA1-lipid complexes are precipitated by the addition of CaCl2 and collected by centrifugation. Elution of the PLA1 is effected by treating the complexes with 40% dimethylformamide, a reversible inhibitor of this enzyme, which is easily removed by dialysis. In combination with DEAE cellulose ion exchange chromatography, PSI yielded homogeneous PLA1 preparations with a 14% recovery and a 416-fold increase in specific activity. This procedure, which can be completed within 1 day, may prove useful for the isolation of phospholipases from other sources. This practical method for the purification of a microbial PLA1 opens the way to large-scale production of these types of enzyme, which are not as yet commercially available.

Monotherapy or polytherapy for epilepsy?

Monotherapy has been promoted as the ideal in epilepsy treatment because of reduced side effects, absence of drug interactions, better compliance, lower cost and, in many cases, improved seizure control compared to polytherapy. The question of monotherapy vs. polytherapy has assumed increasing importance with the availability of multiple new antiepileptic drugs (AEDs), initially tested as add-on agents. The new drugs clobazam, lamotrigine, vigabatrin, gabapentin and topiramate, have also been shown to be effective as monotherapy. These data bring up the possibility of using them as first-line agents. However, a high percentage of patients with resistant epilepsy are treated with polytherapy, which probably benefits only a minority of them. The availability of multiple drugs with different mechanisms of action favours the possibility of "rational polytherapy", taking advantage of possible synergism, a yet unproven concept. This article reviews the theoretical advantages of monotherapy and monotherapy with traditional and newer AEDs.

Clobazam in partial status epilepticus.

Clobazam is the first and only 1,5-benzodiazepine to be used in the management of epilepsy. The use and effectiveness of oral clobazam in patients with status epilepticus has only been previously described in one study of 16 cases, seven of whom were in complex partial status. We have used clobazam in four patients with EEG-proven partial status epilepticus refractory to standard antiepileptic drugs. In all cases, except one, the seizure activity was controlled within 2 hr of administering clobazam. Our four patients responded promptly to an oral loading of clobazam which was approximately twice the usual daily maintenance dose. The drug was well tolerated and no adverse effects were seen. Clobazam's effectiveness as a first-line agent remains to be studied. Further controlled studies are recommended.

Celiac disease, bilateral occipital calcifications and intractable epilepsy: mechanisms of seizure origin.

PURPOSE: To elucidate the mechanisms of seizure origin in patients with celiac disease and bilateral occipital calcifications (CEBOC). Individuals with CEBOC frequently present with occipital lobe seizures, but additional lesions and additional attack patterns may occur. METHODS: We studied two men and one woman who had CEBOC. Villous atrophy was revealed in the two patients who underwent duodenal biopsy. All had a comprehensive presurgical evaluation, including prolonged video-EEG recordings. Two had magnetic resonance imaging (MRI) with volumetric study of mesial temporal structures (MRIV). One patient had undergone stereotactic intracranial depth electrode studies (SEEG). RESULTS: All patients presented with intractable complex partial seizures. Two had partial simple seizures with visual aura. Neurologic examination was normal; one was of normal intelligence, and two were mildly retarded. Neuroimaging studies showed that each had bilateral occipital calcifications as well as epileptiform abnormalities over temporal lobes. In one, MRI showed an additional right frontal lesion, but SEEG demonstrated right occipital lobe seizure origin with anterior spread; this male patient later underwent a right occipital lobe resection. Another with a history of prolonged febrile convulsions had bilateral hippocampal and amygdalar atrophy demonstrated by MRIV. CONCLUSIONS: In one patient, SEEG confirmed that seizures originated in the occipital lobe. The presence of dual pathology was demonstrated in another, raising the possibility of both occipital and temporal seizure onset. The presence of extraoccipital lesions or of mesial temporal atrophy requires SEEG for clarification of seizure onset. In the absence of confounding factors and when laterality can be demonstrated, surgical treatment may be considered.

Late-onset temporal lobe epilepsy and dilatation of the hippocampal sulcus by an enlarged Virchow-Robin space.

MRI signal changes within the hippocampal sulcus have been attributed to a dilated Virchow-Robin space within that sulcus, but no clinical correlates have previously been described. We present a 64-year-old man who developed right temporal seizures. MRI revealed an unusually enlarged Virchow-Robin space within the hippocampus, suggesting space-occupying effect. Such an abnormality should be considered a possible etiology in patients with late-onset temporal lobe epilepsy.

De novo aphasic status epilepticus.

The literature contains only a handful of reports of patients with aphasia as the principal or only obvious manifestation of partial status epilepticus. Even fewer patients of this type have been well documented both clinically and by ictal EEG monitoring. We studied an otherwise healthy woman with abrupt onset of aphasia initially thought to be the result of an infarct of the left temporoparietal area. We were able to document partial status epilepticus involving the left temporoparietal area with EEG/video monitoring and showed rapid reversal of the aphasic disorder with antiepileptic drug (AED) treatment. The case is presented with a review of previous reports to underscore the importance of considering this diagnosis in patients with abrupt onset of aphasia.