RORγt expression in Tregs promotes systemic lupus erythematosus via IL-17 secretion, alteration of Treg phenotype and suppression of Th2 responses.

Systemic lupus erythematosus (SLE) is a common autoimmune disorder with a complex and poorly understood immunopathogenesis. However, a pathogenic role for the T helper type 17 (Th17) axis was demonstrated by many studies, while regulatory T cells (Tregs ) were shown to mediate protection. Recently, we and others characterized a novel and independent T cell population expressing both the Treg characteristic transcription factor forkhead box protein 3 (FoxP3) and the Th17-defining retinoic acid receptor-related orphan nuclear receptor γt (RORγt). Studies in a model of acute glomerulonephritis unveiled potent regulatory, but also proinflammatory, functions of RORγt+ FoxP3+ Tregs . This bi-functional nature prompted us to suggest the name 'biTregs '. Importantly, the pathogenic biTreg effects were dependent upon expression of RORγt. We thus aimed to evaluate the contribution of RORγt+ FoxP3+ biTregs to pristane-induced SLE and explored the therapeutic potential of interference with RORγt activation. Our analyses revealed expansion of IL-17 producing biTregs in a distinctive time-course and organ-specific pattern, coincident with the development of autoimmunity and tissue injury. Importantly, specific ablation of RORγt activation in endogenous biTregs resulted in significant amelioration of pristane-induced pulmonary vasculitis and lupus nephritis. As potential mechanisms underlying the observed protection, we found that secretion of IL-17 by biTregs was abrogated completely in FoxP3Cre  × RORCfl/fl mice. Furthermore, Tregs showed a more activated phenotype after cell-specific inactivation of RORγt signalling. Finally, and remarkably, biTregs were found to potently suppress anti-inflammatory Th2 immunity in a RORγt-dependent manner. Our study thus identifies biTregs as novel players in SLE and advocates RORγt-directed interventions as promising therapeutic strategies.

Influence of lipid saturation grade and headgroup charge: a refined lung surfactant adsorption model.

Rapid adsorption of surfactant material to the air/liquid interface of the lung is essential for maintaining normal lung function. The detailed mechanism of this process, however, remains unclear. In this study, we elucidate the influence of lipid saturation grade and headgroup charge of surface layer lipids on surfactant protein (SP)-induced vesicle insertion into monolayers spread at the air/water interface of a film balance. We used dipalmitoylphosphatidlycholine (DPPC),1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) as monolayer lipids doped with either hydrophobic surfactant-specific protein SP-B or SP-C (0.2 and 0.4 mol %, respectively). Vesicles consisting of DPPC/DPPG (4:1, mol ratio) were injected into a stirred subphase to quantify adsorption kinetics. Based on kinetic film balance and fluorescence measurements, a refined model describing distinct steps of vesicle adsorption to surfactant monolayers is presented. First, in a protein-independent step, lipids from vesicles bridged to the interfacial film by Ca(2+) ions are inserted into defects of a disordered monolayer at low surface pressures. Second, in a SP-facilitated step, active material insertion involving an SP-B- or SP-C-induced flip-flop of lipids occurs at higher surface pressures. Negatively charged lipids obviously influence the threshold pressures at which this second protein-mediated adsorption mechanism takes place.

Genetic structure of populations of Rhizoctonia solani anastomosis group-1 IA from soybean in Brazil.

The Basidiomycete fungus Rhizoctonia solani anastomosis group (AG)-1 IA is a major pathogen of soybean in Brazil, where the average yield losses have reached 30 to 60% in some states in Northern Brazil. No information is currently available concerning levels of genetic diversity and population structure for this pathogen in Brazil. A total of 232 isolates of R. solani AG1 IA were collected from five soybean fields in the most important soybean production areas in central-western, northern, and northeastern Brazil. These isolates were genotyped using 10 microsatellite loci. Most of the multilocus genotypes (MLGTs) were site-specific, with few MLGTs shared among populations. Significant population subdivision was evident. High levels of admixture were observed for populations from Mato Grosso and Tocantins. After removing admixed genotypes, three out of five field populations (Maranhao, Mato Grosso, and Tocantins), were in Hardy-Weinberg (HW) equilibrium, consistent with sexual recombination. HW and gametic disequilibrium were found for the remaining soybean-infecting populations. The findings of low genotypic diversity, departures from HW equilibrium, gametic disequilibrium, and high degree of population subdivision in these R. solani AG-1 IA populations from Brazil are consistent with predominantly asexual reproduction, short-distance dispersal of vegetative propagules (mycelium or sclerotia), and limited long-distance dispersal, possibly via contaminated seed. None of the soybean-infecting populations showed a reduction in population size (bottleneck effect). We detected asymmetric historical migration among the soybean-infecting populations, which could explain the observed levels of subdivision.

Response to Comment on "Permanent human occupation of the central Tibetan Plateau in the early Holocene".

Zhang et al contest that Chusang was part of an annual mobility round that "more likely" included seasonal use of high-elevation environments than permanent use. We show that their probabilistic statement hinges on indefensible claims about hunter-gatherer mobility. In the context of quantitative data from hunter-gatherer ethnography, our travel model shows that seasonal-use models are highly unlikely to explain Chusang.

Response to Comment on "Permanent human occupation of the central Tibetan Plateau in the early Holocene".

We show that Zhang and Li's sedimentological model for the Chusang travertine neglects the three-dimensional information from multiple outcrops and that their optically stimulated luminescence (OSL) age of about 20,000 years for the human imprints is untenable. We highlight the robustness of our chronology and explore reasons why Zhang and Li's OSL age is a gross overestimation of the real depositional age of the imprinted travertine.

Permanent human occupation of the central Tibetan Plateau in the early Holocene.

Current models of the peopling of the higher-elevation zones of the Tibetan Plateau postulate that permanent occupation could only have been facilitated by an agricultural lifeway at ~3.6 thousand calibrated carbon-14 years before present. Here we report a reanalysis of the chronology of the Chusang site, located on the central Tibetan Plateau at an elevation of ~4270 meters above sea level. The minimum age of the site is fixed at ~7.4 thousand years (thorium-230/uranium dating), with a maximum age between ~8.20 and 12.67 thousand calibrated carbon-14 years before present (carbon-14 assays). Travel cost modeling and archaeological data suggest that the site was part of an annual, permanent, preagricultural occupation of the central plateau. These findings challenge current models of the occupation of the Tibetan Plateau.

Phospholipase A2 inhibitors as potential anti-inflammatory agents.

Phospholipase A(2) (PLA(2))-catalyzed hydrolysis of membrane phospholipids results in the stoichiometric production of a free fatty acid, most importantly arachidonic acid, and a lysophospholipid. Both of these phospholipid metabolites serve as precursors for inflammatory mediators such as eicosanoids or platelet-activating factor (PAF). Since it was initially discovered that non-steroidal anti-inflammatory drugs inhibit prostaglandin synthesis, a vast amount of drug development has been performed to selectively inhibit the production of the inflammatory metabolites of arachidonic acid while preserving their protective role. This research has culminated in the development of selective cyclooxygenase-2 (COX-2) inhibitors that act on the inducible, inflammatory COX enzyme, but do not affect the constitutive prostaglandin synthesis in cells that is mediated via COX-1. The development of PLA(2) inhibitors as potential anti-inflammatory agents has also been extensively pursued since the release of arachidonic acid from membrane phospholipids by PLA(3) is one of the rate-limiting factors for eicosanoid production. In addition to the production of eicosanoids, PLA(2)-catalyzed membrane phospholipid hydrolysis is also the initiating step in the generation of PAF, a potent inflammatory agent. Thus, inhibition of PLA(2) activity should, in theory, be a more effective anti-inflammatory approach. However, developing an inhibitor that would be selective for the production of inflammatory metabolites and not inhibit the beneficial properties of PLA(2) has so far proved to be elusive. This review will focus on agents used currently to inhibit PLA(2) activity and will explore their possible therapeutic use.

Pharmacokinetics of chlorpheniramine, phenytoin, glipizide and nifedipine in an individual homozygous for the CYP2C9*3 allele.

Genetic polymorphisms in the cytochrome P450 (CYP) family are widely known to contribute to interindividual differences in the pharmacokinetics of many drugs. Several alleles for the CYP2C9 gene have been reported. Individuals homozygous for the Leu359 variant (CYP2C9*3) have been shown to have significantly lower drug clearances compared with Ile359 (CYP2C9*1) homozygous individuals. A male Caucasian who participated in six bioavailability studies in our laboratory over a period of several years showed extremely low clearance of two drugs: phenytoin and glipizide (both substrates of CYP2C9), but not for nifedipine (a CYP3A4 substrate) and chlorpheniramine (a CYP2D6 substrate). His oral clearance of phenytoin was 21% of the mean of the other 11 individuals participating in the study, and his oral clearance of glipizide, a second generation sulfonylurea structurally similar to tolbutamide, was only 188% of the mean of the other 10 individuals. However, his oral clearance of nifedipine and chlorpheniramine did not differ from individuals in other studies performed at our laboratories. An additional blood sample was obtained from this individual to determine if he possessed any of the known CYP2C9 or CYP2C19 allelic variants that would account for his poor clearance of the CYP2C9 substrates (phenytoin and glipizide) compared with the CYP3A4 (nifedipine) and CYP2D6 (chlorpheniramine) substrates. The results of the genotype testing showed that this individual was homozygous for the CYP2C9*3 allele and did not possess any of the known defective CYP2C19 alleles. This study establishes that the Leu359 mutation is responsible for the phenytoin and glipizide/tolbutamide poor metabolizer phenotype.

Folic acid dosage for chronic hemodialysis patients.

The doses of folic acid, necessary to avoid folic acid deficiency in patients being maintained on hemodialysis, have been estimated to be between 1 and 5 mg daily. To more precisely define an adequate dose of folic acid, 6 anephric patients were studied. The patients were maintained on hemodialysis and received, in a crossover fashion, 1 mg and 5 mg of folic acid for 3 wk. In a second crossover study, 3 anephric patients were first maintained on 0 and then on 1 mg of folic acid after each dialysis for 3 wk periods. Pre- and postdialysis folic acid blood levels were measured and dialyzer clearances of folic acid were determined. The results of this study support the conclusion of the Food and Drug Administration report suggesting that daily doses of 1 mg of folic acid are adequate to sustain therapeutic folate levels. The data further indicate that the administration of 1 mg of folic acid after each dialysis, rather than 1 mg of folic acid daily, can provide adequate folate.

Nonlinear ethotoin kinetics.

Five healthy subjects were given single 500-, 1500-, and 2500-mg doses of ethotoin as 250-mg tablets at 7-day intervals. Plasma samples were collected for 49 hr after dosing and were assayed by HPLC for ethotoin. The drug was more slowly absorbed after the two higher doses. There was a disproportionate increase in AUC in all subjects with the escalating doses. Individual subject data were fitted to a first-order model and one incorporating Michaelis-Menten elimination kinetics. Eleven of the 15 data sets were best described by the nonlinear model. All subjects reported visual disturbances with the two higher doses, and four of five experienced dyscoordination of gait with the 2500-mg dose. These effects did not appear to be related to plasma ethotoin concentration.

Cloning and sequence analysis of laccase-encoding cDNA clones from tobacco.

Three laccase-encoding cDNAs were cloned from a tobacco stem cDNA library. One of them contains a full length sequence coding for a cationic laccase. The predicted polypeptide sequence shows 48% identity with sycamore laccase. Amino acid comparisons with other laccases and ascorbate oxidases have shown that this new plant laccase sequence also contains four potential copper binding regions which are highly conserved among the blue copper oxidases.

United States Food and Drug Administration requirements for approval of generic drug products.

As generic products become more available for the treatment of psychiatric disorders, clinicians must stay abreast of the U.S. Food and Drug Administration (FDA) requirements for the approval of generic drug products. The FDA declares that pharmaceutical equivalents only are therapeutically equivalent, and pharmacokinetic data are all that is usually required to determine therapeutic equivalence. The rationale behind the overall concept of bioequivalence is that if 2 pharmaceutical equivalents provide identical plasma concentration-time profiles in humans, there is no evidence to demonstrate that the 2 identical dosage forms will exhibit a difference in safety and efficacy. This article reviews current terminology used in abbreviated new drug applications for generic products, typical bioequivalence study designs, and FDA bioequivalence guidance for clozapine.

Bioequivalence, dose-proportionality, and pharmacokinetics of naltrexone after oral administration.

Healthy male volunteers (N = 24) participated in a four-way crossover study to compare the rate and extent of absorption of naltrexone after administration of 50 mg tablets as 50, 100, and 200 mg doses and a 10 mg/ml reference syrup. A high-performance liquid chromatographic method was employed to measure naltrexone and 6-beta-naltrexol in plasma and urine. Compared to the syrup, the 50 mg tablets were absorbed more slowly but equally well. There was excellent linearity between the administered dose and the area under the plasma concentration-time profile, as well as total urinary recovery of both drug and metabolite. The mean half-lives for naltrexone and beta-naltrexol were approximately 4 and 12 hours, respectively. The fraction of drug reaching the systemic circulation was estimated to be 5% of the administered dose because of extensive first-pass metabolism. Less than 1% of the dose was excreted in the urine as naltrexone after 48 hours, while 25% was recovered as unconjugated beta-naltrexol. The renal clearance of naltrexone and beta-naltrexol was approximately 127 ml/min and 283 ml/min, respectively. The total systemic clearance for naltrexone was approximately 94 L/hr.

A multiple-dose study of sustained-release theophylline and aminophylline.

This study evaluated the relative bioavailability of a sustained-release capsule of theophylline, an elixir of theophylline, and a sustained-release tablet of aminophylline. Twelve healthy, nonsmoking, adult male subjects received nine doses of each of the three products in a crossover study conducted over a ten-day period. Each dosage form contained approximately 250 mg of theophylline and was administered every eight hours. Concentrations of theophylline in the plasma at steady state demonstrated the equivalence of the three dosage forms in terms of the percent of drug absorbed, including the tablet which had exhibited reduced bioavailability in an earlier single-dose study of only five subjects. The steady-state average concentrations of theopylline in the plasma were 9.8 micrograms/ml, 10.3 micrograms/ml and 10.8 micrograms/ml for the tablet, capsulse, and elixir, respectively. The areas under the curves of the plasma level vs time for the three dosage forms were within 9 percent of each other. These data indicate that a significant reduction in fluctuations of the plasma level of theophylline was achieved with the two sustained-release dosage forms, compared to the elixir.

Intolerance to volume expansion: a theorized mechanism for the development of preeclampsia.

We present a theorized mechanism for the development of preeclampsia, suggesting that one important underlying pathophysiologic mechanism is intolerance to volume expansion. The stage is set for this intolerance by chronic volume constriction, which leads to a requirement for increased basal peripheral vasoconstrictor tone to maintain blood pressure and allow for continued perfusion of the upright hominid head. In pregnancy, volume expansion signaled by the placenta cannot be accommodated by the constricted vascular system. The inability of the normally adaptive endothelial vasodilatory mechanisms to overcome the chronic vasoconstrictor tone leads to endothelial damage, exacerbation of vasoconstriction, and clinical hypertension. Disease resolution, characterized by diuresis, occurs with the elimination of the placenta-derived drive to retain volume. The reason preeclampsia does not recur uniformly with subsequent pregnancy is permanent restructuring of the maternal cardiovascular system with pregnancy that allows for greater plasma volume expansion in future gestations.

Adenosine diphosphate-induced platelet activation inhibited by magnesium in a dose-dependent manner.

OBJECTIVE: To examine the hypothesis that magnesium inhibits platelet activation at concentrations equivalent to therapeutic levels. METHODS: Fifteen subjects were enrolled: five healthy, female donors with regular, spontaneous menstrual cycles; five women with uncomplicated third-trimester pregnancies; and five preeclamptic subjects before magnesium therapy. Anticoagulated whole blood was added to tubes containing 0.5 micromol/L adenosine diphosphate (to activate platelets), HP1-1D (activation-independent platelet antibody), CD62 antibody and CD63 antibody (activation-dependent platelet antibodies), and magnesium sulfate in increasing concentrations (2-100 mg/dL). The percentage of activated platelets (CD62 or CD63 positive) was determined using three-color flow cytometric analysis. Data were analyzed using the Student t test, repeated measures analysis of variance, two-way analysis of variance, and Student-Newman-Keuls for pairwise comparison in appropriate cases. P < .05 was considered significant. RESULTS: Adenosine diphosphate-induced platelet activation was inhibited with increasing magnesium concentration in all subjects (P < .001). Significant inhibition of CD62 and CD63 expression first occurred at a magnesium concentration of 4 mg/dL in the normal pregnant group (P < .05), at 6 mg/dL in the preeclamptic group (P < .05), and at 8 mg/dL in the nonpregnant group (P < .05). CONCLUSION: Magnesium inhibits adenosine diphosphate-induced platelet activation in a dose-dependent manner. This effect initially attains statistical significance at concentrations equivalent to therapeutic levels.

The influence of dosage form on papaverine bioavailability.

The bioavailability of sustained-release papaverine HCl dosage forms were compared to equivalent doses of the drug administered as an elixir and conventional compressed tablets to 12 healthy human subjects. Papaverine plasma levels were determined using a gas-chromatographic procedure. The drug was absorbed more rapidly and completely from the two nonsustained-release formulations. There was a large intersubject variability, and the plasma half-life of the drug was esstimated to be 1 hour. The area under the plasma level-time curve for the nine sustained-release products ranged from 18 to 64% relative to the area achieved by the papaverine elixir. It was concluded that the sustained-release dosage forms of papaverine included in each study group could be considered bioequivalent, but they exhibited inadequate bioavailability relative to either the elixir or the compressed tablet dosage form.

Clinically significant interactions of psychotropic agents with antipsychotic drugs.

Various psychotropic drugs are commonly combined with antipsychotic agents. Such combinations can induce pharmacodynamically based, presumably additive, beneficial (e.g. sedative or mood-altering) effects or adverse autonomic, cardiac depressant and CNS intoxicating effects. Clinically significant interactions also arise through competition with or induction of hepatic microsomal cytochrome P450 (CYP) enzymes, particularly the CYP1A2 and CYP2D6 isozymes by which most antipsychotics are oxidised. Such pharmacokinetic interactions can elevate circulating concentrations of antipsychotics (both typical agents and the atypical antipsychotic clozapine) to potentially toxic ranges, which may lead to increased risks of adverse effects. Such interactions occur particularly with serotonin (5-hydroxytryptamine; 5-HT) reuptake inhibitor antidepressants. Metabolic interactions that lead to lesser increases in antipsychotic concentrations may arise in combining these drugs with other antidepressants, benzodiazepines or propranolol. In contrast, most anticonvulsants, except valproic acid (sodium valproate), induce the oxidative metabolism of antipsychotics and can lower their plasma concentrations to potentially subtherapeutic levels, with unpredictable increases after their discontinuation. Since simultaneous use of multiple psychotropic agents is increasingly common, special caution is required to avoid untoward consequences of interactive adverse effects due to drug interactions, which can sometimes be severe or life-threatening.

Serious bioavailability problems with a generic prolonged-release quinidine gluconate product.

A recently marketed prolonged-release quinidine gluconate tablet was compared with the innovator's tablet in a single-dose bioavailability study with 12 healthy male subjects. The extent of absorption of quinidine from the new marketed product was only 50 per cent of the innovator's product. This finding, as well as projections of steady-state plasma concentrations to be expected during multiple-dose administration, indicated a bioequivalence problem with medically significant implications. The data obtained in this study resulted in a Class I recall of the less completely absorbed product by the U.S. Food and Drug Administration.