Integrated Advanced Molecular Tools Predict In Situ cVOC Degradation Rates: Field Demonstration.

Chlorinated volatile organic compound (cVOC) degradation rate constants are crucial information for site management. Conventional approaches generate rate estimates from the monitoring and modeling of cVOC concentrations. This requires time series data collected along the flow path of the plume. The estimates of rate constants are often plagued by confounding issues, making predictions cumbersome and unreliable. Laboratory data suggest that targeted quantitative analysis of Dehalococcoides mccartyi (Dhc) biomarker genes (qPCR) and proteins (qProt) can be directly correlated with reductive dechlorination activity. To assess the potential of qPCR and qProt measurements to predict rates, we collected data from cVOC-contaminated aquifers. At the benchmark study site, the rate constant for degradation of cis-dichloroethene (cDCE) extracted from monitoring data was 11.0 ± 3.4 yr-1, and the rate constant predicted from the abundance of TceA peptides was 6.9 yr-1. The rate constant for degradation of vinyl chloride (VC) from monitoring data was 8.4 ± 5.7 yr-1, and the rate constant predicted from the abundance of TceA peptides was 5.2 yr-1. At the other study sites, the rate constants for cDCE degradation predicted from qPCR and qProt measurements agreed within a factor of 4. Under the right circumstances, qPCR and qProt measurements can be useful to rapidly predict rates of cDCE and VC biodegradation, providing a major advance in effective site management.

Dehalogenation of Chlorinated Ethenes to Ethene by a Novel Isolate, "Candidatus Dehalogenimonas etheniformans".

Dehalococcoides mccartyi strains harboring vinyl chloride (VC) reductive dehalogenase (RDase) genes are keystone bacteria for VC detoxification in groundwater aquifers, and bioremediation monitoring regimens focus on D. mccartyi biomarkers. We isolated a novel anaerobic bacterium, "Candidatus Dehalogenimonas etheniformans" strain GP, capable of respiratory dechlorination of VC to ethene. This bacterium couples formate and hydrogen (H2) oxidation to the reduction of trichloro-ethene (TCE), all dichloroethene (DCE) isomers, and VC with acetate as the carbon source. Cultures that received formate and H2 consumed the two electron donors concomitantly at similar rates. A 16S rRNA gene-targeted quantitative PCR (qPCR) assay measured growth yields of (1.2 ± 0.2) × 108 and (1.9 ± 0.2) × 108 cells per µmol of VC dechlorinated in cultures with H2 or formate as electron donor, respectively. About 1.5-fold higher cell numbers were measured with qPCR targeting cerA, a single-copy gene encoding a putative VC RDase. A VC dechlorination rate of 215 ± 40 µmol L-1 day-1 was measured at 30°C, with about 25% of this activity occurring at 15°C. Increasing NaCl concentrations progressively impacted VC dechlorination rates, and dechlorination ceased at 15 g NaCl L-1. During growth with TCE, all DCE isomers were intermediates. Tetrachloroethene was not dechlorinated and inhibited dechlorination of other chlorinated ethenes. Carbon monoxide formed and accumulated as a metabolic by-product in dechlorinating cultures and impacted reductive dechlorination activity. The isolation of a new Dehalogenimonas species able to effectively dechlorinate toxic chlorinated ethenes to benign ethene expands our understanding of the reductive dechlorination process, with implications for bioremediation and environmental monitoring. IMPORTANCE Chlorinated ethenes are risk drivers at many contaminated sites, and current bioremediation efforts focus on organohalide-respiring Dehalococcoides mccartyi strains to achieve detoxification. We isolated and characterized the first non-Dehalococcoides bacterium, "Candidatus Dehalogenimonas etheniformans" strain GP, capable of metabolic reductive dechlorination of TCE, all DCE isomers, and VC to environmentally benign ethene. In addition to hydrogen, the new isolate utilizes formate as electron donor for reductive dechlorination, providing opportunities for more effective electron donor delivery to the contaminated subsurface. The discovery that a broader microbial diversity can achieve detoxification of toxic chlorinated ethenes in anoxic aquifers illustrates the potential of naturally occurring microbes for biotechnological applications.

Identification and widespread environmental distribution of a gene cassette implicated in anaerobic dichloromethane degradation.

Anthropogenic activities and natural processes release dichloromethane (DCM, methylene chloride), a toxic chemical with substantial ozone-depleting capacity. Specialized anaerobic bacteria metabolize DCM; however, the genetic basis for this process has remained elusive. Comparative genomics of the three known anaerobic DCM-degrading bacterial species revealed a homologous gene cluster, designated the methylene chloride catabolism (mec) gene cassette, comprising 8-10 genes encoding proteins with 79.6%-99.7% amino acid identities. Functional annotation identified genes encoding a corrinoid-dependent methyltransferase system, and shotgun proteomics applied to two DCM-catabolizing cultures revealed high expression of proteins encoded on the mec gene cluster during anaerobic growth with DCM. In a DCM-contaminated groundwater plume, the abundance of mec genes strongly correlated with DCM concentrations (R2  = 0.71-0.85) indicating their potential value as process-specific bioremediation biomarkers. mec gene clusters were identified in metagenomes representing peat bogs, the deep subsurface, and marine ecosystems including oxygen minimum zones (OMZs), suggesting a capacity for DCM degradation in diverse habitats. The broad distribution of anaerobic DCM catabolic potential infers a role for DCM as an energy source in various environmental systems, and implies that the global DCM flux (i.e., the rate of formation minus the rate of consumption) might be greater than emission measurements suggest.

Metagenome-Guided Proteomic Quantification of Reductive Dehalogenases in the Dehalococcoides mccartyi-Containing Consortium SDC-9.

At groundwater sites contaminated with chlorinated ethenes, fermentable substrates are often added to promote reductive dehalogenation by indigenous or augmented microorganisms. Contemporary bioremediation performance monitoring relies on nucleic acid biomarkers of key organohalide-respiring bacteria, such as Dehalococcoides mccartyi (Dhc). Metagenome sequencing of the commercial, Dhc-containing consortium, SDC-9, identified 12 reductive dehalogenase (RDase) genes, including pceA (two copies), vcrA, and tceA, and allowed for specific detection and quantification of RDase peptides using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Shotgun (i.e., untargeted) proteomics applied to the SDC-9 consortium grown with tetrachloroethene (PCE) and lactate identified 143 RDase peptides, and 36 distinct peptides that covered greater than 99% of the protein-coding sequences of the PceA, TceA, and VcrA RDases. Quantification of RDase peptides using multiple reaction monitoring (MRM) assays with 13C-/15N-labeled peptides determined 1.8 × 103 TceA and 1.2 × 102 VcrA RDase molecules per Dhc cell. The MRM mass spectrometry approach allowed for sensitive detection and accurate quantification of relevant Dhc RDases and has potential utility in bioremediation monitoring regimes.

Proteogenomics Reveals Novel Reductive Dehalogenases and Methyltransferases Expressed during Anaerobic Dichloromethane Metabolism.

Dichloromethane (DCM) is susceptible to microbial degradation under anoxic conditions and is metabolized via the Wood-Ljungdahl pathway; however, mechanistic understanding of carbon-chlorine bond cleavage is lacking. The microbial consortium RM contains the DCM degrader "Candidatus Dichloromethanomonas elyunquensis" strain RM, which strictly requires DCM as a growth substrate. Proteomic workflows applied to DCM-grown consortium RM biomass revealed a total of 1,705 nonredundant proteins, 521 of which could be assigned to strain RM. In the presence of DCM, strain RM expressed a complete set of Wood-Ljungdahl pathway enzymes, as well as proteins implicated in chemotaxis, motility, sporulation, and vitamin/cofactor synthesis. Four corrinoid-dependent methyltransferases were among the most abundant proteins. Notably, two of three putative reductive dehalogenases (RDases) encoded within strain RM's genome were also detected in high abundance. Expressed RDase 1 and RDase 2 shared 30% amino acid identity, and RDase 1 was most similar to an RDase of Dehalococcoides mccartyi strain WBC-2 (AOV99960, 52% amino acid identity), while RDase 2 was most similar to an RDase of Dehalobacter sp. strain UNSWDHB (EQB22800, 72% amino acid identity). Although the involvement of RDases in anaerobic DCM metabolism has yet to be experimentally verified, the proteome characterization results implicated the possible participation of one or more reductive dechlorination steps and methyl group transfer reactions, leading to a revised proposal for an anaerobic DCM degradation pathway.IMPORTANCE Naturally produced and anthropogenically released DCM can reside in anoxic environments, yet little is known about the diversity of organisms, enzymes, and mechanisms involved in carbon-chlorine bond cleavage in the absence of oxygen. A proteogenomic approach identified two RDases and four corrinoid-dependent methyltransferases expressed by the DCM degrader "Candidatus Dichloromethanomonas elyunquensis" strain RM, suggesting that reductive dechlorination and methyl group transfer play roles in anaerobic DCM degradation. These findings suggest that the characterized DCM-degrading bacterium Dehalobacterium formicoaceticum and "Candidatus Dichloromethanomonas elyunquensis" strain RM utilize distinct strategies for carbon-chlorine bond cleavage, indicating that multiple pathways evolved for anaerobic DCM metabolism. The specific proteins (e.g., RDases and methyltransferases) identified in strain RM may have value as biomarkers for monitoring anaerobic DCM degradation in natural and contaminated environments.

A Strengths-Based Case Management Intervention to Reduce HIV Viral Load Among People Who Use Drugs.

Engaging highly marginalized HIV positive people in sustained medical care is vital for optimized health and prevention efforts. Prior studies have found that strengths-based case management helps link people who use drugs to HIV care. We conducted a pilot to assess whether a strengths-based case management intervention may help people who use injection drugs (PWID) or smoke crack cocaine (PWSC) achieve undetectable HIV viral load. PWID and PWSC were recruited in Oakland, California using targeted sampling methods and referral from jails and were tested for HIV. HIV positive participants not receiving HIV care (n = 19) were enrolled in a pilot strengths-based case management intervention and HIV positive participants already in HIV care (n = 29) were followed as comparison participants. The intervention was conducted by a social worker and an HIV physician. Special attention was given to coordinating care as participants cycled through jail and community settings. Surveys and HIV viral load tests were conducted quarterly for up to 11 visits. HIV viral load became undetectable for significantly more participants in the intervention than in the comparison group by their last follow-up (intervention participants: 32% at baseline and 74% at last follow-up; comparison participants: 45% at baseline and 34% at last follow-up; p = 0.008). In repeated measures analysis, PBO intervention participants had higher odds of achieving undetectable viral load over time than comparison participants (p = 0.033). Strengths-based case management may help this highly vulnerable group achieve undetectable HIV viral load over time.

Normalized Quantitative PCR Measurements as Predictors for Ethene Formation at Sites Impacted with Chlorinated Ethenes.

Quantitative PCR (qPCR) targeting Dehalococcoides mccartyi ( Dhc) biomarker genes supports effective management at sites impacted with chlorinated ethenes. To establish correlations between Dhc biomarker gene abundances and ethene formation (i.e., detoxification), 859 groundwater samples representing 62 sites undergoing monitored natural attenuation or enhanced remediation were analyzed. Dhc 16S rRNA genes and the vinyl chloride (VC) reductive dehalogenase genes bvcA and vcrA were detected in 88% and 61% of samples, respectively, from wells with ethene. Dhc 16S rRNA, bvcA, vcrA, and tceA (implicated in cometabolic reductive VC dechlorination) gene abundances all positively correlated with ethene formation. Significantly greater ethene concentrations were observed when Dhc 16S rRNA gene and VC RDase gene abundances exceeded 107 and 106 copies L-1, respectively, and when Dhc 16S rRNA- and bvcA + vcrA-to-total bacterial 16S rRNA gene ratios exceeded 0.1%. Dhc 16S rRNA gene-to- vcrA/ bvcA ratios near unity also indicated elevated ethene; however, no increased ethene was observed in 19 wells where vcrA and/or bvcA gene copy numbers exceeded Dhc cell numbers 10- to 10 000-fold. Approximately one-third of samples with detectable ethene lacked bvcA, vcrA, and tceA, suggesting that comprehensive understanding of VC detoxification biomarkers has not been achieved. Although the current biomarker suite is incomplete, the data analysis corroborates the value of the available Dhc DNA biomarkers for prognostic and diagnostic groundwater monitoring at sites impacted with chlorinated ethenes.

Effects of a novel sodium channel blocker, GSK2339345, in patients with refractory chronic cough
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OBJECTIVE: Voltage-gated sodium channels (VGSC) are important in the initiation and propagation of action potentials in afferent sensory nerve fibers responsible for evoking cough. This study investigated the efficacy of GSK2339345, a VGSC inhibitor, in the treatment of refractory chronic cough (RCC). METHODS: A three-part randomized, double-blind, placebo-controlled, cross-over study was conducted in the UK. In part A, patients with RCC received two inhaled doses of either GSK2339345 or placebo, 4 hours apart during three study periods. Patients were monitored for cough for 8 hours post-first dose using the VitaloJAK, ambulatory cough monitor. In parts B and C, patients underwent full dose-response cough challenges with capsaicin and citric acid respectively following single doses of randomly assigned GSK2339345 or placebo (4 study days). Part A was analyzed using a mixed effects model and parts B and C using population non-linear mixed effects models. RESULTS: Of 16 enrolled patients, 11 completed the study. 8-hour cough counts increased following GSK2339345 treatment compared with placebo (GSK2339345/placebo ratio of adjusted geometric means: 1.26 (90% credible interval 1.10, 1.44), associated with GSK2339345-evoked coughing, recorded during the 2 minutes post-dose. This was not observed with placebo. The effect of GSK2339345 on cough responses during cough challenges was inconclusive. GSK2339345 was well tolerated. CONCLUSIONS: While these data could not determine if GSK2339345 reached the target VGSC, they strongly suggest that GSK2339345 has no anti-tussive effect despite reaching airway sensory nerves as evidenced by the evoked transient cough.
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Once-daily dosing of levocabastine has comparable efficacy to twice-daily dosing in the treatment of allergic rhinitis assessed in an allergen challenge chamber.

OBJECTIVES: To test the hypothesis that intranasal levocabastine (LEVO) may provide benefits as a oncedaily treatment in allergic rhinitis (AR), this non-inferiority study compared the effect at steady state of once- and twice-daily dosing with LEVO on allergen-induced nasal symptoms in AR patients. METHODS: This was a randomized, double-blind, three-way cross over study evaluating the effects of repeat doses of LEVO 200 µg once-daily, LEVO 200 µg twice-daily (total dose 400 µg) and placebo, all via intranasal spray, in 78 AR patients. The primary endpoint was weighted mean total nasal symptom score (TNSS) during a 4-hour allergen exposure in the Environmental Exposure Chamber measured at trough pharmacokinetic levels either 12 (LEVO twice-daily) or 24 (LEVO once-daily) hours post-dose. RESULTS: After 7 days dosing, the difference in weighted mean TNSS (0-4 hours) following LEVO once-daily versus twice-daily was 0.23 units (95% CI -0.36, 0.82), demonstrating noninferiority between the two LEVO dosing regimens by meeting the pre-specified criterion of an upper limit of 95% CI<1.0. Both dosing regimens of LEVO resulted in a statistically significant reduction in mean TNSS compared with placebo (adjusted mean difference from placebo: LEVO once-daily: -1.12 (95% CI -1.71, -0.53); LEVO twice-daily: -1.35 (-1.94, -0.76)), meeting the pre-specified criterion for superiority (upper limit of 95% CI<0). All treatments were well-tolerated. CONCLUSIONS: The results of this study support the hypothesis that at steady state LEVO 200 µg taken once-daily provides similar benefit to LEVO 200 µg dosed twice-daily.

The biotransformation of ibuprofen to trihydroxyibuprofen in activated sludge and by Variovorax Ibu-1.

A bacterium was isolated from activated sewage sludge that has the ability to use ibuprofen as its sole carbon and energy source. Phylogenetic analysis of the 16S rRNA gene sequence placed the strain in the Variovorax genus within the ß-proteobacteria. When grown on ibuprofen it accumulated a transient yellow intermediate that disappeared upon acidification, a trait consistent with meta ring-fission metabolites. GC/MS analysis of derivatized culture supernatant yielded two spectra consistent with trihydroxyibuprofen bearing all three hydroxyl groups on the aromatic ring. These metabolites were only detected when 3-fluorocatechol, a meta ring-fission inhibitor, was added to Ibu-1 cultures and the supernatant was then derivatized with aqueous acetic anhydride and diazomethane. These findings suggest the possibility of ibuprofen metabolism proceeding via a trihydroxyibuprofen meta ring-fission pathway. Identical spectra, consistent with these putative ring-hydroxylated trihydroxyibuprofen metabolites, were also obtained from ibuprofen-spiked sewage sludge, but only when it was poisoned with 3-fluorocatechol. The presence of the same trihydroxylated metabolites in both spiked sewage sludge and culture supernatants suggests that this trihydroxyibuprofen extradiol ring-cleavage pathway for the degradation of ibuprofen may have environmental relevance.

Transient receptor potential vanilloid 1 (TRPV1) antagonism in patients with refractory chronic cough: a double-blind randomized controlled trial.

BACKGROUND: Inhalation of capsaicin, the extract of hot chili peppers, induces coughing in both animals and human subjects through activation of transient receptor potential vanilloid 1 (TRPV1) on airway sensory nerves. Therefore the TRPV1 receptor is an attractive target for the development of antitussive agents. OBJECTIVE: We sought to assess the antitussive effect of TRPV1 antagonism in patients with refractory chronic cough. METHODS: Twenty-one subjects with refractory chronic cough (>8 weeks) attending a specialist clinic were recruited to a randomized, double-blind, placebo-controlled crossover trial assessing a TRPV1 antagonist (SB-705498). Cough reflex sensitivity to capsaicin (concentration of capsaicin inducing at least 5 coughs) and 24-hour cough frequency were coprimary end points assessed after a single dose of SB-705498 (600 mg) and matched placebo. Cough severity and urge to cough were reported on visual analog scales, and cough-specific quality of life data were also collected. RESULTS: Treatment with SB-705498 produced a significant improvement in cough reflex sensitivity to capsaicin at 2 hours and a borderline significant improvement at 24 hours compared with placebo (adjusted mean difference of +1.3 doubling doses at 2 hours [95% CI, +0.3 to +2.2; P = .0049] and +0.7 doubling doses at 24 hours [95% CI, +0.0 to +1.5; P = .0259]). However, 24-hour objective cough frequency was not improved compared with placebo. Patient-reported cough severity, urge to cough, and cough-specific quality of life similarly suggested no effect of SB-705498. CONCLUSION: This study raises important questions about both the role of TRVP1-mediated mechanisms in patients with refractory chronic cough and also the predictive value of capsaicin challenge testing in the assessment of novel antitussive agents.

TRPV1 inhibition does not prevent cold dry air-elicited symptoms in non-allergic rhinitis.

BACKGROUND: The transient receptor potential vanilloid 1 (TRPV1)-expressing sensory C-fibers may play a role in the development of nasal hyper-responsiveness and symptoms of non-allergic rhinitis (NAR). OBJECTIVE: To evaluate the effects of a TRPV1-antagonist, SB-705498, on cold dry air (CDA)-induced symptoms in patients with NAR. METHODS: This randomized, double-blind, placebo-controlled, crossover study evaluated 14 days of once daily, topical intranasal SB-705498 12 mg in 40 patients with NAR using a CDA challenge experimental model in an environmental exposure chamber (EEC, Cetero Research, Mississauga, Ontario). The primary endpoint was total symptom score (TSS), expressed as weighted mean over 60 minutes (WM0-60) or maximum TSS at 1 hour and 24 hours postdosing. RESULTS: Treatment with SB-705498, relative to placebo, did not improve WM0-60 or maximum TSS at 1 hour and 24 hours post-dosing on days 1 or 14. Mean (95% CI) treatment differences (SB-705498 - placebo) on day 14 were, for WM0-60 at 1 hour: -0.12 (-0.60, 0.36); for maximum TSS at 1 hour: -0.03 (-0.58, 0.51). SB-705498 had no impact on any other efficacy parameters. SB-705498 was well tolerated and pharmacokinetics analysis supported the dosing regimen. CONCLUSION: SB-705498 12 mg for 14 days did not alleviate the CDA-induced symptoms of NAR. Despite engagement of the TRPV1 receptor, there was no translation to clinical efficacy, suggesting redundancy in symptom pathways.

Nitrous oxide inhibition of methanogenesis represents an underappreciated greenhouse gas emission feedback.

Methane (CH4) and nitrous oxide (N2O) are major greenhouse gases that are predominantly generated by microbial activities in anoxic environments. N2O inhibition of methanogenesis has been reported, but comprehensive efforts to obtain kinetic information are lacking. Using the model methanogen Methanosarcina barkeri strain Fusaro and digester sludge-derived methanogenic enrichment cultures, we conducted growth yield and kinetic measurements and showed that micromolar concentrations of N2O suppress the growth of methanogens and CH4 production from major methanogenic substrate classes. Acetoclastic methanogenesis, estimated to account for two-thirds of the annual 1 billion metric tons of biogenic CH4, was most sensitive to N2O, with inhibitory constants (KI) in the range of 18-25 µM, followed by hydrogenotrophic (KI, 60-90 µM) and methylotrophic (KI, 110-130 µM) methanogenesis. Dissolved N2O concentrations exceeding these KI values are not uncommon in managed (i.e. fertilized soils and wastewater treatment plants) and unmanaged ecosystems. Future greenhouse gas emissions remain uncertain, particularly from critical zone environments (e.g. thawing permafrost) with large amounts of stored nitrogenous and carbonaceous materials that are experiencing unprecedented warming. Incorporating relevant feedback effects, such as the significant N2O inhibition on methanogenesis, can refine climate models and improve predictive capabilities.

Genetic and chemical characterization of ibuprofen degradation by Sphingomonas Ibu-2.

Sphingomonas Ibu-2 has the unusual ability to cleave the acid side chain from the pharmaceutical ibuprofen and related arylacetic acid derivatives to yield corresponding catechols under aerobic conditions via a previously uncharacterized mechanism. Screening a chromosomal library of Ibu-2 DNA in Escherichia coli EPI300 allowed us to identify one fosmid clone (pFOS3G7) that conferred the ability to metabolize ibuprofen to isobutylcatechol. Characterization of pFOS3G7 loss-of-function transposon mutants permitted identification of five ORFs, ipfABDEF, whose predicted amino acid sequences bore similarity to the large and small units of an aromatic dioxygenase (ipfAB), a sterol carrier protein X (SCPx) thiolase (ipfD), a domain of unknown function 35 (DUF35) protein (ipfE) and an aromatic CoA ligase (ipfF). Two additional ORFs, ipfH and ipfI, which encode putative ferredoxin reductase and ferredoxin components of an aromatic dioxygenase system, respectively, were also identified on pFOS3G7. Complementation of a markerless loss-of-function ipfD deletion mutant restored catechol production as did complementation of the ipfF Tn mutant. Expression of subcloned ipfABDEF alone in E. coli did not impart full metabolic activity unless coexpressed with ipfHI. CoA ligation followed by ring oxidation is common to phenylacetic acid pathways. However, the need for a putative SCPx thiolase (IpfD) and DUF35 protein (IpfE) in aerobic arylacetic acid degradation is unprecedented. This work provides preliminary insights into the mechanism behind this novel arylacetic acid-deacylating, catechol-generating activity.

Objective measurement of cough in otherwise healthy volunteers with acute cough.

Cough is one of the commonest reasons for medical consultation and acute cough associated with upper respiratory tract infections (URTIs) is a global problem. In otherwise healthy volunteers complaining of cough associated with symptoms of URTI, we aimed to assess objective and subjective measures of cough and their repeatability and perform power calculations for the design of future studies to test therapies. We studied 54 otherwise healthy volunteers with acute cough (<3 weeks) (median age 22 yrs (interquartile range 21-26 yrs), 64% female, mean forced expiratory volume in 1 s 97.6±10.5% predicted). All subjects performed 24-h ambulatory cough monitoring and reported cough frequency and severity using visual analogue scales (VAS) on 2 consecutive days. Sample size calculations were performed for crossover and parallel group study designs. Objective cough frequency was high (session 1: geometric mean 12.1 coughs·h(-1) (95%CI 9.7-15.2)) and fell significantly (session 2: 9.0 coughs·h(-1) (95%CI 6.9-11.6); p<0.001). Repeatability was higher for objective cough frequency (intra-class correlation coefficient (ICC)=0.94, p<0.001) than reported cough frequency (daytime VAS ICC=0.784, p<0.001). Crossover/parallel studies require <15 and <41 subjects per arm to detect a 50% reduction in cough frequency with 90% power, respectively. Acute cough frequency is highly repeatable over any 48-h period, allowing small sample sizes to be used when investigating the effectiveness of novel anti-tussives.

The effects of a TRPV1 antagonist, SB-705498, in the treatment of seasonal allergic rhinitis.

BACKGROUND: Current pharmacotherapy for allergic rhinitis (AR) does not totally ameliorate all symptoms for all patients. Residual symptoms could be due to nasal neuronal hyperresponsiveness caused by stimulation of the ion channel transient receptor potential vanilloid 1 (TRPV1). SB-705498 is a TRPV1 antagonist that has been developed in an intranasal formulation for treatment of AR. METHODS: This randomized, double-blind, 3-way incomplete block crossover study evaluated the effects of 8 days treatment with SB-705498 12 mg alone, SB-705498 12 mg plus fluticasone propionate 200 µg (FP), FP 200 µg alone or placebo on allergen-induced symptoms in 70 patients with AR. The primary endpoint was total nasal symptom score (TNSS), expressed as mean over 4 hours or maximum TNSS during allergen challenge in the Vienna Challenge Chamber on 8th day of treatment. RESULTS: At the end of treatment, there were no differences in allergen-induced mean TNSS between SB-705498 alone and placebo or between SB-705498 plus FP and FP alone. Treatment with FP and SB-705498 plus FP resulted in a significant decrease in TNSS vs. placebo. Mean (90% CI) treatment differences in TNSS over 0 - 4 hours were: SB-705498 - placebo: -0.2 (-0.9, 0.4); SB-705498 plus FP - FP: 0.7 (0.2, 1.2); FP - placebo: -2.9 (-3.4, -2.5); SB-705498 plus FP - placebo: -2.3 (-2.8, -1.8). SB-705498 had no impact on diary card symptoms, nasal airflow or Rhinoconjunctivitis Quality of Life Questionnaire scores. SB-705498 was well tolerated and pharmacokinetics exposure results supported the dosing regimen. CONCLUSION: SB-705498 12 mg for 8 days did not alleviate the allergen-induced symptoms of AR, or provide additional relief of symptoms when in combination with FP. Despite engagement of the TRPV1 receptor there was no translation to clinical efficacy, suggesting redundancy in symptom pathways.

Diversity and community analysis of ammonia oxidizing bacteria in a streambed surrounding an artificial dam.

The degree to which small natural dams affect the native bacterial nitrogen cycling community was explored by molecular methods. The identities and relative abundances of ammonia oxidizing bacteria in the sediment surrounding an artificial dam both at the surface and in the hyporheic zone were characterized. Analyses were performed using tRFLP of the conserved amoA gene using a semi-nested degenerate PCR approach. Additionally, an amoA gene library was constructed to characterize the most dominant sediment genotypes. The results of the tRFLP analyses showed clear differences between the upstream and downstream communities at different depths in the sediment column. Non-metric multidimensional scaling ordination of the tRFLP data set produced a stable one-dimensional solution with significant correlations to oxygen, pH, nitrate, and dissolved organic nitrogen levels. The sample corresponding to the hyporheic zone downstream of the dam showed 28-50% higher amoA richness and higher diversity than the other samples. All gene fragments sequenced from the samples grouped with sequences of the Nitrosospira type. Ordination of 16S rDNA tRFLP data revealed a two dimensional data structure, one axis of which had similar chemical correlation characteristics as the amoA model axis. Taken together, the results from this study suggest that the presence of the dam creates physical and chemical heterogeneity that may foster genetic diversity and community changes amongst ammonia oxidizing bacteria.

Pollen starch granules in bronchial inflammation.

BACKGROUND: Pollen grains with a diameter of more than 10 µm preferentially deposit in the upper airways. Their contribution to lower airway inflammation is unclear. One hypothesis is that lower airway inflammation is mainly caused by allergen containing pollen starch granules, which are released from the pollen grains and can easily enter the peripheral airways because of their smaller size. OBJECTIVE: To investigate the differential effect of pollen grains and pollen starch granules on nasal symptoms and lower airway inflammation. METHODS: In a 2-period crossover design, 30 patients with allergic rhinitis and mild intermittent asthma underwent 2 allergen challenges on consecutive days in an environmental challenge chamber with either a mixture of pollen grains plus starch granules or starch granules only. End points were the total nasal symptom score (TNSS), nasal secretion weight, nasal flow, spirometry, and exhaled nitric oxide (eNO). RESULTS: The presence of pollen grains had a significant and considerable effect on increase in TNSS and secretion weight and on decrease in nasal flow. Starch granules alone only had minimal effects on nasal symptoms. Challenges with starch granules significantly increased eNO. Pollen had no effect on eNO. CONCLUSION: Pollen grains cause nasal symptoms but do not augment lower airway inflammation, whereas starch granules trigger lower airway inflammation but hardly induce nasal symptoms.

Provocation of nonallergic rhinitis subjects in response to simulated weather conditions using an environmental exposure chamber model.

Nonallergic rhinitis (NAR) subjects present clinically with similar symptoms to subjects with allergic rhinitis, but which mechanistically are not IgE- mediated. NAR is difficult to study because of multiple, as yet unknown, disease mechanisms and lack of biomarkers and diagnostic tests. The purpose of this proof of concept pilot study was to develop an environmental exposure chamber (EEC) model to simulate weather conditions in a controlled setting to objectively diagnose NAR subjects and ultimately to investigate novel NAR therapies. Thirty-seven subjects with a history of NAR confirmed by negative skin-prick test to a panel of aeroallergens were tested with cold dry air (CDA) and temperature change challenges. Objective (acoustic rhinometry [AcR] and nasal secretions) and subjective measures (total nasal symptom scores [TNSSs]: congestion, rhinorrhea, and postnasal drip [0-3]) were collected. Data was presented as mean ± SEM and statistical significance was assessed by paired t-test. The NAR EEC AcR responders to CDA had a significant decrease in mean minimal cross-sectional area (MCA; a measure of nasal patency) of 22.2 ± 2.43% (p < 0.0001) and 6.7 ± 7.22% (not statistically significant) at 30 and 60 minutes, respectively, with a concomitant increase in TNSS of 1.0 ± 0.24 U and 1.4 ± 0.30 U, respectively. AcR responders to temperature change showed a significant decrease in mean MCA to warm air of 16.0 ± 3.82% (p < 0.001) and 19.4 ± 3.88% (p < 0.0001) at 30 and 60 minutes, respectively, with an increase of TNSS of 0.4 ± 0.25 U and 0.4 ± 0.27 U, respectively. With rapid conversion to cold air, further decrease in mean MCA accompanied by an increase in TNSS was observed at 30 and 60 minutes. Increase in rhinorrhea was highest for CDA and the cold air phase of the temperature change challenge. Using the NAR EEC model, significant symptoms were induced in response to simulated weather changes in NAR patient responders. This proof of concept pilot study shows that the EEC model provides a consistent and reliable method to phenotype weather-induced NAR subjects that could be used to investigate disease mechanisms and novel therapies for NAR.

Anti-allergic drug testing in an environmental challenge chamber is suitable both in and out of the relevant pollen season.

BACKGROUND: An environmental challenge chamber (ECC) is a useful tool to expose allergic patients to relevant allergens in a controlled indoor setting and to test anti-allergic treatment. Hitherto, ECC studies with grass pollen are conducted primarily outside of the pollen season to avoid the influence of natural pollen exposure. OBJECTIVE: To investigate whether an established anti-allergic treatment, a combination of cetirizine (CET) and pseudoephedrine (PSE), shows an equivalent treatment effect within and outside of the grass pollen season when tested in an ECC. METHODS: In a randomized, placebo-controlled, double-blind, four-way crossover study, the effect of a combination of 10 mg CET and 120 mg PSE compared with placebo on nasal symptoms, nasal flow, and nasal secretion was investigated in 70 patients with seasonal allergic rhinitis. Subjects underwent four 6-hour pollen challenges in an ECC with administration of the drugs after 2 hours. Two challenges were conducted within the grass pollen season and two out of the grass pollen season. RESULTS: The active treatment significantly improved nasal symptoms and nasal flow and significantly reduced the amount of nasal secretion compared with placebo both within and outside of the pollen season (P < .0001 each). The treatment effect was not different between the seasons (P > .05). CONCLUSION: Controlled allergen provocation in an ECC can be used to test anti-allergic treatment both within and outside of the grass pollen season.