Estrogen regulated transcription of the non-estrogen-regulated hamster uteroglobin gene in MCF-7 cells.

To study the estrogen regulated transcription of the uteroglobin (UG) gene, the founding member of the secretoglobin family widely expressed in many different mammalian species, we re-created functional estrogen response elements (EREs) in the UG gene promoter from a species where UG expression is not regulated by estrogens: the hamster Mesocricetus auratus (Ma), to ascertain if the lack of functional EREs is the real cause of its estrogen insensitivity. Functional EREs in the hamster promoter, including the consensus ERE (cERE), failed to respond to an appropriate estrogen stimulus compared with its estrogen regulated ortholog from the brown hare Lepus capensis (Lc). As the nucleotide sequence is the only difference between genetic constructs from these two species, we suspected that the UG promoter from the hamster probably contains cis-acting genetic elements that negatively impairs the estrogen-regulated transcription mediated by the functional ERE. Accordingly, we prepared chimeric DNA constructs which eventually allowed to identify a region located 29 base pairs (bp) downstream of the ERE as responsible for the lack of estrogen-responsiveness of the Ma-UG gene in the breast cancer cell line MCF-7. This region contains the sequence ACACCCC which has been identified as the core sequence of the Sp/ Krüppel-like factor (KLF) family of transcription factors. This finding is relevant, not only due to the observation on a novel mechanism that control estrogen-induced transcription, but also because it may encourage further investigation for better defining specific genes with an ERE that do not respond to estrogen signaling in MCF-7 cells, a cell line widely employed as an in vitro model in breast cancer research.

Correlation of Serum Clara Cell Secretory Protein 16, Plasma Fibrinogen and Serum Amyloid A with the Severity of Acute Exacerbated COPD and Their Combination in Prognosis Assessment.

Introduction: Chronic obstructive pulmonary disease (COPD) has tremendous detrimental effects on patients' quality of life, lung function, disease progression and socioeconomic burden. This study aimed to investigate new serum biomarkers for COPD detection. Three recently emerging biomarkers, including Clara cell secretory protein⁃16 (CC16), plasma fibrinogen (FIB) and serum amyloid A (SAA), were investigated for their potential in stratifying the severity of COPD. Methods: A total of 220 patients with AECOPD were recruited. Multivariate logistical regression was used to analyze odds ratios of an array of characteristic of patients, including age, global initiative for chronic obstructive lung disease (GOLD), diabetes mellitus, heart diseases, PaCO2, CC16, FIB, and SAA. Correlations of CC16, FIB and SAA levels to each other, GOLD, and PaCO2 were also measured using Spearman correlation. Receiver operating characteristic (ROC)/curve analysis was used to assess sensitivity and specificity of CC16, FIB, SAA and the combination of the three markers in identifying AECOPD patients with poor prognosis. Results: Our data suggested that age, GOLD, diabetes mellitus, heart diseases, PaCO2, CC16, FIB, and SAA are all significant risk factors for poor prognosis of AECOPD. CC16, FIB and SAA were positively correlated to each other and to GOLD and PaCO2 levels. CC16, FIB and SAA all had a high sensitivity and specificity in identifying patients with a poor prognosis. CC16, FIB and SAA are new markers with potentially high predictive value in AECOPD. Discussion: Our data support further development of these biomarkers to improve clinical management of AECOPD through providing more accurate prognosis of AECOPD patients that enable timely adjustment of treatment plans.

The Impact of CC16 on Pulmonary Epithelial-Driven Host Responses during Mycoplasma pneumoniae Infection in Mouse Tracheal Epithelial Cells.

Club Cell Secretory Protein (CC16) plays many protective roles within the lung; however, the complete biological functions, especially regarding the pulmonary epithelium during infection, remain undefined. We have previously shown that CC16-deficient (CC16-/-) mouse tracheal epithelial cells (MTECs) have enhanced Mp burden compared to CC16-sufficient (WT) MTECs; therefore, in this study, we wanted to further define how the pulmonary epithelium responds to infection in the context of CC16 deficiency. Using mass spectrometry and quantitative proteomics to analyze proteins secreted apically from MTECs grown at an air-liquid interface, we investigated the protective effects that CC16 elicits within the pulmonary epithelium during Mycoplasma pneumoniae (Mp) infection. When challenged with Mp, WT MTECs have an overall reduction in apical protein secretion, whereas CC16-/- MTECs have increased apical protein secretion compared to their unchallenged controls. Following Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) assessment, many of the proteins upregulated from CC16-/- MTECS (unchallenged and during Mp infection) were related to airway remodeling, which were not observed by WT MTECs. These findings suggest that CC16 may be important in providing protection within the pulmonary epithelium during respiratory infection with Mp, which is the major causative agent of community-acquired pneumoniae.

Circulating CC16 and Asthma: A Population-based, Multicohort Study from Early Childhood through Adult Life.

Rationale: Club cell secretory protein (CC16) is an antiinflammatory protein highly expressed in the airways. CC16 deficiency has been associated with lung function deficits, but its role in asthma has not been established conclusively. Objectives: To determine 1) the longitudinal association of circulating CC16 with the presence of active asthma from early childhood through adult life and 2) whether CC16 in early childhood predicts the clinical course of childhood asthma into adult life. Methods: We assessed the association of circulating CC16 and asthma in three population-based birth cohorts: the Tucson Children's Respiratory Study (years 6-36; total participants, 814; total observations, 3,042), the Swedish Barn/Children, Allergy, Milieu, Stockholm, Epidemiological survey (years 8-24; total participants, 2,547; total observations, 3,438), and the UK Manchester Asthma and Allergy Study (years 5-18; total participants, 745; total observations, 1,626). Among 233 children who had asthma at the first survey in any of the cohorts, baseline CC16 was also tested for association with persistence of symptoms. Measurements and Main Results: After adjusting for covariates, CC16 deficits were associated with increased risk for the presence of asthma in all cohorts (meta-analyzed adjusted odds ratio per 1-SD CC16 decrease, 1.20; 95% confidence interval [CI], 1.12-1.28; P < 0.0001). The association was particularly strong for asthma with frequent symptoms (meta-analyzed adjusted relative risk ratio, 1.40; 95% CI, 1.24-1.57; P < 0.0001), was confirmed for both atopic and nonatopic asthma, and was independent of lung function impairment. After adjustment for known predictors of persistent asthma, children with asthma in the lowest CC16 tertile had a nearly fourfold increased risk for having frequent symptoms persisting into adult life compared with children with asthma in the other two CC16 tertiles (meta-analyzed adjusted odds ratio, 3.72; 95% CI, 1.78-7.76; P < 0.0001). Conclusions: Circulating CC16 deficits are associated with the presence of asthma with frequent symptoms from childhood through midadult life and predict the persistence of asthma symptoms into adulthood. These findings support a possible protective role of CC16 in asthma and its potential use for risk stratification.

Role of club cell 16-kDa secretory protein in asthmatic airways.

BACKGROUND: Club cell 16-kDa secretory protein (CC16) is a pneumoprotein and functions as an anti-inflammatory or antioxidant protein. However, altered levels of serum CC16 as well as their effect on airways inflammation have not been fully evaluated. METHODS: We recruited 63 adult asthmatics on maintenance medications and 61 healthy controls (HCs). The asthmatic subjects were divided into two groups according to the result of bronchodilator responsiveness (BDR) test: the present BDR (n = 17) and absent BDR (n = 46) groups. Serum CC16 levels were measured by ELISA. As an in vitro study, the effect of Dermatophagoides pteronyssinus antigen 1 (Der p1) on the production of CC16 in airways epithelial cells (AECs) according to a time-dependent manner was assessed; the effects of CC16 protein on oxidative stress system, airways inflammation and remodelling were tested. RESULTS: Serum CC16 levels showed significantly higher in the asthmatics than in the HCs (p < .001) with a positive correlation with FEV1 % (r = .352, p = .005). The present BDR group had significantly lower levels of serum CC16, FEV1 % and MMEF%, but showed higher level of FeNO than the absent BDR group. Serum CC16 levels (below 496.0 ng/mL) could discriminate the present BDR group from the absent BDR group (area under the curve = 0.74, p = .004). In vitro testing demonstrated that Der p1 exposure significantly induced CC16 release from AECs for 1 h, which was progressively decreased after 6 h and followed by MMP-9 and TIMP-1 production. These findings were associated with oxidant/antioxidant disequilibrium and restored by CC16 treatment (but not dexamethasone). CONCLUSION: Decreased CC16 production contributes to persistent airways inflammation and lung function decline. CC16 may be a potential biomarker for asthmatics with BDR.

Levels of Clara cell secretory protein and surfactant protein A in municipal solid waste management workers in Ibadan, Southwest Nigeria.

Toxic pneumonitis and related respiratory symptoms are common among waste management workers (WMWs). Products of different cellular responses following exposure to toxic components of wastes can lead to the production of a variety of biomolecules. There is a growing recognition of the importance of biomarkers in risk assessment and a strong advocacy for their determination and use as indicators of health and safety. This study assessed the prevalence of respiratory symptoms and the relevance of pulmonary surfactant protein A (SP-A) and Clara cell 16 protein (CC16) as indicators of occupational inhalation exposure to toxic substances and irritants in WMW. A total of 172 subjects consisting of 112 WMWs and 60 Non-WMWs were recruited by purposive sampling. Data on socio-economic and work-related symptoms were collected using structured questionnaire. CC16 and SP-A were determined by ELISA in serum samples. Clinical history reveals a slightly higher prevalence of respiratory symptoms in WMWs relative to control subjects. Increased permeability of the lung-blood barrier, characterized by significant elevation of serum SP-A and serum CC16, was associated with respiratory symptoms in WMWs. Steady increases in SP-A and CC16, respectively, in relation to occupational duration were observed in WMWs relative to control. Receiver operating characteristic curve and multivariate analyses revealed SP-A and CC16 as important lung biomarkers for assessing sub-clinical effects of occupational exposure. Our data suggest SP-A and CC16 may be relevant indicators for assessing occupational inhalation exposure to toxic substances and irritants among WMWs.

Low CC16 mRNA Expression Levels in Bronchial Epithelial Cells Are Associated with Asthma Severity.

Rationale: CC16 is a protein mainly produced by nonciliated bronchial epithelial cells (BECs) that participates in host defense. Reduced CC16 protein concentrations in BAL and serum are associated with asthma susceptibility. Objectives: Few studies have investigated the relationship between CC16 and asthma progression, and none has focused on BECs. In this study, we sought to determine if CC16 mRNA expression levels in BECs are associated with asthma severity. Methods: Association analyses between CC16 mRNA expression levels in BECs (242 asthmatics and 69 control subjects) and asthma-related phenotypes in Severe Asthma Research Program were performed using a generalized linear model. Measurements and Main Results: Low CC16 mRNA expression levels in BECs were significantly associated with asthma susceptibility and asthma severity, high systemic corticosteroids use, high retrospective and prospective asthma exacerbations, and low pulmonary function. Low CC16 mRNA expression levels were significantly associated with high T2 inflammation biomarkers (fractional exhaled nitric oxide and sputum eosinophils). CC16 mRNA expression levels were negatively correlated with expression levels of Th2 genes (IL1RL1, POSTN, SERPINB2, CLCA1, NOS2, and MUC5AC) and positively correlated with expression levels of Th1 and inflammation genes (IL12A and MUC5B). A combination of two nontraditional T2 biomarkers (CC16 and IL-6) revealed four asthma endotypes with different characteristics of T2 inflammation, obesity, and asthma severity. Conclusions: Our findings indicate that low CC16 mRNA expression levels in BECs are associated with asthma susceptibility, severity, and exacerbations, partially through immunomodulation of T2 inflammation. CC16 is a potential nontraditional T2 biomarker for asthma development and progression.

Club cell secretory protein 16 is a potential biomarker for silica-induced pulmonary fibrosis.

This study was conducted to investigate the changes of Club cell protein 16 (CC16) and surfactant protein D (SP-D) levels in serum and bronchoalveolar lavage fluid (BALF) in silicotic rats and to explore their potential as early biomarkers for silicosis. Pulmonary fibrosis models of rats were constructed by exposing them to silica particles. BALF and serum were collected to determine CC16 and SP-D levels using enzyme-linked immunosorbent assay (ELISA) at different times after the exposure. Hydroxyproline (HYP) level in BALF and CC16 level in the lung tissues were also measured immunohistochemistrially. The BALF levels of CC16 decreased from 49.65 to 38.02 ng/mg after the rats were exposed to silica for 3 and 28 days, which were all significantly lower as compared with the controls (P<0.05), where the levels remained barely changed during the same period (61.27 to 56.76 ng/mg). The serum CC16 also showed a similar decrease from 9.8 ng/ml to 8.78 ng/ml during the period, while in the controls, the serum CC16 levels remained constantly between 11.04 and 10.96 ng/ml. The levels of SP-D in the serum of silica-exposed rats did not decrease as compared with the controls and BALF SP-D presented a parabolic curve change with silica exposure. Immunohistochemical examinations showed that the lung Club cells were severely damaged and CC16 expression was obviously decreased after silica exposure. BALF HYP level was higher in silica-exposed rats than in control only when the exposure was at 50 mg/ml. Our work demonstrates that expressions of CC16 and SP-D are pulmonary tissue-specific and CC16 expression is down-regulated as a result of silica-exposure. The significant relationship between CC16 and silica dose indicates that CC16 may be exploited as an early biomarker to assess silica-induced pulmonary fibrosis.

CC16 polymorphisms in asthma, asthma subtypes, and asthma control in adults from the Agricultural Lung Health Study.

BACKGROUND: The club cell secretory protein (CC16) has anti-inflammatory and antioxidant effects and is a potential early biomarker of lung damage. The CC16 single nucleotide polymorphism (SNP) rs3741240 risk allele (A) has been inconsistently linked to asthma; other tagging SNPs in the gene have not been explored. The aim was to determine whether CC16 tagging polymorphisms are associated with adult asthma, asthma subtypes or asthma control in the Agricultural Lung Health Study (ALHS). METHODS: The ALHS is an asthma case-control study nested in the Agricultural Health Study cohort. Asthma cases were individuals with current doctor diagnosed asthma, likely undiagnosed asthma, or asthma-COPD overlap defined by questionnaire. We also examined asthma subtypes and asthma control. Five CC16 tagging SNPs were imputed to 1000 Genomes Integrated phase 1 reference panel. Logistic regression was used to estimate associations between CC16 SNPs and asthma outcomes adjusted for covariates. RESULTS: The sample included 1120 asthma cases and 1926 controls of European ancestry, with a mean age of 63 years. The frequency of the risk genotype (AA) for rs3741240 was 12.5% (n = 382). CC16 rs3741240 was not associated with adult asthma outcomes. A tagging SNP in the CC16 gene, rs12270961 was associated with uncontrolled asthma (n = 208, ORadj= 1.4, 95% CI 1.0, 1.9; p = 0.03). CONCLUSION: This study, the largest study to investigate associations between CC16 tagging SNPs and asthma phenotypes in adults, did not confirm an association of rs3741240 with adult asthma. A tagging SNP in CC16 suggests a potential relationship with asthma control.

Impact of Spirometrically Confirmed Chronic Obstructive Pulmonary Disease on Arterial Stiffness and Surfactant Protein D After Percutaneous Coronary Intervention. The CATEPOC Study.

Background: Several mechanisms have been proposed to explain why chronic obstructive pulmonary disease (COPD) impairs the prognosis of coronary events. We aimed to explore COPD variables related to a worse prognosis in patients undergoing percutaneous coronary intervention (PCI). Methods: Patients with an acute coronary event treated by PCI were prospectively included. One month after discharge, clinical characteristics, comorbidities measured with the Charlson index, and prognostic coronary scales (logistic EuroSCORE; GRACE 2.0) were collected. Post-bronchodilator spirometry, arterial stiffness, and serum inflammatory and myocardial biomarkers were measured. Lung plasmatic biomarkers (Surfactant protein D, desmosine, and Clara cell secretory protein-16) were determined with ELISA. COPD was defined by the fixed ratio (FEV1/FVC <70%). Spirometric values were also analyzed as continuous variables using adjusted and non-adjusted ANCOVA analysis. Finally, we evaluated the presence of a respiratory pattern defined by non-stratified spirometric values and pulmonary biomarkers. Results: A total of 164 patients with a mean age of 65 (±10) years (79% males) were included. COPD was diagnosed in 56 (34%) patients (68% previously undiagnosed). COPD patients had a longer smoking history, higher scores on the EuroSCORE (p < 0.0001) and GRACE 2.0 (p < 0.001) scales, and more comorbidities (p = 0.006). Arterial stiffness determined by pulse wave velocity was increased in COPD patients (7.35 m/s vs 6.60 m/s; p = 0.006). Serum values of high sensitive T troponin (p = 0.007) and surfactant protein D (p = 0.003) were also higher in COPD patients. FEV1% remained significantly associated with arterial stiffness and surfactant protein D in the adjusted ANCOVA analysis. In the cluster exploration, 53% of the patients had a respiratory pattern. Conclusion: COPD affects one-third of patients with an acute coronary event and frequently remains undiagnosed. Several mechanisms, including arterial stiffness and SPD, were increased in COPD patients. Their relationship with the prognosis should be confirmed with longitudinal follow-up of the cohort.

Molecular Mechanisms of RSV and Air Pollution Interaction: A Scoping Review.

RSV is one of the major infectious agents in paediatrics, and its relationship with air pollution is frequently observed. However, the molecular basis of this interaction is sparsely reported. We sought to systematically review the existing body of literature and identify the knowledge gaps to answer the question: which molecular mechanisms are implied in the air pollutants-RSV interaction? Online databases were searched for original studies published before August 2022 focusing on molecular mechanisms of the interaction. The studies were charted and a narrative synthesis was based upon three expected directions of influence: a facilitated viral entry, an altered viral replication, and an inappropriate host reaction. We identified 25 studies published between 1993 and 2020 (without a noticeable increase in the number of studies) that were performed in human (n = 12), animal (n = 10) or mixed (n = 3) models, and analysed mainly cigarette smoke (n = 11), particulate matter (n = 4), nanoparticles (n = 3), and carbon black (n = 2). The data on a damage to the epithelial barrier supports the hypothesis of facilitated viral entry; one study also reported accelerated viral entry upon an RSV conjugation to particulate matter. Air pollution may result in the predominance of necrosis over apoptosis, and, as an effect, an increased viral load was reported. Similarly, air pollution mitigates epithelium function with decreased IFN-γ and Clara cell secretory protein levels and decreased immune response. Immune response might also be diminished due to a decreased viral uptake by alveolar macrophages and a suppressed function of dendritic cells. On the other hand, an exuberant inflammatory response might be triggered by air pollution and provoke airway hyperresponsiveness (AHR), prolonged lung infiltration, and tissue remodeling, including a formation of emphysema. AHR is mediated mostly by increased IFN-γ and RANTES concentrations, while the risk of emphysema was related to the activation of the IL-17 → MCP-1 → MMP-9 → MMP-12 axis. There is a significant lack of evidence on the molecular basics of the RSV-air pollution interaction, which may present a serious problem with regards to future actions against air pollution effects. The major knowledge gaps concern air pollutants (mostly the influence of cigarette smoke was investigated), the mechanisms facilitating an acute infection or a worse disease course (since it might help plan short-term, especially non-pharmacological, interventions), and the mechanisms of an inadequate response to the infection (which may lead to a prolonged course of an acute infection and long-term sequelae). Thus far, the evidence is insufficient regarding the broadness and complexity of the interaction, and future studies should focus on common mechanisms stimulated by various air pollutants and a comparison of influence of the different contaminants at various concentrations.

Brief Research Report: Serum clara cell 16 kDa protein levels are increased in patients hospitalized for severe SARS-CoV-2 or sepsis infection.

Background: Clara cell 16 kDa protein (CC16) is a secretory protein primarily expressed in epithelial cells in the lungs. Previous studies show that CC16 exerts anti-inflammatory and immune-modulatory properties in both acute and chronic pulmonary diseases. However, despite the evidence of CC16's high biomarker potential, evaluation of its role in infectious diseases is yet very limited. Methods: Serum CC16 concentrations were measured by ELISA and assessed in two different types of severe infections. Using a case-control study design, patients treated for either severe SARS-CoV-2 or severe non-pulmonary sepsis infection were compared to age- and sex-matched healthy human subjects. Results: Serum CC16 was significantly increased in both types of infection (SARS-CoV-2: 96.22 ± 129.01 ng/ml vs. healthy controls: 14.05 ± 7.48 ng/ml, p = 0.022; sepsis: 35.37 ± 28.10 ng/ml vs. healthy controls: 15.25 ± 7.51 ng/ml, p = 0.032) but there were no distinct differences between infections with and without pulmonary focus (p = 0.089). Furthermore, CC16 serum levels were positively correlated to disease duration and inversely to the platelet count in severe SARS-CoV-2 infection. Conclusions: Increased CC16 serum levels in both SARS-CoV-2 and sepsis reinforce the high potential as a biomarker for epithelial cell damage and bronchoalveolar-blood barrier leakage in pulmonary as well as non-pulmonary infectious diseases.

Relationships of serum CC16 levels with smoking status and lung function in COPD.

BACKGROUND: The club cell secretory protein (CC16) has anti-inflammatory and antioxidant effects, and low CC16 serum levels have been associated with both risk and progression of COPD, yet the interaction between smoking and CC16 on lung function outcomes remains unknown. METHODS: Utilizing cross-sectional data on United States veterans, CC16 serum concentrations were measured by ELISA and log transformed for analyses. Spirometry was conducted and COPD status was defined by post-bronchodilator FEV1/FVC ratio < 0.7. Smoking measures were self-reported on questionnaire. Multivariable logistic and linear regression were employed to examine associations between CC16 levels and COPD, and lung function with adjustment for covariates. Unadjusted Pearson correlations described relationships between CC16 level and lung function measures, pack-years smoked, and years since smoking cessation. RESULTS: The study population (N = 351) was mostly male, white, with an average age over 60 years. An interaction between CC16 and smoking status on FEV1/FVC ratio was demonstrated among subjects with COPD (N = 245, p = 0.01). There was a positive correlation among former smokers and negative correlation among current or never smokers with COPD. Among former smokers with COPD, CC16 levels were also positively correlated with years since smoking cessation, and inversely related with pack-years smoked. Increasing CC16 levels were associated with lower odds of COPD (ORadj = 0.36, 95% CI 0.22-0.57, Padj < 0.0001). CONCLUSIONS: Smoking status is an important effect modifier of CC16 relationships with lung function. Increasing serum CC16 corresponded to increases in FEV1/FVC ratio in former smokers with COPD versus opposite relationships in current or never smokers. Additional longitudinal studies may be warranted to assess relationship of CC16 with smoking cessation on lung function among subjects with COPD.

Effects of obesity on CC16 and their potential role in overweight/obese asthma.

INTRODUCTION: Club cell secretory protein-16 (CC16) is a major anti-inflammatory protein expressed in the airway; however, the potential role of CC16 on overweight/obese asthma has not been assessed. In this study, we examined whether obesity reduces airway/circulatory CC16 levels using experimental and epidemiological studies. Then, we explored the mediatory role of CC16 in the relationship of overweight/obesity with clinical asthma measures. METHODS: Circulating CC16 levels were assessed by ELISA in three independent human populations, including two groups of healthy and general populations and asthma patients. The percentage of cells expressing club markers in obese vs. non-obese mice and human airways was determined by immunohistochemistry. A causal mediation analysis was conducted to determine whether circulatory CC16 acted as a mediator between overweight/obesity and clinical asthma measures. RESULTS: BMI was significantly and monotonously associated with reduced circulating CC16 levels in all populations. The percentage of CC16-expressing cells was reduced in the small airways of both mice and humans with obesity. Finally, mediation analysis revealed significant contributions of circulatory CC16 in the association between BMI and clinical asthma measures; 21.8% of its total effect in BMI's association with airway hyperresponsiveness of healthy subjects (p = 0.09), 26.4% with asthma severity (p = 0.030), and 23% with the required dose of inhaled corticosteroid (p = 0.042). In logistic regression analysis, 1-SD decrease in serum CC16 levels of asthma patients was associated with 87% increased odds for high dose ICS requirement (p < 0.001). CONCLUSIONS: We demonstrate that airway/circulating CC16, which is inversely associated with BMI, may mediate development and severity in overweight/obese asthma.

Association of inflammation and lung function decline caused by personal PM2.5 exposure: a machine learning approach in time-series data.

Numerous studies focused on the association between lung function impairment and inflammation caused by fine particulate matter (PM2.5), but the causal relationships are difficult to clarify. In the current study, twenty healthy Chinese young adults who participated in 7 days of observation every four seasons were enrolled, and autoregression models (AM) and classification and regression trees (CART) in a machine learning framework were applied to analyze the association among PM2.5 exposure, inflammation, and lung function from a data structure perspective. There were strong cross-correlations between personal dose of PM2.5 (Dw) and lung functions (vital capacity (VC), forced vital capacity (FVC), etc.). These cross-correlation coefficients were associated with inflammatory indicators (uteroglobin (UG), serum amyloid (SAA), and fractional exhaled nitric oxide (FeNO)). CART reported that inflammatory indicators UG and SAA had the predictive ability of the directional association between Dw and FVC at 1-day lag and that high levels of UG and SAA predicted that PM2.5 exposure induced lung function decline. Consistently, lower lung function indicators at a 2-day lag after personal PM2.5 exposure predicted the high value of inflammatory indicator FeNO. Taken together, we applied machine learning algorithms to analyze repeated measurement data, finding that inflammation and lung function decline caused by PM2.5 could affect each other.

Club cell secretory protein and lung function in children with cystic fibrosis.

BACKGROUND: Club cell secretory protein (CC16) exerts anti-inflammatory functions in lung disease. We sought to determine the relation of serum CC16 deficits and genetic variants that control serum CC16 to lung function among children with cystic fibrosis (CF). METHODS: We used longitudinal data from CF children (EPIC Study) with no positive cultures for Pseudomonas aeruginosa prior to enrollment. Circulating levels of CC16 and an inflammatory score (generated from CRP, SAA, calprotectin, G-CSF) were compared between participants with the lowest and highest FEV1 levels in adolescence (LLF and HLF groups, respectively; N = 130-per-group). Single nucleotide variants (SNVs) in the SCGB1A1, EHF-APIP loci were tested for association with circulating CC16 and with decline of FEV1 and FEV1/FVC% predicted levels between ages 7-16 using mixed models. RESULTS: Compared with the HLF group, the LLF group had lower levels of CC16 (geometric means: 8.2 vs 6.5 ng/ml, respectively; p = 0.0002) and higher levels of the normalized inflammatory score (-0.21 vs 0.21, p = 0.0007). Participants in the lowest CC16 and highest inflammation tertile had the highest odds for having LLF (p<0.0001 for comparison with participants in the highest CC16 and lowest inflammation tertile). Among seven SNVs associated with circulating CC16, the top SNV rs3741240 was associated with decline of FEV1/FVC and, marginally, FEV1 (p = 0.003 and 0.025, respectively; N = 611 participants, 20,801 lung function observations). CONCLUSIONS: Serum CC16 deficits are strongly associated with severity of CF lung disease and their effects are additive with systemic inflammation. The rs3741240 A allele is associated with low circulating CC16 and, possibly, accelerated lung function decline in CF.

Urinary CC16, a potential indicator of lung integrity and inflammation, increases in children after short-term exposure to PM2.5/PM10 and is driven by the CC16 38GG genotype.

Particular matter (PM) exposure is a big hazard for public health, especially for children. Serum CC16 is a well-known biomarker of respiratory health. Urinary CC16 (U-CC16) can be a noninvasive alternative, albeit requiring adequate adjustment for renal handling. Moreover, the SNP CC16 G38A influences CC16 levels. This study aimed to monitor the effect of short-term PM exposure on CC16 levels, measured noninvasively in schoolchildren, using an integrative approach. We used a selection of urine and buccal DNA samples from 86 children stored in an existing biobank. Using a multiple reaction monitoring method, we measured U-CC16, as well as RBP4 (retinol binding protein 4) and ß2M (beta-2-microglobulin), required for adjustment. Buccal DNA samples were used for CC16 G38A genotyping. Linear mixed-effects models were used to find relevant associations between U-CC16 and previously obtained data from recent daily PM ≤ 2.5 or 10 µm exposure (PM2.5, PM10) modeled at the child's residence. Our study showed that exposure to low PM at the child's residence (median levels 18.9 µg/m³ (PM2.5) and 23.6 µg/m³ (PM10)) one day before sampling had an effect on the covariates-adjusted U-CC16 levels. This effect was dependent on the CC16 G38A genotype, due to its strong interaction with the association between PM levels and covariates-adjusted U-CC16 (P = 0.024 (PM2.5); P = 0.061 (PM10)). Only children carrying the 38GG genotype showed an increase of covariates-adjusted U-CC16, measured 24h after exposure, with increasing PM2.5 and PM10 (ß = 0.332; 95% CI: 0.110 to 0.554 and ß = 0.372; 95% CI: 0.101 to 0.643, respectively). To the best of our knowledge, this is the first study using an integrative approach to investigate short-term PM exposure of children, using urine to detect early signs of pulmonary damage, and taking into account important determinants such as the genetic background and adequate adjustment of the measured biomarker in urine.

Using intracellular SCGB1A1-sorted, formalin-fixed club cells for successful transcriptomic analysis.

As opposed to surface marker staining, certain cell types can only be recognized by intracellular markers. Intracellular staining for use in cell sorting remains challenging. Fixation and permeabilization steps for intracellular staining and the presence of RNases notably affect preservation of high-quality mRNA. We report the work required for the optimization of a successful protocol for microarray analysis of intracellular target-sorted, formalin-fixed human bronchial club cells. Cells obtained from differentiated air-liquid interface cultures were stained with the most characteristic intracellular markers for club cell (SCGB1A1+) sorting. A benchmarked intracellular staining protocol was carried out before flow cytometry. The primary outcome was the extraction of RNA sufficient quality for microarray analysis as assessed by Bioanalyzer System. Fixation with 4% paraformaldehyde coupled with 0.1% Triton/0.1% saponin permeabilization obtained optimal results for SCGB1A1 staining. Addition of RNase inhibitors throughout the protocol and within the appropriate RNA extraction kit (Formalin-Fixed-Paraffin-Embedded) dramatically improved RNA quality, resulting in samples eligible for microarray analysis. The protocol resulted in successful cell sorting according to specific club cell intracellular marker without using cell surface marker. The protocol also preserved RNA of sufficient quality for subsequent microarray transcriptomic analysis, and we were able to generate transcriptomic signature of club cells.