Plasmapheresis eliminates the negative impact of AAV antibodies on microdystrophin gene expression following vascular delivery.

Duchenne muscular dystrophy is a monogenic disease potentially treatable by gene replacement. Use of recombinant adeno-associated virus (AAV) will ultimately require a vascular approach to broadly transduce muscle cells. We tested the impact of preexisting AAV antibodies on microdystrophin expression following vascular delivery to nonhuman primates. Rhesus macaques were treated by isolated limb perfusion using a fluoroscopically guided catheter. In addition to serostatus stratification, the animals were placed into one of the three immune suppression groups: no immune suppression, prednisone, and triple immune suppression (prednisone, tacrolimus, and mycophenolate mofetil). The animals were analyzed for transgene expression at 3 or 6 months. Microdystrophin expression was visualized in AAV, rhesus serotype 74 sero-negative animals (mean: 48.0 ± 20.8%) that was attenuated in sero-positive animals (19.6 ± 18.7%). Immunosuppression did not affect transgene expression. Importantly, removal of AAV binding antibodies by plasmapheresis in AAV sero-positive animals resulted in high-level transduction (60.8 ± 18.0%), which is comparable with that of AAV sero-negative animals (53.7 ± 7.6%), whereas non-pheresed sero-positive animals demonstrated significantly lower transduction levels (10.1 ± 6.0%). These data support the hypothesis that removal of AAV binding antibodies by plasmapheresis permits successful and sustained gene transfer in the presence of preexisting immunity (natural infection) to AAV.

Factors affecting the caffeine and polyphenol contents of black and green tea infusions.

The effects of product and preparation variables on the in-cup chemical composition of tea extracts is of interest because the appearance and taste characteristics and the possible health effects of a tea liquor arise from the chemical components extracted from the leaf during tea preparation. A comprehensive study was therefore undertaken to determine the contributions of product and preparation variables on the total soluble solids, caffeine, and polyphenol contents of tea extracts. The results of this study show that the variety, growing environment, manufacturing conditions, and grade (particle size) of the tea leaves each influence the tea leaf and final infusion compositions. In addition, the composition of the tea infusion was shown to be influenced by whether the tea was contained in a teabag and, if so, the size and material of construction of the bag. Finally, the preparation method, including the amounts of tea and water used, infusion time, and amount of agitation, was shown to be a major determinant of the component concentrations of tea beverages as consumed. An illustration of the variation introduced by these product and preparation factors is provided by comparing solids, caffeine, and polyphenol contents of green and black tea infusions when commercial products are prepared according to the instructions given on their packaging.

N-acetyllactosamine and the CT carbohydrate antigen mediate agrin-dependent activation of MuSK and acetylcholine receptor clustering in skeletal muscle.

Galbeta1,3GalNAc and Galbeta1,4GIcNAc are the subterminal saccharide structures present on the CT carbohydrate antigen GalNAcbeta1,4[NeuAcalpha2,3]-Galbeta1-(3GalNAc or 4GIcNAc)-R, which is localized at the mammalian neuromuscular junction. Here we show that Galbeta1,3GalNAc, Galbeta1,4GIcNAc, and the CT carbohydrate antigen affect postsynaptic assembly in cultured muscle cells. Treatment of C2C12 myotubes with benzyl-O-alpha-GalNAc or neuraminidase increased peanut agglutinin (PNA) expression and AChR clustering. Induction of AChR clustering was blocked by PNA and by muscle agrin. Addition of Galbeta1,4GIcNAc or Galbeta1,3GalNAc increased AChR clustering in myotubes and muscle-specific kinase (MUSK) autophosphorylation in vitro, while NeuAcalpha2,3Galbeta1,4GIcNAc and Galbeta1,4GIc did not. Neural agrin activated MuSK in vitro if the lactosamine-containing mucin domain was present, and this activation was blocked in large part by Galbeta1,3GalNAc and Galbeta1,4GIcNAc. Agrin fragments and MuSK bound to these disaccharides with differing specificities. Overexpression of the CT carbohydrate antigen also increased AChR clustering and MuSK autophosphorylation in the presence of neural agrin. These data suggest a model in which different portions of the CT carbohydrate structure contribute to agrin-dependent signal transduction.

Comparison of the effects of zaleplon, zolpidem, and triazolam on memory, learning, and psychomotor performance.

Twenty-four healthy male and female subjects, who participated in this randomized, double-blind, crossover study, received single nighttime doses of zaleplon 10 mg (therapeutic dose), zaleplon 20 mg, zolpidem 10 mg (therapeutic dose), zolpidem 20 mg, triazolam 0.25 mg (positive control), and placebo. Subjective behavioral ratings and psychomotor tests were completed before and 1.25 and 8.25 hours after administration of the study drug. The Immediate and Delayed Word Recall tests and the Digit Span Test were used to assess memory. The Digit-Symbol Substitution Test, Paired Associates Learning Test, and Divided Attention Test were used to assess other cognitive skills. Zaleplon 10 mg did not produce any significant changes in memory or learning compared with placebo. All other active treatments, including zolpidem 10 mg, caused psychomotor impairment at the 1.25-hour test battery. Zolpidem 20 mg (twice the therapeutic dose) produced more psychomotor impairment at the 1.25-hour assessment than did any of the other active treatments, including zaleplon 20 mg. At the 8.25-hour time point, test scores for subjects who received zaleplon 10 mg and 20 mg did not differ from the test scores for those who received placebo. However, cognitive impairment persisted up to the 8.25-hour observation for subjects who were administered triazolam 0.25 mg and zolpidem 20 mg. Adverse events associated with the use of zaleplon were transient and mild-to-moderate in severity. Overall, this study shows that zaleplon is a safe hypnotic that does not affect memory, learning, or psychomotor skills associated with vigilance.

Alpha-galactosidase stimulates acetylcholine receptor aggregation in skeletal muscle cells via PNA-binding carbohydrates.

Aggregation of nicotinic acetylcholine receptors (AChRs) in skeletal muscle is an essential step in the formation of the mammalian neuromuscular junction. While proteins that bind to myotube receptors such as agrin and laminin can stimulate AChR aggregation in cultured myotubes, removal of cell surface sialic acids stimulates aggregation in a ligand-independent manner. Here, we show that removal of cell surface alpha-galactosides also stimulates AChR aggregation in the absence of added laminin or agrin. AChR aggregation stimulated by alpha-galactosidase was blocked by peanut agglutinin (PNA), which binds to lactosamine-containing disaccharides, but not by the GalNAc-binding lectin Vicia villosa agglutinin (VVA-B4). AChR aggregation stimulated by alpha-galactosidase potentiated AChR clustering induced by either neural agrin or laminin-1 and could be inhibited by muscle agrin. These data suggest that capping of cell surface lactosamines or N-acetyllactosamines with alpha-galactose affects AChR aggregation much as capping with sialic acids does.

Distribution of ten laminin chains in dystrophic and regenerating muscles.

Using immunohistochemical methods, we assessed the distribution of all 10 known laminin chains (alpha1-5, beta1-3, gamma1 and gamma2) in skeletal muscles from patients with Duchenne, congenital, limb girdle, or Emery-Dreifuss muscular dystrophies. The alpha2, beta1 and gamma1 chains were abundant in the basal lamina surrounding muscle fibers in normal controls; alpha1, alpha3-alpha5, beta3, and gamma2 were undetectable; and beta2 was present at a low level. Compared to controls, levels of the alpha5 chain were increased in muscles from many dystrophic patients; levels of beta1 were reduced and/or levels of beta2 were increased in a minority. However, these changes were neither specific for, nor consistent within, diagnostic categories. In contrast, levels of alpha4 were increased in muscles from all patients with alpha2 laminin (merosin)-deficient congenital muscular dystrophy. Loss of alpha2 laminin in congenital dystrophy is disease-specific but some other changes in laminin isoform expression in dystrophic muscles could be secondary consequences of myopathy, denervation, regeneration or immaturity. To distinguish among these possibilities, we compared the laminins of embryonic, denervated, regenerating, and mutant mouse muscles with those in normal adult muscle. Embryonic muscle basal lamina contained alpha4 and alpha5 along with alpha2, and regenerating muscle re-expressed alpha5 but not alpha4. Levels of alpha5 but not alpha4 were increased in dystrophin (mdx) mutants and in dystrophin/utrophin double mutants (mdx:utrn -/-), models for Duchenne dystrophy. In contrast, laminin alpha4 was upregulated more than alpha5 in muscles of laminin alpha2 mutant mice (dy/dy; a model for alpha2-deficient congenital dystrophy). Based on these results, we hypothesize that the expression of alpha5 in many dystrophies reflects the regenerative process, whereas the selective expression of alpha4 in alpha2-deficient muscle is a specific compensatory response to loss of alpha2.

Lack of pharmacokinetic interaction between oral pantoprazole and cisapride in healthy adults.

Pantoprazole, an irreversible proton pump inhibitor, may be administered with cisapride, a prokinetic agent. As increased cisapride concentrations may result in longer electrocardiogram (ECG) QTc intervals, a crossover study was conducted in healthy subjects to evaluate the oral pharmacokinetic interaction between cisapride (20 mg) and pantoprazole (40 mg). After dosing, serial blood samples and 12-lead ECGs were collected, and cisapride plasma concentrations were quantitated. For cisapride alone, mean parameter values were the following: peak concentration (Cmax), 56 ng/mL; time to Cmax (tmax), 1.7 hours; area under the concentration-time curve (AUC), 426 ng x h/mL; and terminal half-life (t1/2), 5.8 hours. Pantoprazole coadministration did not alter cisapride AUC or other pharmacokinetic parameters except for a slight 17% decrease in Cmax' resulting in 90% confidence limits of 79% to 88%, which were marginally outside strict bioequivalence limits. In addition, cisapride did not affect ECG QTc intervals, with or without pantoprazole. Therefore, no dosage adjustment is needed when pantoprazole and cisapride are coadministered.

Rapid disappearance of zaleplon from breast milk after oral administration to lactating women.

Five lactating mothers were administered the therapeutic dose of zaleplon (10 mg) orally in an open-label, single-dose, pharmacokinetic study. Plasma and breast milk were sampled through 8 hours after dose administration for subsequent determinations of zaleplon and its major, though inactive, plasma metabolite 5-oxo-zaleplon. Zaleplon concentrations peaked in plasma and milk approximately 1 hour after dosing and then disappeared rapidly. The mean terminal half-life was slightly greater than 1 hour. Milk concentrations "mirrored" plasma concentrations closely with no discernible delay between peak times. The average milk-to-plasma (M/P) concentration ratio for zaleplon was approximately 0.50 over the time course. 5-oxo-zaleplon was undetectable in all but one milk sample. The maximum exposure of an infant to zaleplon during a feeding at peak milk concentrations was estimated to range from 1.28 micrograms to 1.66 micrograms, corresponding to 0.013% to 0.017% of the maternal dose or 0.320 microgram/kg to 0.415 microgram/kg for a 4 kg infant. The results indicate that zaleplon taken by a nursing mother is transferred through breast milk to her infant in very small quantities that are unlikely to be clinically important.

Distinct structures and functions of related pre- and postsynaptic carbohydrates at the mammalian neuromuscular junction.

Carbohydrates that terminate in beta-linked N-acetylgalactosamine (betaGalNAc) residues are concentrated in the postsynaptic apparatus of the skeletal neuromuscular junction and have been implicated in the differentiation of the postsynaptic membrane. We now report that distinct synapse-specific betaGalNAc-containing carbohydrates are associated with motor nerve terminals. Two monoclonal antibodies that recognize distinct betaGalNAc-containing epitopes, CT1 and CT2, both stain synaptic sites on skeletal muscle fibers. However, CT1 selectively stains nerve terminal, whereas CT2 selectively stains the postsynaptic apparatus. Likewise, CT1 and CT2 selectively stain motoneuron-like and muscle cell lines, respectively. Using the cell lines, we identify distinct CT1- and CT2-reactive glycolipids and glycoproteins. Finally, we show that GalNAc modulates the adhesion of motoneuron-like cells to recombinant fragments of a synaptic cleft component, laminin beta2. Together, these results show that pre- as well as postsynaptic membranes bear and are affected by distinct but related synapse-specific carbohydrates.

Comparative kinetics and dynamics of zaleplon, zolpidem, and placebo.

PURPOSE: This study evaluated the relationship of dose, plasma concentration, and time to the pharmacodynamics of zaleplon and zolpidem, 2 structurally distinct benzodiazepine receptor agonists. METHOD: Ten healthy male volunteers received single oral doses of placebo, 10 mg zaleplon, 20 mg zaleplon, 10 mg zolpidem, and 20 mg zolpidem in a double-blind, 5-condition crossover study, with 48 hours elapsing between trials. Plasma drug concentrations and pharmacodynamic effects were measured during the 8 to 24 hours after administration. RESULTS: Kinetics of zaleplon and zolpidem were not significantly related to dose. However, zaleplon had more rapid elimination (apparent elimination half-life [t1/2] of 1 hour) and higher apparent oral clearance (approximately 4300 mL/min) than zolpidem (t1/2, 2.0 to 2.2 hours; apparent oral clearance, 340 to 380 mL/min). Active treatments produced pharmacodynamic effects consistent with benzodiazepine agonist activity: self- and observer-rated sedation, impairment of digit symbol substitution test (DSST) performance, impaired memory, and increased electroencephalographic activity in the beta frequency range. The overall order of agonist potency was as follows: placebo < 10 mg zaleplon < 20 mg zaleplon < 10 mg zolpidem < 20 mg zolpidem; on a number of measures, 20 mg zaleplon was comparable to 10 mg zolpidem. Quantitative effects of zolpidem 20 mg far exceeded those of other treatments. Dynamic effects of both drugs were significantly related to plasma concentration. CONCLUSIONS: Benzodiazepine agonist effects of zaleplon and zolpidem were dose and concentration dependent. At the usual clinically effective hypnotic dose (10 mg of either drug), agonist effects of zolpidem exceeded those of zaleplon.

Microscale high-performance liquid chromatography-electrospray tandem mass spectrometry assay for cyclosporin A in blood.

To facilitate quantitative analysis of cyclosporin A in low volume blood samples we developed a sensitive and specific microscale reversed-phase HPLC-electrospray tandem mass spectrometry assay. Blood samples (100 microl) were prepared by acetonitrile precipitation and C18 solid-phase extraction. Detection was by multiple-reactant monitoring. The method was linear over the range 5-1000 microg/l (r> or =0.997) with accuracy between 95.4 and 102.0% over this range. Total imprecision was 11.1% at 10 microg/l and 2.8% at 800 microg/l. Absolute recovery of cyclosporin A and internal standard was 72.5 and 73.3%, respectively. When this method was evaluated against a conventional HPLC with UV detection, in patient samples, they were interchangeable (y=0.988x + 10.0, r=0.996). This HPLC-ESI-MS-MS method will be applicable to therapeutic monitoring in paediatric transplant patients and multiple point pharmacokinetic studies in animals and humans.

Integrins mediate adhesion to agrin and modulate agrin signaling.

Agrin, a basal lamina-associated proteoglycan, is a crucial nerve-derived organizer of postsynaptic differentiation at the skeletal neuromuscular junction. Because integrins serve as cellular receptors for many basal lamina components, we asked whether agrin interacts with integrins. Agrin-induced aggregation of acetylcholine receptors on cultured myotubes was completely blocked by antibodies to the beta1 integrin subunit and partially blocked by antibodies to the alpha(v) integrin subunit. Agrin-induced clustering was also inhibited by antisense oligonucleotides to alpha(v) and a peptide that blocks the alpha(v) binding site. Non-muscle cells that expressed alpha(v) and beta1 integrin subunits adhered to immobilized agrin, and this adhesion was blocked by anti-alpha(v) and anti-beta1 antibodies. Integrin alpha(v)-negative cells that did not adhere to agrin were rendered adherent by introduction of alpha(v). Together, these results implicate integrins, including alpha(v)beta1, as components or modulators of agrin's signal transduction pathway.

Synaptic integrins in developing, adult, and mutant muscle: selective association of alpha1, alpha7A, and alpha7B integrins with the neuromuscular junction.

Differentiation of both pre- and postsynaptic structures at the skeletal neuromuscular junction is organized by the basal lamina that occupies the synaptic cleft. As beta1 integrins are a major class of receptors for basal lamina components, we stained muscles with antibodies to the 10 integrin alpha subunits known to form dimers with beta1, to determine if any of these molecules were concentrated at synaptic sites on muscle fibers. In both developing and adult muscle, the integrin alpha1 chain was selectively associated with presynaptic cells (Schwann cells and/or nerve terminals), while alpha7 was present on both synaptic and extrasynaptic portions of the muscle fiber surface. Thus alpha1 and alpha7 integrins are present in synaptic membranes. Expression of the alpha7 chain was analyzed further by staining with antibodies specific for three alternatively spliced products of the alpha7 gene (A, B, and C), all of which were expressed in muscle. The alpha7A and alpha7B isoforms were confined to synaptic sites in adult muscle, while alpha7C was present both synaptically and extrasynaptically. In developing muscle, alpha7A appeared postnatally and specifically at the synapse; alpha7B was present throughout the muscle fiber perinatally, becoming confined to the synapse in the second postnatal week; and alpha7C was present extrasynaptically both perinatally and in adulthood. Thus, two of the alpha7 integrins are synapse-specific, and all three show distinct spatiotemporal patterns of expression within a single cell type. Finally, we asked whether perturbation of laminin expression affected the distribution of the alpha7 integrins. In normal mice, laminin beta2 is concentrated in synaptic basal lamina. In beta2-null mutant mice, alpha7A was still present at synaptic sites, but alpha7B was absent. This result provides genetic evidence that basal lamina composition is a determinant of integrin distribution.

A synaptic localization domain in the synaptic cleft protein laminin beta 2 (s-laminin)

The basal lamina that ensheaths skeletal muscle fibers traverses the synaptic cleft at the neuromuscular junction. Synaptic and extrasynaptic portions of the basal lamina contain different laminin beta chains: beta 2 (or s) at synapses and beta 1 (or B1) extrasynaptically. Laminin beta 2 is also confined to synapselike patches on myotube surfaces in vitro, whereas beta 1 is present throughout the extracellular matrix. This differential localization of laminin beta chains was analyzed by expression of chimeric beta 1-beta 2 molecules in cultured mouse myotubes. A 16-amino acid carboxyl-terminal sequence in beta 2 was necessary for synaptic localization, and an amino-terminal domain in beta 1 promoted association with extracellular fibrils. The synaptic targeting sequence of beta 2 contains a site previously shown to be adhesive for motor neurons.

Neurosecretory habituation in PC12 cells: modulation during parallel habituation.

PC12 cells habituate during repetitive stimulation with acetylcholine, bradykinin, or high potassium. Interspersing these stimulants did not affect the rate of habituation of the others, but it could modulate the amplitude of the norepinephrine secretion each could achieve. Stimulation with acetylcholine inhibited norepinephrine secretion caused by high potassium and bradykinin stimulation, while high potassium had no effect on acetylcholine or bradykinin, and bradykinin increased secretion caused by acetylcholine. Changes in norepinephrine secretion resulting from any of these stimulants correlated with changes in internal calcium levels. Cyclic AMP-, protein kinase C-, and calmodulin-dependent second messenger pathways all modulated norepinephrine secretion caused by acetylcholine and high potassium and showed a distinct hierarchy in their effectiveness. These data demonstrate that different receptor pathways can change the norepinephrine response of one another while not changing the levels of the molecules responsible for habituation.

Role for a synapse-specific carbohydrate in agrin-induced clustering of acetylcholine receptors.

Lectins such as VVA-B4, which bind N-acetylgalactosaminyl (GalNAc)-terminated saccharides, selectively stain the neuromuscular junction, thus defining a synapse-specific carbohydrate. In seeking roles for this carbohydrate, we asked whether VVA-B4 affected the distribution of acetylcholine receptors (AChRs) on cultured muscle cells. We found that incubation of myotubes with VVA-B4 induced formation of AChR clusters and potentiated the effect of a nerve-derived clustering factor, agrin. Additional experiments implicated GalNAc-terminated glycoconjugates as modulators of agrin-induced AChR clustering. Enzymatic removal of GalNAc residues or treatment with a multivalent protein-GalNAc conjugate blocked agrin-induced clustering, whereas enzymatic unmasking of additional GalNAc residues induced clustering in the absence of added agrin. Moreover, incubation with agrin led to redistribution of VVA-B4-binding material on myotubes. Together, these results suggest that agrin-induced clustering of AChRs involves a GalNAc-dependent step.

Access to the superior mediastinum.

Surgical access for diseases that involve the superior mediastinum can be achieved without thoracotomy by utilizing the suprasternal approach with extension of the head and neck and transection of the interclavicular ligament, median sternotomy, resection of the medial third to one half of the clavicle, or resection of the manubrium sterni. There has been minimal morbidity and no operative or postoperative mortality in a review of 53 consecutive patients. There has been no significant disability. Combined with exposure at the base of the neck, the access to the superior mediastinum is good to excellent.

Regulation of neurosecretory habituation by cAMP. Role of adaptation of cAMP signals.

The role of adaptation in receptors was studied with regard to the processing of neural information by cAMP in PC12 cells. Both secretin and adenosine A2 receptors elevated cAMP levels. During prolonged stimulation, secretin receptors demonstrated adaptation of cAMP levels, while adenosine A2 receptors did not. Incubation of cells with secretin increased the norepinephrine secretion resulting from stimulation by acetylcholine, but not from high potassium depolarization. The difference in the adaptation of cAMP by secretin and adenosine A2 receptors was responsible for the changes observed in norepinephrine secretion. This difference in secretory responses was caused by protein-kinase-C activation, which occurred during acetylcholine stimulation but not during depolarization by high potassium. Secretin receptors also demonstrated a reduced ability to elevate cAMP levels during repetitive stimulation, even for stimulation times where no adaptation of cAMP signals was observed, suggesting that secretin receptors possess the ability to habituate in addition to undergoing adaptation. These results are discussed with regard to the modulatory roles cAMP may play in neurosecretory habituation.

Regulation of neurosecretory habituation in PC12 cells: parallel pathways used by cAMP and calcium.

Habituation of norepinephrine secretion in PC12 cells serves as a model for understanding the molecular mechanisms of simple memory processes in neurons. Elevation of intracellular cAMP levels by incubation with N-ethylcarboxamidoadenosine (NECA) or forskolin increased norepinephrine secretion in response to depolarization by high potassium or stimulation with acetylcholine. The extent to which cAMP altered norepinephrine secretion was dependent on the timing of its elevation, and it also altered the rate of habituation under certain conditions. However, cAMP increased norepinephrine secretion by a pathway distinct from that governing internal calcium levels, which correlates with habituation in the absence of elevated cAMP. An inverse correlation was found between the ability of calcium to lower NECA-induced cAMP levels and the ability of calcium to cause norepinephrine secretion. A model is proposed in which a single calcium-dependent pathway modulates both norepinephrine secretion and cAMP metabolism.