A phase 2a randomized, double-blind, placebo-controlled, sequential dose-escalation study to evaluate the efficacy and safety of ASP015K, a novel Janus kinase inhibitor, in patients with moderate-to-severe psoriasis.

BACKGROUND: Many immune-mediated disorders, including psoriasis, involve cytokine signalling via Janus kinase (JAK) enzymes. ASP015K (also designated JNJ-54781532), a novel oral JAK inhibitor, has shown moderate selectivity for JAK3 over JAK1 and JAK2 in enzyme assays. OBJECTIVES: The objective of this study was to evaluate the efficacy and safety of escalating, sequentially grouped, doses of ASP015K vs. placebo in patients with moderate-to-severe psoriasis. METHODS: This phase 2a multicentre, double-blind, randomized, placebo-controlled study (NCT01096862) enrolled 124 patients with moderate-to-severe plaque psoriasis. Five sequential ASP015K cohorts were enrolled, consisting of four twice-daily dosing groups (10, 25, 60, 100 mg) and one once-daily dosing group (50 mg) for 6 weeks. RESULTS: The primary efficacy end point [mean change in Psoriasis Area and Severity Index score from baseline to end of treatment (EOT; day 42)] significantly favoured ASP015K (overall treatment effect; P < 0.001) vs. placebo, with greater improvements at higher doses. By EOT, the secondary end points [Physician Static Global Assessment (PSGA) score, percentage of patients achieving PSGA success, and change in percentage, body surface area (BSA)] also improved with ASP015K vs. placebo (P < 0.001 for PSGA score and BSA; P < 0.01 for PSGA success). Epidermal thickness and proliferation decreased from baseline with ASP015K vs. placebo. ASP015K was generally well tolerated, with no serious adverse events (AEs) reported. CONCLUSIONS: In patients with moderate-to-severe psoriasis, ASP015K demonstrated dose-dependent improvements in clinical and histological measures of severity over 6 weeks of treatment. At all doses, ASP015K was well tolerated, with no reported serious AEs.

Analytical cytology: applications to neurotoxicology.

This unit describes methods for analyzing the effects of neurotoxicants on cell cycle regulation by dual parameter flow cytometry and on cell signaling by quantifying intracellular calcium concentrations within individual cells by scanning confocal laser microscopy or using the fluorescent calcium probe fluo-3.

Muscarinic receptor-induced calcium responses in astroglia.

BACKGROUND: The objective of this study was to characterize and quantitate the calcium responses to cholinergic stimulation in individual primary rat cortical astrocytes and human 132 1N1 astrocytoma cells. Materials and Methods The fluorescent calcium probe Indo-1 AM and an attached cell analysis and sorting (ACAS) instrument were used to quantitate calcium responses in these cells. RESULTS: A concentration-dependent response to carbachol was seen in both cell types. However, carbachol was more potent and efficacious, and the response was more homogeneous in the cell line. The calcium response was mediated by the M3 subtype of muscarinic receptors. Experiments in the absence of extracellular calcium and with EGTA demonstrated that the initial calcium spike was due to calcium release from intracellular calcium stores, whereas the sustained elevation and oscillations were dependent on calcium influx. Protein kinase C exerts a feedback inhibition of these calcium responses, and appears to be involved in maintaining the elevated calcium concentration and oscillations. CONCLUSIONS: This study provided a detailed quantitation of the changes in intracellular calcium evoked in individual astroglial cells by activation of M3 muscarinic receptors. This will allow for the study of pharmacological and toxicological agents on this response.

Effect of ethanol on muscarinic receptor-induced calcium responses in astroglia.

The effects of ethanol on muscarinic receptor-mediated calcium responses were investigated in individual primary rat astrocytes and human 132 1N1 astrocytoma cells using indo-1/AM and image cytometry. After a 30-min incubation, carbachol-induced calcium responses were inhibited only at 100 or 250 mM ethanol. The effects of ethanol were more pronounced and occurred at lower concentrations with longer exposures, with significant inhibition seen at 10 mM following a 24-hr incubation. Thapsigargin- and glutamate-induced responses were unaffected by ethanol, indicating some selectivity in this inhibition. Upon removal of ethanol, inhibition of calcium responses persisted for up to 6-12 hr, with carbachol responses returning to control levels by 24 hr after washout. Ethanol exposure did not affect muscarinic-receptor binding in astrocytoma cells, but inhibited carbachol-induced IP(3) formation. Inhibition of (3)H-thymidine incorporation by ethanol also persisted upon removal of the alcohol, with a time-dependency similar to that of the calcium responses. These results indicate that ethanol inhibits muscarinic receptor-induced calcium responses in astroglia in a concentration- and duration-dependent manner. They also show that co-incubation with ethanol is not necessary for this effect, suggesting that long-term exposure to ethanol may modify, in a reversible manner, the coupling of muscarinic receptors with its effector. This effect of ethanol may play a role in ethanol's inhibition of carbachol-induced thymidine incorporation.

Effects of ethanol on calcium homeostasis in the nervous system: implications for astrocytes.

Ethanol is a major health concern, with neurotoxicity occurring after both in utero exposure and adult alcohol abuse. Despite a large amount of research, the mechanism(s) underlying the neurotoxicity of ethanol remain unknown. One of the cellular aspects that has been investigated in relationship to the neuroteratogenicity and neurotoxicity of ethanol is the maintenance of calcium homeostasis. Studies in neuronal cells and other cells have shown that ethanol can alter intracellular calcium levels and affect voltage and receptor-operated calcium channels, as well as G protein-mediated calcium responses. Despite increasing evidence of the important roles of glial cells in the nervous systems, few studies exist on the potential effects of ethanol on calcium homeostasis in these cells. This brief review discusses a number of reported effects of alcohol on calcium responses that may be relevant to astrocytes' functions.

Rapid and inexpensive approaches to managing abnormal vaginal discharge or lower abdominal pain: an evaluation in women attending gynaecology and family planning clinics in Peru.

OBJECTIVES: To assess low abdominal pain, yellow vaginal discharge, other symptoms and signs, and demographic and behavioural variables as predictors for cervical or vaginal infection. METHODS: A cross sectional study of women attending gynaecology and family planning clinics in Lima, Peru was undertaken. 630 consecutive eligible female patients with chief or elicited complaints of yellow vaginal discharge, low abdominal pain, or both were interviewed and examined, together with a comparable reference group without these complaints. Vaginal specimens were tested for trichomoniasis and bacterial vaginosis. Endocervical specimens were tested for Neisseria gonorrhoeae and Chlamydia trachomatis using the ligase chain reaction. RESULTS: Infections found included chlamydial infection in 69 women (10.9%), gonorrhoea in 10 (1.6%), and either infection in 77 (12.2%); trichomoniasis in 46 (7.3%), bacterial vaginosis in 189 (30%), and either infection in 209 (33.2%). Cervical infection with C trachomatis and/or N gonorrhoeae was independently associated with history of a new sex partner within the last 3 months, more than one sex partner within the last year, use of condoms never or in less than 50% of sex acts, history of sex partner with STD within the last year; with symptoms of persistent low abdominal pain and of yellow vaginal discharge; and with signs of profuse and yellow vaginal discharge, cervical ectopy, easily induced endocervical bleeding, or brown cervical secretion. Using these findings, an algorithm was created that had a positive predictive value (PPV) of 36% for cervical infection among women reporting chief or elicited complaint of this abnormal vaginal discharge and a PPV of 25% among those without a complaint. A chief complaint of yellow vaginal discharge had a PPV of 50% for trichomoniasis or bacterial vaginosis. Among women without a chief complaint of yellow vaginal discharge, clinical findings of yellow vaginal discharge had a PPV of 55%. CONCLUSIONS: Where economic and technical constraints preclude testing, clinical findings and risk assessment are helpful in detecting vaginal and cervical infections. Several demographic, behavioural, clinical, and laboratory variables were predictive of infection in this population.

The effects of ethanol on glial cell proliferation: relevance to the fetal alcohol syndrome.

Exposure to ethanol during pregnancy is detrimental to brain development. Individuals affected by the Fetal Alcohol Syndrome present a number of central nervous system dysfunctions including microencephaly and mental retardation. Studies on the mechanisms of ethanol's developmental neurotoxicity have focused on its interaction with neurons; however, emerging evidence is suggesting that ethanol can significantly affect glial cells as well. A number of in vitro studies have shown that ethanol can inhibit the proliferation of various glial cells (mostly primary astrocytes or astrocytoma cells) at relatively high concentrations (100-200 mM). On the other hand, proliferation induced by some, but not all mitogens, is inhibited by low concentrations (10-50 mM) of ethanol. These inhibitory effects of ethanol may contribute to its developmental neurotoxicity observed following in vivo exposure. Animal models have indeed shown that ethanol causes microencephaly when given during the brain growth spurt, a period of brain development characterized by astroglial proliferation and maturation.

Xestospongins: potent membrane permeable blockers of the inositol 1,4,5-trisphosphate receptor.

Xestospongins (Xe's) A, C, D, araguspongine B, and demethylxestospongin B, a group of macrocyclic bis-1-oxaquinolizidines isolated from the Australian sponge, Xestospongia species, are shown to be potent blockers of IP3-mediated Ca2+ release from endoplasmic reticulum vesicles of rabbit cerebellum. XeC blocks IP3-induced Ca2+ release (IC50 = 358 nM) without interacting with the IP3-binding site, suggesting a mechanism that is independent of the IP3 effector site. Analysis of Pheochromocytoma cells and primary astrocytes loaded with Ca2+-sensitive dye reveals that XeC selectively blocks bradykinin- and carbamylcholine-induced Ca2+ efflux from endoplasmic reticulum stores. Xe's represent a new class of potent, membrane permeable IP3 receptor blockers exhibiting a high selectivity over ryanodine receptors. Xe's are a valuable tool for investigating the structure and function of IP3 receptors and Ca2+ signaling in neuronal and nonneuronal cells.

Effect of exposure in vitro to ethanol and hypoxia on gamma-aminobutyric acid efflux in the hippocampus of the fetal and adult guinea-pig.

The objective of this study was to determine the effects of acute direct exposure to ethanol, hypoxia or ethanol plus hypoxia on K+-stimulated gamma-aminobutyric acid (GABA) efflux (neuronal release minus uptake) in the hippocampus of the near-term fetal and adult guinea-pig. Transverse hippocampal slices were studied in a static-interface system. Exposure in vitro to ethanol or hypoxia involved 10-min incubation with 50 mM ethanol or 10-min incubation in a 95% N2/5% CO2 environment. GABA was quantitated by HPLC. Ethanol did not alter K+-stimulated GABA efflux; hypoxia augmented K+-stimulated GABA efflux three-fold in the near-term fetus and seven-fold in the adult; concurrent exposure to ethanol did not alter the effect of hypoxia. The data demonstrate that, for acute direct exposure to hypoxia and/or ethanol, only hypoxia increases K+-stimulated GABA efflux, the magnitude of which is dependent on the extent of development of the GABA system.

Ethanol neuro-behavioural teratogenesis in the guinea pig: behavioural dysfunction and hippocampal morphologic change.

Ethanol neuro-behavioural teratogenesis was studied in the guinea pig because of its extensive prenatal brain development. Our objective was to study, in the offspring of the guinea pig, behavioural and hippocampal morphologic effects produced by chronic maternal administration of 3 and 4 g ethanol.kg body weight-1 x day-1. Pregnant guinea pig received one of the following chronic treatments via intubation into the oral cavity: 3 or 4 g ethanol.kg-1 x day-1 as two equally divided doses 2 h apart: isocaloric sucrose and pair feeding; or water. Five litters were obtained for each treatment. Locomotor activity rate was determined on postnatal days 10, 20, and 60, and testing of the spontaneous alteration task was conducted beginning on postnatal days 22 and 62. After behavioural testing, the hippocampus of the brain of randomly selected guinea pig offspring of each treatment group was assessed histologically by light microscopic examination of cresyl violet stained coronal sections. The 3 and 4 g ethanol.kg-1 x day-1 regimens did not restrict maternal body weight gain or growth of the offspring. Both regimens increased locomotor activity rate in the offspring, which persisted into adulthood for the 4 g ethanol.kg-1 x day-1 regimen. Neither ethanol regimen impaired spontaneous alternation, but the 4 g ethanol.kg-1 x day-1 regimen increased the percent completed trials. Only the 4 g ethanol.kg-1 x day-1 regimen produced structural injury in the hippocampus, consisting of a 25% decrease in the number of CA1 pyramidal neurons. The data demonstrate that the 4 g ethanol.kg-1 x day-1 regimen produces more behavioural dysfunction and hippocampal morphologic change compared with the 3 g ethanol.kg-1 x day-1 regimen.

Dose-dependent effects of prenatal ethanol exposure in the guinea pig.

The guinea pig is an appropriate animal for studying ethanol central nervous system (CNS) teratogenesis due to its extensive prenatal CNS development. In order to establish an ethanol dosage regimen that produces CNS teratogenesis, the objective of this study was to characterize the dose-dependent effects of chronic ethanol administration on pregnancy outcome and locomotor activity of the offspring. Pregnant guinea pigs received one of the following oral treatments, via intubation into the oral cavity, throughout gestation: 3, 4, 5 or 6 g ethanol/kg maternal body weight/day; isocaloric sucrose and pair feeding; or water. The 5 and 6 g ethanol/kg/day regimens produced maternal death, spontaneous abortion, and perinatal death with at least 75% incidence; the 3 and 4 g ethanol/kg/day regimens produced little or no maternal, embryonic/fetal, or perinatal lethality. The 3 and 4 g ethanol/kg/day regimens did not affect other indices of pregnancy outcome compared with the respective isocaloric-sucrose pair-fed control animals and water-treated animals. The 3, 4, and 5 g ethanol/kg/day regimens increased spontaneous locomotor activity in the offspring, and there was a direct relationship between the magnitude of hyperactivity at days 10 and 60 of age and each of the ethanol dosage regimens and the maternal blood ethanol concentration on day 56 of gestation. The data demonstrate that, in the guinea pig, chronic oral administration of ethanol produces: (a) dose-dependent effects on pregnancy outcome, (b) hyperactivity in the offspring that is dose- (and maternal blood ethanol concentration-) and age-related, and (c) persistent hyperactivity into adulthood with minimal toxicity on pregnancy outcome for the 4 g ethanol/kg/day regimen.