A Randomized, Double-Blind, Placebo-Controlled, Parallel-Group Phase 2b Trial of P2X3 Receptor Antagonist Sivopixant for Refractory or Unexplained Chronic Cough.

INTRODUCTION: To determine the optimal dose of sivopixant, a highly selective P2X3 receptor antagonist, for refractory or unexplained chronic cough (RCC/UCC). METHODS: In this phase 2b, randomized, double-blind, placebo-controlled, parallel-group, multicenter trial, patients received sivopixant 50, 150, or 300 mg or placebo once daily for 4 weeks. The primary endpoint was a change from baseline in 24-h cough frequency (coughs/h) with sivopixant vs placebo. RESULTS: Overall, 390/406 randomized patients completed the study. Placebo-adjusted changes in hourly cough count over 24 h were 13.17% (P = 0.3532), - 1.77% (P = 0.8935), and - 12.47% (P = 0.3241) and in cough severity (visual analog scale) were 1.75 mm (P = 0.5854), - 1.21 mm (P = 0.7056), and - 6.55 mm (P = 0.0433) with sivopixant 50, 150, and 300 mg, respectively. Placebo-adjusted changes from baseline in Leicester Cough Questionnaire total scores were - 0.37 (P = 0.4207), - 0.07 (P = 0.8806), and 0.69 (P = 0.1473) with sivopixant 50, 150, and 300 mg, respectively. Additionally, 61.3%, 78.3%, 86.8%, and 71.4% of patients receiving sivopixant 50, 150, and 300 mg and placebo, respectively, reported any improvements in Patient Global Impression of Change. The incidence of treatment-emergent adverse events (TEAEs) was 25.7%, 32.0%, 49.0%, and 20.6% in sivopixant 50, 150, and 300 mg and placebo groups, respectively; all TEAEs in the sivopixant group were mild-to-moderate. CONCLUSION: Sivopixant did not demonstrate a statistically significant difference vs placebo in change from baseline in 24-h cough frequency. The dose of 300 mg has potential for RCC/UCC, showing the greatest improvements in cough frequency and patient-reported outcomes and dose-related mild to moderate reversible taste disturbance, although further trials are needed. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT04110054; registered September 26, 2019.

Iron serum levels and iron homeostasis parameters in patients with nosocomial pneumonia treated with cefiderocol: post hoc analysis of the APEKS-NP study.

Critically ill patients often present with low serum iron levels or anemia. We evaluated the impact of iron levels and iron homeostasis on the efficacy and safety of cefiderocol, an iron-chelator siderophore cephalosporin, in patients with nosocomial pneumonia in a post hoc analysis of the randomized, double-blind, Phase 3 APEKS-NP study (NCT03032380). Patients with Gram-negative nosocomial pneumonia received cefiderocol 2 g, 3-h infusion, q8h, or high-dose, extended-infusion meropenem 2 g, 3-h infusion, q8h, for 7-14 days. Efficacy and safety parameters, including specific iron homeostasis parameters (i.e., hepcidin, iron, total iron binding capacity, transferrin saturation), were analyzed according to baseline iron levels. In the cefiderocol and meropenem arms, 79.1% (117/148) and 83.3% (125/150) randomized patients, respectively, had low baseline serum iron levels. Rates of 14-day (12.3% [14/114] vs 11.6% [14/121]) and 28-day all-cause mortality (20.5% [23/112] vs 19.0% [23/121]), clinical cure (63.2% [72/114] vs 67.2% [82/122]), and microbiological eradication (43.6% [41/94] vs 48.1% [51/106]) at test of cure were similar in cefiderocol vs meropenem arms, respectively. In the overall safety population, rates of anemia-related adverse events were similar (cefiderocol arm 18.2% [27/148], meropenem arm 18.7% [28/150]). Changes from baseline to test of cure in hepcidin, iron, total iron binding capacity, and transferrin saturation were similar between treatment arms. Cefiderocol treatment did not affect iron homeostasis, and its efficacy and safety were not influenced by baseline serum iron levels. Clinicaltrials.gov registration: NCT03032380. Date of registration: 26 January 2017.

Mechanisms of Reduced Susceptibility to Cefiderocol Among Isolates from the CREDIBLE-CR and APEKS-NP Clinical Trials.

The objective of this study was to characterize isolates with reduced susceptibility to cefiderocol in patients receiving cefiderocol for nosocomial pneumonia or carbapenem-resistant infections in the Phase 3 APEKS-NP and CREDIBLE-CR studies. Susceptibility testing of isolates was conducted at a central laboratory, and post-treatment changes were evaluated according to available breakpoints for cefiderocol. Whole-genome sequencing and multilocus sequence typing were performed for isolates to confirm their origin and identify mutations. Five (APEKS-NP) and nine (CREDIBLE-CR) isolates demonstrated a ≥ 4-fold minimum inhibitory concentration (MIC) increase compared with genetically related baseline isolates; most remained susceptible to cefiderocol despite the ≥4-fold MIC increase. Mutations in ß-lactamases or penicillin-binding protein (PBP) were identified in 4/14 isolates: one Enterobacter cloacae (amino acid [AA] substitution [A313P] in ACT-17); two Acinetobacter baumannii (one PBP3 AA substitution [H370Y], one with OXA-23 substitutions [N85I and P225S]); and one Pseudomonas aeruginosa (PDC-30 [4AA deletion "TPMA" position 316-319]). Cloning experiments using isogenic Escherichia coli strains containing wild-type and those mutant cephalosporinase enzymes show that the mutant enzymes may contribute to decreased susceptibility to cefiderocol. Pharmacokinetic data were available for nine patients, for whom cefiderocol exposures exceeded 100% fT > 4 × MIC. No clear pattern between mutations and development or extent of MIC increases was observed. No mutations were identified in genes related to iron transport, including fiu, cirA, piuA/C, and pirA, among recovered Gram-negative isolates. Clinicaltrials.gov: APEKS-NP: NCT03032380; CREDIBLE-CR: NCT02714595.

Intrapulmonary pharmacokinetic profile of cefiderocol in mechanically ventilated patients with pneumonia.

OBJECTIVES: Lung penetration of cefiderocol, a novel siderophore cephalosporin approved for treatment of nosocomial pneumonia, has previously been evaluated in healthy subjects. This study assessed the intrapulmonary pharmacokinetic profile of cefiderocol at steady state in hospitalized, mechanically ventilated pneumonia patients. METHODS: Patients received cefiderocol 2 g (or ≤1.5 g if renally impaired), administered IV q8h as a 3 h infusion, or 2 g q6h if patients had augmented renal function (estimated CLCR > 120 mL/min). After multiple doses, each patient underwent a single bronchoalveolar lavage (BAL) procedure either at the end of the infusion or at 2 h after the end of infusion. Plasma samples were collected at 1, 3, 5 and 7 h after the start of infusion. After correcting for BAL dilution, cefiderocol concentrations in epithelial lining fluid (ELF) for each patient and the ELF/unbound plasma concentration ratio (RC, E/P) were calculated. Safety was assessed up to 7 days after the last cefiderocol dose. RESULTS: Seven patients received cefiderocol. Geometric mean ELF concentration of cefiderocol was 7.63 mg/L at the end of infusion and 10.40 mg/L at 2 h after the end of infusion. RC, E/P was 0.212 at the end of infusion and 0.547 at 2 h after the end of infusion, suggesting delayed lung distribution. There were no adverse drug reactions. CONCLUSIONS: The results suggest that cefiderocol penetrates the ELF in critically ill pneumonia patients with concentrations that are sufficient to treat Gram-negative bacteria with an MIC of ≤4 mg/L.

Efficacy and safety of cefiderocol or best available therapy for the treatment of serious infections caused by carbapenem-resistant Gram-negative bacteria (CREDIBLE-CR): a randomised, open-label, multicentre, pathogen-focused, descriptive, phase 3 trial.

BACKGROUND: New antibiotics are needed for the treatment of patients with life-threatening carbapenem-resistant Gram-negative infections. We assessed the efficacy and safety of cefiderocol versus best available therapy in adults with serious carbapenem-resistant Gram-negative infections. METHODS: We did a randomised, open-label, multicentre, parallel-group, pathogen-focused, descriptive, phase 3 study in 95 hospitals in 16 countries in North America, South America, Europe, and Asia. We enrolled patients aged 18 years or older admitted to hospital with nosocomial pneumonia, bloodstream infections or sepsis, or complicated urinary tract infections (UTI), and evidence of a carbapenem-resistant Gram-negative pathogen. Participants were randomly assigned (2:1 by interactive web or voice response system) to receive either a 3-h intravenous infusion of cefiderocol 2 g every 8 h or best available therapy (pre-specified by the investigator before randomisation and comprised of a maximum of three drugs) for 7-14 days. For patients with pneumonia or bloodstream infection or sepsis, cefiderocol treatment could be combined with one adjunctive antibiotic (excluding polymyxins, cephalosporins, and carbapenems). The primary endpoint for patients with nosocomial pneumonia or bloodstream infection or sepsis was clinical cure at test of cure (7 days [plus or minus 2] after the end of treatment) in the carbapenem-resistant microbiological intention-to-treat population (ITT; ie, patients with a confirmed carbapenem-resistant Gram-negative pathogen receiving at least one dose of study drug). For patients with complicated UTI, the primary endpoint was microbiological eradication at test of cure in the carbapenem-resistant microbiological ITT population. Safety was evaluated in the safety population, consisting of all patients who received at least one dose of study drug. Mortality was reported through to the end of study visit (28 days [plus or minus 3] after the end of treatment). Summary statistics, including within-arm 95% CIs calculated using the Clopper-Pearson method, were collected for the primary and safety endpoints. This trial is registered with ClinicalTrials.gov (NCT02714595) and EudraCT (2015-004703-23). FINDINGS: Between Sept 7, 2016, and April 22, 2019, we randomly assigned 152 patients to treatment, 101 to cefiderocol, 51 to best available therapy. 150 patients received treatment: 101 cefiderocol (85 [85%] received monotherapy) and 49 best available therapy (30 [61%] received combination therapy). In 118 patients in the carbapenem-resistant microbiological ITT population, the most frequent carbapenem-resistant pathogens were Acinetobacter baumannii (in 54 patients [46%]), Klebsiella pneumoniae (in 39 patients [33%]), and Pseudomonas aeruginosa (in 22 patients [19%]). In the same population, for patients with nosocomial pneumonia, clinical cure was achieved by 20 (50%, 95% CI 33·8-66·2) of 40 patients in the cefiderocol group and ten (53%, 28·9-75·6) of 19 patients in the best available therapy group; for patients with bloodstream infection or sepsis, clinical cure was achieved by ten (43%, 23·2-65·5) of 23 patients in the cefiderocol group and six (43%, 17·7-71·1) of 14 patients in the best available therapy group. For patients with complicated UTIs, microbiological eradication was achieved by nine (53%, 27·8-77·0) of 17 patients in the cefiderocol group and one (20%, 0·5-71·6) of five patients in the best available therapy group. In the safety population, treatment-emergent adverse events were noted for 91% (92 patients of 101) of the cefiderocol group and 96% (47 patients of 49) of the best available therapy group. 34 (34%) of 101 patients receiving cefiderocol and nine (18%) of 49 patients receiving best available therapy died by the end of the study; one of these deaths (in the best available therapy group) was considered to be related to the study drug. INTERPRETATION: Cefiderocol had similar clinical and microbiological efficacy to best available therapy in this heterogeneous patient population with infections caused by carbapenem-resistant Gram-negative bacteria. Numerically more deaths occurred in the cefiderocol group, primarily in the patient subset with Acinetobacter spp infections. Collectively, the findings from this study support cefiderocol as an option for the treatment of carbapenem-resistant infections in patients with limited treatment options. FUNDING: Shionogi.

Cefiderocol versus high-dose, extended-infusion meropenem for the treatment of Gram-negative nosocomial pneumonia (APEKS-NP): a randomised, double-blind, phase 3, non-inferiority trial.

BACKGROUND: Nosocomial pneumonia due to multidrug-resistant Gram-negative pathogens poses an increasing challenge. We compared the efficacy and safety of cefiderocol versus high-dose, extended-infusion meropenem for adults with nosocomial pneumonia. METHODS: We did a randomised, double-blind, parallel-group, phase 3, non-inferiority trial in 76 centres in 17 countries in Asia, Europe, and the USA (APEKS-NP). We enrolled adults aged 18 years and older with hospital-acquired, ventilator-associated, or health-care-associated Gram-negative pneumonia, and randomly assigned them (1:1 by interactive response technology) to 3-h intravenous infusions of either cefiderocol 2 g or meropenem 2 g every 8 h for 7-14 days. All patients also received open-label intravenous linezolid (600 mg every 12 h) for at least 5 days. An unmasked pharmacist prepared the assigned treatments; investigators and patients were masked to treatment assignment. Only the unmasked pharmacist was aware of the study drug assignment for the infusion bags, which were administered in generic infusion bags labelled with patient and study site identification numbers. Participants were stratified at randomisation by infection type and Acute Physiology and Chronic Health Evaluation II (APACHE II) score (≤15 and ≥16). The primary endpoint was all-cause mortality at day 14 in the modified intention-to-treat (ITT) population (ie, all patients receiving at least one dose of study drug, excluding patients with Gram-positive monomicrobial infections). The analysis was done for all patients with known vital status. Non-inferiority was concluded if the upper bound of the 95% CI for the treatment difference between cefiderocol and meropenem groups was less than 12·5%. Safety was investigated to the end of the study in the safety population, which included all patients who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov, NCT03032380, and EudraCT, 2016-003020-23. FINDINGS: Between Oct 23, 2017, and April 14, 2019, we randomly assigned 148 participants to cefiderocol and 152 to meropenem. Of 292 patients in the modified ITT population, 251 (86%) had a qualifying baseline Gram-negative pathogen, including Klebsiella pneumoniae (92 [32%]), Pseudomonas aeruginosa (48 [16%]), Acinetobacter baumannii (47 [16%]), and Escherichia coli (41 [14%]). 142 (49%) patients had an APACHE II score of 16 or more, 175 (60%) were mechanically ventilated, and 199 (68%) were in intensive care units at the time of randomisation. All-cause mortality at day 14 was 12·4% with cefiderocol (18 patients of 145) and 11·6% with meropenem (17 patients of 146; adjusted treatment difference 0·8%, 95% CI -6·6 to 8·2; p=0·002 for non-inferiority hypothesis). Treatment-emergent adverse events were reported in 130 (88%) of 148 participants in the cefiderocol group and 129 (86%) of 150 in the meropenem group. The most common treatment-emergent adverse event was urinary tract infection in the cefiderocol group (23 patients [16%] of 148) and hypokalaemia in the meropenem group (23 patients [15%] of 150). Two participants (1%) of 148 in the cefiderocol group and two (1%) of 150 in the meropenem group discontinued the study because of drug-related adverse events. INTERPRETATION: Cefiderocol was non-inferior to high-dose, extended-infusion meropenem in terms of all-cause mortality on day 14 in patients with Gram-negative nosocomial pneumonia, with similar tolerability. The results suggest that cefiderocol is a potential option for the treatment of patients with nosocomial pneumonia, including those caused by multidrug-resistant Gram-negative bacteria. FUNDING: Shionogi.

Designing A Pathogen-Focused Study To Address The High Unmet Medical Need Represented By Carbapenem-Resistant Gram-Negative Pathogens - The International, Multicenter, Randomized, Open-Label, Phase 3 CREDIBLE-CR Study.

Carbapenem-resistant (CR) Gram-negative infections, including those caused by Enterobacteriaceae and the non-fermenters, represent the greatest unmet need for new effective treatments. The clinical development of new antibiotics for the treatment of CR infections is challenging and should focus on the individual pathogens irrespective of the infection site. However, the drug approval pathway is generally infection-site specific and rarely includes such drug-resistant pathogens. To overcome this limitation, a streamlined clinical development program may include a pathogen-focused clinical study, such as the CREDIBLE-CR study, to meet the expectations of some health authorities (ie, the European Medicines Agency [EMA]) and the medical community. Cefiderocol is a novel siderophore cephalosporin designed to target CR pathogens, including CR strains of Enterobacteriaceae (CRE), Pseudomonas aeruginosa, Acinetobacter baumannii, and also Stenotrophomonas maltophilia, which is intrinsically CR. The CREDIBLE-CR study was planned to evaluate cefiderocol in patients with CR Gram-negative infections regardless of species or infection-site source. Rapid diagnostic testing and/or selective media were provided to facilitate detection of CR pathogens to rapidly enroll patients with nosocomial pneumonia, bloodstream infection/sepsis, or complicated urinary tract infection. Patients were randomized 2:1 to receive cefiderocol or best available therapy. There were no pre-specified statistical hypotheses for this study, as the sample size was driven by enrollment feasibility and not based on statistical power calculations. The objective of the CREDIBLE-CR study was to provide descriptive evidence of the efficacy and safety of cefiderocol for the target population of patients with CR infections, including the non-fermenters. The CREDIBLE-CR study is currently the largest pathogen-focused, randomized, open-label, prospective, Phase 3 clinical study to investigate a new antibiotic in patients with CR Gram-negative infections. Here we describe the design of this pathogen-focused study and steps taken to aid patient enrollment into the study within an evolving regulatory environment. CLINICALTRIALSGOV REGISTRATION: NCT02714595. EUDRA-CT REGISTRATION: 2015-004703-23.

[Relevance of the five-repetition sit-to-stand test for diagnosing sarcopenia in elderly inpatients with cardiac disease].

PURPOSE: The aim of this study was to determine the reference values for diagnosing sarcopenia using the five-repetition sit-to-stand test in elderly inpatients with cardiac disease. METHODS: We studied 71 inpatients with cardiac disease ≥65 years of age (mean age 78.0±7.9 years, 42.3% women) who were admitted between April 2015 and March 2016. Patients were assessed for sarcopenia, and we performed the five-repetition sit-to-stand test. We defined sarcopenia using the Asian Working Group for Sarcopenia-suggested diagnostic algorithm. A logistic regression analysis was performed to estimate the odds ratios (ORs) and 95% confidence intervals (CIs) of the relationship between sarcopenia and the five-repetition sit-to-stand test. A multivariate analysis showed that the age, admission diagnosis, the New York Heart Association classification, the Charlson comorbidity index, and the ratio of extracellular to total body water were relevant covariates. The cut-off value of the five-repetition sit-to-stand test to diagnose sarcopenia was determined using a receiver operating characteristic curve. RESULTS: Sarcopenia was diagnosed in 25 patients (35.2%). A multivariate logistic regression analysis showed that the five-repetition sit-to-stand test was significantly associated with sarcopenia (p=0.024), and the OR (95% CI) was 1.31 (1.04-1.65). The cut-off value of the five-repetition sit-to-stand test to diagnose sarcopenia was 10.9 s (sensitivity 80.0%, specificity 70.0%, area under the curve 0.83). CONCLUSIONS: The five-repetition sit-to-stand test is a useful screening tool for sarcopenia in elderly inpatients with cardiac disease. The cut-off value to diagnose sarcopenia was 10.9 s in this study.

Patients with MAC Lung Disease Have a Low Visceral Fat Area and Low Nutrient Intake.

OBJECTIVE: This study aimed to examine the nutritional status and nutrient intake of patients with MAC lung disease with a focus on visceral fat area. PATIENTS AND METHODS: Among 116 patients of our hospital with nontuberculous mycobacteriosis who were registered between May 2010 and August 2011, 103 patients with MAC lung disease were included in this study. In all patients, nutritional status and nutrient intake were prospectively examined. RESULTS: Patients were 23 men and 80 women (mean age, 72.3±10.9 years). BMI (kg/m2) at the time of registration was 20.4±2.7 in men and 19.2±2.9 in women. Visceral fat area (cm2) was significantly lower in women (35.7±26.6) than in men (57.5±47.4) (p=0.0111). The comparison with general healthy adults according to age revealed a markedly reduced visceral fat area among patients with MAC lung disease. With respect to nutrient intake, energy adequacy (86.1±15.7%), protein adequacy (82.4±18.2%), lipid adequacy (78.1±21.8%), and carbohydrate adequacy (89.6±19.2%) ratios were all low at the time of registration. BMI was significantly correlated with protein adequacy (p=0.0397) and lipid adequacy (p=0.0214) ratios, while no association was found between visceral fat area and nutrient intake. CONCLUSION: Patients with MAC lung disease had a low visceral fat area and low nutrient intake.

Prevalence of asthma with airflow limitation, COPD, and COPD with variable airflow limitation in older subjects in a general Japanese population: the Hisayama Study.

BACKGROUND: Elucidating the prevalence of asthma and chronic obstructive pulmonary disease (COPD) is important for designing a public health strategy. Recent studies have discriminated a phenotype of COPD with variable airflow limitation (COPD-VAL) associated with asthma-COPD overlap syndrome. Its prevalence remains uncertain. The age and occupational distributions in the town of Hisayama and in Japan are nearly identical. Each disease's prevalence was estimated for the town's residents. METHODS: In 2008, town residents (≥ 40 years) were solicited to participate in a health checkup. Individuals with abnormal spirometry (forced expiratory volume in 1s/forced vital capacity [FEV1/FVC]<70% and/or %FVC<80%) were recommended for further evaluations. Two pulmonologists in a blinded fashion reviewed their medical records, including bronchodilator reversibility. Individuals with airflow limitation were classified as having asthma, COPD, COPD-VAL, or other diseases. The prevalence of each disease was then estimated. RESULTS: A total of 2100 residents (43.4% of residents in the age group) completed spirometry. In 455 residents with abnormal spirometry, 190 residents had further evaluations, and the medical records of 174 residents were reviewed. The prevalence of asthma with airflow limitation, COPD, and COPD-VAL, were 2.0%, 8.4%, and 0.9%, respectively. The prevalence of COPD and COPD-VAL were higher in men and smokers than in women and never-smokers. The prevalence of COPD, but not COPD-VAL or asthma, increased with age. CONCLUSION: The prevalence of asthma with airflow limitation, COPD, and COPD-VAL were estimated in a population of residents (≥ 40 years) in Hisayama.

Secondhand smoke exposure within semi-open air cafes and tobacco specific 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) concentrations among nonsmoking employees.

OBJECTIVES: Secondhand smoke (SHS) is a defined occupational hazard. The association though between SHS exposure in semi-open air venues and tobacco specific carcinogen uptake is an area of debate. MATERIAL AND METHODS: A cross sectional survey of 49 semi-open air cafes in Athens, Greece was performed during the summer of 2008, prior to the adoption of the national smoke free legislation. All venues had at least 1 entire wall open to allow for free air exchange. Indoor concentrations of particulate matter smaller than 2.5 microns (PM2.5) attributable to SHS were assessed during a work shift, while 1 non-smoking employee responsible for indoor and outdoor table service from each venue provided a post work shift urine sample for analysis of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). RESULTS: Post work shift NNAL concentrations were correlated with work shift PM2.5 concentrations attributable to SHS (r = 0.376, p = 0.0076). Urinary NNAL concentrations among employees increased by 9.5%, per 10 µg/m(3) increase in PM2.5 concentrations attributable to SHS after controlling for the time of day and day of week. CONCLUSIONS: These results indicate that the commonly proposed practice of maintaining open sliding walls as a means of free air exchange does not lead to the elimination of employee exposure to tobacco specific carcinogens attributable to workplace SHS.

The association between cigarette rod length, slim design, and blood cadmium levels among U.S. smokers: NHANES 1999-2010.

BACKGROUND: Previous research has shown that long or slim cigarette design can mitigate perception of harmfulness. Cadmium (Cd) is a toxicant in cigarettes and is associated with adverse outcomes. We assessed the relationship between cigarette rod length/circumference and blood Cd levels among U.S. smokers using data from the 1999-2010 National Health and Nutrition Examination Survey. METHODS: The analyses were performed on 4486 current cigarette smokers aged ≥20years old. Cigarette rod length included regular (68-72mm), king (79-88mm), long (94-101mm), and ultra-long (110-121mm) cigarettes currently smoked. Overall and gender stratified multivariate linear regression analyses were performed, adjusting for other influential covariates. RESULTS: Gender stratified analysis indicated that female smokers of long and ultra-long cigarette had 20% and 27% higher mean Cd levels compared to smokers of regular sized cigarettes respectively, despite the fact that all the ultra-long cigarettes were slim cigarettes. Furthermore, among females, slim cigarette users did not reduce blood cadmium levels compared to non-slim users. CONCLUSIONS: Female smokers of long or ultra-long cigarettes had higher mean blood Cd levels compared to smokers of regular cigarettes independent of slim design. Further research into this association is warranted.

The relationship between cotinine concentrations and inflammatory markers among highly secondhand smoke exposed non-smoking adolescents.

BACKGROUND: Secondhand smoke (SHS) exposure is a risk factor of respiratory, cardiovascular and inflammatory diseases, however its association with inflammatory markers among highly SHS exposed adolescents has not yet been explored. METHODS: Participants included in this study were a subset of 68 non-smoking adolescents, aged 12.5-17.5 from the Healthy Lifestyle in Europe by Nutrition in Adolescence (HELENA) study, recruited from Crete Greece. Smoking and SHS exposure was assessed via serum cotinine concentrations. Cytokines (Interleukin-1ß, 2, 4, 5 and 6, tumor necrosis factor-α, interferon-γ, tumor growth factor-ß1), immunoglobulins IgG, IgA, IgM, complement factors C3, C4, high sensitivity C-reactive protein, and endothelial inflammatory markers [soluble E-selectin, soluble L-selectin, soluble intercellular adhesion molecules (sICAM-1) and soluble vascular cell adhesion molecules-1 (sVCAM-1)] were assessed. Inflammatory markers in the lower 25th percentile and upper 75th percentile groups of cotinine levels were compared and multivariate linear regression analysis was performed controlling for age, sex and BMI. RESULTS: Cotinine concentrations were notably elevated (geometric mean 0.82ng/ml, 95%CI 0.62-1.07) in this study population. A significant decrease in IL-4 (130.09 vs. 25.77pg/ml, p=0.014) and IL-6 (19.52 vs. 5.52pg/ml, p=0.008) concentrations between the upper 75th percentile cotinine level group and lower 25th percentile cotinine level group was observed. In a multivariate linear regression analysis, cotinine concentrations had a weak inverse association with IL-4 and IL-6 (p=0.028 and p=0.06) which was not statistically significant when adjusted for multiple comparisons (modified Bonferroni, p>0.016). No differences in the other variables was noted. CONCLUSIONS: Among highly SHS exposed adolescents, cotinine levels had weak inverse association with IL-4 and IL-6, which did not achieve statistical significance. However, our results potentially indicate an immunosuppressive role of SHS. Further research is warranted to explore this hypothesis.

The acute effect of cigarette smoking on the respiratory function and FENO production among young smokers.

INTRODUCTION: Smoking is known to have a long-term impact on lung function; however, the acute physiological response of smoking a single cigarette and the influential role of pack years and cigarettes per day on pulmonary indices remains an area of interest, especially among young smokers. METHODS: 50 naive smokers (ages: 18-26, 24 males: mean pack years 3.8) participated in this experimental study. Respiratory resistance (R), reactance (X), and impedance (Z) were assessed through impulse oscillometry. The participants' fraction of exhaled nitric oxide (FENO) was measured. All tests were performed immediately before and after smoking one single cigarette. RESULTS: Smoking a single cigarette was found to immediately increase airway impedance (Z 5 Hz) by 0.024 kPa/(L/s) (P = .002), airway resistance at R 5 Hz, R 10 Hz, and R 20 Hz by 0.024 kPa/(L/s)(P < .001), 0.016 kPa/(L/s)(P = .019), and 0.023 kPa/(L/s) (P = .007), respectively, after adjusting for BMI, age, gender, and pack years. FENO concentrations also decreased from 11.70 ppb to 9.85 ppb, P < .001. Sensitivity analyses indicated that the participants' number of pack years and cigarettes per day influenced pulmonary reactance at 10 Hz and 20 Hz, however only at baseline with these differences found to disappear immediately after smoking. CONCLUSIONS: The present study indicates that the consumption of a single cigarette may alter lung mechanics and FENO production among young smokers. Further research is needed to assess the mechanisms and washout period after which these parameters return to normal.

Cigarette smoke impairs phagocytosis of apoptotic neutrophils by alveolar macrophages via inhibition of the histone deacetylase/Rac/CD9 pathways.

Efferocytosis, which is the homeostatic phagocytosis of apoptotic cells, prevents the release of toxic intracellular contents and subsequent tissue damage. Impairment of efferocytosis was reported in alveolar macrophages (AMs) of patients with chronic obstructive pulmonary disease (COPD), a common disease caused by smoking. In COPD, histone deacetylase (HDAC) activity is reduced in AMs. We investigated whether the reduction of HDAC activity is associated with the impairment of efferocytosis. Murine AMs were collected by bronchoalveolar lavage and their ability to efferocytose apoptotic human polymorphonuclear leukocytes was assessed. Pre-treatment of AMs with cigarette smoke extract (CSE) or trichostatin A (TSA), an HDAC inhibitor, suppressed efferocytosis and CSE reduced HDAC activity. TSA inhibited the activity of Rac, a key mediator of efferocytosis. These TSA-induced impairments were restored by treatment of AMs with aminophylline, a potent activator of HDAC. To further elucidate the underlying mechanism, we explored a role of CD9 in TSA-induced impairment of efferocytosis. CD9 is a transmembrane protein of the tetraspanin family that facilitates the uptake of several pathogens and other material. TSA profoundly down-regulated the expression of CD9 on AMs. The expression of CD9 was partly down-regulated by the Rac inhibitor. Pretreatment with an anti-CD9 mAb or CD9 small interfering RNA inhibited efferocytosis, which was attributable to the reduced binding of AMs to apoptotic cells. These results suggest that smoking impairs efferocytosis via inhibition of HDAC/Rac/CD9 pathways. Aminophylline/theophylline is effective in restoring the impairment of efferocytosis and might have benefit for the treatment of patients with COPD.

Leukotriene B4 receptor BLT2 negatively regulates allergic airway eosinophilia.

Leukotriene B4 (LTB4) has been implicated in the pathogenesis of allergic diseases. BLT2, a low-affinity LTB4 receptor, is activated by LTB4 and 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT). Although the high-affinity LTB4 receptor BLT1 has been shown to exert proinflammatory roles, the role of BLT2 in allergic inflammation has not been clarified. To study the function of BLT2 in development of asthma, we used mice model of ovalbumin (OVA)-induced allergic airway disease. The 12-HHT levels were elevated in bronchoalveolar lavage (BAL) fluids of OVA-sensitized/challenged wild-type mice. BLT2-deficient mice exhibited enhanced eosinophilia in BAL fluids after OVA exposure. Interleukin (IL)-13 levels in BAL fluids and IL-13-producing CD4(+) T cells in the lungs were elevated in BLT2-deficient mice compared to wild-type mice, whereas the levels of IL-4, IL-5, and interferon (IFN)-γ in BAL fluids and serum OVA-specific IgE were comparable. Transfection of BLT2-specific small interfering RNA enhanced IL-13 production in CD4(+) T cells in vitro. Expression of BLT2 mRNA in CD4(+) T cells was significantly reduced in patients with asthma compared to healthy control subjects. These findings indicate that BLT2 has a protective role in allergic airway inflammation and that diminished BLT2 expression in CD4(+) T cells may contribute to the pathophysiology of asthma.

Mast cells contribute to double-stranded RNA-induced augmentation of airway eosinophilia in a murine model of asthma.

BACKGROUND: Clinical studies showed the contribution of viral infection to the development of asthma. Although mast cells have multiple roles in the pathogenesis of allergic asthma, their role of in the virus-associated pathogenesis of asthma remains unknown. Most respiratory viruses generate double-stranded (ds) RNA during their replication. dsRNA provokes innate immune responses. We recently showed that an administration of polyinocinic polycytidilic acid (poly IC), a mimetic of viral dsRNA, during allergen sensitization augments airway eosinophilia and hyperresponsiveness in mice via enhanced production of IL-13. METHODS: The effect of poly IC on allergen-induced airway eosinophilia was investigated for mast cell-conserved Kit+/+ mice and -deficient KitW/KitW-v mice. The outcome of mast cell reconstitution was further investigated. RESULTS: Airway eosinophilia and IL-13 production were augmented by poly IC in Kit+/+ mice but not in KitW/KitW-v mice. When KitW/KitW-v mice were reconstituted with bone marrow-derived mast cells (BMMCs), the augmentation was restored. The augmentation was not induced in the mice systemically deficient for TIR domain-containing adaptor-inducing IFN-ß (TRIF) or interferon regulatory factor (IRF)-3, both mediate dsRNA-triggered innate immune responses. The augmentation was, however, restored in KitW/KitW-v mice reconstituted with TRIF-deficient or IRF-3-deficient BMMCs. Although leukotriene B4 and prostaglandin D2 are major lipid mediators released from activated mast cells, no their contribution was shown to the dsRNA-induced augmentation of airway eosinophilia. CONCLUSIONS: We conclude that mast cells contribute to dsRNA-induced augmentation of allergic airway inflammation without requiring direct activation of mast cells with dsRNA or involvement of leukotriene B4 or prostaglandin D2.

Design, synthesis, and evaluation of novel 4-thiazolylimidazoles as inhibitors of transforming growth factor-ß type I receptor kinase.

A novel series of 4-thiazolylimidazoles was synthesized as transforming growth factor-ß (TGF-ß) type I receptor (also known as activin receptor-like kinase 5 or ALK5) inhibitors. These compounds were evaluated for their ALK5 inhibitory activity in an enzyme assay and their TGF-ß-induced Smad2/3 phosphorylation inhibitory activity in a cell-based assay. N-{[5-(1,3-benzothiazol-6-yl)-4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazol-2-yl]methyl}butanamide 20, a potent and selective ALK5 inhibitor, exhibited good enzyme inhibitory activity (IC(50)=8.2nM) as well as inhibitory activity against TGF-ß-induced Smad2/3 phosphorylation at a cellular level (IC(50)=32nM).

Role of mast cells and basophils in IgE responses and in allergic airway hyperresponsiveness.

We established a diphtheria toxin (DT)-based conditional deletion system using Il4 enhancer elements previously shown to be specific for IL-4 production in mast cells (MCs) or basophils (Mas-TRECK and Bas-TRECK mice). DT treatment of Bas-TRECK mice resulted in specific deletion of basophils, whereas both MCs and basophils were deleted in Mas-TRECK mice. DT-treated Mas-TRECK mice had impaired passive cutaneous anaphylaxis, IgE-mediated passive systemic anaphylaxis, and IgE-mediated chronic allergic inflammation, whereas DT-treated Bas-TRECK mice had impaired IgE-mediated chronic allergic inflammation. Using these mice, we also sought to tease out the role of MCs and basophils in airway hyperresponsiveness (AHR). Although MC deletion resulted in a slight increase in basal Ag-specific IgE levels and significant increases in basal IgE levels, we found that this deletion markedly impaired the AHR effector phase and was accompanied by decreased histamine levels. By contrast, basophil deletion had no effect on the AHR effector phase or on IgE production induced by systemic OVA immunization. Our results, using these newly established Mas-TRECK and Bas-TRECK models, demonstrated an indispensable role for MCs as effector cells in AHR.