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.

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.

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.

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.

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.

Vitamin D3 resolved human and experimental asthma via B lymphocyte-induced maturation protein 1 in T cells and innate lymphoid cells.

Background: Vitamin D3 (VitD3) is known to have immunomodulatory functions, and VitD3 deficiency is associated with more severe asthma. Objective: We aimed to assess the immunoregulatory effects of VitD3 food supplementation on asthma manifestation, with particular focus on T cells and type 2 innate lymphoid cells. Methods: Preschool children and adult asthmatic cohorts were analyzed in the context of VitD3 supplementation and serum levels. In a murine model of ovalbumin-induced asthma, effects of diet VitD3 sufficiency and deficiency on T cells and type 2 innate lymphoid cells immune mechanisms were investigated. Results: We found less severe and better-controlled asthma phenotypes along with reduced need for steroid medication in preschool children and asthmatic adults with VitD3 supplementation. VitD3 serum levels correlated with B lymphocyte-induced maturation protein 1 (Blimp-1) expression in blood peripheral mononuclear cells. VitD3-supplement-fed mice showed decreased asthmatic traits, with a decrease in IgE serum levels, reduced airway mucus, and increased IL-10 production by lung cells. Furthermore, we discovered an upregulation of effector T cells and Blimp-1+ lung tissue-resident memory T cells as well as induction of anti-inflammatory Blimp-1+ lung innate lymphoid cells producing IL-10. Conclusion: Supplementing VitD3 resulted in amelioration of clinical asthma manifestations in human studies as well as in experimental allergic asthma, indicating that VitD3 shifts proinflammatory immune responses to anti-inflammatory immune responses via upregulating Blimp-1 in lung innate lymphoid cells and tissue-resident memory cells.

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.

Rhinovirus Suppresses TGF-ß-GARP Presentation by Peripheral NK Cells.

Asthma is a chronic airway disease whose exacerbations are often triggered by rhinovirus infection. TGF-ß1 induces rhinovirus replication in infected cells. Moreover, TGF-ß1 is a pleiotropic mediator that is produced by many immune cells in the latent, inactive form bound to the latency-associated peptide (LAP) and to the transmembrane protein glycoprotein A repetitions predominant (GARP). In this study we wanted to investigate the effect of rhinovirus infection on the TGF-ß secretion and the downstream signaling via TGF-ßRI/RII in peripheral blood mononuclear cells from control and asthmatic patients after rhinovirus infection ex vivo. Here, we found a significant upregulation of TGF-ßRII in untouched PBMCs of asthmatics as well as a suppression of TGF-ß release in the rhinovirus-infected PBMC condition. Moreover, consistent with an effect of TGF-ß on Tregs, PBMCs infected with RV induced Tregs, and TGF-ßRII directly correlated with RV1b mRNA. Finally, we found via flow cytometry that NK cells expressed less GARP surface-bound TGF-ß, while cytokine-producing NKbright cells were induced. In summary, we show that rhinovirus infection inhibits TGF-ß release in PBMCs, which results in the activation of both Treg and NK cells.

Leptin, obestatin and apelin levels in patients with obstructive sleep apnoea syndrome.

BACKGROUND: Recent studies suggest that adipose tissue hormones are involved in the pathogenesis of obstructive sleep apnoea syndrome (OSAS). The role of leptin, obestatin and apelin still needs to be established. MATERIAL/METHODS: Ten patients with newly diagnosed OSAS (AHI >10/h and ESS >10 points) were enrolled in the study as well as ten healthy volunteers as controls. All underwent measurements for Leptin, Obestatin and Apelin in four hour intervals during diagnostic polysomnography for 24 h and the patients also three months after onset of CPAP treatment. Furthermore the HOMA-index and body composition were quantified. RESULTS: Plasma apelin levels in the patients decreased under CPAP therapy, but showed no significant difference in patients and volunteers. We found a positive correlation to AHI, BMI in the therapy group at all observation points. Leptin plasma levels were higher in the patient group and decreased after onset of CPAP therapy. Leptin plasma levels were positively correlated to the BMI, min. 02 and AHI in the patient group before therapy. Plasma obestatin levels did not differ significantly in these three observation groups, but were partly correlated to AHI and weight in the newly diagnosed OSAS group. CONCLUSIONS: In agreement with previous investigations, we could demonstrate a difference in leptin plasma levels between healthy volunteers and patients with newly diagnosed OSAS. Apelin decreases under CPAP therapy, but not significantly. Obestatin remains unchanged after onset of CPAP. We further found a linkage between leptin plasma levels and BMI, AHI and weight in the untreated patient group.

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.

Regulation and Function of Interferon-Lambda (IFNλ) and Its Receptor in Asthma.

Asthma is a chronic respiratory disease affecting people of all ages, especially children, worldwide. Origins of asthma are suggested to be placed in early life with heterogeneous clinical presentation, severity and pathophysiology. Exacerbations of asthma disease can be triggered by many factors, including viral respiratory tract infections. Rhinovirus (RV) induced respiratory infections are the predominant cause of the common cold and also play a crucial role in asthma development and exacerbations. Rhinovirus mainly replicates in epithelial cells lining the upper and lower respiratory tract. Type III interferons, also known as interferon-lambda (IFNλ), are potent immune mediators of resolution of infectious diseases but they are known to be involved in autoimmune diseases as well. The protective role of type III IFNs in antiviral, antibacterial, antifungal and antiprotozoal functions is of major importance for our innate immune system. The IFNλ receptor (IFNλR) is expressed in selected types of cells like epithelial cells, thus orchestrating a specific immune response at the site of viruses and bacteria entry into the body. In asthma, IFNλ restricts the development of TH2 cells, which are induced in the airways of asthmatic patients. Several studies described type III IFNs as the predominant type of interferon increased after infection caused by respiratory viruses. It efficiently reduces viral replication, viral spread into the lungs and viral transmission from infected to naive individuals. Several reports showed that bronchial epithelial cells from asthmatic subjects have a deficient response of type III interferon after RV infection ex vivo. Toll like Receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) expressed on infectious agents, and induce the development of antiviral and antibacterial immunity. We recently discovered that activation of TLR7/8 resulted in enhanced IFNλ receptor mRNA expression in PBMCs of healthy and asthmatic children, opening new therapeutic frontiers for rhinovirus-induced asthma. This article reviews the recent advances of the literature on the regulated expression of type III Interferons and their receptor in association with rhinovirus infection in asthmatic subjects.

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.

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.

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.

Lung volumes and mean apnea duration in obstructive sleep apnea.

Former studies suggested that lung volumes might play a role in pathomechanisms of obstructive sleep apnea (OSA). Mean apnea duration (MAD) is a rarely investigated parameter in OSA but is possibly a surrogate of arousal threshold. The aim of this study was to evaluate the influence of lung volumes to MAD in OSA. In 69 patients with obstructive sleep apnea (51 male und 18 female, BMI 34.2+/-6.0 kg/m(2), age 53.6+/-9.7 years, AHI 43.1+/-21.1/h) we performed a polysomnography and pulmonary function testing in daytime. There was a significant correlation between MAD and residual volume (RV) (r=0.51; p<0.001), which was the highest correlation we found. In linear regression analysis RV remained the only independent variable with significant influence on MAD (p<0.001). We could show that RV seems to play a role in the mechanisms of apnea termination in terms of MAD. MAD reflects the time until a specific negative intrathoracic pressure is reached to induce an arousal. In this process dependency on RV could explain our results. Despite some limitations these results provide some new aspects in understanding pathophysiology of OSA.