Rationale: Neutrophils are recruited to the airways of individuals with cystic fibrosis (CF). In adolescents and adults with CF, airway neutrophils actively exocytose the primary granule protease elastase (NE), whose extracellular activity correlates with lung damage. During childhood, free extracellular NE activity is measurable only in a subset of patients, and the exocytic function of airway neutrophils is unknown.Objectives: To measure NE exocytosis by airway neutrophils in relation to free extracellular NE activity and lung damage in children with CF.Methods: We measured lung damage using chest computed tomography coupled with the Perth-Rotterdam Annotated Grid Morphometric Analysis for Cystic Fibrosis scoring system. Concomitantly, we phenotyped blood and BAL fluid leukocytes by flow and image cytometry, and measured free extracellular NE activity using spectrophotometric and Förster resonance energy transfer assays. Children with airway inflammation linked to aerodigestive disorder were enrolled as control subjects.Measurements and Main Results: Children with CF but not disease control children harbored BAL fluid neutrophils with high exocytosis of primary granules, before the detection of bronchiectasis. This measure of NE exocytosis correlated with lung damage (R = 0.55; P = 0.0008), whereas the molecular measure of free extracellular NE activity did not. This discrepancy may be caused by the inhibition of extracellular NE by BAL fluid antiproteases and its binding to leukocytes.Conclusions: NE exocytosis by airway neutrophils occurs in all children with CF, and its cellular measure correlates with early lung damage. These findings implicate live airway neutrophils in early CF pathogenesis, which should instruct biomarker development and antiinflammatory therapy in children with CF.

Numerous changes in epigenetic mechanisms have been described in various types of tumors. In search for new biomarkers, we investigated the expression of Polycomb-group (PcG) proteins EZH2, BMI1 and SUZ12 and associated histone modification H3K27me3 in colorectal cancer. Nuclear expression of PcG proteins and histone modification H3K27me3 were immunohistochemically (IHC) stained on a tissue microarray (TMA), including 247 tumor tissues and 47 normal tissues, and scored using the semi-automated Ariol system. Tumor tissues showed higher expression of EZH2 (p = 0.05) and H3K27me3 (p<0.001) as compared to their normal counterparts. Combined marker trend analyses indicated that an increase in the number of markers showing high expression was associated with better prognosis. High expression of all four markers in the combined marker analyses was correlated with the best patient survival and the longest recurrence-free survival, with overall survival (p = 0.01, HR 0.42(0.21–0.84)), disease-free survival (p = 0.007, HR 0.23(0.08–0.67) and local recurrence-free survival (p = 0.02, HR 0.30(0.11–0.84)). In conclusion, we found that expression of PcG proteins and H3K27me3 showed prognostic value in our study cohort. Better stratification of patients was obtained by combining the expression data of the investigated biomarkers as compared to the individual markers, underlining the importance of investigating multiple markers simultaneously.

Background: Post-translational modification of histone tails by methylation plays an important role in tumorigenesis. In this study, we investigated the nuclear expression of H3K4me3, H3K9me3 and H4K20me3 in early-stage colon cancer in relation to clinical outcome. Methods: Tumor tissue cores of 254 TNM stage I-III colorectal cancer patients were immunohistochemically stained for H3K4me3, H3K9me3 and H4K20me3 and scored using the semi-automated Ariol system. Cox proportional hazard trend analyses were performed to assess the prognostic value of the combined markers with respect to patient survival and tumor recurrence.

Cystic fibrosis (CF) lung disease progressively worsens from infancy to adulthood. Disease-driven changes in early CF airway fluid metabolites may identify therapeutic targets to curb progression.CF patients aged 12-38 months (n=24; three out of 24 later denoted as CF screen positive, inconclusive diagnosis) received chest computed tomography scans, scored by the Perth-Rotterdam Annotated Grid Morphometric Analysis for CF (PRAGMA-CF) method to quantify total lung disease (PRAGMA-%Dis) and components such as bronchiectasis (PRAGMA-%Bx). Small molecules in bronchoalveolar lavage fluid (BALF) were measured with high-resolution accurate-mass metabolomics. Myeloperoxidase (MPO) was quantified by ELISA and activity assays.Increased PRAGMA-%Dis was driven by bronchiectasis and correlated with airway neutrophils. PRAGMA-%Dis correlated with 104 metabolomic features (p<0.05, q<0.25). The most significant annotated feature was methionine sulfoxide (MetO), a product of methionine oxidation by MPO-derived oxidants. We confirmed the identity of MetO in BALF and used reference calibration to confirm correlation with PRAGMA-%Dis (Spearman's ρ=0.582, p=0.0029), extending to bronchiectasis (PRAGMA-%Bx; ρ=0.698, p=1.5×10), airway neutrophils (ρ=0.569, p=0.0046) and BALF MPO (ρ=0.803, p=3.9×10).BALF MetO associates with structural lung damage, airway neutrophils and MPO in early CF. Further studies are needed to establish whether methionine oxidation directly contributes to early CF lung disease and explore potential therapeutic targets indicated by these findings.

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