Poly I:C Pre-Treatment Induced the Anti-Viral Interferon Response in Airway Epithelial Cells.

Type I and III interferons are among the most important antiviral mediators. Increased susceptibility to infections has been described as being associated with impaired interferon response in asthmatic patients. In this work, we focused on the modulation of interferon dysfunction after the rhinovirus infection of airway epithelial cells. Therefore, we tested polyinosinic:polycytidylic acid (poly I:C), a TLR3 agonist, as a possible preventive pre-treatment to improve this anti-viral response. In our human study on asthma, we found a deficiency in interferon levels in the nasal epithelial cells (NEC) from asthmatics at homeostatic level and after RV infection, which might contribute to frequent airway infection seen in asthmatic patients compared to healthy controls. Finally, pre-treatment with the immunomodulatory substance poly I:C before RV infection restored IFN responses in airway epithelial cells. Altogether, we consider poly I:C pre-treatment as a promising strategy for the induction of interferon response prior to viral infections. These results might help to improve current therapeutic strategies for allergic asthma exacerbations.

Increase of liver stiffness and altered bile acid metabolism after triple CFTR modulator initiation in children and young adults with cystic fibrosis.

BACKGROUND: Novel cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies (elexacaftor/tezacaftor/ivacaftor-ETI) promise clinically significant and sustained improvements for patients with cystic fibrosis (CF). In this study, we investigated the impact of ETI therapy on liver stiffness and bile acid metabolism in a cohort of children and young adults with CF. METHODS: A prospective observational study (NCT05576324) was conducted from September 2020 to November 2021 enrolling CF patients naive to ETI. Standard laboratory chemistry, sweat test, lung function, share wave velocity (SWV) derived by acoustic radiation force impulse imaging (ARFI) and serum bile acid profiles were assessed before and 6 months after induction of ETI therapy. RESULTS: A total of 20 patients (10 aged <20 years) completed the study. While lung function and BMI improved after ETI therapy, ARFI SWV increased in CF patients <20 years of age (from 1.27 to 1.43 m/s, p = 0.023). Bile acid (BA) profiles revealed a decrease in unconjugated (5.75 vs 1.46, p = 0.007) and increase in glycine-conjugated derivatives (GCDCA) (4.79 vs 6.64 p = 0.016). There was a positive correlation between ARFI SWV values and GCDCA (r = 0.80, p < 0.0001). Glycine-conjugated BA provided high diagnostic accuracy to predict increased ARFI measurements (AUC 0.90) and clinical (Colombo) CFLD grading (AUC 0.97). CONCLUSIONS: ARFI SWV and bile acid profiles provide evidence for early increase in liver stiffness and altered bile acid metabolism in young CF patients after initiation of ETI and may serve as synergistic measures for detection of hepatic complications during ETI therapy.

ILC2 Lung-Homing in Cystic Fibrosis Patients: Functional Involvement of CCR6 and Impact on Respiratory Failure.

Cystic fibrosis patients suffer from a progressive, often fatal lung disease, which is based on a complex interplay between chronic infections, locally accumulating immune cells and pulmonary tissue remodeling. Although group-2 innate lymphoid cells (ILC2s) act as crucial initiators of lung inflammation, our understanding of their involvement in the pathogenesis of cystic fibrosis remains incomplete. Here we report a marked decrease of circulating CCR6+ ILC2s in the blood of cystic fibrosis patients, which significantly correlated with high disease severity and advanced pulmonary failure, strongly implicating increased ILC2 homing from the peripheral blood to the chronically inflamed lung tissue in cystic fibrosis patients. On a functional level, the CCR6 ligand CCL20 was identified as potent promoter of lung-directed ILC2 migration upon inflammatory conditions in vitro and in vivo using a new humanized mouse model with light-sheet fluorescence microscopic visualization of lung-accumulated human ILC2s. In the lung, blood-derived human ILC2s were able to augment local eosinophil and neutrophil accumulation and induced a marked upregulation of pulmonary type-VI collagen expression. Studies in primary human lung fibroblasts additionally revealed ILC2-derived IL-4 and IL-13 as important mediators of this type-VI collagen-inducing effect. Taken together, the here acquired results suggest that pathologically increased CCL20 levels in cystic fibrosis airways induce CCR6-mediated lung homing of circulating human ILC2s. Subsequent ILC2 activation then triggers local production of type-VI collagen and might thereby drive extracellular matrix remodeling potentially influencing pulmonary tissue destruction in cystic fibrosis patients. Thus, modulating the lung homing capacity of circulating ILC2s and their local effector functions opens new therapeutic avenues for cystic fibrosis treatment.

Confocal Laser Endomicroscopy for Diagnosing Malignant Pleural Effusions.

BACKGROUND Confocal laser endomicroscopy (CLE) enables "in vivo" microscopic tissue diagnosis based on tissue reflectance or tissue fluorescence upon application of fluorescence agents. The aim of the present study was to evaluate CLE as a new diagnostic approach for differentiation between malignant versus non-malignant pleural effusions. MATERIAL AND METHODS In 100 patients with pleural effusions, thoracentesis was performed. Cresyl violet and acriflavine were used as contrast agents for probe-based CLE of effusions. CLE video sequences were assessed by 4 independent investigators (2 experienced in this technique, 2 with only basic knowledge). In addition, all CLE samples were evaluated by an expert pathologist (p). Results were compared with conventional cytology of effusions and histology of cell blocks. RESULTS CLE reliably permitted identification of malignant cells in pleural effusions. Sensitivity for detection of malignant effusions was 87% (p: 87%) and 81% (p: 72%) for acriflavine and cresyl violet, respectively. With regard to specificity, acriflavine and cresyl violet yielded a mean value of 99% (p: 100%) and 92% (p: 100%). CONCLUSIONS In this pilot study, CLE permitted simple and rapid detection of malignant pleural effusions. Larger prospective studies are warranted to corroborate our findings.

Identification of Bronchoalveolar Lavage Components Applying Confocal Laser Endomicroscopy.

BACKGROUND In many studies, confocal laser endomicroscopy (CLE) has proven to be a useful tool in pulmonology; nevertheless, the application in this field is still experimental. By contrast, CLE is almost a standard technique in gastroenterology. The aim of the present study was to demonstrate the identification of bronchoalveolar lavage (BAL) components applying CLE, using a dye. MATERIAL AND METHODS In 21 patients with various underlying diseases a bronchoscopy with BAL was performed. As in routine clinical practice common, BAL fluid (BALF) was analyzed in terms of cytologic, virologic, and microbiologic aspects. To one fraction of BALF, we added acriflavine. After centrifugation CLE was applied and the video sequences were analyzed by an experienced investigator. RESULTS Using CLE, BALF components (such as alveolar macrophages or leucocytes) could be easily identified. A further subdivision of leucocytes (neutrophilic, eosinophilic granulocytes, and lymphocytes) was not possible. Analogous to conventional cytology, a precise distinction of lymphocyte subpopulation (cd 4/cd 8 ratio) was not feasible. In terms of quantification, this is still the application field of flow cytometry and immunohistochemistry. CONCLUSIONS Using CLE, alveolar macrophages and leucocytes in stained BALF can be differentiated independent of smoking status. Further studies should be initiated in order to subclassify leucocytes in eosinophilic, neutrophilic granulocytes, and lymphocytes, which is important for routine clinical practice.

Public spirometry for primary prevention of COPD.

BACKGROUND: The most effective action for primary prevention of chronic obstructive lung disease is smoking cessation early enough. In secondary prevention, smokers with airway obstruction were more likely to quit smoking. The aim of this study was to evaluate the impact of a public spirometry on smoking habits in terms of primary prevention. METHODS: Spirometry with its medical analysis was offered to visitors of a local public event called 'Lange Nacht der Wissenschaften' ('Long night of sciences'). The impact of results on smoking habits was evaluated in all smokers with an anonymized questionnaire afterwards. RESULTS: Two hundred fifty-seven people with the median age of 30 years (interquartile range 22-46) were examined. Out of 44 current smokers (17.1%), only two individuals showed a prebronchodilator FEV1/forced vital capacity-value <0.7. Fourteen smokers stated to have an increased motivation to quit smoking whereas 28 smokers declared that their motivation to quit smoking was independent of spirometry result. These smokers were significantly younger (median age 28 vs. 40 years, P = 0.025) without differences in spirometry results or smoking habits. CONCLUSION: In an unselected population with a high amount of younger adults, normal spirometry did not show a short-term benefit for primary prevention of chronic obstructive lung disease in terms of increasing motivation to quit smoking.

The validation of estrogen receptor 1 mRNA expression as a predictor of outcome in patients with metastatic non-small cell lung cancer.

The prognostic role of estrogen receptors in lung cancer is not validated. Results from patients with early stage non-small lung cancer patients indicate a prognostic role of estrogen receptor 1 (ESR1) mRNA expression in these patients. Automated RNA extraction from paraffin and RT-quantitative PCR was used for evaluation of tumoral ESR1 and progesterone receptor (PGR) mRNA expression. The test cohort consisted of 31 patients with advanced or metastatic non-small cell lung cancer (NSCLC) patients, treated in a first-line registry trial. For validation, 53 patients from a randomized multicentre first-line study with eligible tumor samples were evaluated. There was no significant correlation of ESR1 expression with clinical characteristics. ESR1 high expression was of significant positive prognostic value in the training set with a median overall survival (OS) of 15.9 versus 6.2 months for high versus low ESR1 expression patients (p = 0.0498, HR 0.39). This could be confirmed in the validation cohort with a median OS of 10.9 versus 5.0 months in ESR1 high versus low patients, respectively (p = 0.0321, HR 0.51). In the multivariate analysis adjusted for histological subtype, gender, age and performance status, ESR1 expression remained an independent prognostic parameter for survival in both cohorts. In contrast to ESR1, PGR expression was not able to separate prognostic groups or to predict outcome significantly (for OS; p = 0.94). Our study shows that ESR1 mRNA as assessed by qPCR represents a reliable method for detecting ESR1 expression in NSCLC and that ESR1 expression is an independent prognostic factor in metastatic NSCLC.

Confocal laser endomicroscopy for diagnosing lung cancer in vivo.

Confocal laser endomicroscopy is a novel endoscopic technique that may allow imaging of living cells in lung tissue in vivo. We assessed the potential of this technique for the detection of histology during screening bronchoscopy for lung cancer. 32 patients with suspected malignancies underwent bronchoscopy with endomicroscopy using acriflavine hydrochloride. Standardised areas and localised lesions were analysed by in vivo confocal imaging during bronchoscopy and biopsies were taken. Confocal images were graded and correlated prospectively with conventional histology from biopsies. Acriflavine hydrochloride yielded high-quality confocal images and strongly labelled airway epithelial cells. No side-effects were noted. 75,522 confocal images from 56 different locations were compared prospectively with histological data from biopsy specimens. Endomicroscopy allowed subsurface imaging with detailed analysis of cellular and subcellular structures. Neoplastic changes could be predicted with high accuracy (sensitivity 96.0%, specificity 87.1%, accuracy 91.0%). Confocal laser endomicroscopy with acriflavine is a novel diagnostic tool for the analysis of living cells during bronchoscopy and permits virtual histology of neoplastic changes in the airways with high accuracy. This technique may enable the rapid diagnosis of neoplasia during ongoing endoscopy in patients with suspected lung cancer.

Methylene blue-aided in vivo staining of central airways during flexible bronchoscopy.

BACKGROUND: The early diagnosis of malignant and premalignant changes of the bronchial mucosa remains a major challenge during bronchoscopy. Intravital staining techniques are not new. Previous small case series suggested that analysis of the bronchial mucosal surface using chromoendoscopy allows a prediction between neoplastic and nonneoplastic lesions. OBJECTIVES: The aim of the present study was to evaluate chromobronchoscopy as a method to identify malignant and premalignant lesions in the central airways in a prospective manner. METHODS: In 26 patients we performed chromoendoscopy with 0.1% methylene blue during ongoing flexible white light bronchoscopy. Circumscribed lesions in central airways were further analyzed by biopsies and histopathologic examination. RESULTS: In the majority of cases neither flat nor polypoid lesions in the central airways were stained by methylene blue. In particular, exophytic growth of lung cancer did not show any specific pattern in chromobronchoscopy. However, a specific dye staining was detected in one case where exophytic growth of metastatic colorectal cancer was present in the right upper lobe. In two other cases, a circumscribed staining was noted in unsuspicious mucosa. But histology revealed inflammation only. CONCLUSIONS: In contrast to previous studies, the present findings clearly indicate that chromobronchoscopy is not useful for early detection of malignant or premalignant lesions of the central airways.

Fluorescein-aided confocal laser endomicroscopy of the lung.

BACKGROUND: There are only few reports about confocal laser endomicroscopy (CLE) for pulmonary imaging. In these studies, in contrast to gastrointestinal endoscopy, CLE was performed without fluorescein. OBJECTIVES: The aim of the present study was to evaluate the value of fluorescein usage for CLE of the lung. METHODS: Fluorescein-aided CLE was performed in 15 consecutively recruited patients and in 4 young healthy volunteers with a miniprobe during flexible bronchoscopy. Before and after intravenous administration of fluorescein, central airways and alveolar structures were evaluated. RESULTS: Fluorescein administration did not permit imaging of epithelial cells in the central airways. In the lung periphery, alveolar walls and partially macrophages could be seen in native imaging, as expected. After administration of fluorescein, alveoli were almost filled with foam in areas with normal lung tissue. The origin of this foam was shown to be artificial. Furthermore, in patients with pathologies of the lung parenchyma, dark neoplastic and inflammatory cells adjacent to the alveolar walls were identified. No relevant side effects of fluorescein administration could be observed. CONCLUSIONS: Fluorescein-aided CLE of the lung appeared to be safe and well tolerated. While the lack of staining of cells in the central airways was a major limitation, it permitted analysis of the lung interstitium and alveolar space and thus emerges as a new approach for the in vivo analysis of interstitial lung diseases.

Predictive and prognostic factors in small cell lung carcinoma (SCLC)--analysis from routine clinical practice.

BACKGROUND: Prognostic and predictive factors of routine clinical practice among patients with small cell lung carcinoma (SCLC) were evaluated. PATIENTS AND METHODS: Data from 106 patients with SCLC treated by first-line adriamycin, cyclophosphamide and etoposide (ACE) chemotherapy were analyzed. Multivariate analysis was performed. RESULTS: The median overall survival (mOS) of patients was 9.36 months with mOS of 31%, 8% and 3% after 1, 2 and 5 years, respectively. Using multivariate analysis ECOG performance status (p =0.008) and white blood count (WBC) (p=0.022) were independent prognostic factors for mOS. With both, three groups of outcome (good, intermediate, poor) resulting in mOS of 15.8 months, 6.87 months and 3.35 months (p<0.0001) could be established, respectively. The absence of brain metastases (p=0.002), dose reduction (p=0.002) and LDH value (p=0.017) were independent predictive markers. Additionally, female gender was predictive (p=0.025) for complete response (CR). CONCLUSION: Patients with a poor prediction profil might not benefit from ACE chemotherapy. As a consequence, prognostic/predictive factors should be included as stratification criteria in prospective clinical studies.

Butyrate inhibits leukocyte adhesion to endothelial cells via modulation of VCAM-1.

BACKGROUND: Leukocyte recruitment to areas of inflammation depends on Integrin-VCAM/ICAM interaction. Blocking the vascular cell adhesion molecule (VCAM-1) and the intracellular adhesion molecule (ICAM-1) may have therapeutic benefit for the inflammatory component of bowel disease. Notably, the induction of ICAM and VCAM is mediated by a nuclear factor kappaB (NF-kappaB)-dependent mechanism. We investigated whether the anti-inflammatory properties of butyrate are mediated via the modulation of VCAM and ICAM on human endothelial cells. METHODS: VCAM-1 and ICAM-1 expression on human endothelial cells upon tumor necrosis factor-alpha (TNF-alpha) stimulation was assessd by FACS analysis. A monocyte adhesion assay was performed to evaluate the relevance of a modulated CAM-expression. Electrophoretic mobility shift assays were applied to investigate NF-kappaB activation. RESULTS: The observed butyrate-associated inhibition of monocyte adhesion to endothelial cells is associated with an inhibition of NF-kappaB activation in human endothelial cells. In this context, the observed suppression of the TNF-alpha induced VCAM-1 expression is likely to play an essential role. CONCLUSIONS: Butyrate inhibits VCAM-1 mediated leukocyte adhesion to human endothelial cells. This inhibition may contribute to the anti-inflammatory effects of butyrate in patients with distal ulcerative colitis.