Serum and Cerebrospinal Fluid Pharmacokinetics of Intravenous and Oral Lamivudine in Human Immunodeficiency Virus-Infected Children

We studied the pharmacokinetics of intravenously and orally administered lamivudine at six dose levels ranging from 0.5 to 10 mg/kg of body weight in 52 children with human immunodeficiency virus infection. A two-compartment model with first-order elimination from the central compartment was simultaneously fitted to the serum drug concentration-time data obtained after intravenous and oral administration. The maximal concentration at the end of the 1-h intravenous infusion and the area under the concentration-time curve after oral and intravenous administration increased proportionally with the dose. The mean clearance of lamivudine (± standard deviation) in the children was 0.53 ± 0.19 liter/kg/h (229 ± 77 ml/min/m² of body surface area), and the mean half-lives at the distribution and elimination phases were 0.23 ± 0.18 and 2.2 ± 2.1 h, respectively. Clearance was age dependent when normalized to body weight but age independent when normalized to body surface area. Lamivudine was rapidly absorbed after oral administration, and 66% ± 25% of the oral dose was absorbed. Serum lamivudine concentrations were maintained above 1 μM for ≥8 h of 24 h on the twice daily oral dosing schedule with doses of ≥2 mg/kg. The cerebrospinal fluid drug concentration measured 2 to 4 h after the dose was 12% (range, 0 to 46%) of the simultaneously measured serum drug concentration. A limited-sampling strategy was developed to estimate the area under the concentration-time curve for concentrations in serum at 2 and 6 h.     

Syntheses of Novel Pyridazinomorphinans by Inverse Electron Demand Cycloaddition and their Binding to μ and κ Receptors

A number of novel pyridazinomorphinans have been synthesized by the inverse electron demand Diels-Alder reaction of various 3,6-disubstituted 1,2,4,5-tetrazines with enamines derived from dihydrocodeinone and with codeinone. Reduction of some of the pyridazinomorphinans did not furnish the expected pyrroloepoxymorphinans; in all cases investigated reductive cleavage of the epoxybridge was observed to yield dihydropyridazino- or pyrrolomorphinans. The structures of all new compounds were assigned by the spectral data, that of the cycloadduct of codeinone was additionally verified by X-ray crystallography. Compounds 5a, 8, 11a , and 16 have been evaluated for their affinity at μ and κ opioid receptors in radioligand binding assays. Their ability to inhibit [³H]DAMGO binding at μ and [³H]U 69.593 binding at κ receptors, respectively as compared to codeine has been found to be lower.     

Affective and physiological responses to environmental noises and music.

Research suggests that respiratory patterns may reflect general dimensions of emotional response. In this study, we investigated the relationships between judgments of affective valence (pleasantness) and arousal and respiratory responses to acoustic stimuli. Sixteen environmental noises and 16 musical fragments of 30 s duration were presented to 31 participants, while respiration, skin conductance level and heart rate were recorded. Judgments of valence and arousal were registered using the 9-point Self-Assessment Manikin. For noises, breathing accelerated and minute ventilation augmented with decreases in pleasantness for low-arousal stimuli and with increases in arousal for positive stimuli. For music, breathing accelerated and minute ventilation augmented with increases both in rated valence and arousal. Skin conductance level increased with arousal ratings for music but not for noises, whereas mean heart rate increased with rated arousal for noises but not for music. Although both noises and music are sound-vibrations, differences in the relationships between affective judgments and physiological responses were found suggesting differences in the processing of the two types of acoustic stimuli.     

Vasopressin-induced taurine efflux from rat pituicytes: a potential negative feedback for hormone secretion

Previous work on the whole neurohypophysis has shown that hypotonic conditions increase release of taurine from neurohypophysial astrocytes (pituicytes). The present work confirms that taurine is present in cultured pituicytes, and that its specific release increases in response to a hypotonic shock. We next show that vasopressin (VP) and oxytocin (OT) also specifically release taurine from pituicytes. With an EC₅₀ of ∼2 n m , VP is much more potent than OT, and the effects of both hormones are blocked by SR 49059, a V_1a receptor antagonist. This pharmacological profile matches the one for VP- and OT-evoked calcium signals in pituicytes, consistent with the fact that VP-induced taurine efflux is blocked by BAPTA-AM. However, BAPTA-AM also blocks the taurine efflux induced by a 270 mosmol l⁻¹ challenge, which per se does not evoke any calcium signal, suggesting a permissive role for calcium in this case. Nevertheless, the fact that structurally unrelated calcium-mobilizing agents and ionomycin are able to induce taurine efflux suggests that calcium may also play a signalling role in this event. It is widely accepted that in hypotonic conditions taurine exits cells through anionic channels. Antagonism by the chloride channel inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) suggests the same pathway for VP-induced taurine efflux, which is also blocked in hypertonic conditions (330 mosmol l⁻¹). Moreover, it is likely that the osmosensitivity of the taurine channel is up-regulated by calcium. These results, together with our in situ experiments showing stimulation of taurine release by endogenous VP, strengthen the concept of a glial control of neurohormone output.     

3-Methylglutaconic aciduria type III in a non-Iraqi-Jewish kindred: clinical and molecular findings

Type III 3-methylglutaconic aciduria (MGA) (MIM 258501) consists of early bilateral optic atrophy, later development of spasticity, extrapyramidal dysfunction and occasionally cognitive deficits, and urinary excretion of 3-methylglutaconic acid and 3-methylglutaric acid. The presence of the disorder in an Iraqi-Jewish genetic isolate led to mapping of the OPA3 gene to chromosome 19q13.2-q13.3, followed by isolation of the gene itself. OPA3 consists of two exons and codes for a peptide of 179 amino acids. Iraqi-Jewish patients with type III MGA are homozygous for a splice site founder mutation in OPA3 (IVS1-1G>C) which abolishes mRNA expression in fibroblasts. Here we report a novel mutation in OPA3 (320-337del) in a Kurdish-Turkish patient with optic atrophy and 3-methylglutaconic and 3-methylglutaric aciduria, previously carrying the diagnosis of type IV MGA. We conclude that type III MGA occurs in patients of non-Iraqi-Jewish ancestry, and should be considered in patients with type IV MGA that have optic atrophy and ataxia.     

Corneal crystals in nephropathic cystinosis: natural history and treatment with cysteamine eyedrops

Although renal disease is the most prominent feature of the lysosomal storage disease cystinosis, corneal cystine crystal formation remains a major complication, leading to photophobia, corneal erosions, and keratopathies. Moreover, the extent of corneal crystal accumulation reflects the course and severity of the disease itself, and the cornea is accessible to direct examination. Therefore, we employed a scoring system, based on a library of slit-lamp photographs of corneas with increasing crystal densities (0.00-3.00), to assess the degree of crystal accumulation in 170 patients with nephropathic cystinosis examined at the National Institutes of Health between 1976 and 2000. None of the patients had received topical cystine-depleting therapy at the time of the evaluation. In this natural history study, infants in the first year of life had absent or minimal corneal crystals, i.e., a corneal cystine crystal score (CCCS) of 0 or 0.25. However, the CCCS increased linearly with age, such that every patient had visible crystals by 16 months of age, and plateaued at approximately 3.00 by early adolescence. Longitudinal studies in representative patients support the cross-sectional results. Individuals homozygous for the common 57-kb deletion involving the cystinosis gene (CTNS) displayed the same course of corneal crystal accumulation as did individuals not bearing the large deletion. Patients with ocular or nonnephropathic cystinosis had CCCSs that were, in general, half those expected for patients with nephropathic cystinosis of the same age. Administration of 0.55% cysteamine eyedrops, given 6 to 12 times per day, dissolved corneal cystine crystals in 10 representative patients with nephropathic cystinosis aged 1 to 32 years within 8 to 41 months.     

Localization of self antigen: implications for antigen presentation and induction of tolerance

The fifth component of complement (C5) is a self antigen expressed in serum of normal mice at a concentration of about 50 μg/ml. We have previously shown that C5 is constitutively processed and presented by antigen-presenting cells (APC) in normal mice to induce and maintain complete tolerance in major histocompatibility complex (MHC) class II-restricted T cells. This report addresses the question of whether C5 presentation involves exogenous antigen which has been internalized for processing or whether intracellular, biosynthesized C5 is being presented with MHC class II. Macrophages were found to synthesize, but not secrete C5 in bone marrow chimeras made from irradiated C5-deficient [C5(?)] hosts reconstituted with C5-sufficient [C5(+)] bone marrow [C5(+) ← C5(?)]. In these mice, macrophages are the only source of C5. [C5(+) ← C5(?)] chimeras are not tolerant of C5 and generate C5-specific T and B cell responses upon immunization indistinguishable from those of C5(-) mice. Macrophages from [C5(+) ← C5(-)] chimeras are unable to activate C5-specific T cell hybrids in vitro unlike macrophages from a C5(?) strain that has matured in a C5-expressing environment [C5(?) ← C5(+) chimeras]. This shows that under physiological conditions in vivo intracellular C5 does not get access to the class II presentation pathway and thus, does not induce tolerance in class II-restricted T cells.     

A Randomized Clinical Trial of Topical Cysteamine Disulfide (Cystamine) versus Free Thiol (Cysteamine) in the Treatment of Corneal Cystine Crystals in Cystinosis

In nephropathic cystinosis, corneal cystine crystals cause severe photophobia and corneal erosions. Topical cysteamine dissolves these crystals, but cannot be marketed because it rapidly oxidizes to the disulfide form, cystamine, at room temperature. Since cystamine itself could be used commercially, we compared the efficacy of cystamine and cysteamine with respect to cystine crystal dissolution in a randomized, double-masked clinical trial. One eye each of 14 patients with cystinosis was randomized to either cystamine or cysteamine, 0.5%, with 0.01% benzalkonium chloride; the companion eye was treated with the alternate preparation. Corneal crystals were photographed and a density score was assigned to each slide based on 13 standard slides. After 8–20 months, 6 patients showed significant reduction of the corneal crystal score in only one eye. In each case, the improved eye was the cysteamine-treated eye. Theoretically, cysteamine should dissolve both intracellular and extracellular crystals, whereas cystamine should dissolve only intracellular crystals because it must first be reduced to the free thiol by the cytoplasmic-reducing environment. Hence, the lack of efficacy of the disulfide cystamine suggests that some corneal cystine crystals in cystinosis patients are extracellular, and that another form of stable, topical cysteamine must be developed for cystinosis patients.     

In vitro negative selection of αβ T cell receptor transgenic thymocytes by conditionally immortalized thymic cortical epithelial cell lines and dendritic cells

We have established conditionally immortalized thymic cortical epithelial cell lines from transgenic mice carrying a temperature-sensitive SV40 large Tantigen. One of these cell lines expresses cortical markers and produces IL-1α, IL-6, IL-7, and TGF-β1. These cells express class I major histocompatibility complex (MHC) constitutively and class II MHC upon induction with IFN-μ. The cells appear to have a normal class I antigen presenting pathway since messages for both peptide transporter genes (TAP1, TAP2) were detected. The ability of these cortical epithelial cells to present peptide antigen was compared to that of thymic dendritic cells. In suspension culture with αβ Tcell receptor (TcR) transgenic thymocytes, these epithelial cells and dendritic cells (pre-pulsed with peptide cognate for the transgenic TcR) caused down-regulation of CD4, CD8, and TcR in an antigen dose-dependent and MHC-restricted manner. CD4^dullCD8^dull cells were taken as evidence for negative selection because these cells contained apoptotic DNA. Concentration of peptide required for negative selection of thymocytes was similar between dendritic cells and cortical epithelial cells. In contrast, αβ transgenic spleen cells were activated only by dendritic cells but not by cortical epithelial cells.