Enhancing pre-clinical research with simplified intestinal cell line models.

Two-dimensional culture remains widely employed to determine the bioavailability of orally delivered drugs. To gain more knowledge about drug uptake mechanisms and risk assessment for the patient after oral drug admission, intestinal in vitro models demonstrating a closer similarity to the in vivo situation are needed. In particular, Caco-2 cell-based Transwell® models show advantages as they are reproducible, cost-efficient, and standardized. However, cellular complexity is impaired and cell function is strongly modified as important transporters in the apical membrane are missing. To overcome these limitations, primary organoid-based human small intestinal tissue models were developed recently but the application of these cultures in pre-clinical research still represents an enormous challenge, as culture setup is complex as well as time- and cost-intensive. To overcome these hurdles, we demonstrate the establishment of primary organoid-derived intestinal cell lines by immortalization. Besides exhibiting cellular diversity of the organoid, these immortalized cell lines enable a standardized and more cost-efficient culture. Further, our cell line-based Transwell®-like models display an organ-specific epithelial barrier integrity, ultrastructural features and representative transport functions. Altogether, our novel model systems are cost-efficient with close similarity to the in vivo situation, therefore favoring their use in bioavailability studies in the context of pre-clinical screenings.

The Path from Nasal Tissue to Nasal Mucosa on Chip: Part 2-Advanced Microfluidic Nasal In Vitro Model for Drug Absorption Testing.

The nasal mucosa, being accessible and highly vascularized, opens up new opportunities for the systemic administration of drugs. However, there are several protective functions like the mucociliary clearance, a physiological barrier which represents is a difficult obstacle for drug candidates to overcome. For this reason, effective testing procedures are required in the preclinical phase of pharmaceutical development. Based on a recently reported immortalized porcine nasal epithelial cell line, we developed a test platform based on a tissue-compatible microfluidic chip. In this study, a biomimetic glass chip, which was equipped with a controlled bidirectional airflow to induce a physiologically relevant wall shear stress on the epithelial cell layer, was microfabricated. By developing a membrane transfer technique, the epithelial cell layer could be pre-cultivated in a static holder prior to cultivation in a microfluidic environment. The dynamic cultivation within the chip showed a homogenous distribution of the mucus film on top of the cell layer and a significant increase in cilia formation compared to the static cultivation condition. In addition, the recording of the ciliary transport mechanism by microparticle image velocimetry was successful. Using FITC-dextran 4000 as an example, it was shown that this nasal mucosa on a chip is suitable for permeation studies. The obtained permeation coefficient was in the range of values determined by means of other established in vitro and in vivo models. This novel nasal mucosa on chip could, in future, be automated and used as a substitute for animal testing.

Comparison of Tumour-Specific Phenotypes in Human Primary and Expandable Pancreatic Cancer Cell Lines.

There is an ongoing need for patient-specific chemotherapy for pancreatic cancer. Tumour cells isolated from human tissues can be used to predict patients' response to chemotherapy. However, the isolation and maintenance of pancreatic cancer cells is challenging because these cells become highly vulnerable after losing the tumour microenvironment. Therefore, we investigated whether the cells retained their original characteristics after lentiviral transfection and expansion. Three human primary pancreatic cancer cell lines were lentivirally transduced to create expandable (Ex) cells which were then compared with primary (Pri) cells. No obvious differences in the morphology or epithelial-mesenchymal transition (EMT) were observed between the primary and expandable cell lines. The two expandable cell lines showed higher proliferation rates in the 2D and 3D models. All three expandable cell lines showed attenuated migratory ability. Differences in gene expression between primary and expandable cell lines were then compared using RNA-Seq data. Potential target drugs were predicted by differentially expressed genes (DEGs), and differentially expressed pathways (DEPs) related to tumour-specific characteristics such as proliferation, migration, EMT, drug resistance, and reactive oxygen species (ROS) were investigated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. We found that the two expandable cell lines expressed similar chemosensitivity and redox-regulatory capability to gemcitabine and oxaliplatin in the 2D model as compared to their counterparts. In conclusion, we successfully generated expandable primary pancreatic cancer cell lines using lentiviral transduction. These expandable cells not only retain some tumour-specific biological traits of primary cells but also show an ongoing proliferative capacity, thereby yielding sufficient material for drug response assays, which may provide a patient-specific platform for chemotherapy drug screening.

The Path from Nasal Tissue to Nasal Mucosa on Chip: Part 1-Establishing a Nasal In Vitro Model for Drug Delivery Testing Based on a Novel Cell Line.

In recent years, there has been a significant increase in the registration of drugs for nasal application with systemic effects. Previous preclinical in vitro test systems for transmucosal drug absorption studies have mostly been based on primary cells or on tumor cell lines such as RPMI 2650, but both approaches have disadvantages. Therefore, the aim of this study was to establish and characterize a novel immortalized nasal epithelial cell line as the basis for an improved 3D cell culture model of the nasal mucosa. First, porcine primary cells were isolated and transfected. The P1 cell line obtained from this process was characterized in terms of its expression of tissue-specific properties, namely, mucus expression, cilia formation, and epithelial barrier formation. Using air-liquid interface cultivation, it was possible to achieve both high mucus formation and the development of functional cilia. Epithelial integrity was expressed as both transepithelial electrical resistance and mucosal permeability, which was determined for sodium fluorescein, rhodamine B, and FITC-dextran 4000. We noted a high comparability of the novel cell culture model with native excised nasal mucosa in terms of these measures. Thus, this novel cell line seems to offer a promising approach for developing 3D nasal mucosa tissues that exhibit favorable characteristics to be used as an in vitro system for testing drug delivery systems.

The Effects of Noise and Simulated Conductive Hearing Loss on Physiological Response Measures During Interactive Conversations.

PURPOSE: The purpose of this work was to study the effects of background noise and hearing attenuation associated with earplugs on three physiological measures, assumed to be markers of effort investment and arousal, during interactive communication. METHOD: Twelve pairs of older people (average age of 63.2 years) with age-adjusted normal hearing took part in a face-to-face communication to solve a Diapix task. Communication was held in different levels of babble noise (0, 60, and 70 dBA) and with two levels of hearing attenuation (0 and 25 dB) in quiet. The physiological measures obtained included pupil size, heart rate variability, and skin conductance. In addition, subjective ratings of perceived communication success, frustration, and effort were obtained. RESULTS: Ratings of perceived success, frustration, and effort confirmed that communication was more difficult in noise and with approximately 25-dB hearing attenuation and suggested that the implemented levels of noise and hearing attenuation resulted in comparable communication difficulties. Background noise at 70 dBA and hearing attenuation both led to an initial increase in pupil size (associated with effort), but only the effect of the background noise was sustained throughout the conversation. The 25-dB hearing attenuation led to a significant decrease of the high-frequency power of heart rate variability and a significant increase of skin conductance level, measured as the average z value of the electrodermal activity amplitude. CONCLUSION: This study demonstrated that several physiological measures appear to be viable indicators of changing communication conditions, with pupillometry and cardiovascular as well as electrodermal measures potentially being markers of communication difficulty.

A Data-driven Distance Metric for Evaluating the effects of Dynamic Range Compression in Adverse Conditions.

Dynamic range compression is a crucial component in hearing aids, aiming to restore audibility for hearing-impaired listeners. However, determining suitable compression parameters, such as the time constants for the level estimation stage, remains a topic of debate, as the perceptual benefit of different parameter configurations varies depending on the acoustic conditions. In this study, a data-driven distance metric based on physical metrics was developed to evaluate and compare the performance of various compression systems. This analysis encompassed fast-acting and slow-acting compression, as well as a "scene-aware" compression that dynamically adjusted the release time constant based on the presence of the target. A reference system called "source-independent compression" was also considered, which had access to individual speech and noise signals. Multiple physical metrics were employed to assess the effects of these different compression systems under diverse acoustic conditions, including varying levels of interfering noise and degrees of room reverberation. Factor analysis was applied to derive a concise set of interpretable features representing the impact of compression, expressed as linear combinations of carefully selected objective metrics. The reduced dimensional representation enabled the use of the Manhattan distance to measure the similarity between the compression systems. Results demonstrated that the scene-aware compressor generally exhibited a smaller distance to the reference system compared to both the fast-acting and slow-acting compressors. This finding suggests that an adaptive compression system holds potential benefits across a range of acoustic conditions.

Development of a chemically defined medium for Paenibacillus polymyxa by parallel online monitoring of the respiration activity in microtiter plates.

BACKGROUND: One critical parameter in microbial cultivations is the composition of the cultivation medium. Nowadays, the application of chemically defined media increases, due to a more defined and reproducible fermentation performance than in complex media. In order, to improve cost-effectiveness of fermentation processes using chemically defined media, the media should not contain nutrients in large excess. Additionally, to obtain high product yields, the nutrient concentrations should not be limiting. Therefore, efficient medium optimization techniques are required which adapt medium compositions to the specific nutrient requirements of microorganisms. RESULTS: Since most Paenibacillus cultivation protocols so far described in literature are based on complex ingredients, in this study, a chemically defined medium for an industrially relevant Paenibacillus polymyxa strain was developed. A recently reported method, which combines a systematic experimental procedure in combination with online monitoring of the respiration activity, was applied and extended to identify growth limitations for Paenibacillus polymyxa. All cultivations were performed in microtiter plates. By systematically increasing the concentrations of different nutrient groups, nicotinic acid was identified as a growth-limiting component. Additionally, an insufficient buffer capacity was observed. After optimizing the growth in the chemically defined medium, the medium components were systematically reduced to contain only nutrients relevant for growth. Vitamins were reduced to nicotinic acid and biotin, and amino acids to methionine, histidine, proline, arginine, and glutamate. Nucleobases/-sides could be completely left out of the medium. Finally, the cultivation in the reduced medium was reproduced in a laboratory fermenter. CONCLUSION: In this study, a reliable and time-efficient high-throughput methodology was extended to investigate limitations in chemically defined media. The interpretation of online measured respiration activities agreed well with the growth performance of samples measured in parallel via offline analyses. Furthermore, the cultivation in microtiter plates was validated in a laboratory fermenter. The results underline the benefits of online monitoring of the respiration activity already in the early stages of process development, to avoid limitations of medium components, oxygen limitation and pH inhibition during the scale-up.

Cytological, molecular, cytogenetic, and physiological characterization of a novel immortalized human enteric glial cell line.

Enteric glial cells (EGCs), the major components of the enteric nervous system (ENS), are implicated in the maintenance of gut homeostasis, thereby leading to severe pathological conditions when impaired. However, due to technical difficulties associated with EGCs isolation and cell culture maintenance that results in a lack of valuable in vitro models, their roles in physiological and pathological contexts have been poorly investigated so far. To this aim, we developed for the first time, a human immortalized EGC line (referred as ClK clone) through a validated lentiviral transgene protocol. As a result, ClK phenotypic glial features were confirmed by morphological and molecular evaluations, also providing the consensus karyotype and finely mapping the chromosomal rearrangements as well as HLA-related genotypes. Lastly, we investigated the ATP- and acetylcholine, serotonin and glutamate neurotransmitters mediated intracellular Ca2+ signaling activation and the response of EGCs markers (GFAP, SOX10, S100ß, PLP1, and CCL2) upon inflammatory stimuli, further confirming the glial nature of the analyzed cells. Overall, this contribution provided a novel potential in vitro tool to finely characterize the EGCs behavior under physiological and pathological conditions in humans.

[Incapacity to work in prehospital German emergency medical services staff]. / Arbeitsunfähigkeit bei präklinischen Rettungskräften in Deutschland: Ergebnisse der EMS-Health-Studie.

Background: The determination of incapacity to work is a central approach for analyses of absence due to sickness. Nevertheless, no data are yet available for incapacity to work and associated factors in the German prehospital emergency medical services (EMS) staff. Objective: The aim of this analysis was to identify the proportion of EMS staff with at least one incapacity for work (AU) in the previous 12 months and associated factors. Material and methods: This was a nationwide survey study with rescue workers. Factors associated with work disability were identified using multivariable logistic regression, calculating odds ratios (OR) and associated 95% confidence intervals (95% CI). Results: Included in this analysis were 2298 employees of the German emergency medical services (female 42.6%, male 57.2%). Overall, 60.10% of female participants and 58.98% of male participants reported an incapacity for work in the previous 12 months. Incapacity for work was significantly associated with having a high school diploma (high school diploma: OR: 0.51, 95% CI 0.30; 0.88, p = 0.016; reference: secondary school diploma), working in a rural environment (OR: 0.65, 95% CI 0.50; 0.86, p = 0.003) or urban environment (OR: 0.72, 95% CI: 0.53; 0.98, p = 0.037). Furthermore, hours worked per week (OR: 1.01, 95% CI: 1.00; 1.02, p = 0.003) and 5-< 10 years of service (OR: 1.40, 95% CI: 1.04; 1.89, p = 0.025) were associated with higher odds of work disability. Neck and back pain, depression, osteoarthritis, and asthma in the previous 12 months also showed a significant association with work disability in the same time period. Conclusion: This analysis shows that chronic diseases, educational attainment, area of assignment, years of service, and hours worked per week, among others, were associated with incapacity for work in the previous 12 months in German EMS staff.

Human-Based New Approach Methodologies in Developmental Toxicity Testing: A Step Ahead from the State of the Art with a Feto-Placental Organ-on-Chip Platform.

Developmental toxicity testing urgently requires the implementation of human-relevant new approach methodologies (NAMs) that better recapitulate the peculiar nature of human physiology during pregnancy, especially the placenta and the maternal/fetal interface, which represent a key stage for human lifelong health. Fit-for-purpose NAMs for the placental-fetal interface are desirable to improve the biological knowledge of environmental exposure at the molecular level and to reduce the high cost, time and ethical impact of animal studies. This article reviews the state of the art on the available in vitro (placental, fetal and amniotic cell-based systems) and in silico NAMs of human relevance for developmental toxicity testing purposes; in addition, we considered available Adverse Outcome Pathways related to developmental toxicity. The OECD TG 414 for the identification and assessment of deleterious effects of prenatal exposure to chemicals on developing organisms will be discussed to delineate the regulatory context and to better debate what is missing and needed in the context of the Developmental Origins of Health and Disease hypothesis to significantly improve this sector. Starting from this analysis, the development of a novel human feto-placental organ-on-chip platform will be introduced as an innovative future alternative tool for developmental toxicity testing, considering possible implementation and validation strategies to overcome the limitation of the current animal studies and NAMs available in regulatory toxicology and in the biomedical field.

A New Immortalized Human Alveolar Epithelial Cell Model to Study Lung Injury and Toxicity on a Breathing Lung-On-Chip System.

The evaluation of inhalation toxicity, drug safety and efficacy assessment, as well as the investigation of complex disease pathomechanisms, are increasingly relying on in vitro lung models. This is due to the progressive shift towards human-based systems for more predictive and translational research. While several cellular models are currently available for the upper airways, modelling the distal alveolar region poses several constraints that make the standardization of reliable alveolar in vitro models relatively difficult. In this work, we present a new and reproducible alveolar in vitro model, that combines a human derived immortalized alveolar epithelial cell line (AXiAEC) and organ-on-chip technology mimicking the lung alveolar biophysical environment (AXlung-on-chip). The latter mimics key features of the in vivo alveolar milieu: breathing-like 3D cyclic stretch (10% linear strain, 0.2 Hz frequency) and an ultrathin, porous and elastic membrane. AXiAECs cultured on-chip were characterized for their alveolar epithelial cell markers by gene and protein expression. Cell barrier properties were examined by TER (Transbarrier Electrical Resistance) measurement and tight junction formation. To establish a physiological model for the distal lung, AXiAECs were cultured for long-term at air-liquid interface (ALI) on-chip. To this end, different stages of alveolar damage including inflammation (via exposure to bacterial lipopolysaccharide) and the response to a profibrotic mediator (via exposure to Transforming growth factor ß1) were analyzed. In addition, the expression of relevant host cell factors involved in SARS-CoV-2 infection was investigated to evaluate its potential application for COVID-19 studies. This study shows that AXiAECs cultured on the AXlung-on-chip exhibit an enhanced in vivo-like alveolar character which is reflected into: 1) Alveolar type 1 (AT1) and 2 (AT2) cell specific phenotypes, 2) tight barrier formation (with TER above 1,000 Ω cm2) and 3) reproducible long-term preservation of alveolar characteristics in nearly physiological conditions (co-culture, breathing, ALI). To the best of our knowledge, this is the first time that a primary derived alveolar epithelial cell line on-chip representing both AT1 and AT2 characteristics is reported. This distal lung model thereby represents a valuable in vitro tool to study inhalation toxicity, test safety and efficacy of drug compounds and characterization of xenobiotics.

Insights in the Complex DegU, DegS, and Spo0A Regulation System of Paenibacillus polymyxa by CRISPR-Cas9-Based Targeted Point Mutations.

Despite being unicellular organisms, bacteria undergo complex regulation mechanisms which coordinate different physiological traits. Among others, DegU, DegS, and Spo0A are the pleiotropic proteins which govern various cellular responses and behaviors. However, the functions and regulatory networks between these three proteins are rarely described in the highly interesting bacterium Paenibacillus polymyxa. In this study, we investigate the roles of DegU, DegS, and Spo0A by introduction of targeted point mutations facilitated by a CRISPR-Cas9-based system. In total, five different mutant strains were generated, the single mutants DegU Q218*, DegS L99F, and Spo0A A257V, the double mutant DegU Q218* DegS L99F, and the triple mutant DegU Q218* DegS L99F Spo0A A257V. Characterization of the wild-type and the engineered strains revealed differences in swarming behavior, conjugation efficiency, sporulation, and viscosity formation of the culture broth. In particular, the double mutant DegU Q218* DegS L99F showed a significant increase in conjugation efficiency as well as a stable exopolysaccharides formation. Furthermore, we highlight similarities and differences in the roles of DegU, DegS, and Spo0A between P. polymyxa and related species. Finally, this study provides novel insights into the complex regulatory system of P. polymyxa DSM 365. IMPORTANCE To date, only limited knowledge is available on how complex cellular behaviors are regulated in P. polymyxa. In this study, we investigate several regulatory proteins which play a role in governing different physiological traits. Precise targeted point mutations were introduced to their respective genes by employing a highly efficient CRISPR-Cas9-based system. Characterization of the strains revealed some similarities, but also differences, to the model bacterium Bacillus subtilis with regard to the regulation of cellular behaviors. Furthermore, we identified several strains which have superior performance over the wild-type. The applicability of the CRISPR-Cas9 system as a robust genome editing tool, in combination with the engineered strain with increased genetic accessibility, would boost further research in P. polymyxa and support its utilization for biotechnological applications. Overall, our study provides novel insights, which will be of importance in understanding how multiple cellular processes are regulated in Paenibacillus species.

The prevalence of diseases in German emergency medical services staff: A survey study.

For the general German population, the GEDA study is the main health monitoring study. Since nothing comparable is existing regarding the health of emergency medical services (EMS) staff, the aim of the present study was to estimate the 12-months prevalence of socially relevant diseases and symptoms among German pre-hospital EMS staff. This was a nationwide survey study. For retrieval of information on the health status of participating EMS staff items from the GEDA2014/2015 study were used. Afterwards, 12-months prevalence or proportions and corresponding 95% confidence intervals (95% CI) were calculated. Overall, 2,313 German pre-hospital EMS staff (42.6% female/57.2% male) with a median age of 25.0 (min. 18.0; max. 63.0) years were included into the final analysis. Obesity based on BMI was present in 25.9% (95% CI: 23.5%; 28.4%) of male compared to 15.7% (95% CI: 13.5%; 18.1%) of female study participants. Highest 12-months prevalence in the full study sample were calculated for lower back pain (41.1% [95%: 39.1%; 43.2%]), cervical pain (32.9% [95% CI: 30.9%; 34.8%]), allergies (32.6% [95% CI: 30.7%; 34.6%], depression (13.7% [95% CI: 12.4%; 15.2%]), and hypertension (11.5% [95% CI: 10.3%; 12.9%]). The 12-months prevalence in specific age-/gender-groups were higher for asthma, depression, hypertension, increased blood lipids, and osteoarthritis among study participants compared to the general German population (GEDA study) with the same age and gender (ratio ≥ 1.5). This study indicates that the prevalence of obesity, depression, hypertension, and other disorders is high among study participants and disease prevention measures for pre-hospital EMS staff are needed.

The effect of hearing aid dynamic range compression on speech intelligibility in a realistic virtual sound environment.

Measures of "aided" speech intelligibility (SI) for listeners wearing hearing aids (HAs) are commonly obtained using rather artificial acoustic stimuli and spatial configurations compared to those encountered in everyday complex listening scenarios. In the present study, the effect of hearing aid dynamic range compression (DRC) on SI was investigated in simulated real-world acoustic conditions. A spatialized version of the Danish Hearing In Noise Test was employed inside a loudspeaker-based virtual sound environment to present spatialized target speech in background noise consisting of either spatial recordings of two real-world sound scenarios or quadraphonic, artificial speech-shaped noise (SSN). Unaided performance was compared with results obtained with a basic HA simulator employing fast-acting DRC. Speech reception thresholds (SRTs) with and without DRC were found to be significantly higher in the conditions with real-world background noise than in the condition with artificial SSN. Improvements in SRTs caused by the HA were only significant in conditions with real-world background noise and were related to differences in the output signal-to-noise ratio of the HA signal processing between the real-world versus artificial conditions. The results may be valuable for the design, development, and evaluation of HA signal processing strategies in realistic, but controlled, acoustic settings.

Dexamethasone-mediated inhibition of Notch signalling blocks the interaction of leukaemia and mesenchymal stromal cells.

Acute myeloid leukaemia (AML) is a haematological malignancy characterized by a poor prognosis. Bone marrow mesenchymal stromal cells (BM MSCs) support leukaemic cells in preventing chemotherapy-induced apoptosis. This encouraged us to investigate leukaemia-BM niche-associated signalling and to identify signalling cascades supporting the interaction of leukaemic cells and BM MSC. Our study demonstrated functional differences between MSCs originating from leukaemic (AML MSCs) and healthy donors (HD MSCs). The direct interaction of leukaemic and AML MSCs was indispensable in influencing AML cell proliferation. We further identified an important role for Notch expression and its activation in AML MSCs contributing to the enhanced proliferation of AML cells. Supporting this observation, overexpression of the intracellular Notch domain (Notch ICN) in AML MSCs enhanced AML cells' proliferation. From a therapeutic point of view, dexamethasone treatment impeded Notch signalling in AML MSCs resulting in reduced AML cell proliferation. Concurrent with our data, Notch inhibitors had only a marginal effect on leukaemic cells alone but strongly influenced Notch signalling in AML MSCs and abrogated their cytoprotective function on AML cells. In vivo, dexamethasone treatment impeded Notch signalling in AML MSCs leading to a reduced number of AML MSCs and improved survival of leukaemic mice. In summary, targeting the interaction of leukaemic cells and AML MSCs using dexamethasone or Notch inhibitors might further improve treatment outcomes in AML patients.

Guided ecological momentary assessment in real and virtual sound environments.

Ecological momentary assessment (EMA) outcome measures can relate people's subjective auditory experience to their objective acoustical reality. While highly realistic, EMA data often contain considerable variability, such that it can be difficult to interpret the results with respect to differences in people's hearing ability. To address this challenge, a method for "guided" EMA is proposed and evaluated. Accompanied and instructed by a guide, normal-hearing participants carried out specific passive and active listening tasks inside a real-world public lunch scenario and answered EMA questionnaires related to aspects of spatial hearing, listening ability, quality, and effort. In situ speech and background noise levels were tracked, allowing the guided EMA task to be repeated inside two acoustically matched, loudspeaker-based laboratory environments: a 64-channel virtual sound environment (VSE) and a three-channel audiology clinic setup. Results showed that guided EMA provided consistent passive listening assessments across participants and conditions. During active listening, the clinic setup was found to be less challenging than the real-world and the VSE conditions. The proposed guided EMA approach may provide more focused real-world assessments and can be applied in realistic laboratory settings to aid the development of ecologically valid hearing testing.

Airway Administration of Flagellin Regulates the Inflammatory Response to Pseudomonas aeruginosa.

Excessive lung inflammation and airway epithelial damage are hallmarks of human inflammatory lung diseases, such as cystic fibrosis (CF). Enhancement of innate immunity provides protection against pathogens while reducing lung-damaging inflammation. However, the mechanisms underlying innate immunity-mediated protection in the lung remain mysterious, in part because of the lack of appropriate animal models for these human diseases. TLR5 (Toll-like receptor 5) stimulation by its specific ligand, the bacterial protein flagellin, has been proposed to enhance protection against several respiratory infectious diseases, although other cellular events, such as calcium signaling, may also control the intensity of the innate immune response. Here, we investigated the molecular events prompted by stimulation with flagellin and its role in regulating innate immunity in the lung of the pig, which is anatomically and genetically more similar to humans than rodent models. We found that flagellin treatment modulated NF-κB signaling and intracellular calcium homeostasis in airway epithelial cells. Flagellin pretreatment reduced the NF-κB nuclear translocation and the expression of proinflammatory cytokines to a second flagellin stimulus as well as to Pseudomonas aeruginosa infection. Moreover, in vivo administration of flagellin decreased the severity of P. aeruginosa-induced pneumonia. Then we confirmed these beneficial effects of flagellin in a pathological model of CF by using ex vivo precision-cut lung slices from a CF pigz model. These results provide evidence that flagellin treatment contributes to a better regulation of the inflammatory response in inflammatory lung diseases such as CF.

Speech intelligibility in a realistic virtual sound environment.

In the present study, speech intelligibility was evaluated in realistic, controlled conditions. "Critical sound scenarios" were defined as acoustic scenes that hearing aid users considered important, difficult, and common through ecological momentary assessment. These sound scenarios were acquired in the real world using a spherical microphone array and reproduced inside a loudspeaker-based virtual sound environment (VSE) using Ambisonics. Speech reception thresholds (SRT) were measured for normal-hearing (NH) and hearing-impaired (HI) listeners, using sentences from the Danish hearing in noise test, spatially embedded in the acoustic background of an office meeting sound scenario. In addition, speech recognition scores (SRS) were obtained at a fixed signal-to-noise ratio (SNR) of -2.5 dB, corresponding to the median conversational SNR in the office meeting. SRTs measured in the realistic VSE-reproduced background were significantly higher for NH and HI listeners than those obtained with artificial noise presented over headphones, presumably due to an increased amount of modulation masking and a larger cognitive effort required to separate the target speech from the intelligible interferers in the realistic background. SRSs obtained at the fixed SNR in the realistic background could be used to relate the listeners' SI to the potential challenges they experience in the real world.

A method for realistic, conversational signal-to-noise ratio estimation.

The analysis of real-world conversational signal-to-noise ratios (SNRs) can provide insight into people's communicative strategies and difficulties and guide the development of hearing devices. However, measuring SNRs accurately is challenging in everyday recording conditions in which only a mixture of sound sources can be captured. This study introduces a method for accurate in situ SNR estimation where the speech signal of a target talker in natural conversation is captured by a cheek-mounted microphone, adjusted for free-field conditions and convolved with a measured impulse response to estimate its power at the receiving talker. A microphone near the receiver provides the noise-only component through voice activity detection. The method is applied to in situ recordings of conversations in two real-world sound scenarios. It is shown that the broadband speech level and SNR distributions are estimated more accurately by the proposed method compared to a typical single-channel method, especially in challenging, low-SNR environments. The application of the proposed two-channel method may render more realistic estimates of conversational SNRs and provide valuable input to hearing instrument processing strategies whose operating points are determined by accurate SNR estimates.

Characterization of Novel Human Immortalized Thyroid Follicular Epithelial Cell Lines.

Introduction: Investigation of normal human thyroid function using in vitro culture systems is dependent on cells that recapitulate physiology of differentiated thyrocytes. Primary thyrocytes retain features of the native organ but have limited lifespan in culture. Immortalized thyrocytes offer an alternative if challenges maintaining phenotypic stability can be overcome to retain functional features of primary cells. Materials and Methods: CI-SCREEN immortalization technology was applied to normal human thyroid tissue to generate four cell line variants. The lines were characterized for transgene integration, biomarker expression, genomic stability, and proliferation rates. Thyroid Stimulating Hormone (TSH)-dependent morphology, thyroglobulin production, thyroxine hormone synthesis, and viability were assessed using conventional 2D monolayer and 3D microtissue culture formats in huThyrEC or h7H medium. Results: Despite differential transgene profiles, the lines had similar biomarker expression patterns and proliferation rates. In 2D culture there was no thyroxine synthesis or changes in viability, but TSH-dependent thyroglobulin production was more significant for several lines in h7H than huThyrEC medium. Comparatively, in 3D microtissues, TSH-dependent thyroglobulin induction was greater for cell lines in h7H medium. Synthesis of thyroxine in one cell line was higher than background with TSH exposure, but not significantly different than control. Discussion: Immortalization of primary human thyrocytes yielded transgenic lines of epithelial origin. When evaluated in 2D or 3D culture formats, h7H medium supported thyroglobulin production to a greater magnitude than huThyrEC medium. One cell line cultured in 3D microtissue format marginally recapitulated T4 synthesis under continuous TSH exposure. Conclusion: Select human thyroid cell lines exhibited morphological and functional features of primary thyrocytes and are a novel resource for in vitro disease modeling and toxicity testing that will enable reproducible culture models more representative of normal human thyroid function.