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During a three-year field campaign of measuring waterfall generated ions, we monitored five different waterfalls in the Austrian Alps. Most measurements were performed at the Krimml waterfall (Salzburg, Austria), which is the biggest waterfall in Europe, and the Gartl waterfall (Mölltal, Austria). We characterized spatial, time and size distributions of waterfall-generated ions under the influence of surrounding topography. The smallest ions with boundary diameters of 0.9, 1.5 and 2 nm, were measured with a cylindrical air ion detector (CDI-06), while ion sizes from 5.5 to 350 nm were measured using a modified Grimm SMPS aerosol spectrometer. High negative ion concentration gradients are detected in the vicinity of the waterfalls, whereas the increase of positive ions was only moderate. Ions in the nano range were the most abundant at 2 nm, and at 120 nm in the sub-micrometer range.

Objective: The study aim was to investigate the course of pain in rest and motion in seven different rheumatic diseases (RMD), prior and after multimodal spa therapy including low-dose radon treatment and at 3-, 6-; and 9-month follow up. Methods: Complete data from the radon indication registry including information on 561 subjects with RMD were analysed to explore the association of timepoint of measurement with pain in rest and motion. For this purpose, linear regression models adjusted for RMD-type, age, sex and body mass index (BMI) were applied. Results: The mean age of the sample was 55 years, the average body mass index was 26.8, and 275 subjects were women. Pain scores were significantly improved at all-time points compared to baseline. Pain courses were different for each RMD with the largest improvement seen in fibromyalgia. Conclusion: Timing spa facility visits according to RMD-specific pain courses may result in sustained pain reduction., Competing Interests: The authors declare that they do not have any conflicts of interest., (Copyright © 2023 van der Zee-Neuen, Fuchs, Wildburger, Gaisberger, Kloppenburg, Fioravanti, Stamm, Offenbaecher, Radlmueller, Foisner, Hoelzl and Ritter.)

Objective: To study whether layer-specific cartilage transverse relaxation time (T2) and/or longitudinal change is associated with clinically relevant knee osteoarthritis (OA) disease progression., Methods: The Foundation for the National Institutes of Health Biomarker Consortium was a nested case-control study on 600 knees from 600 Osteoarthritis Initiative participants. Progressor knees had both medial tibiofemoral radiographic joint space width (JSW) loss (≥0.7 mm) and a persistent increase in Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain score (≥9 on a 0-100 scale) at 24-48 months from baseline (n = 194). Multiecho spin-echo (MESE) magnetic resonance images (MRIs) for cartilage T2 analysis had been acquired in the right knees only (97 progressor knees). These were compared to 104 control knees without JSW or pain progression. Fifty-three knees had JSW progression, and 57 pain progression only. Cartilage thickness segmentations obtained from double-echo steady-state MRI were matched to MESE MRI to extract superficial and deep femorotibial cartilage T2. Superficial medial femorotibial compartment (MFTC) T2 at baseline was the primary, and change in deep MFTC T2 between baseline and 12 months was the secondary analytic outcome of this post hoc exploratory study., Results: Baseline superficial MFTC T2 was significantly elevated in progressor knees (adjusted mean 47.2 msec [95% confidence interval (95% CI) 46.5, 48.0]) and JSW progression only knees (adjusted mean 47.3 msec [95% CI 46.3, 48.3]), respectively, versus non-progressor knees (45.8 msec [95% CI 45.0, 46.5]) after adjustment for age, sex, body mass index, WOMAC pain score, and medial joint space narrowing grade (analysis of covariance). Change in T2 was not significantly associated with case status., Conclusion: Baseline superficial, but not deep, medial cartilage T2 is associated with clinically relevant disease progression in knee OA., (© 2021 The Authors. Arthritis Care & Research published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

Background: Patients with ankylosing spondylitis (AS) have significantly lower quality of life (QoL) than the general population. Holistic interventions addressing QoL comprise spa- or balneotherapy including radon. These interventions have shown to be beneficial in reducing pain and improving QoL in AS-patients. We explored the association of spa-therapy including low-dose radon with QoL in AS-patients over an extended time period., Methods: Registry data collected for the "Radon indication registry" in the Austrian Gastein valley comprising data on QoL (EuroQol EQ-5D) directly before the treatment (baseline), directly(t1), 3 (t2); 6(t3) and 9(t4) months after the treatment, age, sex and body mass index (BMI) were analysed. Linear regression models explored the association of measurement time with 1) EQ-5D-5L utilities and 2) EuroQol visual analogue scale (VAS) score. Alterations of 0.05 (utilities) and 5.00 (VAS) were considered clinically relevant., Results: Two-hundred-ninety-one AS-patients were included in the analyses. Forty-four percent (n = 128) were women, the mean age was 52 (SD 10) and the average BMI was 26 (SD 4). Utilities (t1: 0.09 [0.07;0.11]; t2: 0.08 [0.06; 0.10]; t3: 0.06 [0.05;0.09]; t4: 0.04 [0.02;0.06]) and VAS (t1: 11.68 [9.38; 13.97]; t2: 12.20 [9.78; 14.61]; t3: 9.70 [7.24; 12.17]; t4: 6.11 [3.57; 8.65]) were significantly higher at all timepoints compared to baseline. Improvements were clinically relevant at all timepoints in case of the VAS and until 6 months after treatment for the utilities., Conclusion: AS-patients who received spa therapy including radon show significantly and clinically relevant improvements in Qol until 6-9 months after treatment., (© 2022. The Author(s).)

Objective: To study the longitudinal performance of fully automated cartilage segmentation in knees with radiographic osteoarthritis (OA), we evaluated the sensitivity to change in progressor knees from the Foundation for the National Institutes of Health OA Biomarkers Consortium between the automated and previously reported manual expert segmentation, and we determined whether differences in progression rates between predefined cohorts can be detected by the fully automated approach., Methods: The OA Initiative Biomarker Consortium was a nested case-control study. Progressor knees had both medial tibiofemoral radiographic joint space width loss (≥0.7 mm) and a persistent increase in Western Ontario and McMaster Universities Osteoarthritis Index pain scores (≥9 on a 0-100 scale) after 2 years from baseline (n = 194), whereas non-progressor knees did not have either of both (n = 200). Deep-learning automated algorithms trained on radiographic OA knees or knees of a healthy reference cohort (HRC) were used to automatically segment medial femorotibial compartment (MFTC) and lateral femorotibial cartilage on baseline and 2-year follow-up magnetic resonance imaging. Findings were compared with previously published manual expert segmentation., Results: The mean ± SD MFTC cartilage loss in the progressor cohort was -181 ± 245 μm by manual segmentation (standardized response mean [SRM] -0.74), -144 ± 200 μm by the radiographic OA-based model (SRM -0.72), and -69 ± 231 μm by HRC-based model segmentation (SRM -0.30). Cohen's d for rates of progression between progressor versus the non-progressor cohort was -0.84 (P < 0.001) for manual, -0.68 (P < 0.001) for the automated radiographic OA model, and -0.14 (P = 0.18) for automated HRC model segmentation., Conclusion: A fully automated deep-learning segmentation approach not only displays similar sensitivity to change of longitudinal cartilage thickness loss in knee OA as did manual expert segmentation but also effectively differentiates longitudinal rates of loss of cartilage thickness between cohorts with different progression profiles., (© 2020 The Authors. Arthritis Care & Research published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

Background: The present study aimed to predict the expected number of patients with osteoarthritis (OA) in Austria up to the year 2080., Methods: Demographic data and population projections between 2019 and 2080 were obtained from European authorities. Information about recent age- and sex-stratified prevalence of patients with self-reported physician-diagnosed OA was obtained from the Austrian Health Interview Survey (n = 15,771). Projections were stratified by age and sex; sensitivity analyses were performed based on aging, main (most likely), and growth scenarios of the population., Results: Based on the projection, the overall increase in the total number of patients with OA from 2019 to 2080 will be 38% for men and women. In 2019, the highest number of OA-patients nested in the groups of persons aged 70-79 (n = 238,749) and 60-69 (n = 237,729) years. In 2080, the 80+ age group is predicted to have the highest number of OA with 421,548 individuals (i.e. factor 3.45 and factor 2.48 increase in the male and female group, respectively, compared to 2019), followed by the group aged 70-79 with 314,617 individuals (factor 1.45 and factor 1.28 increase in the male and female group, respectively, compared to 2019). Similar trends were found in the ageing and growing scenarios., Conclusions: The projected increase in the occurrence of OA will likely lead to a substantial socioeconomic burden for the Austrian healthcare system in the near and far future. The current findings plead for the development of sustainable concepts for the treatment and prevention of OA by European authorities., (© 2022. The Author(s).)

In a variety of physiological and pathophysiological conditions, cells are exposed to acidic environments. Severe synovial fluid acidification also occurs in a progressive state of osteoarthritis (OA) affecting articular chondrocytes. In prior studies extracellular acidification has been shown to protect cells from apoptosis but the underlying mechanisms remain elusive. In the present study, we demonstrate that the inhibition of Cl - currents plays a significant role in the antiapoptotic effect of acidification in human articular chondrocytes. Drug-induced apoptosis was analyzed after exposure to staurosporine by caspase 3/7 activity and by annexin-V/7-actinomycin D (7-AAD) staining, followed by flow cytometry. Cell viability was assessed by resazurin, CellTiter-Glo and CellTiter-Fluor assays. Cl - currents and the mean cell volume were determined using the whole cell patch clamp technique and the Coulter method, respectively. The results reveal that in C28/I2 cells extracellular acidification decreases caspase 3/7 activity, enhances cell viability following staurosporine treatment and gradually deactivates the volume-sensitive outwardly rectifying (VSOR) Cl - current. Furthermore, the regulatory volume decrease (RVD) as well as the apoptotic volume decrease (ADV), which represents an early event during apoptosis, were absent under acidic conditions after hypotonicity-induced cell swelling and staurosporine-induced apoptosis, respectively. Like acidosis, the VSOR Cl - current inhibitor DIDS rescued chondrocytes from apoptotic cell death and suppressed AVD after induction of apoptosis with staurosporine. Similar to acidosis and DIDS, the VSOR channel blockers NPPB, niflumic acid (NFA) and DCPIB attenuated the staurosporine-induced AVD. NPPB and NFA also suppressed staurosporine-induced caspase 3/7 activation, while DCPIB and Tamoxifen showed cytotoxic effects per se . From these data, we conclude that the deactivation of VSOR Cl - currents impairs cell volume regulation under acidic conditions, which is likely to play an important role in the survivability of human articular chondrocytes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kittl, Winklmayr, Preishuber-Pflügl, Strobl, Gaisberger, Ritter and Jakab.)

Multimodal therapies comprising spa applications are widely used as non-pharmaceutical treatment options for musculoskeletal diseases. The purpose of this randomized, controlled, open pilot study was to elucidate the involvement of the endocannabinoid system in a multimodal therapy approach. Twenty-five elderly patients with knee osteoarthritis (OA) received a 2-week spa therapy with or without combination of low-dose radon therapy in the Bad Gastein radon gallery. A 10-point numerical rating scale (pain in motion and at rest), WOMAC questionnaire, and the EuroQol-5D (EQ-5D) questionnaire were recorded at baseline, and during treatment period at weeks one and two, and at 3-month and 6-month follow-ups. Plasma levels of the endocannabinoid anandamide (AEA) were determined at baseline and at 2 weeks, and serum levels of several cartilage metabolism markers at all five time-points. A significant and sustained reduction of self-reported knee pain was observed in the study population, but no further significant effect of the additional radon therapy up and above base therapy. This pain reduction was accompanied by a significant reduction of AEA plasma levels during treatment in both groups. No significant differences were seen in serum marker concentrations between the groups treated with or without radon, but a small reduction of serum cartilage degradation markers was observed during treatment in both groups. This is the first study investigating AEA levels in the context of a non-pharmacological OA treatment. Since the endocannabinoid system represents a potential target for the development of new therapeutics, further studies will have to elucidate its involvement in OA pain.

Background: Short-term, indoor exposure to environmental tobacco smoke (ETS) is still highly prevalent; however, little is known about the acute lung response in adult asthma., Objectives: We investigated whether acute, experimental ETS exposure influences symptoms, lung function, and inflammatory parameters., Methods: Human subjects with asthma (n = 23) were exposed for 180 min to either room air or ETS at 250, 450, or 850 µg/m3. Respiratory symptoms, lung function, and exhaled nitric oxide (FeNO) were measured. Additionally, blood samples were analyzed for pro- and anti-inflammatory cytokines., Results: Humans with asthma demonstrate an increase in respiratory symptoms at all levels of ETS exposure, while the forced expiratory volume in 1 s (FEV1) and FeNO decrease with increasing ETS. The anti-inflammatory cytokine interleukin (IL)-10 increases at intermediate ETS concentrations, whereas tumor necrosis factor (TNF)-α and IL-8 increase only at the highest ETS concentration., Conclusion: Following 180 min of acute, experimental ETS exposure, we observed a significant increase in respiratory symptoms, a decrease in lung function, and an increase in inflammatory cytokines, indicating an acute lung response in asthma., (© 2020 S. Karger AG, Basel.)

Chondrocytes face extreme alterations of extracellular osmolarity and pH, which force them to appropriately regulate their cell volume (CV) and cellular pH. Perturbations of these mechanisms lead to chondrocyte death and ultimately to osteoarthritis (OA), the most common chronic joint diseases worldwide. OA hallmarks are altered cartilage hydration and severe fluid acidification. Impaired CV regulation and acidotoxicity contribute to disease progression and volume-sensitive anion channels are upregulated in OA. This study assessed the effect of hypotonicity and extracellular acidification on chondrocyte Cl - conductances and CV regulation. Cl - currents and membrane potentials were measured in human C28/I2 cells and primary human chondrocytes using the patch clamp technique. Intracellular pH was assessed by BCECF fluorescence, CV measurements were performed using the Coulter method, and cell viability/cell death by a resazurin assay. Hypotonic cell swelling caused activation of a volume-sensitive outwardly rectifying (VSOR) Cl - current followed by a regulatory volume decrease (RVD), which was attenuated by the Cl - channel blocker DCPIB. Extracellular, but not intracellular acidification to pH ≤ 5.0 elicited an acid-sensitive outwardly rectifying (ASOR) Cl - conductance. Activation of either current depolarized the cell membrane potential. Under simultaneous hypotonic and acidic stimulation, VSOR and ASOR currents transiently coactivated, giving rise to a mixed current phenotype. Over time the VSOR current gradually vanished and the residual conductance showed a pure ASOR current phenotype. Extracellular acidification caused an isotonic CV gain and a complete suppression of RVD under hypotonic conditions. The results suggest that deactivation of the VSOR current under acidic conditions impairs CV regulation in chondrocytes, which is likely to compromise chondrocyte viability., (Copyright © 2020 Kittl, Winklmayr, Helm, Lettner, Gaisberger, Ritter and Jakab.)

Introduction: Patient education is an important part of the management of rheumatic and musculoskeletal diseases. Given that patients with diverse diseases do not have the same needs, it is crucial to assess the educational requirements of targeted groups to provide tailored educational interventions. The aim of our study was to assess educational needs of a large cohort of patients with different rheumatic and musculoskeletal diseases attending a health facility in Austria., Methods: We assessed educational needs, via an online survey of patients with fibromyalgia (FMS), rheumatoid arthritis (RA), and ankylosing spondylitis (AS) recruited from an Austrian health-care facility, using the Austrian version of the Educational Needs Assessment Tool (OENAT)., Results: For our sample of 603 patients, AS (62%), RA (15%), and FMS (24%), there were no educational need differences for the domains of movements, disease process, and self-help measures. Patients with FMS had less need for pain management education and greater need for education about feelings, than other disease groups. Patients with RA had a greater need for education related to treatments than other groups, and patients with AS had a greater need for treatment education than patients with FMS. Patients with AS reported greater need for support system education than other patient groups., Conclusion: Educational needs vary by disease groups, suggesting that health-care professionals should assess disease-specific needs for education to provide optimal assistance in disease management for patients., (© 2020 John Wiley & Sons, Ltd.)

The term long non-coding RNA (lncRNA) describes non protein-coding transcripts with a length greater than 200 base pairs. The ongoing discovery, characterization and functional categorization of lncRNAs has led to a better understanding of the involvement of lncRNAs in diverse biological and pathological processes including cancer. Aberrant expression of specific lncRNA species was demonstrated in various cancer types and associated with unfavorable clinical characteristics. Recent studies suggest that lncRNAs are also involved in the development and progression of biliary tract cancer, a rare disease with high mortality and limited therapeutic options. In this review, we summarize current findings regarding the manifold roles of lncRNAs in biliary tract cancer and give an overview of the clinical and molecular consequences of aberrant lncRNA expression as well as of underlying regulatory functions of selected lncRNA species in the context of biliary tract cancer.

Microglia are first-line defense antigen-presenting phagocytes in the central nervous system. Activated microglial cells release pro-inflammatory cytokines and can trigger an oxidative burst. The amino acid glycine exerts anti-inflammatory, immunomodulatory and cytoprotective effects and influences cell volume regulation. This study aimed to investigate the role of glycine in the modulation of inflammatory processes in mouse BV-2 microglial cells. Inflammatory stress was induced by lipopolysaccharide/interferon-γ (LPS/IFN-γ) treatment for 24 h in the absence or presence of 1 or 5 mM glycine. Cells were analyzed by flow cytometry for cell volume, side scatter, apoptosis/necrosis and expression of activation-specific surface markers. Apoptosis progression was monitored by life cell imaging. Reduced glutathione/oxidized glutathione (GSH/GSSG) ratios and release of the pro-inflammatory cytokines IL-6 and TNF-α were measured using luminescence-based assays and ELISA, respectively. We found that LPS/IFN-γ-induced apoptosis was decreased and the fraction of living cells was increased by glycine. Expression of the surface markers CD11b, CD54 and CD80 was dose-dependently increased, while IL-6 and TNF-α release was not altered compared to LPS/IFN-γ-treated cells. We showed that in BV-2 microglial cells glycine improves viability and counteracts deleterious responses to LPS/IFN-γ, which might be relevant in neurodegenerative processes associated with inflammation, like Alzheimer's or Parkinson's disease., Competing Interests: The authors declare no conflict of interest.

The SARS-CoV‑2 has infected millions of humans worldwide in the past few months and hundreds of thousands have died as a result of an infection. The end of the pandemic is not in sight and many people are anxious of becoming infected in different settings. The Gastein Healing Gallery (GHG) is a unique outpatient facility combining heat, high humidity and mild radon radiation. Every year approximately 12,000 patients with inflammatory rheumatic, degenerative diseases and chronic pain are treated. We have therefore reviewed and analyzed the literature with respect to a possible increased risk of infection for patients during treatment in the GHG. On the one hand the climatic and physical conditions in the GHG can be viewed as hostile to viruses and on the other hand the mild radon hyperthermia and the geographic location of the GHG lead to positive effects on the patient's health via complex physiological processes. We therefore consider the likelihood of infection with viruses in the GHG in no way increased, in contrast, it is probably considerably lower compared to other settings., (© The Author(s) 2020.)

Cannabidiol (CBD) is the most abundant non-psychoactive compound of Cannabis sativa extracts. Cannabinoids have been shown to exhibit anti-inflammatory, analgesic, antioxidant, neuroprotective, and anti-tumorigenic effects. In the present study, we investigated the effects of CBD on human articular chondrocytes. Cell viability was determined by Resazurin assays. Apoptosis was analyzed by annexin-V/7-actinomycin D (7-AAD) staining followed by flow cytometry. Caspase 3/7 activity was measured with caspase assays. Intracellular Ca 2+ ([Ca 2+ ] i ) was monitored by time-lapse fluorescence imaging. The perforated whole-cell patch-clamp technique was used for measuring the cell membrane potential. Erk1/2 phosphorylation was assessed by western blot analysis. The chondrocyte cell line C28/I2 and primary chondrocytes showed a reduced viability after treatment with concentrations of CBD greater than 4 µM. This apoptotic effect was accompanied by an increase of caspase 3/7 activity and an increase in the early apoptotic cell population. CBD elevated [Ca 2+ ] i , which was accompanied by depolarization of the cell membrane potential. The increase of [Ca 2+ ] i was abrogated, when Ca 2+ was omitted from the bath solution, indicating an influx of extracellular Ca 2+ . The cannabinoid receptor 1 (CB1) antagonist AM251 inhibited the Ca 2+ influx triggered by CBD. Preincubation with AM251 reduced the toxic effects of CBD. By looking for mediators of the apoptotic CBD effect downstream of the CB1 receptor, enhanced Erk1/2 phosphorylation could be detected after CBD treatment. However, this Erk1/2 activation proved to be unaffected by CB1 receptor blockage. The present study demonstrates that CBD promotes apoptosis and [Ca 2+ ] i elevation in human articular chondrocytes via a CB1-receptor-mediated mechanism. © 2019 The Authors. Journal of Orthopaedic Research ® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society J Orthop Res 37:2540-2549, 2019., (© 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society.)

Cellular migration is essential in diverse physiological and pathophysiological processes. Here, we present a protocol for quantitative analysis of migration using confluence detection allowing continuous, non-endpoint measurement with minimal hands-on time under cell incubator conditions. Applicability was tested using substances which enhance (EGF) or inhibit (cytochalasin D, ouabain) migration. Using a gap-closure assay we demonstrate that automated confluence detection monitors cellular migration in the 96-well microplate format. Quantification by % confluence, % cell free-area or % confluence in cell-free area against time, allows detailed analysis of cellular migration. The study describes a practicable approach for continuous, non-endpoint measurement of migration in 96-well microplates and for detailed data analysis, which allows for medium/high-throughput analysis of cellular migration in vitro.

The transfer of radon from thermal water via the skin to different human organs in radon therapy can experimentally be determined by measuring the radon activity concentration in the exhaled air. In this study, six volunteers were exposed to radon-rich thermal water in a bathtub, comprising eleven measurements. Exhaled activity concentrations were measured intermittently during the 20 min bathing and 20 min resting phases. Upon entering the bathtub, the radon activity concentration in the exhaled breath increased almost linearly with time, reaching its maximum value at the end of the exposure, and then decreased exponentially with time in the subsequent resting phase. Although for all individuals the time-dependence of exhaled radon activity was similar during bathing and resting, significant inter-subject variations could be observed, which may be attributed to individual respiratory parameters and body characteristics. The simulation of the transport of radon through the skin, its distribution among the organs, and the subsequent exhalation via the lungs were based on the biokinetic model of Leggett and co-workers, extended by a skin and a subcutaneous fat compartment. The coupled linear differential equations describing the radon activity concentrations in different organs as a function of time were solved numerically with the program package Mathcad. An agreement between model simulations and experimental results could only be achieved by expressing the skin permeability coefficient and the arterial blood flow rates as a function of the water temperature and the swelling of the skin.

Many cell types express an acid-sensitive outwardly rectifying (ASOR) anion current of an unknown function. We characterized such a current in BV-2 microglial cells and then studied its interrelation with the volume-sensitive outwardly rectifying (VSOR) Cl - current and the effect of acidosis on cell volume regulation. We used patch clamp, the Coulter method, and the pH-sensitive dye BCECF to measure Cl - currents and cell membrane potentials, mean cell volume, and intracellular pH, respectively. The ASOR current activated at pH ≤ 5.0 and displayed an I - > Cl - > gluconate - permeability sequence. When compared to the VSOR current, it was similarly sensitive to DIDS, but less sensitive to DCPIB, and insensitive to tamoxifen. Under acidic conditions, the ASOR current was the dominating Cl - conductance, while the VSOR current was apparently inactivated. Acidification caused cell swelling under isotonic conditions and prevented the regulatory volume decrease under hypotonicity. We conclude that acidification, associated with activation of the ASOR- and inactivation of the VSOR current, massively impairs cell volume homeostasis. ASOR current activation could affect microglial function under acidotoxic conditions, since acidosis is a hallmark of pathophysiological events like inflammation, stroke or ischemia and migration and phagocytosis in microglial cells are closely related to cell volume regulation.

Biliary tract cancer is a devastating disease with limited therapeutic options. The involvement of cancer stem cells in biliary tract cancer is likely. Napabucasin is a previously described cancer stem cell inhibitor that is currently being used in clinical trials. However, data regarding napabucasin and biliary tract cancer are not available yet. We tested the general cytotoxic effect of napabucasin on a comprehensive biliary tract cancer in vitro model, using resazurin assay and Annexin V/7-AAD staining. The effect of napabucasin on functional cancer stem cell characteristics was analyzed via soft agar assay, aldehyde-dehydrogenase-1 assay, measurement of surface CD326 expression, and measurement of clonogenic growth. The evaluation of the effect of napabucasin on cancer stem cell protein and gene expression was performed using Western blot and reverse transcription-PCR-based human cancer stem cell array. Napabucasin showed a concentration- and cell line-dependent cytotoxic effect, and increased the apoptotic and necrotic cell fractions. Treatment with napabucasin significantly reduced the formation of tumor spheres and clonogenic growth, as well as CD326 surface expression. Expression of cancer stem cell markers were reduced following napabucasin treatment on the protein and mRNA levels. Our study provides first data regarding napabucasin as a promising substance for the treatment of biliary tract cancer.

Histone deacetylases (HDACs) play a key role in epigenetic mechanisms in health and disease and their dysfunction is implied in several cancer entities. Analysis of expression patterns in pancreatic neuroendocrine tumors (pNETs) indicated HDAC5 to be a potential target for future therapies. As a first step towards a possible treatment, the aim of this study was to evaluate the in vitro cellular and molecular effects of HDAC5 inhibition in pNET cells. Two pNET cell lines, BON-1 and QGP-1, were incubated with different concentrations of the selective class IIA HDAC inhibitor, LMK-235. Effects on cell viability were determined using the resazurin-assay, the caspase-assay, and Annexin-V staining. Western Blot and immunofluorescence microscopy were performed to assess the effects on HDAC5 functionality. LMK-235 lowered overall cell viability by inducing apoptosis in a dose- and time-dependent manner. Furthermore, acetylation of histone-H3 increased with higher LMK-235 concentrations, indicating functional inhibition of HDAC4/5. Immunocytochemical analysis showed that proliferative activity (phosphohistone H3 and Ki-67) decreased at highest concentrations of LMK-235 while chromogranin and somatostatin receptor 2 (SSTR2) expression increased in a dose-dependent manner. HDAC5 expression was found to be largely unaffected by LMK-235. These findings indicate LMK-235 to be a potential therapeutic approach for the development of an effective and selective pNET treatment.

The clonogenic assay is a widely used method to study the ability of cells to 'infinitely' produce progeny and is, therefore, used as a tool in tumor biology to measure tumor-initiating capacity and stem cell status. However, the standard protocol of using 6-well plates has several disadvantages. By miniaturizing the assay to a 96-well microplate format, as well as by utilizing the confluence detection function of a multimode reader, we here describe a new and modified protocol that allows comprehensive experimental setups and a non-endpoint, label-free semi-automatic analysis. Comparison of bright field images with confluence images demonstrated robust and reproducible detection of clones by the confluence detection function. Moreover, time-resolved non-endpoint confluence measurement of the same well showed that semi-automatic analysis was suitable for determining the mean size and colony number. By treating cells with an inhibitor of clonogenic growth (PTC-209), we show that our modified protocol is suitable for comprehensive (broad concentration range, addition of technical replicates) concentration- and time-resolved analysis of the effect of substances or treatments on clonogenic growth. In summary, this protocol represents a time- and cost-effective alternative to the commonly used 6-well protocol (with endpoint staining) and also provides additional information about the kinetics of clonogenic growth., Competing Interests: The authors declare no conflict of interest.

Background/aims: The neutral, non-essential amino acid glycine has manifold functions and effects under physiological and pathophysiological conditions. Besides its function as a neurotransmitter in the central nervous system, glycine also exerts immunomodulatory effects and as an osmolyte it participates in cell volume regulation. During phagocytosis, glycine contributes to (local) cell volume-dependent processes like lamellipodium formation. Similar to the expansion of the lamellipodium we assume that glycine also affects the migration of microglial cells in a cell volume-dependent manner., Methods: Mean cell volume (MCV) and cell migration were determined using flow cytometry and trans-well migration assays, respectively. Electrophysiological recordings of the cell membrane potential (Vmem) and swelling-dependent chloride (Cl-) currents (IClswell, VSOR, VRAC) were performed using the whole-cell patch clamp technique., Results: In the murine microglial cell line BV-2, flow cytometry analysis revealed that glycine (5 mM) increases the MCV by ∼9%. The glycine-dependent increase in MCV was suppressed by the partial sodium-dependent neutral amino acid transporter (SNAT) antagonist MeAIB and augmented by the Cl- current blocker DCPIB. Electrophysiological recordings showed that addition of glycine activates a Cl- current under isotonic conditions resembling features of the swelling-activated Cl- current (IClswell). The cell membrane potential (Vmem) displayed a distinctive time course after glycine application; initially, glycine evoked a rapid depolarization mediated by Na+-coupled glycine uptake via SNAT, followed by a further gradual depolarization, which was fully suppressed by DCPIB. Interestingly, glycine significantly increased migration of BV-2 cells, which was suppressed by MeAIB, suggesting that SNAT is involved in the migration process of microglial cells., Conclusion: We conclude that glycine acts as a chemoattractant for microglial cells presumably by a cell volume-dependent mechanism involving SNAT-mediated cell swelling., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

In the Gastein valley, numerous facilities use radon for the treatment of various diseases either by exposure to radon in air or in radon rich thermal water. In this study, six test persons were exposed to radon thermal water in a bathtub and the time-dependent radon activity concentration in the exhaled air was recorded. At temperatures between 38°C and 40°C, the radon activity concentration in the water was about 900 kBq/m3 in a total volume of 600 l, where the patients were exposed for 20 min, while continuously sampling the exhaled air during the bathing and 20 min thereafter. After entering the bath, the exhaled radon activity concentration rapidly increased, reaching some kind of saturation after 20 min exposure. The radon activity concentration in the exhaled air was about 8000 Bq/m3 at the maximum, with higher concentrations for male test persons. The total radon transfer from water to the exhaled air was between 480 and 1000 Bq, which is equivalent to 0.08% and 0.2% of the radon in the water., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: The specific microclimate of alpine waterfalls with high levels of ionized water aerosols has been suggested to trigger beneficial immunological and psychological effects. In the present three-armed randomized controlled clinical study, we focused on effects on (i) immunological reagibility, on (ii) physiological stress responses, and on (iii) stress-related psychological parameters., Methods: People with moderate to high stress levels (n = 65) spent an active sojourn with daily hiking tours in the National Park Hohe Tauern (Großkirchheim, Austria). Half of the group was exposed to water aerosol of an alpine waterfall for 1 h/day (first arm, n = 33), whereas the other half spent the same time at a distant site (second arm, n = 32). A third arm (control, n = 26) had no intervention (except vaccination) and stayed at home, maintaining their usual lifestyle. The effect of the interventions on the immune system was tested by oral vaccination with an approved cholera vaccine and measuring specific salivary IgA antibody titers. Lung function was determined by peak expiratory flow measurement. Electric skin conductance, heart rate, and adaption of respiration rate were assessed as physiological stress parameters. Psychological stress-related parameters were analyzed by questionnaires and scales., Results: Compared to the control group, both intervention groups showed improvement of the lung function and of most physiological stress test parameters. Analysis of the mucosal immune response revealed a waterfall-specific beneficial effect with elevated IgA titers in the waterfall group. In line with these results, exposure to waterfall revealed an additional benefit concerning psychological parameters such as subjective stress perception (measured via visual analog scale), the Global Severity Index (GSI), and the Positive Symptom Total (PST)., Conclusions: Our study provides new data, which strongly support an "added value" of exposure to waterfall microclimate when combined with a therapeutic sojourn at high altitude including regular physical activity.

Background/aims: Glucose-stimulated insulin secretion (GSIS) of pancreatic β-cells involves glucose uptake and metabolism, closure of KATP channels and depolarization of the cell membrane potential (Vmem), activation of voltage-activated Ca2+ currents (ICav) and influx of Ca2+, which eventually triggers hormone exocytosis. Beside this classical pathway, KATP-independent mechanisms such as changes in intracellular pH (pHi) or cell volume, which also affect β-cell viability, can elicit or modify insulin release. In β-cells the regulation of pHi is mainly accomplished by Na+/H+ exchangers (NHEs). To investigate if other proton extrusion mechanisms than NHEs are involved in pH regulation, we tested for the presence of the non-gastric H+/K+ ATPase in rat insulinoma cells and assessed effects of the H+/K+ ATPase inhibitor SCH-28080 on insulin secretion, cell viability and apoptosis., Methods: In INS-1E cell cultures, H+/K+ ATPase gene and protein expression was analyzed by reverse transcription PCR and Western blotting. Intracellular pH (pHi) recovery after acute acidic load was measured by NH4Cl prepulsing using BCECF. Insulin secretion was determined by ELISA from the cell culture supernatant. Vmem, K+ and Ca2+ currents were recorded using patch clamp. Overall cell responses were determined using resazurin (viability) and cytotoxicity assays. The mean cell volume (MCV), cell granularity (side-scatter; SSC), phosphatidylserine (PS) exposure, cell membrane integrity, caspase activity and the mitochondrial membrane potential (ΔΨm) were measured by flow cytometry., Results: We found that the α-subunit of the non-gastric H+/K+ ATPase (HKα2) is expressed on mRNA and protein level. However, compared to rat colon tissue, in INS-1E cells mRNA abundance was very low. In NH4Cl prepulsing experiments no K+-dependent pHi recovery was observed under Na+-free extracellular conditions. Nonetheless within 1 h, 20 µM SCH-28080 inhibited GSIS by ∼50%, while basal release was unaffected. The L-type ICav blocker nifedipine caused a full inhibition of GSIS at 10 and 20 µM. At 20 µM, SCH-28080 inhibited ICav comparable to 20 µM nifedipine and in addition augmented IKATP recorded at -60 mV and hyperpolarized Vmem by ∼15 mV. Cell viability 2 and 24 h post treatment with SCH-28080 was dose-dependently inhibited with IC50 values of 22.9 µM and 15.3 µM, respectively. At 20 µM the percentages of Annexin-V+, caspase+ and propidium iodide+ cells were significantly increased after 24 and 48 h. Concurrently, the MCV was significantly decreased (apoptotic volume decrease, AVD) and the SSC signal was increased. At concentrations >40-50 µM, SCH-28080 became progressively cytotoxic causing a steep increase in necrotic cells already 2 h post treatment and a breakdown of ΔΨm within 4 h under 50 and 100 µM while 10 and 20 µM had no effect on ΔΨm within 24 h., Conclusion: We demonstrate expression of HKα2 in rat INS-1E cells. However, the pump is apparently non-functional under the given conditions. Nonetheless the H+/K+ ATPase blocker SCH-28080 inhibits insulin secretion and induces cell death. Importantly, we show that SCH-28080 inhibits ICav - and activates KATP channels identifying them as novel "off-targets" of the inhibitor, causing hyperpolarization of Vmem and inhibition of insulin secretion., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

Background/aims: Previously we described insulinotropic effects of Leonurus sibiricus L. plant extracts used for diabetes mellitus treatment in Traditional Mongolian Medicine. The flavonoid quercetin and its glycoside rutin, which exert anti-diabetic properties in vivo by interfering with insulin signaling in peripheral target tissues, are constituents of these extracts. This study was performed to better understand short- and long-term effects of quercetin and rutin on beta-cells., Methods: Cell viability, apoptosis, phospho-protein abundance and insulin release were determined using resazurin, annexin-V binding assays, Western blot and ELISA, respectively. Membrane potentials (Vmem), whole-cell Ca2+ (ICa)- and ATP-sensitive K+ (IKATP) currents were measured by patch clamp. Intracellular Ca2+ (Cai) levels were measured by time-lapse imaging using the ratiometric Ca2+ indicator Fura-2., Results: Rutin, quercetin and the phosphoinositide-3-kinase (PI3K) inhibitor LY294002 caused a dose-dependent reduction in cell viability with IC50 values of ∼75 µM, ∼25 µM and ∼3.5 µM, respectively. Quercetin (50 µM) significantly increased the percentage of Annexin-V+ cells within 48 hrs. The mean cell volume (MCV) of quercetin-treated cells was significantly lower. Within 2 hrs, quercetin significantly decreased basal- and insulin-stimulated Akt(T308) phosphorylation and increased Erk1/2 phosphorylation, without affecting P-Akt(S473) abundance. Basal- and glucose-stimulated insulin release were significantly stimulated by quercetin. Quercetin significantly depolarized Vmem by ∼25 mV which was prevented by the KATP-channel opener diazoxide, but not by the L-type ICa inhibitor nifedipine. Quercetin significantly stimulated ICa and caused a 50% inhibition of IKATP. The effects on Vmem, ICa and IKATP rapidly reached peak values and then gradually diminished to control values within ∼1 minute. With a similar time-response quercetin induced an elevation in Cai which was completely abolished in the absence of Ca2+ in the bath solution. Rutin (50 µM) did not significantly alter the percentage of Annexin-V+ cells, MCV, Akt or Erk1/2 phosphorylation, insulin secretion, or the electrophysiological behavior of INS-1 cells., Conclusion: We conclude that quercetin acutely stimulates insulin release, presumably by transient KATP channel inhibition and ICa stimulation. Long term application of quercetin inhibits cell proliferation and induces apoptosis, most likely by inhibition of PI3K/Akt signaling., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Objective: Ionized water aerosols have been suggested to exert beneficial health effects on pediatric allergic asthma. Their effect was evaluated in a randomized controlled clinical trial as part of a summer asthma camp., Methods: Asthmatic allergic children (n = 54) spent 3 weeks in an alpine asthma camp; half of the group was exposed to water aerosol of an alpine waterfall for 1 hour per day, whereas the other half spent the same time at a "control site". Immunological analysis, lung function testing, and fractional exhaled nitric oxide (FeNO) testing were performed during the stay, and sustaining effects were evaluated 2 months later. Symptom score testing was done over a period of 140 days., Results: The water aerosol group showed a significant improvement in all lung function parameters, whereas only the peak expiratory flow improved in the control group. All patients showed a significant improvement in symptom score and a significant decrease in FeNO after the camp. Only the water aerosol group exhibited a long-lasting effect on asthma symptoms, lung function, and inflammation in the follow-up examination. Induction of interleukin (IL)-10 and regulatory T (Treg) cells was measured in both groups, with a pronounced increase in the water aerosol group. IL-13 was significantly decreased in both groups, whereas IL-5 and eosinophil cationic protein were decreased only in the water aerosol group., Conclusions: Our findings confirm the induction of Treg cells and reduction in inflammation by climate therapy. They indicate a synergistic effect of water aerosols resulting in a long-lasting beneficial effect on asthma symptoms, lung function, and airway inflammation.

Chromatin modifications, such as reversible histone acetylation, play a key role in the regulation of T cell development and function. However, the role of individual histone deacetylases (HDACs) in T cells is less well understood. In this article, we show by conditional gene targeting that T cell-specific loss of HDAC1 led to an increased inflammatory response in an in vivo allergic airway inflammation model. Mice with HDAC1-deficient T cells displayed an increase in all critical parameters in this Th2-type asthma model, such as eosinophil recruitment into the lung, mucus hypersecretion, parenchymal lung inflammation, and enhanced airway resistance. This correlated with enhanced Th2 cytokine production in HDAC1-deficient T cells isolated from diseased mice. In vitro-polarized HDAC1-deficient Th2 cells showed a similar enhancement of IL-4 expression, which was evident already at day 3 of Th2 differentiation cultures and restricted to T cell subsets that underwent several rounds of cell divisions. HDAC1 was recruited to the Il4 gene locus in ex vivo isolated nonstimulated CD4(+) T cells, indicating a direct control of the Il4 gene locus. Our data provide genetic evidence that HDAC1 is an essential HDAC that controls the magnitude of an inflammatory response by modulating cytokine expression in effector T cells.