A genome-wide analysis of the response to inhaled ß2-agonists in chronic obstructive pulmonary disease.

Short-acting ß2-agonist bronchodilators are the most common medications used in treating chronic obstructive pulmonary disease (COPD). Genetic variants determining bronchodilator responsiveness (BDR) in COPD have not been identified. We performed a genome-wide association study (GWAS) of BDR in 5789 current or former smokers with COPD in one African-American and four white populations. BDR was defined as the quantitative spirometric response to inhaled ß2-agonists. We combined results in a meta-analysis. In the meta-analysis, single-nucleotide polymorphisms (SNPs) in the genes KCNK1 (P=2.02 × 10(-7)) and KCNJ2 (P=1.79 × 10(-7)) were the top associations with BDR. Among African Americans, SNPs in CDH13 were significantly associated with BDR (P=5.1 × 10(-9)). A nominal association with CDH13 was identified in a gene-based analysis in all subjects. We identified suggestive association with BDR among COPD subjects for variants near two potassium channel genes (KCNK1 and KCNJ2). SNPs in CDH13 were significantly associated with BDR in African Americans.The Pharmacogenomics Journal advance online publication, 27 October 2015; doi:10.1038/tpj.2015.65.

Computed tomographic measures of airway morphology in smokers and never-smoking normals.

Bronchial wall area percent (WA% = 100 × wall area/total bronchial cross sectional area) is a standard computed tomographic (CT) measure of central airway morphology utilized in smokers with chronic obstructive pulmonary disease (COPD). Although it provides significant clinical correlations, the range of reported WA% is narrow. This suggests limited macroscopic change in response to smoking or that remodeling proportionally affects the airway wall and lumen dimensions such that their ratio is preserved. The objective of this study is to assess central airway wall area (WA), lumen area (Ai), and total bronchial area (Ao) from CT scans of 5,179 smokers and 92 never smoking normal subjects. In smokers, WA, Ai, and Ao were positively correlated with forced expiratory volume in 1 s (FEV1) expressed as a percent of predicted (FEV1%), and the WA% was negatively correlated with FEV1% (P < 0.0001 for all comparisons). Importantly, smokers with lower FEV1% tended to have airways of smaller cross-sectional area with lower WA. The increases in the WA% across GOLD stages of chronic obstructive pulmonary disease (COPD) can therefore not be due to increases in WA. The data suggest two possible origins for the WA% increases: 1) central airway remodeling resulting in overall reductions in airway caliber in excess of the decreased WA or 2) those with COPD had smaller native airways before they began smoking. In both cases, these observations provide an explanation for the limited range of values of WA% across stages of COPD.

Bone mineral density and fractures in older men with chronic obstructive pulmonary disease or asthma.

SUMMARY: In 5,541 community dwelling men, chronic obstructive pulmonary disease, or asthma was associated with lower bone mineral density (BMD) at the spine and total hip and an increased risk of vertebral and nonvertebral fractures independent of age, body mass index, and smoking. Men prescribed with corticosteroids had the lowest BMD. INTRODUCTION: It is unclear whether chronic obstructive pulmonary disease (COPD) is independently associated with BMD and fractures. METHODS: In 5,541 men from the Osteoporotic Fractures in Men Study, history of COPD or asthma, current treatment with corticosteroids, BMD, bone loss after 4.5 years and fractures were ascertained. RESULTS: Seven hundred fourteen (13%) men reported COPD or asthma, of which 103 were prescribed an oral steroid and 177 an inhaled steroid. Independent of confounders, men prescribed corticosteroids for COPD or asthma had the lowest BMD and a 2-fold increased risk of vertebral osteoporosis compared to men with no history of COPD or asthma (OR 2.13, 95% CI (confidence interval) 1.15-3.93 oral steroids; OR 2.05, 95% CI 1.27-3.31 inhaled steroids). During follow-up, BMD increased at the spine, but there was no difference in bone loss at the hip. However, men with COPD or asthma had a 2.6- and 1.4-fold increased risk of vertebral and nonvertebral fractures, respectively. CONCLUSION: Chronic obstructive pulmonary disease or asthma was associated with lower BMD at the spine and hip and increased risk of vertebral and nonvertebral fractures independent of age, clinic site, BMI, and smoking. A history of COPD or asthma may be a useful clinical risk factor to identify patients with osteoporosis.

Predicting worsening asthma control following the common cold.

The asthmatic response to the common cold is highly variable, and early characteristics that predict worsening of asthma control following a cold have not been identified. In this prospective multicentric cohort study of 413 adult subjects with asthma, the mini-Asthma Control Questionnaire (mini-ACQ) was used to quantify changes in asthma control and the Wisconsin Upper Respiratory Symptom Survey-21 (WURSS-21) to measure cold severity. Univariate and multivariable models were used to examine demographic, physiological, serological and cold-related characteristics for their relationship to changes in asthma control following a cold. Clinically significant worsening of asthma control was observed following a cold (mean+/-SD increase in mini-ACQ score of 0.69+/-0.93). Univariate analysis demonstrated that season, centre location, cold duration and cold severity measurements were all associated with a change in asthma control. Multivariable analysis of the covariates available within the first 2 days of cold onset revealed that the day 2 and cumulative sum of day 1 and 2 WURSS-21 scores were significant predictors of the subsequent changes in asthma control. In asthmatic subjects, cold severity within the first 2 days can be used to predict subsequent changes in asthma control. This information may help clinicians prevent deterioration in asthma control following a cold.

Ultrastructural damage to heart tissue from repeated oral exposure to yessotoxin resolves in 3 months.

Yessotoxin (YTX), an algal toxin contaminating edible shellfish, was previously shown to induce ultrastructural changes in some cardiac muscle cells of mice after acute (1 and 2mg/kg) or daily repeated oral exposure (1 and 2mg/kg/day, for 7 days). Therefore, the temporal evolution of the ultrastructural myocardial alterations and the development of other signs of toxicity induced by a repeated daily oral administration of YTX (1mg/kg/day, for 7 days) to mice were evaluated within 3 months after the treatment. Symptoms, food consumption, body weight, gross pathology and histopathology of the main organs and tissues were observed, and plasma levels of transaminases, lactate dehydrogenase, creatinine and creatinine phosphokinase were measured. Heart, liver, kidneys and cerebellum were also analysed by transmission electron microscopy. In addition, the blood concentration of YTX was determined by a direct enzyme linked immunosorbent assay (ELISA) 24h after the last toxin administration. No mortality or other treatment-related changes, including histological or hematoclinical parameters, were recorded in mice administered with YTX. Similarly, electron microscopy did not reveal any ultrastructural alteration in the liver, kidneys, and cerebellum associated with YTX treatment. In contrast, changes in cardiac muscle cells near to the capillaries (clusters of rounded mitochondria and disorganization of myofibrils) were observed 24h after the treatment. These changes were also noted 30 days after the toxin administration, while after 90 days no differences in cardiac muscle cells between control and YTX-treated mice were observed, which indicated a recovery of the ultrastructural alterations induced by the toxin.

Acute phase gene expression in mice exposed to the marine neurotoxin domoic acid.

Domoic acid is a rigid analog of the neurotransmitter glutamate and a potent agonist of kainate subtype glutamate receptors. Persistent activation of these receptor subtypes results in rapid excitotoxicity, calcium dependent cell death and neuronal lesions in areas of the brain where kainate pathways are concentrated. To better understand responses to domoic acid induced excitotoxicity, microarrays were used to profile gene expression in mouse brain following domoic acid exposure. Adult female mice were subjected intraperitoneally to domoic acid at the lethal dose 50, killed and dissected at 30, 60 and 240 min post-injection. Total brain RNA from treated mice was compared with time-matched controls on Agilent 22K feature microarrays. Real-time PCR was performed on selected genes. For the 30, 60 and 240 min time points, 3.96%, 3.94% and 4.36% of the genes interrogated were differentially expressed (P-value < or = 0.01), respectively. Rigorous filtering of the data resulted in a set of 56 genes used for trending analysis and K-medians and agglomerative clustering. The earliest genes induced consisted primarily of early response gene families (Jun, Fos, Ier, Egr, growth arrest and DNA damage 45) and the inflammatory response element cyclooxygenase 2. Some later responding genes involved glucocorticoid responses (Gilz, Sgk), cold inducible proteins (Cirbp, Rbm3), Map kinases (Map3k6) and NF-kappaB inhibition. Real-time PCR in male mice from an additional study confirmed the expression of several of these genes across gender. The transcriptional profile induced by domoic acid shared similarity with expression profiles of brain ischemia and other excitotoxins, suggesting a common transcriptional response.

Mapping and reconstruction of domoic acid-induced neurodegeneration in the mouse brain.

Domoic acid, a potent neurotoxin and glutamate analog produced by certain species of the marine diatom Pseudonitzschia, is responsible for several human and wildlife intoxication events. The toxin characteristically damages the hippocampus in exposed humans, rodents, and marine mammals. Histochemical studies have identified this, and other regions of neurodegeneration, though none have sought to map all brain regions affected by domoic acid. In this study, mice exposed (i.p.) to 4 mg/kg domoic acid for 72 h exhibited behavioral and pathological signs of neurotoxicity. Brains were fixed by intracardial perfusion and processed for histochemical analysis. Serial coronal sections (50 microm) were stained using the degeneration-sensitive cupric silver staining method of DeOlmos. Degenerated axons, terminals, and cell bodies, which stained black, were identified and the areas of degeneration were mapped onto Paxinos mouse atlas brain plates using Adobe Illustrator CS. The plates were then combined to reconstruct a 3-dimensional image of domoic acid-induced neurodegeneration using Amira 3.1 software. Affected regions included the olfactory bulb, septal area, and limbic system. These findings are consistent with behavioral and pathological studies demonstrating the effects of domoic acid on cognitive function and neurodegeneration in rodents.

Review and assessment of in vitro detection methods for algal toxins.

Algal toxins produced by marine and freshwater microalgae present a significant analytical challenge because of their complex structures and frequent occurrence as mixtures of structural congeners, which differ in toxic potencies and are present at varying proportions in contaminated samples. Rapid, sensitive in vitro detection methods specific for each class of algal toxins have been developed over the past decade, including immunoassays, enzyme inhibition assays, receptor assays, and cell assays. This review discusses the conceptual approaches to assay development and provides a detailed assessment of the use of in vitro detection methods for marine and freshwater algal toxins.

Microfluorimetric analysis of a purinergic receptor (P2X7) in GH4C1 rat pituitary cells: effects of a bioactive substance produced by Pfiesteria piscicida.

Pfiesteria piscicida Steidinger & Burkholder is a toxic dinoflagellate that leads to fish and human toxicity. It produces a bioactive substance that leads to cytotoxicity of GH4C1 rat pituitary cells. Extracellular adenosine 5'-triphosphate (ATP) acting on P2X7 purinergic receptors induces the formation of a nonselective cation channel, causing elevation of the cytosolic free calcium followed by a characteristic permeabilization of the cell to progressively larger ions and subsequent cell lysis. We investigated whether GH4C1 rat pituitary cells express functional P2X7 receptors, and if so, are they activated by a bioactive substance isolated from toxic P. piscicida cultures. We tested the selective agonist 2'-3'-O-(benzoyl-4-benzoyl)-ATP (BzATP) and antagonists piridoxalphosphate-6-azophenyl-2'-4'-disulfonic acid (PPADS) and oxidized-ATP (oxATP) using elevated cytosolic free calcium in Fura-2 loaded cells, and induced permeability of these cells to the fluorescent dye YO-PRO-1 as end points. We demonstrated that in GH4C1 cells, BzATP induces both the elevation of cytosolic free calcium and the permeabilization of the cell membrane. ATP-induced membrane permeabilization was inhibited by PPADS reversibly and by oxATP irreversibly. The putative Pfiesteria toxin (pPfTx) also elevated cytosolic free calcium in Fura-2 in GH4C1 cells and increased the permeability to YO-PRO-1 in a manner inhibited fully by oxATP. This study indicates that GH4C1 cells express a purinoceptor with characteristics consistent with the P2X7 subtype, and that pPfTx mimics the kinetics of cell permeabilization by ATP.

Current progress in isolation and characterization of toxins isolated from Pfiesteria piscicida.

The isolation and partial purification of toxic substances derived from Pfiesteria piscicida Steidinger & Burkholder extracts is described. Four distinct bioassay systems were used to monitor bioactivity of the P. piscicida extracts, including a high throughput cell cytotoxicity assay and a reporter gene assay as well as assays using brine shrimp and fish. Using these bioassays to guide fractionation, we have isolated two distinct, active fractions from Pfiesteria culture medium and cell mass extracts on the basis of their solubility characteristics. We have identified and characterized a bioactive lipophilic substance from Pfiesteria-derived extracts as di(2-ethylhexyl)phthalate, a commonly used plasticizer. The source of this typically man-made substance has been identified as originating from Instant Ocean (Aquarium Systems, Mentor, OH, USA), a commercially available seawater salt mixture used to prepare our mass culture growth medium. We have developed chromatographic methodology to isolate a bioactive polar compound isolated from extracts of Pfiesteria culture and presently report the characterization of the activity of this substance. The molecular structural analysis of the polar active component(s) using mass spectrometry and nuclear magnetic resonance spectroscopy is currently under way.

Modification of the cell based assay for brevetoxins using human cardiac voltage dependent sodium channels expressed in HEK-293 cells.

Assays using living cells provide an effective means to generate activity measurements of toxins, especially in situations where the toxins are part of a complex mixture or in an unfamiliar form such as natural or synthetic derivatives or bioactive metabolites. An important step in the refinement of cell based assays is to simplify the cellular reactions needed or required to generate the functional response of interest. Advances in the engineering of functional responses in cells provide a means to direct the response to given toxins. In this report, we describe the homogeneous high level expression of the initial target for brevetoxin, the voltage dependent sodium channel in human embryonic kidney cells (HEK-293). HEK cells stably transfected with a 6.208 kb cDNA of human heart voltage-dependent Na(+) channel (hH1a) were examined as an alternative to mouse neuroblastoma cells (N2A). The HEK-hH1a cells showed a reduced dependence on cofactors, increased sensitivity to brevetoxin and a useful means to assure absolute selectivity to the sodium channel. We next assessed the assay in a reporter gene format. Expression of a panel of minimal response elements as well as the c-fos promoter failed to provide a response to brevetoxin, indicating that the HEK cells lack a necessary intermediate signaling component. The expression of voltage dependent sodium channels in HEK cells is anticipated to provide enhanced performance for cell-based detection of toxins for drug and natural product discovery, biomonitoring and environmental monitoring.

Biomonitoring brevetoxin exposure in mammals using blood collection cards.

A method has been tested in laboratory mice to monitor for the presence of brevetoxins in blood after exposure. The use of blood collection cards is an adaptation of a method employed for routine diagnostic and genetic testing of newborns. Blood is collected and applied to a 0.5-inch diameter circle on a specially prepared blood collection card and allowed to dry. The blood spots are then extracted and the presence of toxin activity is first screened using a high throughput receptor binding assay. Positive samples are then examined for specific brevetoxin congeners by liquid chromatography-tandem mass spectrometry. Preliminary experiments tested the efficiency and linearity of toxin extraction from blood spiked with brevetoxin-3 (PbTx-3). Blood from treated mice was tested for the presence of brevetoxin at different times following exposure to a sublethal dose (180 microg/kg PbTx-3). Brevetoxin activity determined by receptor assay increased to 25 +/- 7.4 nM PbTx-3 equivalents within 4 hr after exposure and was still detectable in three of four animals 24 hr after exposure. Tandem mass spectrometry provided confirmation of PbTx-3, which also increased for the time points between 0.5 and 4.0 hr exposure. However, PbTx-3 was not detected at 24 hr, which suggested the formation of a biologically active metabolite. We anticipate that this approach will provide a method to biomonitor brevetoxins in living marine resources (e.g., finfish), protected species, and humans.

Identification of a P2X7 receptor in GH(4)C(1) rat pituitary cells: a potential target for a bioactive substance produced by Pfiesteria piscicida.

We examined the pharmacologic activity of a putative toxin (pPfTx) produced by Pfiesteria piscicida by characterizing the signaling pathways that induce the c-fos luciferase construct in GH(4)C(1) rat pituitary cells. Adenosine-5'-triphosphate (ATP) was determined to increase and, at higher concentrations, decrease luciferase activity in GH(4)C(1) rat pituitary cells that stably express c-fos luciferase. The inhibition of luciferase results from cytotoxicity, characteristic of the putative P. piscicida toxin (pPfTx). The actions of both pPfTx and ATP to induce c-fos luciferase were inhibited by the purinogenic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Further characterization of a P2X receptor on the GH(4)C(1) cell was determined by the analog selectivity of P2X agonists. The P2X1/P2X3 agonist alpha,beta-methylene ATP (alpha,beta-MeATP) failed to increase or decrease c-fos luciferase. However, the P2X7 agonist 2',3'-(4-benzoyl)benzoyl ATP (BzATP), which had a predominant cytotoxic effect, was more potent than ATP. Immunoblot analysis of GH(4)C(1) cell membranes confirmed the presence of a 70-kDa protein that was immunoreactive to an antibody directed against the carboxy-terminal domain unique to the P2X7 receptor. The P2X7 irreversible antagonist oxidized-ATP (oxATP) inhibited the action of ATP, BzATP, and pPfTx. These findings indicate that GH(4)C(1) cells express purinogenic receptors with selectivity consistent with the P2X7 subtype and that this receptor pathway mediates the induction of the c-fos luciferase reporter gene by ATP and the putative Pfiesteria toxin

Acute and delayed thermoregulatory response of mice exposed to brevetoxin.

Thermal dysthesia, characterized by a painful sensation of warm and cool surfaces, is one of many ailments in humans exposed to various marine algal toxins such as brevetoxin (PbTx). There is no animal model to study thermal dysthesia and little is known of the mechanism of action. There is also little known on the acute and delayed thermoregulatory effects of PbTx. In this study, we developed a behavioral system to assess the possible development of thermal dysthesia in mice exposed to PbTx. Female mice were implanted with radiotransmitters to monitor core temperature (Tc) and motor activity (MA). In one experiment, mice were dosed with the control vehicle or 180 microg/kg PbTx and placed on a floor temperature gradient to measure the selected foot temperature (SFT) while air temperature was kept constant. PbTx-treated mice underwent a 10 degrees C reduction in SFT concomitant with a 3 degrees C reduction in Tc within 30 min after exposure. In another study, Tc and MA were monitored in mice maintained in their home cages after dosing with 180 microg/kg PbTx. Tc but not MA increased for 2-5 days after exposure. SFT was unaffected by PbTx when tested 1-12 days after exposure. However, PbTx-treated mice underwent an increase in Tc when placed in the temperature gradient for up to 12 days after exposure. This suggests that PbTx augments the stress-induced hyperthermia from being placed in a novel environment. Overall, acute PbTx exposure leads to a regulated reduction in Tc as characterized by a preference for cooler SFTs and a reduced Tc. Thermal dysthesia was not apparent, but the exaggerated hyperthermic response with a normal SFT in the temperature gradient may suggest an altered processing of thermal stimuli in mice treated with PbTx.

Sexual dimorphism of brain aromatase activity in medaka: induction of a female phenotype by estradiol.

In this study we identified sex-dependent dimorphism of brain aromatase in the teleost medaka and examined its regulation by sex steriods. We first investigated differential distribution of brain aromatase activity in sexually mature male and female medaka in serial coronal sections of the brain and identified the hypothalamic nuclei contained in each section using the brain atlas of medaka. In the brain of male medaka, high levels of activity are localized in sections containing the preoptic (POA) and suprachiasmatic nuclei (SC) (63-75 fmol/hr) and low levels in the nuclei periventricular dorsalis (HD), ventralis (HV), and caudalis (Hc), nuclei diffusus of lobulus inferiores (NDIL), and nuclei tuberi anteriores (TA) and posteriores (TP) (< 25 fmol/hr). In the brain of female medaka high aromatase activity is localized in sections containing the HD, HV, Hc, NDIL, TA, and TP (85-80 fmol/hr) and highly variable levels in the POA and SC (23-70 fmol/hr). The concentration and time dependency of the exposure of male medaka to estradiol on the total brain aromatase activity and morphologic sex characteristics were determined next. Estradiol increased the activity of brain aromatase in a concentration-dependent manner at 2.5 and 25 microg/L, but the increase was lower at higher concentrations of the hormone. The effect was time dependent, gradually increasing up to the fifth day of exposure, after which it reached a plateau. Estradiol induction of brain aromatase analyzed using Lineweaver-Burke plots of saturation assays revealed a non-first-order reaction. The results indicate that a positive feedback mechanism regulates brain aromatase and imply that the sexual dimorphic distribution of aromatase may be highly sensitive to physiologic cues and environmental perturbations in fish.

The red tide toxin, brevetoxin, induces embryo toxicity and developmental abnormalities.

Brevetoxins are lipophilic polyether toxins produced by the red tide dinoflagellate Gymnodinium breve, and their neurotoxic effects on adult animals have been documented. In this study, we characterized adverse developmental effects of brevetoxin-1 (PbTx-1) using an exposure paradigm that parallels the maternal oocyte transfer of toxin. Medaka fish (Oryzias latipes) embryos were exposed to PbTx-1 via microinjection of toxin reconstituted in a triolein oil droplet. Embryos microinjected with doses of 0.1-8.0 ng/egg (ppm) of brevetoxin-1 exhibited pronounced muscular activity (hyperkinesis) after embryonic day 4. Upon hatching, morphologic abnormalities were commonly found in embryos at the following lowest adverse effect levels: 1.0-3.0 ppm, lateral curvature of the spinal column; 3.1-3.4 ppm, herniation of brain meninges through defects in the skull; and 3.4-4.0 ppm, malpositioned eye. Hatching abnormalities were also commonly observed at brevetoxin doses of 2.0 ppm and higher with head-first, as opposed to the normal tail-first, hatching, and doses > 4.1 ng/egg produced embryos that developed but failed to hatch. Given the similarity of developmental processes found between higher and lower vertebrates, teratogenic effects of brevetoxins have the potential to occur among different phylogenetic classes. The observation of developmental abnormalities after PbTx-1 exposure identifies a new spectrum of adverse effects that may be expected to occur following exposure to G. breve red tide events.

Effect of maitotoxin on guanine nucleotide interaction with G-protein alpha subunits.

Maitotoxin is a potent water-soluble polyether toxin produced by the marine dinofiagellate Gambierdiscus toxicus. Although associated with increased calcium uptake, mobilization of internal calcium stores, and enhanced phosphoinositide metabolism, the primary molecular mechanism underlying its actions remains unclear. In this study, we evaluated the effects of maitotoxin (MTX) on the interaction of guanine nucleotides with G-protein alpha subunits. Equilibrium binding of the nonhydrolyzable GTP analog, GTPgammaS, to alpha subunits (Go, Gs, Gi1, Gi2, and Gi3) was decreased in the presence of MTX. Furthermore, reconstitution of Galpha with Gbetagamma dimer showed a reversal of the inhibition elicited by MTX. GDP/GTP exchange rate for Galpha subunits was significantly inhibited in the presence of MTX. MTX had no effect on the rate of GDP or GTP dissociation from alpha subunits. Also, the mastoparan-induced component of nucleotide exchange is not effected by MTX. These results suggest that MTX acts on Galpha subunits to modulate their interaction with guanine nucleotides, perhaps by stabilizing an empty state of the alpha subunit. Accordingly, MTX may disrupt the normal signal transduction pathways by inhibiting GTP binding to Galpha subunits and interfering with the GDP/GTP exchange.

Comparison of once-daily to twice-daily treatment with mometasone furoate dry powder inhaler.

BACKGROUND: Once-daily dosing with an effective inhaled corticosteroid (ICS) would likely enhance compliance and, therefore, aid in the management of asthma. OBJECTIVE: Several once-daily dosing regimens of mometasone furoate (MF) administered by dry powder inhaler (DPI) were compared with a twice-daily dosing regimen in 286 patients with mild to moderate persistent asthma who were previously being treated with ICS. METHODS: During a 2-week open-label phase, patients received MF-DPI, 200 microg twice daily. They were then randomized to continue MF-DPI, 200 microg twice-daily treatment or to receive MF-DPI, 200 microg once daily in the morning (AM), 200 microg once daily in the evening (PM), 400 microg once daily AM, or placebo as part of the 12-week, double-blind phase. The primary efficacy variable was the mean change from the baseline to endpoint (last evaluable observation) for FEV1. RESULTS: Once-daily MF-DPI, 400 microg, AM maintained FEV1, and morning peak expiratory flow rate, FVC, FEF25%-75%, and asthma symptom scores, at levels similar to those for MF-DPI, 200 microg twice daily and significantly better than placebo. Once-daily MF-DPI, 200 microg, PM was effective in maintaining pulmonary function, but was less effective on other efficacy measures. In comparison to the other MF-DPI groups, once-daily MF-DPI, 200 microg, AM was not as effective overall. The incidence of local adverse events, including oral candidiasis, was low with all dosages. CONCLUSIONS: Once-daily MF-DPI, 400 microg, AM was as effective as MF-DPI, 200 microg twice daily, whereas once-daily MF-DPI, 200 microg, was more effective when administered in the evening compared with morning, for patients receiving ICS therapy. Once-daily dosing offers an effective and convenient treatment that could aid compliance in the treatment of asthma.

Inhaled mometasone furoate reduces oral prednisone requirements while improving respiratory function and health-related quality of life in patients with severe persistent asthma.

BACKGROUND: Inhaled corticosteroid therapy in severe persistent asthma has been shown to reduce or eliminate oral corticosteroid (OCS) use while retaining effective asthma control. OBJECTIVE: We sought to evaluate the ability of mometasone furoate (MF) delivered by means of dry powder inhaler to reduce daily oral prednisone requirements in OCS-dependent patients with severe persistent asthma. METHODS: We performed a 12-week, double-blind, placebocontrolled trial (21 centers, 132 patients) comparing 2 doses of MF (400 and 800 microg administered twice daily) with placebo, followed by a 9-month open-label phase in which 128 patients received treatment with MF. RESULTS: At the endpoint of the double-blind trial, MF 400 and 800 mg twice daily reduced daily OCS requirements by 46.0% and 23.9%, respectively, whereas placebo increased OCS requirements by 164.4% (P <.01). Oral steroids were eliminated in 40%, 37%, and 0% of patients in the MF 400 and 800 mg twice daily and placebo groups, respectively. Pulmonary function and quality of life significantly increased for MF-treated patients. Further reductions in OCS requirements were achieved with long-term MF treatment in the open-label phase. CONCLUSION: MF inhaled orally as a dry powder is an effective alternative to systemic corticosteroids in patients with severe persistent asthma.

Permanent and functional male-to-female sex reversal in d-rR strain medaka (Oryzias latipes) following egg microinjection of o,p'-DDT.

Complete sex reversal of fish is accomplished routinely in aquaculture practices by exposing fish to exogenous sex steroids during gonadal differentiation. A variety of environmental chemicals are also active at sex steroid receptors and theoretically possess the potential to alter normal sexual differentiation in fish. However, in controlled environmental chemical exposures to date, only partial alterations of fish sexual phenotype have been observed. Here we report complete, permanent, and functional male-to-female sex reversal in the Japanese medaka (Oryzias latipes, d-rR strain) after a onetime embryonic exposure to the xenoestrogen o, p'-DDT. d-rR strain medaka are strict gonochorists that possesses both sex-linked pigmentation, which distinguishes genotypic sex, and sexually dimorphic external secondary sexual characteristics, which distinguish phenotypic sex. We directly microinjected the xenoestrogen o, p'-DDT into the egg yolks of medaka at fertilization to parallel the maternal transfer of lipophilic contaminants to the embryo. At 10 weeks of age, microinjected medaka were examined for mortality and sex reversal. A calculated embryonic dose of 511 +/- 22 ng/egg o, p'-DDT (mean +/- standard error) resulted in 50% mortality. An embryonic exposure of 227 +/- 22 ng/egg o, p'-DDT resulted in 86% (6 of 7) sex reversal of genetic males to a female phenotype (XY females). XY females were distinguished by sex-linked male pigmentation accompanying female secondary sexual characteristics. Histologic examination of the gonads confirmed active ovaries in 100% of the XY females. In 10-day breeding trials in which XY females were paired with normal XY males, 50% of the XY females produced fertilized embryos; this represents a comparable breeding success rate to normal XX females. Fertilized eggs produced from XY females hatched to viable larvae. These results clearly indicate that a weakly estrogenic pesticide, o, p'-DDT, when presented during the critical period of gonadal development, can profoundly alter sexual differentiation.