Airway inflammation underlies cystic fibrosis (CF) pulmonary exacerbations. In a prospective multicenter study of randomly selected, clinically stable adolescents and adults, we assessed relationships between 24 inflammation-associated molecules and the future occurrence of CF pulmonary exacerbation using proportional hazards models. We explored relationships for potential confounding or mediation by clinical factors and assessed sensitivities to treatments including CF transmembrane regulator (CFTR) protein synthesis modulators. Results from 114 participants, including seven on ivacaftor or lumacaftor-ivacaftor, representative of the US CF population during the study period, identified 10 biomarkers associated with future exacerbations mediated by percent predicted forced expiratory volume in 1 s. The findings were not sensitive to anti-inflammatory, antibiotic, and CFTR modulator treatments. The analyses suggest that combination treatments addressing RAGE-axis inflammation, protease-mediated injury, and oxidative stress might prevent pulmonary exacerbations. Our work may apply to other airway inflammatory diseases such as bronchiectasis and the acute respiratory distress syndrome.

Introduction: Patent foramen ovale (PFO) affects about 25% of the population. We studied outcomes in cystic fi brosis (CF). Methods: We conducted a case-control study of patients with CF (PwCF) and age- and sex-matched controls who underwent agitated saline contrast (bubble) echocardiography, 1998-2020. We assessed PFO impacts using linear, logistic, quasi-Poisson, and Cox proportional hazards models. Result: Fifty-nine of 64 PwCF and 88 of 93 controls underwent bubble studies to investigate unexplained hypoxemia or dyspnea. PwCF had higher mean pulmonary artery pressure (PAP: 6.9 mm Hg, 95% confidence interval [CI] = 2.35-11.4), reduced tricuspid annular plane systolic excursion (TAPSE: - 3.78 mm, CI = - 5.64 to - 1.93) and similar right ventricular diastolic sizes. Without hypoxemia, the PFO incidence was similar between PwCF and controls; with hypoxemia, PFO was more common in CF (odds ratio = 5.00, CI = 1.32-19.0). In CF, oxygen supplementation occurred at a percent-predicted forced expiratory volume in 1 s (FEV1%), 22.5 points higher with PFO. Adjusted for FEV1%, PFO was associated with 0.59 more prior- year pulmonary exacerbations (CI = 0.20-0.98) and shorter time to next exacerbation (hazard ratio = 1.86, CI = 1.06-3.26). Associations between PFO and hypoxemia or exacerbations were insensitive to PAP, TAPSE, and CF transmembrane regulator protein modulator treatments. PFO was not associated with CF time to death or lung transplantation (median 1.87 years) adjusted for age, sex, FEV1%, and prior-year exacerbation counts. Conclusion: PFO in CF is associated with hypoxemia at higher FEV1% and more pulmonary exacerbations but not survival.

Background Airway inflammation promotes bronchiectasis and lung injury in cystic fibrosis (CF). Amplification of inflammation underlies pulmonary exacerbations of disease. We asked whether sputum inflammatory biomarkers provide explanatory information on pulmonary exacerbations. Patients and Methods We collected sputum from randomly chosen stable adolescents and adults and prospectively observed time to next exacerbation, our primary outcome. We evaluated relationships between potential biomarkers of inflammation, clinical characteristics and outcomes and assessed clinical variables as potential confounders or mediators of explanatory models. We assessed associations between the markers and time to next exacerbation using proportional hazard models adjusting for confounders. Results We enrolled 114 patients, collected data on clinical variables [December 8, 2014 to January 16, 2016; 46% male, mean age 28 years (SD 12), mean percent predicted forced expiratory volume in 1 s (FEV1%) 70 (SD 22)] and measured 24 inflammatory markers. Half of the inflammatory markers were plausibly associated with time to next exacerbation. Age and sex were confounders while we found that FEV1% was a mediator. Three potential biomarkers of RAGE axis inflammation were associated with time to next exacerbation while six potential neutrophil-associated biomarkers indicate associations between protease activity or reactive oxygen species with time to next exacerbation. Conclusion Pulmonary exacerbation biomarkers are part of the RAGE proinflammatory axis or reflect neutrophil activity, specifically implicating protease and oxidative stress injury. Further investigations or development of novel anti-inflammatory agents should consider RAGE axis, protease and oxidant stress antagonists. Tweetable abstract Sputum from 114 randomly chosen people with CF show RAGE axis inflammation, protease and oxidative stress injury are associated with time to next pulmonary exacerbation and may be targets for bench or factorial design interventional studies. (242 characters) ### Competing Interest Statement TGL, JAF, JLJ, YL, KAP and JBV received other support from the CFF (CC132-16AD, LIOU14Y0, LIOU14P0) and the National Heart Lung and Blood Institute (NHLBI) of the National Institutes of Health (NIH) (R01 HL125520) and received support during the current study for performing clinical trials from Abbvie, Calithera Biosciences, Corbus Pharmaceuticals, Gilead Sciences, Laurent Pharmaceuticals, Nivalis Therapeutics, Novartis, Proteostasis, Savara Pharmaceuticals, Translate Bio and Vertex Pharmaceuticals. FRA received additional other support from the NHLBI/NIH (R01 HL125520), the National Science Foundation (EMSW21-RTG) and the Margolis Foundation of Utah. PSB received other support from the CFF (Center and TDC grants) and the NHLBI/NIH (U01 HL114623) and received support for a clinical trial from Alcresta Therapeutics. BAC received other support from the CFF (C112-12, C112-TDC09Y, 10063SUB, 41339154.s132P010379SUB) and received support for clinical trials from Genentech, Novartis and Vertex Pharmaceuticals. CLD received other support from the CFF (C004-11, C004-TDC09Y, DAINES11Y3) and from the Health Resources and Services Administration (T72MC00012). JAF is now an employee of ICON plc, a clinical research organization involved in various trials pertinent to CF; She and ICON had no direct involvement in performance of the study following the change in affiliation. TH received other support from the CFF (PACE, Center Grant) and received support for clinical trials from Celtaxsys and Vertex Pharmaceuticals. JRH received other support from the NHLBI/NIH (HHSN268200900018C) and the Veterans Administration Healthcare System (I01 BX001533). JL received other support from the CFF (C017-11AF). CN received other support from the CFF (C138-12). PR received other support from the CFF (C003-12, C003-TDC09Y). SDS received other support from the CFF (AQUADEK12K1, SAGEL11CS0, GOAL13K2, NICK13A0, SAGEL14K1, NICK15R0) and the NHLBI/NIH (U54 HL096458) and the NCATS/NIH (Colorado CTSA Grant Number UL1 TR002535). JLT-C received other support from the CFF (TDC) and the NHLBI/NIH (HL103801) and received support for clinical trials from Vertex Pharmaceuticals. KNO is funded by the intramural research program of the NHLBI, NIH. ### Clinical Protocols ### Funding Statement This project was supported by the CF Foundation (CFF) (LIOU13A0, LIOU14Y4), the National Center for Advancing Translational Science at the National Institutes of Health (NCATS/NIH 8UL1TR000105 [formerly UL1RR025764]), the Ben B and Iris M Margolis Foundation of Utah and the Claudia Ruth Goodrich Stevens Endowment Fund. Neither the project sponsors nor any sources of other support had direct roles in development and conduct of the study. None of the sponsors of clinical trials mentioned in the competing interests and other support section that follows participated in any way with this trial. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The IRB of the University of Utah gave ethical approval for this work. The Institutional Review Board of St Luke's Health System gave ethical approval for this work. The Western Institutional Review Board for the Las Vegas CF Center gave ethical approval for this work. The Institutional Review Board of National Jewish Health gave ethical approval for this work. The Colorado Multiple Institutional Review Board for Children's Hospital Colorado gave ethical approval for this work. The Institutional Review Board of Phoenix Children's Hospital gave ethical approval for this work. The IRB of the University of Arizona gave ethical approval for this work. The Human Research Review Committee in the Human Research Protections Office of the University of New Mexico gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study will be available upon reasonable request to the authors after peer-reviewed publication.

To examine innate immune responses in early SARS-CoV-2 infection that may change clinical outcomes, we compared nasopharyngeal swab data from 20 virus-positive and 20 virus-negative individuals. Multiple innate immune-related and ACE-2 transcripts increased with infection and were strongly associated with increasing viral load. We found widespread discrepancies between transcription and translation. Interferon proteins were unchanged or decreased in infected samples suggesting virally-induced shut-off of host anti-viral protein responses. However, IP-10 and several interferon-stimulated gene proteins increased with viral load. Older age was associated with modifications of some effects. Our findings may characterize the disrupted immune landscape of early disease.

Background: Biomarkers of inflammation predictive of cystic fibrosis (CF) disease outcomes would increase the power of clinical trials and contribute to better personalization of clinical assessments. A representative patient cohort would improve searching for believable, generalizable, reproducible and accurate biomarkers. Methods: We recruited patients from Mountain West CF Consortium (MWCFC) care centers for prospective observational study of sputum biomarkers of inflammation. After informed consent, centers enrolled randomly selected patients with CF who were clinically stable sputum producers, 12years of age and older, without previous organ transplantation. Results: From December 8, 2014 through January 16, 2016, we enrolled 114 patients (53 male) with CF with continuing data collection. Baseline characteristics included mean age 27 years (SD = 12), 80% predicted forced expiratory volume in 1s (SD = 23%), 1.0 prior year pulmonary exacerbations (SD = 1.2), home elevation 328 m (SD=112) above sea level. Compared with other patients in the US CF Foundation Patient Registry (CFFPR) in 2014, MWCFC patients had similar distribution of sex, age, lung function, weight and rates of exacerbations, diabetes, pancreatic insufficiency, CF-related arthropathy and airway infections including methicillin-sensitive or -resistant Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia complex, fungal and non-tuberculous Mycobacteria infections. They received CF-specific treatments at similar frequencies. Conclusions: Randomly-selected, sputum-producing patients within the MWCFC represent sputum-producing patients in the CFFPR. They have similar characteristics, lung function and frequencies of pulmonary exacerbations, microbial infections and use of CF-specific treatments. These findings will plausibly make future interpretations of quantitative measurements of inflammatory biomarkers generalizable to sputum-producing patients in the CFFPR.

Diesel exhaust particles (DEPs) are major air pollutants that lead to numerous human disorders, especially pulmonary diseases, partly through the induction of oxidative stress. Resveratrol is a polyphenol that ameliorates the production of reactive oxygen species (ROS) and delays aging-related processes. Herein we studied the cytoprotective effect of resveratrol on DEP-exposed human lung cells in a factorial experimental design. This work investigates biophysical features including cellular compositions and biomechanical properties, which were measured at the single-cell level using confocal Raman microspectroscopy (RM) and atomic force microscopy (AFM), respectively. Principal component analysis (PCA), hierarchical cluster analysis (HCA) and partial least square regression (PLS) analysis were applied to analyze Raman spectra with and without resveratrol protection. The health status of individual cells could be effectively predicted using an index derived from characteristic Raman spectral peak (e.g., 1006 cm −1 ) based on PLS model. AFM measurements indicated that cellular adhesion force was greatly reduced, while Young’s modulus was highly elevated in resveratrol treated DEP-exposed cells. Anti-oxidant resveratrol reduced DEP-induced ROS production and suppressed releases of several cytokines and chemokines. These findings suggest resveratrol may enhance resistance of human lung cells (e.g., SAEC) to air pollutants (e.g. DEPs).

BACKGROUND:Cystic fibrosis (CF) transmembrane regulator (CFTR) protein dysfunction causes CF. Improving survival allows detection of increasingly subtle disease manifestations. CFTR dysfunction in the central nervous system (CNS) may disturb circadian rhythm and thus sleep phase. We studied sleep in adults to better understand potential CNS CFTR dysfunction. METHODS:We recruited participants from April 2012 through April 2015 and administered the Munich Chronotype Questionnaire (MCTQ). We compared free-day sleep measurements between CF and non-CF participants and investigated associations with CF survival predictors. RESULTS:We recruited 23 female and 22 male adults with CF aged 18 to 46 years and 26 female and 22 male volunteers aged 18 to 45 years. Compared with volunteers without CF, patients with CF had delayed sleep onset (0.612 h; P = .015), midsleep (1.11 h; P < .001), and wake (1.15 h; P < .001) times and prolonged sleep latency (7.21 min; P = .05) and duration (0.489 h; P = .05). Every hour delay in sleep onset was associated with shorter sleep duration by 0.29 h in patients with CF and 0.75 h in subjects without CF (P = .007) and longer sleep latency by 7.51 min in patients with CF and 1.6 min in volunteers without CF (P = .035). Among patients with CF, FEV1 % predicted, prior acute pulmonary exacerbations, and weight were independent of all free-day sleep measurements. CONCLUSIONS:CF in adults is associated with marked delays in sleep phase consistent with circadian rhythm phase delays. Independence from disease characteristics predictive of survival suggests that sleep phase delay is a primary manifestation of CFTR dysfunction in the CNS.

Lung function, acute pulmonary exacerbations (APE), and weight are the best clinical predictors of survival in cystic fibrosis (CF); however, underlying mechanisms are incompletely understood. Biomarkers of current disease state predictive of future outcomes might identify mechanisms and provide treatment targets, trial endpoints and objective clinical monitoring tools. Such CF-specific biomarkers have previously been elusive. Using observational and validation cohorts comprising 97 non-transplanted consecutively-recruited adult CF patients at the Intermountain Adult CF Center, University of Utah, we identified biomarkers informative of current disease and predictive of future clinical outcomes. Patients represented the majority of sputum producers. They were recruited March 2004-April 2007 and followed through May 2011. Sputum biomarker concentrations were measured and clinical outcomes meticulously recorded for a median 5.9 (interquartile range 5.0 to 6.6) years to study associations between biomarkers and future APE and time-to-lung transplantation or death. After multivariate modeling, only high mobility group box-1 protein (HMGB-1, mean = 5.84 [log ng/ml], standard deviation [SD] = 1.75) predicted time-to-first APE (hazard ratio [HR] per log-unit HMGB-1 = 1.56, p-value = 0.005), number of future APE within 5 years (0.338 APE per log-unit HMGB-1, p<0.001 by quasi-Poisson regression) and time-to-lung transplantation or death (HR = 1.59, p = 0.02). At APE onset, sputum granulocyte macrophage colony stimulating factor (GM-CSF, mean 4.8 [log pg/ml], SD = 1.26) was significantly associated with APE-associated declines in lung function (-10.8 FEV1% points per log-unit GM-CSF, p, 0.001 by linear regression). Evaluation of validation cohorts produced similar results that passed tests of mutual consistency. In CF sputum, high HMGB-1 predicts incidence and recurrence of APE and survival, plausibly because it mediates long-term airway inflammation. High APE-associated GM-CSF identifies patients with large acute declines in FEV1%, possibly providing a laboratory-based objective decision-support tool for determination of an APE diagnosis. These biomarkers are potential CF reporting tools and treatment targets for slowing long-term progression and reducing short-term severity.

In this study, confocal Raman spectroscopy, atomic force microscope (AFM) and multiplex ELISA were applied to analyze the biophysical responses (biomechanics and biospectroscopy) of normal human primary small airway epithelial cells (SAECs) and human lung carcinoma epithelial A549 cells to in vitro short term DEP exposure (up to 2h). Raman spectra revealed the specific cellular biomolecular changes in cells induced by DEP compared to unexposed control cells. Principal component analysis was successfully applied to analyze spectral differences between control and treated groups from multiple individual cells, and indicated that cell nuclei are more sensitive than other cell locations. AFM measurements indicated that 2h of DEP exposure induced a significant decrease in cell elasticity and a dramatic change in membrane surface adhesion force. Cytokine and chemokine production measured by multiplex ELISA demonstrated DEP-induced inflammatory responses in both cell types.