Revisiting the bis(dimethylamido) metallocene complexes of thorium and uranium: improved syntheses, structure, spectroscopy, and redox energetics of (C5Me5)2An(NMe2)2 (An = Th, U).

The reaction of (C5Me5)2AnCl2 (An = Th, U) with 2.8 or 4 equivalents of LiNMe2, respectively, affords (C5Me5)2An(NMe2)2 in high yields. In addition to improved syntheses, the solid-state structures, voltammetric data, and UV-visible-NIR spectra for these classic actinide bis(dimethylamido) complexes are presented for the first time.

Naturally occurring tolerance acquisition to foods in previously allergic children is characterized by antigen specificity and associated with increased subsets of regulatory T cells.

BACKGROUND: Food allergy affects approximately 6-8% of children, and increasing in prevalence. Some children naturally outgrow their food allergy without intervention, but the mechanisms by which this occurs remain poorly understood. We sought to investigate the role of regulatory T cells in the development of naturally acquired tolerance. METHODS: Fifty-eight children (1-18 years) with either egg or peanut allergy, recent acquisition of natural tolerance to egg or peanut, or no food allergy were studied. Peripheral blood mononuclear cells (PBMC) from these groups were stimulated with relevant antigen for 48 h and flow cytometry performed to characterize both surface (CD3, CD4, CD25, CD14, CD19, and CD127) and intracellular markers (IL-10, Foxp3, and IL-5). RESULTS: Resting PBMC from naturally tolerant patients had significantly increased CD3+CD4+CD25+CD127loFoxp3+ cells, when compared to allergic or control patients (mean 6.36 vs. 2.37 vs. 2.62%, respectively, P < 0.05). Upon stimulation with relevant antigen, naturally tolerant patients also had increased IL-10-expressing CD25+CD127lo cells (6.33 vs. 1.65 vs. 0.7, P < 0.01), Foxp3+ cells (mean 12.6 vs. 5.42 vs. 3%, P < 0.01), and CD4+ cells (mean 4.48 vs. 1.59 vs. 0.87%, P < 0.01); the increase was not observed in PBMCs from allergic or control patients. Additionally, this upregulation was only seen with relevant antigen stimulation and not upon stimulation with unrelated antigen. CONCLUSION: The increased CD3+CD4+CD25+CD127lo cells at baseline and upon stimulation and increased induction of IL-10-producing cells of several types, including Tr1 cells, from naturally tolerant patients suggests an important role for regulatory T cell subsets in the acquisition of natural tolerance.

The involvement of the tetrodotoxin-resistant sodium channel Na(v)1.8 (PN3/SNS) in a rat model of visceral pain.

The present study investigated the effect of inhibiting the expression of Na(v)1.8 (PN3/SNS) sodium channels by an antisense oligodeoxynucleotide (ODN) on bladder nociceptive responses induced by intravesical acetic acid infusion in rats. Animals were injected intrathecally with either Na(v)1.8 antisense or mismatch ODN. Control cystometrograms under urethane anesthesia during intravesical saline infusion exhibited intercontraction intervals (ICIs) that were significantly longer in antisense-treated rats than in mismatch ODN-treated rats. Intravesical infusion of 0.1% acetic acid induced bladder hyperactivity as reflected by a 68% reduction in ICIs in mismatch ODN-treated rats but did not significantly reduce ICIs in antisense-treated rats. The number of Fos-positive cells after acetic acid administration were significantly reduced in the L6 spinal cord from antisense-treated animals, compared with mismatch ODN-treated animals. In addition, Na(v)1.8 immunoreactivity was reduced in L6 dorsal root ganglion neurons in the antisense-treated rat. In patch-clamp recordings, the conductance density of TTX-resistant sodium currents in dissociated bladder afferent neurons that were labeled by axonal transport of a fluorescent dye, Fast Blue, injected into the bladder wall was also smaller in antisense-treated rats than in mismatch ODN-treated rats, whereas no changes were observed in TTX-sensitive currents. These results indicate that the Na(v)1.8 TTX-resistant sodium channels are involved in the activation of afferent nerves after chemical irritation of the bladder. These channels represent a new target for the treatment of inflammatory pain from visceral organs such as the urinary bladder.

Storybook-based communication intervention for girls with Rett syndrome and their mothers.

PURPOSE: Storybook reading provides a natural language learning context in which to support early symbolic communication. In this study, we explored the impact of (1) resting hand splints, (2) light tech augmentative communication systems such as voice-output devices and symbols, and (3) very basic parent training on the symbolic communication and labelling behaviours of six girls with Rett syndrome. METHOD: Mothers and daughters were videotaped as they read familiar and unfamiliar storybooks in their homes. RESULTS: Group and individual data collected from the six girls indicated that they became more active and successful participants in the interactions during storybook reading. The girls employed a wider range of communication modes and increased the frequency of their labelling. Familiar storybook reading encouraged greater symbolic communication than unfamiliar storybooks in half the girls. CONCLUSION: This study suggests that motivated parents may not require expensive technologies or lengthy training in order to enhance their children's early communication and participation in storybook reading.

All children are ready to learn: an emergent versus readiness perspective in early literacy assessment.

Assessment of emergent literacy is relatively new to the field of communication disorders. Traditional approaches to reading assessment evaluated mastery of reading readiness skills. By contrast, emergent literacy assessment evaluates the increased awareness and understanding of print that begin early in development. One of the most influential figures in emergent literacy assessment has been Marie M. Clay. She has defined critical components of emergent literacy and, in so doing, has played an integral role in the paradigm shift from a reading readiness to an emergent literacy perspective. This article is intended to distinguish emergent literacy from reading readiness, explicate Marie Clay's contribution to our current understanding of effective emergent literacy assessment, and provide some guidance in using her assessment techniques with children with significant disabilities.

Effect of calcium channel blockers alone and in combination with antiglaucoma medications on intraocular pressure in the primate eye.

PURPOSE: To determine the effect of representative members from six classes of calcium channel blockers on intraocular pressure in the primate eye. Other antiglaucoma medications were administered with verapamil to determine their combined effect on intraocular pressure. METHODS: Six healthy cynomolgus monkeys were anesthetized, and baseline intraocular pressure was measured. Drug-containing solution (50 microL) was instilled in one eye and intraocular pressure was measured in both eyes 90 minutes later. RESULTS: All classes of calcium channel blockers significantly lowered intraocular pressure in the treated eye. The percent reduction in intraocular pressure compared with the baseline pressure was 10% for verapamil (P < 0.002), 18% for nifedipine (P < 0.001), 15% for diltiazem (P < 0.001), 17% for flunarizine (P < 0.001), 19% for prenylamine (P < 0.001), and 6% for perhexiline (P < 0.01). In the fellow eye, a significant reduction in intraocular pressure was also seen with all calcium channel blockers except perhexiline, which suggested a crossover effect. In contrast, neither vehicle treated nor contralateral eyes showed a lowering of intraocular pressure when tested under the same conditions. In the treated eye, 0.5% timolol (P < 0.01) and 0.05% clonidine (P < 0.02) combined with 0.25% verapamil each appeared to produce an additive effect, with a significantly greater pressure-lowering effect than either agent alone. In addition, 0.005% pilocarpine (P < 0.001) and 0.00125% demecarium (P < 0.01) combined with 0.25% verapamil each appeared to produce a synergistic effect, with a significantly greater reduction in intraocular pressure than both agents combined. CONCLUSIONS: Topical calcium channel blockers and combinations of verapamil with antiglaucoma medications may provide a useful alternative for reducing intraocular pressure in patients with ocular hypertension or primary open-angle glaucoma.

Direct effects of muscarinic agents on the outflow pathways in human eyes.

PURPOSE: Recent studies demonstrating the presence of muscarinic receptors and contractile-like cells in the trabecular meshwork tissue and/or cell cultures from human eyes suggest the possibility that there may be a direct effect of muscarinic agonists on outflow facility. The present studies were conducted to determine whether muscarinic agonists could change outflow facility in perfused human ocular anterior segments, which lack an intact ciliary muscle. METHODS: Human eyes were dissected and perfused according to previously described methods. A steady state baseline facility was established for 90 minutes, after which up to four sequential concentrations ranging from 10(-9) to 10(-3) M of pilocarpine, aceclidine, or carbachol were added to the perfusion medium. In other studies, 10(-6) M atropine was perfused alone followed by 10(-7) M carbachol with 10(-6) M atropine, whereas fellow control eyes received carbachol alone. Outflow facility was measured for 60 minutes after each drug addition. The outflow facility measurement in each eye after drug administration was compared with the baseline measurement. RESULTS: Outflow facility increased from baseline facility in eyes treated with pilocarpine, aceclidine, or carbachol at lower concentrations (10(-9) to 10(-6) M) but remained unchanged at higher concentrations (10(-4) to 10(-2) M). The effects of carbachol at 10(-7) M were completely blocked by atropine. CONCLUSIONS: Muscarinic agonists increase outflow facility in human eyes by a direct stimulation of the outflow tissues in the absence of an intact ciliary muscle. This effect is biphasic, occurring at concentrations of 10(-6) M and lower with no effect at higher concentrations.

Expression of adenylate cyclase subtypes II and IV in the human outflow pathway.

PURPOSE: It has been demonstrated that low doses of pilocarpine and other muscarinics substantially increase outflow facility in the isolated human outflow system devoid of ciliary muscle. These cholinergic-induced facility responses were thought possibly to be due to elevation of cAMP as a result of the presence of adenylate cyclases II (AC-II) and IV (AC-IV). Therefore, whether these isoforms are present in outflow tissues was examined. METHODS: Human anterior segments were perfused with carbachol (10(-9)-10(-5) M), and outflow facility and cAMP levels in the perfusate were measured simultaneously. Isolated trabecular meshwork (TM) were incubated with carbachol (10(-7) M), and the subsequent changes in cAMP were measured by radioimmunoassay. AC-II and AC-IV were characterized in ocular tissue with reverse transcription-polymerase chain reaction and in situ hybridization. RESULTS: Outflow facility increased, in a dose-dependent manner, by 10%, 16%, and 27% in response to 10(-9), 10(-7), and 10(-5) M carbachol, respectively. Similarly, cAMP increased by 9%, 70%, and 210% in response to 10(-9), 10(-7), and 10(-5) M carbachol, respectively. In addition, cAMP levels significantly increased by 39% in isolated TM strips incubated with 10(-7) M carbachol. AC-II was detected in most normal tissue examined, but not in any cultured cell lines or any glaucomatous tissue. AC-IV was also widely expressed in most normal tissues, faintly detected in some glaucoma tissue, but not detected in most cultured cells. CONCLUSIONS: The presence of AC-II and AC-IV in outflow tissues supports the hypothesis that cholinergics may indeed exert an effect on outflow facility, mediated by cAMP, which is independent of muscle contraction.

Effects of Na-K-2Cl cotransport regulators on outflow facility in calf and human eyes in vitro.

PURPOSE: Cultured human trabecular meshwork (TM) cells possess substantial Na-K-Cl activity, which is involved in the regulation of TM cell volume. The hypothesis in the present study was that drugs that affect the cotransporter might alter aqueous humor outflow facility (C) in the intact eye. The effects of agents and conditions known to modulate Na-K-CI cotransport activity and/or TM cell volume on C in perfused anterior segments were investigated. METHODS: Human and calf eyes were dissected and perfused, and C was determined according to standard published methods. Perfusates with modified osmolarity were used to cause alterations in TM cell volume. Cl-free perfusate and/or bumetanide (10(-5) M) was used to inhibit Na-K-Cl cotransport activity, and vasopressin (10(-7) M, 10(-8) M) was used to stimulate cotransport activity. RESULTS: In human eyes, hypo-osmotic perfusate decreased C 12%, whereas hyper-osmotic perfusate increased C 44%. These changes lasted approximately 30 minutes, after which C began to normalize. Inhibition of Na-K-Cl cotransport using Cl-free medium or bumetanide resulted in facility increases of 27% and 22%, respectively. There was an additive increase in C with bumetanide plus Cl-free media. Stimulating Na-K-Cl cotransport with 10(-8)M and 10(-7)M vasopressin resulted in 28% and 35% decreases in C, respectively. The results were similar in calf eyes: Cl-free medium or bumetanide resulted in 41% and 52% increases in C, whereas 10(-8) M and 10(-7) M vasopressin resulted in 14% and 19% decreases in C, respectively. CONCLUSIONS: Modulation of Na-K-Cl cotransport results in changes in C that may be mediated in part by cell volume changes.

Effects of N-methyl-D-aspartate (dizocilpine) and alpha-amino-3-hydroxy-4-isoxazolepropionate (LY215490) receptor antagonists on the voiding reflex induced by perineal stimulation in the neonatal rat.

The present study was undertaken to examine the role of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and N-methyl-D-aspartate glutamate receptors in the regulation of voiding reflexes induced by perineal stimulation in the neonatal rat. Four-, six- and 10-day-old awake rats were used in the experiments and perineal stimulation was applied using the tip of a 1-ml syringe to evoke voiding. Voided volume and residual volume were measured. In 10-day-old rats, LY215490 (3-10 mg/kg, i.p.), a competitive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor antagonist, significantly inhibited reflex voiding, increasing the residual volume 34-53-fold. A 3 mg/kg dose decreased the urine release by 55%, whereas 10 mg/kg totally suppressed the voiding reflex induced by the perineal stimulation. LY215490 (10 mg/kg, i.p.) produced similar effects in four- and six-day-old rats. Dizocilpine (1-3 mg/kg, i.p.), a non-competitive N-methyl-D-aspartate receptor antagonist, also significantly decreased the urine release (62-82%) and increased residual volume (180-230-fold). Combined administration of LY215490 (1 mg/kg, i.p.) and dizocilpine (0.3 mg/kg, i.p.) to 10-day-old rats, in doses that individually had no effect on perineal stimulation-evoked voiding, depressed voided volume by 65%. These results indicate that, in neonatal rats, glutamatergic transmission in the spinal cord has an essential role in reflex micturition induced by perineal stimulation, and that facilitatory interactions between alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and N-methyl-D-aspartate glutamatergic mechanisms are important for voiding, as noted previously in adult rats.

Effect of continuous administration of a cholinergic agonist on [3H]4-DAMP binding and m3 mRNA expression in cultured human ciliary muscle cells.

It is well known that chronic topical administration of cholinergic agonists results in a subsensitization in ciliary muscle-mediated increases in outflow facility and accommodation in monkey eyes in vivo. These physiologic changes are apparently mediated by the M3 subtype receptor. However, the nature of this subsensitization remains unclear. This study investigated the effect of the continuous presence of carbachol, a muscarinic agonist, on the expression of the muscarinic receptor subtype m3 and the binding of [3H]4-DAMP in cultured human ciliary muscle cells (H7CM). The H7CM cell line, derived from the ciliary muscle of a one-day-old human infant, was used in this study. Confluent monolayers were treated individually with 1 mM carbachol for 2, 6, 24 and 48 hours. The level of mRNA encoding muscarinic receptor subtype m3 was measured by RNase protection. For confirmation, a receptor binding assay was done using [3H]4-DAMP, a radioligand selective for M3 subtype receptors. At each timepoint, results were compared with untreated controls. Treatment with carbachol resulted in a down regulation ranging from 23.4% to 34.8% of m3 mRNA expression at all time points. All [3H]4-DAMP binding assay results also decreased, ranging from 24.5% to 31.0%.

Distribution of verapamil and norverapamil in the eye and systemic circulation after topical administration of verapamil in rabbits.

Our purpose was to determine the pharmacokinetics of verapamil and its active metabolite norverapamil after topical administration of verapamil in rabbits. New Zealand white albino rabbits were given 50 microl of verapamil ophthalmic formulation topically in each eye. Samples obtained at various time points were analyzed with high performance liquid chromatography and tandem mass spectrometry. The elimination half-life of verapamil after treatment with 0.5% verapamil was 0.76 hour for aqueous, 4.34 hours for vitreous, and 1.82 hours for serum. The peak concentrations for aqueous, vitreous, and serum were 2.34 x 10(-6) M by 0.5 hour, 1.57 x 10(-7) M at 2 hours, and 3.39 x 10(-8) M by 0.5 hour following instillation of one drop of 0.5% verapamil; and 1.41 x 10(-6) M by 0.5 hour, 5.48 x 10(-8) M at 4 hours, and 1.20 x 10(-8) M by 0.5 hour following 0.25% verapamil, respectively. The metabolite norverapamil was found at peak concentrations of 8.65 x 10-(8) M by 0.5 hour in aqueous, 1.65 x 10(-8) M at 2 hours in vitreous, and 1.30 x 10(-9) M by 0.5 hr in serum following administration of 0.5% verapamil. The elimination half-life of norverapamil for aqueous, vitreous, and serum following treatment with 0.5% verapamil was 0.91 hour, 1.43 hours, and 3.60 hours, respectively. We conclude that topical verapamil, administered to the rabbit eye, is rapidly absorbed in the aqueous, vitreous, and blood. Norverapamil, which is an active metabolite of verapamil, can be detected in the aqueous and vitreous of the rabbit eye after topical administration, suggesting enzymatic degradation of verapamil within the eye.

Verapamil increases outflow facility in the human eye.

Verapamil, a calcium channel antagonist, causes a reduction of intraocular pressure in humans. This study investigated whether verapamil's effect on intraocular pressure was related to increased outflow facility. Six pairs of human eyes were dissected and anterior segments were perfused under constant pressure with concentrations of verapamil HCl ranging from 10(-10)-10(-3) M. The maximal increase of outflow facility over baseline was 64% at 10(-9) M. These results indicate that verapamil causes a dose related increase in outflow facility in human eyes, which may account for the reduced intraocular pressure following topical administration of verapamil.

Obstruction of aqueous humor outflow by cross-linked polyacrylamide microgels in bovine, monkey, and human eyes.

PURPOSE: Orcolon, a synthetic viscoelastic, may have contributed to refractory intraocular pressure (IOP) elevation after intracameral injection in some patients. Cross-linked polyacrylamide (microgels), an altered form of the polymer, was investigated as an etiologic candidate. METHODS: Four adult rhesus monkeys underwent anterior chamber exchange with mock aqueous humor containing microgels in one eye and a vehicle in the other. Outflow facility (perfusion) and IOP (applanation) were determined before and at various times thereafter. Facility also was determined before and after microgel or vehicle infusion into organ-cultured individual human (n = 9) and paired calf (n = 6) anterior segments. Representative monkey and human eyes were examined by light and electron microscopy. RESULTS: In the microgel-infused monkey eyes, IOP was consistently higher, by approximately 5 mmHg for approximately 1 month. In all three species, microgel infusion acutely decreased facility by approximately 50% to 80%. In the living monkeys where longer-term observation and retesting were possible, a facility reduction of approximately 40% to 50% persisted for at least 1 to 2 months, and rechallenge again produced an acute 80% facility decrease and subsequent 10-mmHg IOP rise. Results of electron microscopic examination in human and monkey eyes showed accumulation of microgels in the cribriform meshwork and beneath the inner wall of Schlemm's canal, with no cellular alterations or inflammatory infiltrate. CONCLUSIONS: Cross-linked polyacrylamide microgels can produce an acute and longstanding obstruction of trabecular drainage experimentally, and might therefore do so clinically.

Evaluation of a novel point-of-care system, the i-STAT portable clinical analyzer.

We evaluated a novel system designed for rapid, point-of-care measurement of sodium, potassium, chloride, urea nitrogen, glucose, and hematocrit. The i-STAT Portable Clinical Analyzer (PCA) system is composed of a hand-held analyzer and disposable cartridges. Sample analysis takes place in the cartridge, which contains a series of thin-film electrodes microfabricated on silicon chips. The PCA was evaluated for precision, accuracy, and utility in emergency department and stat laboratory settings. Precision did not differ significantly between these two locations, the CVs being as follows: sodium, 0.46-0.89%; potassium, 1.06-1.45%; chloride, 0.69-2.76%; urea nitrogen, 2.54-6.12%; and glucose, 4.39-5.19%. The assessment of accuracy was based on comparison of patients' sample values analyzed by the PCA and the Kodak Ektachem 700 (or the Coulter ST for hematocrit). Regression statistics were acceptable for all analytes except chloride, for which the regression data were influenced by the limited range of results. A difference plot of the chloride comparison showed that the bias rarely exceeded 5 mmol/L. Mean hematocrit values significantly differed between the PCA and the Coulter ST, apparently because of different calibration procedures.

Intercomparison of seven radioimmunoassay kits and a fluorescence polarization immunoassay kit for digoxin.

Samples from 33 patients being treated with digoxin and three concentrations of control material were analyzed for this drug by use of seven radioimmunoassay (RIA) kits and an automated fluorescence polarization immunoassay (FPIA) system. CVs ranged from 2.4 to 6.4% for the FPIA system and from 4.6 to 7.4% for two RIA methods. In the analysis of between-method variability of RIA kits, CVs ranged from 6.1 to 33.2%. We compared each RIA kit with the other six RIA kits (I), as well as each RIA kit with the FPIA system (II). Correlation coefficients were greater than or equal to 0.96 in all cases. Slopes ranged from 0.82 to 1.20 for comparison I and from 0.86 to 1.01 for comparison II. For the FPIA system, analytical recovery of digoxin ranged from 94 to 104%. For the RIA methods we examined, analytical recoveries ranged from 113 to 135%. Analysis time is shorter and precision is greater for the FPIA system than for the RIA methods.

The cynomolgus monkey as a model for orbital research. I. Normal anatomy.

Significant differences in anatomy preclude the use of the more common small research animals in orbital studies. One must look to the anthropoid apes to find a species comparable to man in both bony skeleton and soft tissue contents. While the orbit of the cynomolgus monkey is remarkably similar to that of man, differences still do exist. In this monkey, the ethmoid, frontal and sphenoid sinuses are absent, and the os planum of the ethmoid is represented in only a minority of younger animals. A technique for microscopic examination of the soft tissue contents of the orbit has likewise shown a basic similarity to man, with minor, yet possibly significant, differences in extraocular muscles, connective tissue septae and the lacrimal gland. A recognition of these similarities and differences allows utilization of this animal as a useful model in orbital research.