Sweat induction using Pilocarpine microneedle patches for sweat testing in healthy adults.

BACKGROUND: The sweat test using pilocarpine iontophoresis remains the gold standard for diagnosing cystic fibrosis, but access and reliability are limited by specialized equipment and insufficient sweat volume collected from infants and young children. These shortcomings lead to delayed diagnosis, limited point-of-care applications, and inadequate monitoring capabilities. METHODS: We created a skin patch with dissolvable microneedles (MNs) containing pilocarpine that eliminates the equipment and complexity of iontophoresis. Upon pressing the patch to skin, the MNs dissolve in skin to release pilocarpine for sweat induction. We conducted a non-randomized pilot trial among healthy adults (clinicaltrials.gov, NCT04732195) with pilocarpine and placebo MN patches on one forearm and iontophoresis on the other forearm, followed by sweat collection using Macroduct collectors. Sweat output and sweat chloride concentration were measured. Subjects were monitored for discomfort and skin erythema. RESULTS: Fifty paired sweat tests were conducted in 16 male and 34 female healthy adults. MN patches delivered similar amounts of pilocarpine into skin (1.1 ± 0.4 mg) and induced equivalent sweat output (41.2 ± 25.0 mg) compared to iontophoresis (1.2 ± 0.7 mg and 43.8 ± 32.3 mg respectively). Subjects tolerated the procedure well, with little or no pain, and only mild transient erythema. Sweat chloride concentration measurements in sweat induced by MN patches (31.2 ± 13.4 mmol/L) were higher compared to iontophoresis (24.0 ± 13.2 mmol/L). Possible physiological, methodological, and artifactual causes of this difference are discussed. CONCLUSIONS: Pilocarpine MN patches present a promising alternative to iontophoresis to enable increased access to sweat testing for in-clinic and point-of-care applications.

Pulmonary exacerbations in early cystic fibrosis lung disease are marked by strong modulation of CD3 and PD-1 on luminal T cells.

Background: In chronic cystic fibrosis (CF) lung disease, neutrophilic inflammation and T-cell inhibition occur concomitantly, partly due to neutrophil-mediated release of the T-cell inhibitory enzyme Arg1. However, the onset of this tonic inhibition of T cells, and the impact of pulmonary exacerbations (PEs) on this process, remain unknown. Methods: Children with CF aged 0-5 years were enrolled in a longitudinal, single-center cohort study. Blood (n = 35) and bronchoalveolar lavage (BAL) fluid (n = 18) were collected at stable outpatient clinic visits or inpatient PE hospitalizations and analyzed by flow cytometry (for immune cell presence and phenotype) and 20-plex chemiluminescence assay (for immune mediators). Patients were categorized by PE history into (i) no prior PE, (ii) past history of PE prior to stable visit, or (iii) current PE. Results: PEs were associated with increased concentration of both pro- and anti-inflammatory mediators in BAL, and increased neutrophil frequency and G-CSF in circulation. PE BAL samples showed a trend toward an increased frequency of hyperexocytic "GRIM" neutrophils, which we previously identified in chronic CF. Interestingly, expression levels of the T-cell receptor associated molecule CD3 and of the inhibitory programmed death-1 (PD-1) receptor were respectively decreased and increased on T cells from BAL compared to blood in all patients. When categorized by PE status, CD3 and PD-1 expression on blood T cells did not differ among patients, while CD3 expression was decreased, and PD-1 expression was increased on BAL T cells from patients with current PE. Conclusions: Our findings suggest that airway T cells are engaged during early-life PEs, prior to the onset of chronic neutrophilic inflammation in CF. In addition, increased blood neutrophil frequency and a trend toward increased BAL frequency of hyperexocytic neutrophils suggest that childhood PEs may progressively shift the balance of CF airway immunity towards neutrophil dominance.

Clinical Features of COVID-19 in Patients with Congenital Central Hypoventilation Syndrome.

Background: The clinical course of COVID-19 in patients with congenital central hypoventilation syndrome (CCHS) is unknown. Methods: We conducted a cross-sectional questionnaire study in 43 patients with CCHS who had COVID-19. Results: The median age of patients was 11 [interquartile range (IQR) 6-22] years and 53.5% required assisted ventilation (AV) through tracheostomy. Disease severity ranged from asymptomatic infection (12%) to severe illness with hypoxemia (33%) and hypercapnia requiring emergency care/hospitalization (21%), increased AV duration (42%), increased ventilator settings (12%), and supplemental oxygen demand (28%). The median duration to return to baseline AV (n = 20) was 7 (IQR 3-10) days. Patients with polyalanine repeat mutations required increased AV duration compared with those with nonpolyalanine repeat mutations (P = 0.048). Patients with tracheostomy required increased oxygen during illness (P = 0.02). Patients aged ≥18 years took longer to return to baseline AV (P = 0.04). Conclusions: Our study suggests that all patients with CCHS should be vigilantly monitored during COVID-19 illness.

Impact of viral respiratory infections on pulmonary exacerbations in children with cystic fibrosis.

BACKGROUND: Viral respiratory infections trigger pulmonary exacerbations (PEs) in children with cystic fibrosis (CF), but their clinical impact is not well understood. METHODS: A retrospective review of pediatric patients with CF who underwent nasopharyngeal respiratory viral panel testing during hospitalization for a PE between 2011 and 2018 was conducted. Patients were dichotomized into viral-positive and viral-negative groups. The results of spirometry, respiratory cultures, duration of hospitalization, and risk for subsequent PEs were analyzed. RESULTS: Ninety-five patients had 210 hospitalizations for PE (viral-positive = 71/210, 34%) during the study period. Rhinovirus/enterovirus was the most common virus (52/71, 73%) identified. Viral-positive patients were younger (p < 0.001), had higher baseline forced expiratory volume in 1 s (FEV1) (p = 0.037), continued to maintain higher FEV1 at 3 and 6 months following PE (p = 0.003 and 0.002, respectively), and had a shorter duration of hospitalization (p = 0.006) compared to the viral-negative group. There was no difference between the two groups in the rate of recovery of FEV1 at 3 and 6 months following PE (p = 0.71 and 0.405, respectively), time to the next PE (hazard ratio = 1.34, p = 0.157), number of subsequent PEs in 6 months (p = 0.99), or Pseudomonas aeruginosa (PA) acquisition (p = 0.707). CONCLUSIONS: In this single pediatric CF center cohort, one-third of PEs requiring hospitalization were associated with a viral infection, with rhinovirus/enterovirus being the most common. Viral-positive PEs were not associated with a greater decline or delayed recovery of lung function, increased risk for PA acquisition, shortened duration to next PE, longer hospital stay, or an increase in the frequency of subsequent PEs in 6 months compared to viral-negative PEs.

Macrophage PD-1 associates with neutrophilia and reduced bacterial killing in early cystic fibrosis airway disease.

BACKGROUND: Macrophages are the major resident immune cells in human airways coordinating responses to infection and injury. In cystic fibrosis (CF), neutrophils are recruited to the airways shortly after birth, and actively exocytose damaging enzymes prior to chronic infection, suggesting a potential defect in macrophage immunomodulatory function. Signaling through the exhaustion marker programmed death protein 1 (PD-1) controls macrophage function in cancer, sepsis, and airway infection. Therefore, we sought to identify potential associations between macrophage PD-1 and markers of airway disease in children with CF. METHODS: Blood and bronchoalveolar lavage fluid (BALF) were collected from 45 children with CF aged 3 to 62 months and structural lung damage was quantified by computed tomography. The phenotype of airway leukocytes was assessed by flow cytometry, while the release of enzymes and immunomodulatory mediators by molecular assays. RESULTS: Airway macrophage PD-1 expression correlated positively with structural lung damage, neutrophilic inflammation, and infection. Interestingly, even in the absence of detectable infection, macrophage PD-1 expression was elevated and correlated with neutrophilic inflammation. In an in vitro model mimicking leukocyte recruitment into CF airways, soluble mediators derived from recruited neutrophils directly induced PD-1 expression on recruited monocytes/macrophages, suggesting a causal link between neutrophilic inflammation and macrophage PD-1 expression in CF. Finally, blockade of PD-1 in a short-term culture of CF BALF leukocytes resulted in improved pathogen clearance. CONCLUSION: Taken together, these findings suggest that in early CF lung disease, PD-1 upregulation associates with airway macrophage exhaustion, neutrophil takeover, infection, and structural damage.

Pilot study of inflammatory biomarkers in matched induced sputum and bronchoalveolar lavage of 2-year-olds with cystic fibrosis.

BACKGROUND: In this pilot study, we investigated whether induced sputum (IS) could serve as a viable alternative to bronchoalveolar lavage (BAL) and yield robust inflammatory biomarkers in toddlers with cystic fibrosis (CF) featuring minimal structural lung disease. METHODS: We collected IS, BAL (right middle lobe and lingula), and blood, and performed chest computed tomography (CT) scans from 2-year-olds with CF (N = 11), all within a single visit. Inflammatory biomarkers included 20 soluble immune mediators and neutrophil elastase (NE), as well as frequency and phenotype of T cells, monocytes/macrophages, and neutrophils. RESULTS: At the molecular level, nine mediators showed similar levels in IS and BAL (CXCL1, CXCL8, IL-1α, IL-1RA, IL-6, CCL2, CXCL10, M-CSF, VEGF-A), four were higher in IS than in BAL (CXCL5, IL-1ß, CXCL11, TNFSF10), and two were present in IS, but undetectable in BAL (IL-10, IFN-γ). Meanwhile, soluble NE had lower activity in IS than in BAL. At the cellular level, T-cell frequency was lower in IS than in BAL. Monocytes/macrophages were dominant in IS and BAL with similar frequencies, but differing expression of CD16 (lower in IS), CD115, and surface-associated NE (higher in IS). Neutrophil frequency and phenotype did not differ between IS and BAL. Finally, neutrophil frequency in IS correlated positively with air trapping. CONCLUSIONS: IS collected from 2-year-olds with CF yields biomarkers of early airway inflammation with good agreement with BAL, notably with regard to molecular and cellular outcomes related to neutrophils and monocytes/macrophages.