Human epididymis protein 4 (HE4) plasma concentration inversely correlates with the improvement of cystic fibrosis lung disease in p.Phe508del-CFTR homozygous cases treated with the CFTR modulator lumacaftor/ivacaftor combination.

BACKGROUND: We previously documented that elevated HE4 plasma concentration decreased in people with CF (pwCF) bearing the p.Gly551Asp-CFTR variant in response to CFTR modulator (CFTRm) ivacaftor (IVA), and this level was inversely correlated with the FEV1% predicted values (ppFEV1). Although the effectiveness of lumacaftor (LUM)/IVA in pwCF homozygous for the p.Phe508del-CFTR variant has been evaluated, plasma biomarkers were not used to monitor treatment efficacy thus far. METHODS: Plasma HE4 concentration was examined in 68 pwCF drawn from the PROSPECT study who were homozygous for the p.Phe508del-CFTR variant before treatment and at 1, 3, 6 and 12 months after administration of LUM/IVA therapy. Plasma HE4 was correlated with ppFEV1 using their absolute and delta values. The discriminatory power of delta HE4 was evaluated for the detection of lung function improvements based on ROC-AUC analysis and multiple regression test. RESULTS: HE4 plasma concentration was significantly reduced below baseline following LUM/IVA administration during the entire study period. The mean change of ppFEV1 was 2.6% (95% CI, 0.6 to 4.5) by 6 months of therapy in this sub-cohort. A significant inverse correlation between delta values of HE4 and ppFEV1 was observed especially in children with CF (r=-0.7053; p<0.0001). Delta HE4 predicted a 2.6% mean change in ppFEV1 (AUC: 0.7898 [95% CI 0.6823-0.8972]; P < 0.0001) at a cut-off value of -10.7 pmol/L. Moreover, delta HE4 independently represented the likelihood of being a responder with ≥ 5% delta ppFEV1 at 6 months (OR: 0.89, 95% CI: 0.82-0.95; P = 0.001). CONCLUSIONS: Plasma HE4 level negatively correlates with lung function improvement assessed by ppFEV1 in pwCF undergoing LUM/IVA CFTRm treatment.

Enhanced Expression of Human Epididymis Protein 4 (HE4) Reflecting Pro-Inflammatory Status Is Regulated by CFTR in Cystic Fibrosis Bronchial Epithelial Cells.

Decreased human epididymis protein 4 (HE4) plasma levels were reported in cystic fibrosis (CF) patients under CFTR potentiator ivacaftor therapy, which inversely correlated with lung function improvement. In this study, we investigated whether HE4 expression was affected via modulation of CFTR function in CF bronchial epithelial (CFBE) cells in vitro. HE4 protein levels were measured in the supernatants of CFBE 41o- cells expressing F508del-CFTR or wild-type CFTR (wt-CFTR) after administration of lumacaftor/ivacaftor or tezacaftor/ivacaftor, while HE4 expression in CFBE 41o- cells were also analyzed following application of adenylate cyclase activators Forskolin/IBMX or CFTRinh172. The effect of all of these compounds on CFTR function was monitored by the whole-cell patch-clamp technique. Induced HE4 expression was studied with interleukin-6 (IL-6) in F508del-CFTR CFBE 41o- cells under TNF-α stimulation for 1 h up to 1 week in duration. In parallel, plasma HE4 was determined in CF subjects homozygous for p.Phe508del-CFTR mutation receiving lumacaftor/ivacaftor (Orkambi®) therapy. NF-κB-mediated signaling was observed via the nuclear translocation of p65 subunit by fluorescence microscopy together with the analysis of IL-6 expression by an immunoassay. In addition, HE4 expression was examined after NF-κB pathway inhibitor BAY 11-7082 treatment with or without CFTR modulators. CFTR modulators partially restored the activity of F508del-CFTR and reduced HE4 concentration was found in F508del-CFTR CFBE 41o- cells that was close to what we observed in CFBE 41o- cells with wt-CFTR. These data were in agreement with decreased plasma HE4 concentrations in CF patients treated with Orkambi®. Furthermore, CFTR inhibitor induced elevated HE4 levels, while CFTR activator Forskolin/IBMX downregulated HE4 in the cell cultures and these effects were more pronounced in the presence of CFTR modulators. Higher activation level of baseline and TNF-α stimulated NF-κB pathway was detected in F508del-CFTR vs. wt-CFTR CFBE 41o- cells that was substantially reduced by CFTR modulators based on lower p65 nuclear positivity and IL-6 levels. Finally, HE4 expression was upregulated by TNF-α with elevated IL-6, and both protein levels were suppressed by combined administration of NF-κB pathway inhibitor and CFTR modulators in CFBE 41o- cells. In conclusion, CFTR dysfunction contributes to abnormal HE4 expression via NF-κB in CF.

Laboratory biomarkers for lung disease severity and progression in cystic fibrosis.

Although the clinical outcomes of cystic fibrosis (CF) have been markedly improved through the recent implementation of novel CF transmembrane conductance regulator (CFTR) modulator drugs, robust and reliable biomarkers are still demanded for the early detection of CF lung disease progression, monitoring treatment efficacy and predicting life-threatening clinical complications. Thus, there is an unmet need to identify and validate novel, ideally blood based biomarkers with strong correlations to the severity of CF lung disease, which represents a major contribution to overall CF morbidity and mortality. In this review, we aim to summarize the utility of thus far studied blood-, sputum- and bronchoalveolar lavage (BAL)-based biomarkers to evaluate inflammatory conditions in the lung and to follow treatment efficacy in CF. Measurements of sweat chloride concentrations and the spirometric parameter FEV1 are currently utilized to monitor CFTR function and the effect of various CF therapies. Nonetheless, both have inherent pitfalls and limitations, thus routinely analyzed biomarkers in blood, sputum or BAL samples are required as surrogates for lung disorders. Recent discovery of new protein (e.g. HE4) and RNA-based biomarkers, such as microRNAs may offer a higher efficacy, which in aggregate may be valuable to evaluate disease prognosis and to substantiate CF drug efficacy.

Human epididymis protein 4 (HE4) levels inversely correlate with lung function improvement (delta FEV1) in cystic fibrosis patients receiving ivacaftor treatment.

BACKGROUND: We have recently shown that human epididymis protein 4 (HE4) levels correlate with the severity of cystic fibrosis (CF) lung disease. However, there are no data on how HE4 levels alter in patients receiving CFTR modulating therapy. METHODS: In this retrospective clinical study, 3 independent CF patient cohorts (US-American: 29, Australian: 12 and Irish: 19 cases) were enrolled carrying at least one Class III CFTR CF-causing mutation (p.Gly551Asp) and being treated with CFTR potentiator ivacaftor. Plasma HE4 was measured by immunoassay before treatment (baseline) and 1-6 months after commencement of ivacaftor, and were correlated with FEV1 (% predicted), sweat chloride, C-reactive protein (CRP) and body mass index (BMI). RESULTS: After 1 month of therapy, HE4 levels were significantly lower than at baseline and remained decreased up to 6 months. A significant inverse correlation between absolute and delta values of HE4 and FEV1 (r = -0.5376; P < .001 and r = -0.3285; P < .001), was retrospectively observed in pooled groups, including an independent association of HE4 with FEV1 by multiple regression analysis (ß = -0.57, P = .019). Substantial area under the receiver operating characteristic curve (ROC-AUC) value was determined for HE4 when 7% mean change of FEV1 (0.722 [95% CI 0.581-0.863]; P = .029) were used as classifier, especially in the first 2 months of treatment (0.806 [95% CI 0.665-0.947]; P < .001). CONCLUSIONS: This study shows that plasma HE4 levels inversely correlate with lung function improvement in CF patients receiving ivacaftor. Overall, this potential biomarker may be of value for routine clinical and laboratory follow-up of CFTR modulating therapy.

Impaired Immunosuppressive Effect of Bronchoalveolar Mesenchymal Stem Cells in Hypersensitivity Pneumonitis: Preliminary Findings.

BACKGROUND: Bronchoalveolar mesenchymal stem cells (MSCs) play an important role in the maintenance of lung integrity. Therapeutic application of bone marrow-derived MSCs reduced chronic bronchial inflammation in idiopathic pulmonary fibrosis, and improved the ratio of survivors in sepsis with pneumonia. This study investigated the effect of MSCs from bronchoalveolar lavage fluid (BALF) of hypersensitivity pneumonitis (HP) on T-cell function under in vitro conditions. METHODS: Bronchoalveolar MSCs were obtained via bronchoscopy with BAL from children with severe subacute HP. As control, BALF MSCs were assessed from children without any inflammatory lung disease. Isolated MSCs were characterized via immunophenotyping by flow cytometry and confocal laser scanning microscopy. HP-derived and healthy separated peripheral blood mononuclear cells (PBMCs) were stimulated by 5 µg/mL phytohemagglutinin in the presence of HP-derived or control MSCs in 5-day cultures. Proliferation and activation of T-cells were characterized by the mean fluorescence intensity (MFI) of 5,6-carboxyfluorescein-diacetat succinimidyl ester (CFSE) and CD25, CD69 as well as HLA-DR surface positivities, respectively. RESULTS: HP-derived MSCs showed significantly lower level of CD73, CD90, and CD105 expression compared to control MSCs in both flow cytometric and confocal microscopic experiments. MSCs from HP did not reduce T-cell proliferation based on CFSE MFI values, while the level of CD25 expression on both control and HP-derived CD4+ and CD8+ T-cells was significantly reduced by normal MSCs, while HP-derived MSCs did not have any significant effect. The level of other activation markers was not markedly modulated by MSCs. CONCLUSIONS: BALF MSCs from HP are unable to downregulate the proliferation and activation of T-cells that may support the development of recurrent intrapulmonary inflammation in HP. © 2016 International Clinical Cytometry Society.

Human Epididymis Protein 4: A Novel Serum Inflammatory Biomarker in Cystic Fibrosis.

BACKGROUND: Increased expression of the human epididymis protein 4 (HE4) was previously described in lung biopsy samples from patients with cystic fibrosis (CF). It remains unknown, however, whether serum HE4 concentrations are elevated in CF. METHODS: Seventy-seven children with CF from six Hungarian CF centers and 57 adult patients with CF from a Czech center were enrolled. In addition, 94 individuals with non-CF lung diseases and 117 normal control subjects with no pulmonary disorders were analyzed. Serum HE4 levels were measured by using an immunoassay, and their expression was further investigated via the quantification of HE4 messenger RNA by using quantitative reverse transcription polymerase chain reaction in CF vs non-CF respiratory epithelium biopsy specimens. The expression of the potential regulator miR-140-5p was analyzed by using an UPL-based quantitative reverse transcription polymerase chain reaction assay. HE4 was measured in the supernatants from unpolarized and polarized cystic fibrosis bronchial epithelial cells expressing wild-type or F508del-CFTR. RESULTS: Median serum HE4 levels were significantly elevated in children with CF (99.5 [73.1-128.9] pmol/L) compared with control subjects (36.3 [31.1-43.4] pmol/L; P < .0001). This observation was replicated in adults with CF (115.7 [77.8-148.7] pmol/L; P < .0001). In contrast, abnormal but lower HE4 concentrations were found in cases of severe bronchitis, asthma, pneumonia, and bronchiectasis. In patients with CF, the concentrations of HE4 were positively correlated with overall disease severity and C-reactive protein concentrations, whereas a significant inverse relationship was found between HE4 and the spirometric FEV1 value. Relative HE4 mRNA levels were significantly upregulated (P = .011) with a decreased miR-140-5p expression (P = .020) in the CF vs non-CF airway biopsy specimens. Twofold higher HE4 concentrations were recorded in the supernatant of polarized F508del-CF transmembrane conductance regulator/bronchial epithelial cells compared with wild-type cells. CONCLUSIONS: HE4 serum levels positively correlate with the overall severity of CF and the degree of pulmonary dysfunction. HE4 may thus be used as a novel inflammatory biomarker and possibly also as a measure of treatment efficacy in CF lung disease.

Decreased VEGF Level Is Associated with Elevated Ferritin Concentration in Bronchoalveolar Lavage Fluid of Children with Interstitial Lung Diseases.

BACKGROUND: A decreased level of vascular endothelial growth factor (VEGF) was previously described in bronchoalveolar lavage fluid (BALF) of adults with interstitial lung diseases (ILD) due to bronchial epithelial cell apoptosis and its proteolytic degradation. Elevated intrapulmonary ferritin was produced by alveolar cells that promoted oxidative injury in such patients. OBJECTIVES: In this study, we analyzed the concentrations of VEGF and ferritin in BALF samples of ILD children and studied the relationship between their levels and the degree of inflammation. METHODS: BALF and serum concentration of VEGF as well as ferritin and albumin in BALF samples were measured using enzyme-linked immunosorbent assay in children with idiopathic interstitial pneumonia (n = 16), hypersensitivity pneumonitis (n = 11) and idiopathic pulmonary hemosiderosis (n = 3). Twenty-four age- and gender-matched subjects with suspicious foreign body aspiration served as a control group. RESULTS: VEGF per albumin levels in BALF were significantly decreased in ILD children compared to controls (1,075 [784-1,415] pg/mg albumin vs. 2,741 [1,131-4,660] pg/mg albumin, p = 0.0008). These values showed a significant negative correlation with inflammatory markers of total immune cell count in BALF (r = -0.411, p = 0.002) and serum C-reactive protein (r = -0.367, p = 0.006). Although serum VEGF was augmented in ILD children versus controls, no difference was observed among the ILD groups. In addition, BALF ferritin/albumin level (688 [188-1,571] ng/mg albumin vs. 256 [178-350] ng/mg albumin, p = 0.022) was significantly higher than normal in ILD individuals, especially in idiopathic pulmonary hemosiderosis. CONCLUSION: Depressed VEGF and increased ferritin in BALF may reflect the severity of chronic pulmonary inflammation in altered respiratory epithelium of childhood ILD.

Efficacy of methylprednisolone in children with severe community acquired pneumonia.

BACKGROUND: The clinical value of adjuvant corticosteroid treatment in community-acquired pneumonia (CAP) seemed to be controversial in adults, and even less data are available on the use of corticosteroids in children with CAP. MATERIALS AND METHODS: In this study, we investigated the efficacy of a 5-day adjuvant methylprednisolone therapy to imipenem in 29 children with severe CAP. In parallel, 30 subjects with the same disease were treated with imipenem and placebo, and the two study groups were compared based on the different parameters of the primary and secondary end points. The primary end points were the duration of fever, the levels of white blood cells (WBC) and high sensitive C-reactive protein (hsCRP). Secondary end points were the length of hospital stay, and the number of severe complications with or without surgical interventions. RESULTS: The additive methylprednisolone treatment significantly reduced the duration of fever with 2.5 days, the WBC counts (P = 0.014), the hsCRP levels showing a 48.7% decrease, and the length of hospital stay with 5.2 days versus the placebo group. Moreover, patients treated on imipenem alone had twice more complications and four times more invasive interventions compared to those on the combined therapy. CONCLUSIONS: The 5-day methylprednisolone therapy with imipenem was found effective in children having severe CAP. However, trials with larger cohorts are needed to study further beneficial effects of corticosteroids in children with CAP.