Respiratory Responses to Ozone Exposure. MOSES (The Multicenter Ozone Study in Older Subjects).

RATIONALE: Acute respiratory effects of low-level ozone exposure are not well defined in older adults. OBJECTIVES: MOSES (The Multicenter Ozone Study in Older Subjects), although primarily focused on acute cardiovascular effects, provided an opportunity to assess respiratory responses to low concentrations of ozone in older healthy adults. METHODS: We performed a randomized crossover, controlled exposure study of 87 healthy adults (59.9 ± 4.5 yr old; 60% female) to 0, 70, and 120 ppb ozone for 3 hours with intermittent exercise. Outcome measures included spirometry, sputum markers of airway inflammation, and plasma club cell protein-16 (CC16), a marker of airway epithelial injury. The effects of ozone exposure on these outcomes were evaluated with mixed-effect linear models. A P value less than 0.01 was chosen a priori to define statistical significance. MEASUREMENTS AND MAIN RESULTS: The mean (95% confidence interval) FEV1 and FVC increased from preexposure values by 2.7% (2.0-3.4) and 2.1% (1.3-2.9), respectively, 15 minutes after exposure to filtered air (0 ppb). Exposure to ozone reduced these increases in a concentration-dependent manner. After 120-ppb exposure, FEV1 and FVC decreased by 1.7% (1.1-2.3) and 0.8% (0.3-1.3), respectively. A similar concentration-dependent pattern was still discernible 22 hours after exposure. At 4 hours after exposure, plasma CC16 increased from preexposure levels in an ozone concentration-dependent manner. Sputum neutrophils obtained 22 hours after exposure showed a marginally significant increase in a concentration-dependent manner (P = 0.012), but proinflammatory cytokines (IL-6, IL-8, and tumor necrosis factor-α) were not significantly affected. CONCLUSIONS: Exposure to ozone at near ambient levels induced lung function effects, airway injury, and airway inflammation in older healthy adults. Clinical trial registered with www.clinicaltrials.gov (NCT01487005).

Temporal structure/function variation in cultured differentiated human nasal epithelium associated with acute single exposure to tobacco smoke or E-cigarette vapor.

OBJECTIVE: Mucociliary clearance sustains a baseline functionality and an "on demand" capability to upregulate clearance upon irritant exposure involving mucus hypersecretion and accelerated ciliary beat frequency (CBF) modulated by nitric oxide (NO). This study characterized these elements as well as cellular and exogenous NO concentrations subsequent to a single exposure to tobacco smoke (TS) or e-cigarette vapor (EV) on cultured human airway epithelium. MATERIALS AND METHODS: Air-liquid interface (ALI) airway epithelial cultures per nonsmoking human subjects were subjected to single TS or EV exposures. Measures of ciliary function and secretion were performed and cellular and exogenous NO concentrations under control and experimental conditions were assessed. RESULTS: Both TS and EV exposures resulted similar patterns of decline in CBF within 1 min of the completion of exposure followed by a gradual return often exceeding baseline within 1 h. Post-exposure examination of exposed cultures suggested morphologic differences in secretory function relative to controls. The relative NO concentrations of TS and EV chamber air were sharply different with EV NO being only slightly elevated relative to cellular NO production. DISCUSSION AND CONCLUSIONS: Epithelial remodeling and mucociliary dysfunction have been clearly associated with TS exposure. However, information contrasting epithelial structure/function following a single acute TS or EV exposure is limited. This study demonstrates a similar pattern of epithelial response to acute TS or EV exposure. Inasmuch as NO may contribute to an inflammatory milieu and generation of toxic metabolites, it is plausible that recurrent exposures over time may be contributory to chronic pathologies.

Exome sequencing identifies mutations in CCDC114 as a cause of primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal-recessive disorder, characterized by oto-sino-pulmonary disease and situs abnormalities. PCD-causing mutations have been identified in 14 genes, but they collectively account for only ~60% of all PCD. To identify mutations that cause PCD, we performed exome sequencing on six unrelated probands with ciliary outer dynein arm (ODA) defects. Mutations in CCDC114, an ortholog of the Chlamydomonas reinhardtii motility gene DCC2, were identified in a family with two affected siblings. Sanger sequencing of 67 additional individuals with PCD with ODA defects from 58 families revealed CCDC114 mutations in 4 individuals in 3 families. All 6 individuals with CCDC114 mutations had characteristic oto-sino-pulmonary disease, but none had situs abnormalities. In the remaining 5 individuals with PCD who underwent exome sequencing, we identified mutations in two genes (DNAI2, DNAH5) known to cause PCD, including an Ashkenazi Jewish founder mutation in DNAI2. These results revealed that mutations in CCDC114 are a cause of ciliary dysmotility and PCD and further demonstrate the utility of exome sequencing to identify genetic causes in heterogeneous recessive disorders.

Mutations in SPAG1 cause primary ciliary dyskinesia associated with defective outer and inner dynein arms.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal-recessive disorder, characterized by oto-sino-pulmonary disease and situs abnormalities. PCD-causing mutations have been identified in 20 genes, but collectively they account for only ∼65% of all PCDs. To identify mutations in additional genes that cause PCD, we performed exome sequencing on three unrelated probands with ciliary outer and inner dynein arm (ODA+IDA) defects. Mutations in SPAG1 were identified in one family with three affected siblings. Further screening of SPAG1 in 98 unrelated affected individuals (62 with ODA+IDA defects, 35 with ODA defects, 1 without available ciliary ultrastructure) revealed biallelic loss-of-function mutations in 11 additional individuals (including one sib-pair). All 14 affected individuals with SPAG1 mutations had a characteristic PCD phenotype, including 8 with situs abnormalities. Additionally, all individuals with mutations who had defined ciliary ultrastructure had ODA+IDA defects. SPAG1 was present in human airway epithelial cell lysates but was not present in isolated axonemes, and immunofluorescence staining showed an absence of ODA and IDA proteins in cilia from an affected individual, thus indicating that SPAG1 probably plays a role in the cytoplasmic assembly and/or trafficking of the axonemal dynein arms. Zebrafish morpholino studies of spag1 produced cilia-related phenotypes previously reported for PCD-causing mutations in genes encoding cytoplasmic proteins. Together, these results demonstrate that mutations in SPAG1 cause PCD with ciliary ODA+IDA defects and that exome sequencing is useful to identify genetic causes of heterogeneous recessive disorders.

Clinical features of childhood primary ciliary dyskinesia by genotype and ultrastructural phenotype.

RATIONALE: The relationship between clinical phenotype of childhood primary ciliary dyskinesia (PCD) and ultrastructural defects and genotype is poorly defined. OBJECTIVES: To delineate clinical features of childhood PCD and their associations with ultrastructural defects and genotype. METHODS: A total of 118 participants younger than 19 years old with PCD were evaluated prospectively at six centers in North America using standardized procedures for diagnostic testing, spirometry, chest computed tomography, respiratory cultures, and clinical phenotyping. MEASUREMENTS AND MAIN RESULTS: Clinical features included neonatal respiratory distress (82%), chronic cough (99%), and chronic nasal congestion (97%). There were no differences in clinical features or respiratory pathogens in subjects with outer dynein arm (ODA) defects (ODA alone; n = 54) and ODA plus inner dynein arm (IDA) defects (ODA + IDA; n = 18) versus subjects with IDA and central apparatus defects with microtubular disorganization (IDA/CA/MTD; n = 40). Median FEV1 was worse in the IDA/CA/MTD group (72% predicted) versus the combined ODA groups (92% predicted; P = 0.003). Median body mass index was lower in the IDA/CA/MTD group (46th percentile) versus the ODA groups (70th percentile; P = 0.003). For all 118 subjects, median number of lobes with bronchiectasis was three and alveolar consolidation was two. However, the 5- to 11-year-old IDA/CA/MTD group had more lobes of bronchiectasis (median, 5; P = 0.0008) and consolidation (median, 3; P = 0.0001) compared with the ODA groups (median, 3 and 2, respectively). Similar findings were observed when limited to participants with biallelic mutations. CONCLUSIONS: Lung disease was heterogeneous across all ultrastructural and genotype groups, but worse in those with IDA/CA/MTD ultrastructural defects, most of whom had biallelic mutations in CCDC39 or CCDC40.

Mutations in RSPH1 cause primary ciliary dyskinesia with a unique clinical and ciliary phenotype.

RATIONALE: Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder of motile cilia, but the genetic cause is not defined for all patients with PCD. OBJECTIVES: To identify disease-causing mutations in novel genes, we performed exome sequencing, follow-up characterization, mutation scanning, and genotype-phenotype studies in patients with PCD. METHODS: Whole-exome sequencing was performed using NimbleGen capture and Illumina HiSeq sequencing. Sanger-based sequencing was used for mutation scanning, validation, and segregation analysis. MEASUREMENTS AND MAIN RESULTS: We performed exome sequencing on an affected sib-pair with normal ultrastructure in more than 85% of cilia. A homozygous splice-site mutation was detected in RSPH1 in both siblings; parents were carriers. Screening RSPH1 in 413 unrelated probands, including 325 with PCD and 88 with idiopathic bronchiectasis, revealed biallelic loss-of-function mutations in nine additional probands. Five affected siblings of probands in RSPH1 families harbored the familial mutations. The 16 individuals with RSPH1 mutations had some features of PCD; however, nasal nitric oxide levels were higher than in patients with PCD with other gene mutations (98.3 vs. 20.7 nl/min; P < 0.0003). Additionally, individuals with RSPH1 mutations had a lower prevalence (8 of 16) of neonatal respiratory distress, and later onset of daily wet cough than typical for PCD, and better lung function (FEV1), compared with 75 age- and sex-matched PCD cases (73.0 vs. 61.8, FEV1 % predicted; P = 0.043). Cilia from individuals with RSPH1 mutations had normal beat frequency (6.1 ± Hz at 25°C), but an abnormal, circular beat pattern. CONCLUSIONS: The milder clinical disease and higher nasal nitric oxide in individuals with biallelic mutations in RSPH1 provides evidence of a unique genotype-phenotype relationship in PCD, and suggests that mutations in RSPH1 may be associated with residual ciliary function.

Founder mutation in RSPH4A identified in patients of Hispanic descent with primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a rare, autosomal recessive, genetically heterogeneous disorder characterized by ciliary dysfunction resulting in chronic oto-sino-pulmonary disease, respiratory distress in term neonates, laterality (situs) defects, and bronchiectasis. Diagnosis has traditionally relied on ciliary ultrastructural abnormalities seen by electron microscopy. Mutations in radial spoke head proteins occur in PCD patients with central apparatus defects. Advances in genetic testing have been crucial in addressing the diagnostic challenge. Here, we describe a novel splice-site mutation (c.921+3_6delAAGT) in RSPH4A, which leads to a premature translation termination signal in nine subjects with PCD (seven families). Loss-of-function was confirmed with quantitative ciliary ultrastructural analysis, measurement of ciliary beat frequency and waveform, and transcript analysis. All nine individuals carrying c.921+3_6delAAGT splice-site mutation in RSPH4A were Hispanic with ancestry tracing to Puerto Rico. This mutation is a founder mutation and a common cause of PCD without situs abnormalities in patients of Puerto Rican descent.

Primary ciliary dyskinesia-causing mutations in Amish and Mennonite communities.

OBJECTIVE: To determine whether individuals with primary ciliary dyskinesia (PCD) from unrelated Amish and Mennonite families harbor a single and unique founder mutation. STUDY DESIGN: Subjects from Amish and Mennonite communities in several states were enrolled in the study. All subjects were clinically characterized, and nasal nitric oxide levels were measured. Nasal epithelial scrapings were collected from several subjects for ciliary ultrastructural analyses. DNA was isolated from patients with PCD and their unaffected first- and second-degree relatives. Genome-wide homozygosity mapping, linkage analyses, targeted mutation analyses, and exome sequencing were performed. RESULTS: All subjects from Old-Order Amish communities from Pennsylvania were homozygous for a nonsense mutant DNAH5 allele, c.4348C>T (p.Q1450X). Two affected siblings from an unrelated Mennonite family in Arkansas were homozygous for the same nonsense DNAH5 mutation. Children with PCD from an Amish family from Wisconsin had biallelic DNAH5 mutations, c.4348C>T (p.Q1450X) and c.10815delT (p.P3606HfsX23), and mutations in other genes associated with PCD were also identified in this community. CONCLUSION: The Amish and Mennonite subjects from geographically dispersed and socially isolated communities had the same founder DNAH5 mutation, owing to the common heritage of these populations. However, disease-causing mutations in other PCD-associated genes were also found in affected individuals in these communities, illustrating the genetic heterogeneity in this consanguineous population.

Pulmonary responses in current smokers and ex-smokers following a two hour exposure at rest to clean air and fine ambient air particles.

BACKGROUND: Increased susceptibility of smokers to ambient PM may potentially promote development of COPD and accelerate already present disease. OBJECTIVES: To characterize the acute and subacute lung function response and inflammatory effects of controlled chamber exposure to concentrated ambient fine particles (CAFP) with MMAD ≤ 2.5 microns in ex-smokers and lifetime smokers. METHODS: Eleven subjects, aged 35-74 years, came to the laboratory 5 times; a training day and two exposure days separated by at least 3 weeks, each with a post-exposure visit 22 h later. Double-blind and counterbalanced exposures to "clean air" (mean 1.5 ± 0.6 µg/m3) or CAFP (mean 108.7 ± 24.8 µg/m3 ) lasted 2 h with subjects at rest. RESULTS: At 3 h post-exposure subjects' DTPA clearance half-time significantly increased by 6.3 min per 100 µg/m3 of CAFP relative to "clean air". At 22 h post-exposure they showed significant reduction of 4.3% per 100 µg/m3 in FEV1 and a significant DLCO decrease by 11.1% per 100 µg/m3 of CAFP relative to "clean air". At both 3 h and 22 h the HDL cholesterol level significantly decreased by 4.5% and 4.1%, respectively. Other blood chemistries and markers of lung injury, inflammation and procoagulant activity were within the normal range of values at any condition. CONCLUSIONS: The results suggest that an acute 2 h resting exposure of smokers and ex-smokers to fine ambient particulate matter may transiently affect pulmonary function (spirometry and DLCO) and increase DTPA clearance half-time. Except for a post exposure decrease in HDL no other markers of pulmonary inflammation, prothrombotic activity and lung injury were significantly affected under the conditions of exposure.

Mutations of DNAH11 in patients with primary ciliary dyskinesia with normal ciliary ultrastructure.

RATIONALE: Primary ciliary dyskinesia (PCD) is an autosomal recessive, genetically heterogeneous disorder characterised by oto-sino-pulmonary disease and situs abnormalities (Kartagener syndrome) due to abnormal structure and/or function of cilia. Most patients currently recognised to have PCD have ultrastructural defects of cilia; however, some patients have clinical manifestations of PCD and low levels of nasal nitric oxide, but normal ultrastructure, including a few patients with biallelic mutations in dynein axonemal heavy chain 11 (DNAH11). OBJECTIVES: To test further for mutant DNAH11 as a cause of PCD, DNAH11 was sequenced in patients with a PCD clinical phenotype, but no known genetic aetiology. METHODS: 82 exons and intron/exon junctions in DNAH11 were sequenced in 163 unrelated patients with a clinical phenotype of PCD, including those with normal ciliary ultrastructure (n=58), defects in outer and/or inner dynein arms (n=76), radial spoke/central pair defects (n=6), and 23 without definitive ultrastructural results, but who had situs inversus (n=17), or bronchiectasis and/or low nasal nitric oxide (n=6). Additionally, DNAH11 was sequenced in 13 subjects with isolated situs abnormalities to see if mutant DNAH11 could cause situs defects without respiratory disease. RESULTS: Of the 58 unrelated patients with PCD with normal ultrastructure, 13 (22%) had two (biallelic) mutations in DNAH11; and two patients without ultrastructural analysis had biallelic mutations. All mutations were novel and private. None of the patients with dynein arm or radial spoke/central pair defects, or isolated situs abnormalities, had mutations in DNAH11. Of the 35 identified mutant alleles, 24 (69%) were nonsense, insertion/deletion or loss-of-function splice-site mutations. CONCLUSIONS: Mutations in DNAH11 are a common cause of PCD in patients without ciliary ultrastructural defects; thus, genetic analysis can be used to ascertain the diagnosis of PCD in this challenging group of patients.

Interferon γ stimulates accumulation of gas phase nitric oxide in differentiated cultures of normal and cystic fibrosis airway epithelial cells.

BACKGROUND: Exhaled nitric oxide (NO) levels have been reported to be lower in patients with cystic fibrosis (CF) than in controls; however the mechanism(s) responsible and the effect on pathogenesis are unclear. The objective of these studies was to determine if the low levels of gas phase NO (gNO) could be reproduced in well-differentiated air-liquid interface (ALI) cultures of normal and CF cells. METHODS: Human bronchial epithelial (HBE) cells from CF and control tissues were cultured under ALI conditions that promote differentiation into a mostly ciliated, pseudostratified epithelium similar to that of the in vivo airway. Cultures were incubated in gas tight chambers and the concentration of gNO was measured using a Sievers nitric oxide analyzer. RESULTS: In CF and control cultures the level of accumulated gNO under baseline conditions was low (<20 ppb). Treatment with interferon gamma (IFNγ) induced iNOS expression and increased gNO significantly in differentiated cultures, while having no significant effect on undifferentiated cultures. Submersion of the apical surface with fluid drastically reduced the level of gNO. Importantly, the average level of gNO measured after IFNγ treatment of control cells (576 ppb) was threefold greater than that from CF cells (192 ppb). CONCLUSIONS: The results demonstrate that the lower level of exhaled NO observed in CF patients is reproduced in well-differentiated primary cultures of HBE cells treated with IFNγ, supporting the hypothesis that the regulation of NO production is altered in CF. The results also demonstrate that IFNγ treatment of differentiated cells results in higher levels of gNO than treatment of undifferentiated cells, and that a layer of fluid on the apical surface drastically reduces the amount of gNO, possibly by limiting the availability of oxygen.

Lung function and inflammatory responses in healthy young adults exposed to 0.06 ppm ozone for 6.6 hours.

RATIONALE: Exposure to ozone causes a decrease in spirometric lung function and an increase in airway inflammation in healthy young adults at concentrations as low as 0.08 ppm, close to the National Ambient Air Quality Standard for ground level ozone. OBJECTIVES: To test whether airway effects occur below the current ozone standard and if they are more pronounced in potentially susceptible individuals, such as those deficient in the antioxidant gene glutathione S-transferase mu 1 (GSTM1). METHODS: Pulmonary function and subjective symptoms were measured in 59 healthy young adults (19-35 yr) immediately before and after exposure to 0.0 (clean air, CA) and 0.06 ppm ozone for 6.6 hours in a chamber while undergoing intermittent moderate exercise. The polymorphonuclear neutrophil (PMN) influx was measured in 24 subjects 16 to 18 hours postexposure. MEASUREMENTS AND MAIN RESULTS: Subjects experienced a significantly greater (P = 0.008) change in FEV(1) (± SE) immediately after exposure to 0.06 ppm ozone compared with CA (-1.71 ± 0.50% vs. -0.002 ± 0.46%). The decrement in FVC was also greater (P = 0.02) after ozone versus CA (-2.32 ± 0.41% vs. -1.13 ± 0.34%). Similarly, changes in %PMN were greater after ozone (54.0 ± 4.6%) than CA (38.3 ± 3.7%) exposure (P < 0.001). Symptom scores were not different between ozone versus CA. There were no significant differences in changes in FEV(1), FVC, and %PMN between subjects with GSTM1-positive and GSTM1-null genotypes. CONCLUSIONS: Exposure of healthy young adults to 0.06 ppm ozone for 6.6 hours causes a significant decrement of FEV(1) and an increase in neutrophilic inflammation in the airways. GSTM1 genotype alone appears to have no significant role in modifying the effects.

Nasal nitric oxide and lifestyle exposure to tobacco smoke.

OBJECTIVES: Nitric oxide (NO) is a reactive gas generated by inflammatory cells and mucosal epithelial cells of the nose and paranasal sinuses and is an important mediator in nonspecific host defense against infectious agents. However, NO also mediates physiologic events such as vasodilation, mucus hypersecretion, and mucosal disruption that are associated with inflammatory conditions, and it is a regulator of ciliary beat frequency. In the present study, we hypothesized that lifestyle exposure to tobacco smoke, whether through active smoking or by inadvertent exposure to secondhand tobacco smoke, would result in higher detectable levels of nasal NO (nNO) than are found in well-documented nonsmokers. METHODS: Nasal NO measurements were obtained concomitant with assays of urine cotinine from well-documented nonsmokers, active smokers, and individuals exposed by lifestyle to secondhand smoke. These parameters were statistically analyzed to determine whether increasing levels of tobacco smoke exposure yield higher concentrations of nNO. RESULTS: Our results and subsequent statistical analyses imply that active smokers who exhibit high urine cotinine levels exhibit significant increases in nNO levels in comparison to both nonsmokers and nonsmokers exposed to secondhand smoke. CONCLUSIONS: There is an increased level of nNO associated with tobacco smoke exposure that may contribute to the inflammatory processes characteristic of disease pathogenesis in smokers.

Primary ciliary dyskinesia in Amish communities.

Primary ciliary dyskinesia is an autosomal recessive multigenic disease that results in impaired mucociliary clearance. We have diagnosed 9 subjects with primary ciliary dyskinesia from geographically dispersed Amish communities, on the basis of clinical characteristics and ciliary ultrastructural defects. Despite consanguinity, affected individuals had evidence of genetic heterogeneity.

Concentrated ambient ultrafine particle exposure induces cardiac changes in young healthy volunteers.

RATIONALE: Exposure to ambient ultrafine particles has been associated with cardiopulmonary toxicity and mortality. Adverse effects specifically linked to ultrafine particles include loss of sympathovagal balance and altered hemostasis. OBJECTIVES: To characterize the effects of acute exposure to ambient ultrafine particles in young healthy humans. METHODS: Nineteen healthy nonsmoking male and female subjects between the ages of 18 and 35 were exposed to filtered air or to an atmosphere in which captured ultrafine (<0.16 microm) particles were concentrated by a factor of up to 20-fold over ambient levels with the use of particle concentrators fitted with size-selective outlets (ultrafine concentrated ambient particles [UFCAPs]). Subjects underwent bronchoalveolar lavage 18 hours after each exposure. Cardiovascular endpoints measured included pulmonary function, clinical chemistry, and hematological parameters, as well as heart rate variability and repolarization indices. MEASUREMENTS AND MAIN RESULTS: Exposure to UFCAPs was statistically associated with an increase in frequency domain markers of heart rate variability, specifically indicative of elevated vagal input to the heart. Consistent with this finding were increases in the variance associated with the duration of the QT interval. In addition, UFCAP exposure resulted in a significant increase in blood levels of the fibrin degradation product D-dimer as well as a modest elevation in the inflammatory chemokine IL-8 recovered in the lavage fluid. CONCLUSIONS: These findings show mild inflammatory and prothrombic responses and are suggestive of alterations in cardiac repolarization induced by UFCAP inhalation.

An alternative form and level of the human health ozone standard.

Controlled human laboratory studies have shown that there is a disproportionately greater pulmonary function response from higher hourly average ozone (O3) concentrations than from lower hourly average values and thus, a nonlinear relationship exists between O3 dose and pulmonary function (FEV1) response. The nonlinear dose-response relationship affects the efficacy of the current 8-h O3 standard to describe adequately the observed spirometric response to typical diurnal O3 exposure patterns. We have reanalyzed data from five controlled human response to O3 health laboratory experiments as reported by Hazucha et al. (1992), Adams (2003, 2006a, 2006b), and Schelegle et al. (2009). These investigators exposed subjects to multi-hour variable/stepwise O3 concentration profiles that mimicked typical diurnal patterns of ambient O3 concentrations. Our findings indicate a common response pattern across most of the studies that provides valuable information for the development of a lung function (FEV1)-based alternate form for the O3 standard. Based on our reanalysis of the realistic exposure profiles used in these experiments, we suggest that an alternative form of the human health standard, similar to the proposed secondary (i.e., vegetation) standard form, be considered. The suggested form is an adjusted 5-h cumulative concentration weighted O3 exposure index, which addresses both the delay associated with the onset of response (FEV1 decrement) and the nonlinearity of response (i.e., the greater effect of higher concentrations over the mid- and low-range values) on an hourly basis.

Low-level ozone exposure induces airways inflammation and modifies cell surface phenotypes in healthy humans.

The effects of low-level ozone exposure (0.08 ppm) on pulmonary function in healthy young adults are well known; however, much less is known about the inflammatory and immunomodulatory effects of low-level ozone in the airways. Techniques such as induced sputum and flow cytometry make it possible to examine airways inflammatory responses and changes in immune cell surface phenotypes following low-level ozone exposure. The purpose of this study was to determine if exposure to 0.08 parts per million ozone for 6.6 h induces inflammation and modifies immune cell surface phenotypes in the airways of healthy adult subjects. Fifteen normal volunteers underwent an established 0.08 part per million ozone exposure protocol to characterize the effect of ozone on airways inflammation and immune cell surface phenotypes. Induced sputum and flow cytometry were used to assess these endpoints 24 h before and 18 h after exposure. The results showed that exposure to 0.08 ppm ozone for 6.6 h induced increased airway neutrophils, monocytes, and dendritic cells and modified the expression of CD14, HLA-DR, CD80, and CD86 on monocytes 18 h following exposure. Exposure to 0.08 parts per million ozone is associated with increased airways inflammation and promotion of antigen-presenting cell phenotypes 18 hours following exposure. These findings need to be replicated in a similar experiment that includes a control air exposure.

Nasal Nitric Oxide Measurement in Primary Ciliary Dyskinesia. A Technical Paper on Standardized Testing Protocols.

Nasal nitric oxide concentrations are extremely low in primary ciliary dyskinesia (PCD), and measurement of this nasal gas is recommended as a PCD diagnostic test in cooperative patients aged 5 years and older. However, nasal nitric oxide measurements must be performed with chemiluminescence analyzers using a standardized protocol to ensure proper results, because nasal nitric oxide values can be influenced by various internal and external factors. Repeat nasal nitric oxide testing on separate visits is required to ensure that low diagnostic values are persistent and consistent with PCD. This technical paper presents the standard operating procedures for nasal nitric oxide measurement used by the PCD Foundation Clinical and Research Centers Network at various specialty centers across North America. Adherence to this document ensures reliable nasal nitric oxide testing and high diagnostic accuracy when employed in a population with appropriate clinical phenotypes for PCD.

Do Ambient Ozone or Other Pollutants Modify Effects of Controlled Ozone Exposure on Pulmonary Function?

Rationale: In a previous trial (MOSES [Multicenter Ozone Study of oldEr Subjects]), 3 hours of controlled ozone (O3) exposure caused concentration-related reductions in lung function with evidence of airway inflammation and injury, but without convincing evidence of effects on cardiovascular function. However, the subjects' exposures to indoor and outdoor air pollution in the hours and days before each controlled O3 exposure may have modified biomarker responses to the controlled O3 exposures.Objectives: We sought to determine whether personal measures of nitrogen dioxide (NO2) and O3, or ambient concentrations of O3, particulate matter ≤2.5 µm in aerodynamic diameter, NO2, carbon monoxide (CO), and sulfur dioxide (SO2) in the 72 and 96 hours before the exposure visit modified biomarker responses to controlled O3 exposure.Methods: MOSES subjects were exposed for 3 hours in random order to clean air containing 0 ppb O3, 70 ppb O3, or 120 ppm O3, alternating 15 minutes of moderate exercise with 15 minutes of rest. Cardiovascular and pulmonary endpoints (biomarkers of autonomic function, repolarization, ST segment change, arrhythmia, prothrombotic vascular status, systemic inflammation, vascular function, pulmonary function, oxidative stress, and lung injury) were measured on the day before, the day of, and up to 22 hours after each exposure. We evaluated whether ambient pollutant concentrations in the 96 hours before the pre-exposure visit modified pre- to post-exposure lung function biomarker responses to the controlled O3 exposures, using tertiles of passive personal exposure samplers (PES) of O3 and NO2, ambient air pollutant concentrations, and mixed effects linear regression. We also similarly explored the effect modification of controlled O3 effects on biomarkers of other MOSES outcome groups in the same way. Although we used P < 0.01 to define statistical significance, we did not formally correct for multiple comparisons.Results: The effects of MOSES controlled O3 exposures on forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) were modified by ambient NO2 and CO, and PES NO2. Reductions in FEV1 and FVC were observed only when these concentrations were in the "medium" or "high" tertile in the 72 hours before the pre-exposure visit. There was no such modification of the effect of controlled O3 exposure on any other cardiopulmonary outcome group.Conclusions: Reductions in markers of lung function, but not other pathways, by the MOSES controlled O3 exposure were modified by ambient NO2 and CO, and PES NO2, and these reductions were observed only when these pollutant concentrations were elevated in the hours and days before the pre-exposure visit.Clinical trial registered with ClinicalTrials.gov (NCT01487005).

Increased nasal epithelial ciliary beat frequency associated with lifestyle tobacco smoke exposure.

The ciliated epithelium of the respiratory airways is one of the first vital systemic surfaces in contact with the ambient air. Ex vivo nasal epithelial ciliary beat frequency (CBF) at room temperature is on the order of 7-8 Hz but may be stimulated by irritant exposure. The upregulation of CBF in response to acute irritant exposure is generally considered to be a transient event with eventual return to baseline. However, studies of CBF dynamics in response to typical lifestyle exposures are limited. This study assessed nasal epithelial CBF among human subjects as a function of quantifiable lifestyle tobacco smoke exposure. Nasal epithelial biopsies were obtained from human subjects with well documented histories of tobacco smoke exposure. CBF was determined using a digital photometric technique and concurrent assays of nasal nitric oxide and urine cotinine and creatinine were performed. Mean CBF among active smokers and non-smokers exposed to environmental tobacco smoke (ETS) was elevated over non-smokers. Although there were dramatic differences in relative levels of tobacco smoke exposure, CBF values among tobacco smoke-exposed groups were comparable. Parallel in vitro studies of cultured nasal epithelium exposed to cigarette smoke condensate further supported these observations. These studies suggest that persistent elevation in nasal epithelial CBF is an early, subtle, physiologic effect associated with lifestyle tobacco smoke exposure. The molecular mechanisms that upregulate CBF may also create a cell molecular milieu capable of provoking the eventual emergence of more overt adverse health effects and the pathogenesis of chronic airway disease.