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Primary airway epithelial cells (pAECs) cultivated at air-liquid interface (ALI) conditions are widely used as surrogates for human in vivo epithelia. To extend the proliferative capacity and to enable serially passaging of pAECs, conditional reprogramming (cr) has been employed in recent years. However, ALI epithelia derived from cr cells often display functional changes with increasing passages. This highlights the need for thorough validation of the ALI cultures for the respective application. In our study, we evaluated the use of serially passaged cr nasal epithelial cells (crNECs) as a model to study SARS-CoV-2 infection and effects on ion and water transport. NECs were obtained from healthy individuals and cultivated as ALI epithelia derived from passages 1, 2, 3, and 5. We compared epithelial differentiation, ion and water transport, and infection with SARS-CoV-2 between passages. Our results show that epithelia maintained major differentiation characteristics and physiological ion and water transport properties through all passages. However, the frequency of ciliated cells, short circuit currents reflecting epithelial Na+ channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) activity and expression of aquaporin 3 and 5 decreased gradually over passages. crNECs also expressed SARS-CoV-2 receptors angiotensin converting enzyme 2 (ACE2) and transmembrane serin2 protease 2 (TMPRSS2) across all passages and allowed SARS-CoV-2 replication in all passages. In summary, we provide evidence that passaged crNECs provide an appropriate model to study SARS-CoV-2 infection and also epithelial transport function when considering some limitations that we defined herein.
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COVID-19 , Diferenciación Celular , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/metabolismo , Humanos , Recién Nacido , SARS-CoV-2RESUMEN
Neutrophil granulocytes are the vanguard of innate immunity in response to numerous pathogens. Their activity drives the clearance of microbe- and damage-associated molecular patterns, thereby contributing substantially to the resolution of inflammation. However, excessive stimulation during sepsis leads to cellular unresponsiveness, immunological dysfunction, bacterial expansion, and subsequent multiple organ dysfunction. During the short lifespan of neutrophils, they can become significantly activated by complement factors, cytokines, and other inflammatory mediators. Following stimulation, the cells respond with a defined (electro-)physiological pattern, including depolarization, calcium influx, and alkalization as well as with increased metabolic activity and polarization of the actin cytoskeleton. Activity of ion transport proteins and aquaporins is critical for multiple cellular functions of innate immune cells, including chemotaxis, generation of reactive oxygen species, and phagocytosis of both pathogens and tissue debris. In this review, we first describe the ion transport proteins and aquaporins involved in the neutrophil ion-water fluxes in response to chemoattractants. We then relate ion and water flux to cellular functions with a focus on danger sensing, chemotaxis, phagocytosis, and oxidative burst and approach the role of altered ion transport protein expression and activity in impaired cellular functions and cell death during systemic inflammation as in sepsis.
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Granulocitos/patología , Inmunidad Innata/inmunología , Inflamación/complicaciones , Neutrófilos/patología , Estallido Respiratorio , Sepsis/patología , Animales , Humanos , Especies Reactivas de Oxígeno , Sepsis/etiologíaRESUMEN
Mucus clearance provides an essential innate defense mechanism to keep the airways and lungs free of particles and pathogens. Baseline and stimulated mucin secretion from secretory airway epithelial cells need to be tightly regulated to prevent mucus hypersecretion and mucus plugging of the airways. It is well established that extracellular ATP is a potent stimulus for regulated mucus secretion. Previous studies revealed that ATP acts via metabotropic P2Y2 purinoreceptors on goblet cells. Extracellular ATP, however, is also a potent agonist for ionotropic P2X purinoreceptors. Expression of several P2X isoforms has been reported in airways, but cell type-specific expression and the function thereof remained elusive. With this study, we now provide evidence that P2X4 is the predominant P2X isoform expressed in secretory airway epithelial cells. After IL-13 treatment of either human primary tracheal epithelial cells or mice, P2X4 expression is upregulated in vitro and in vivo under conditions of chronic inflammation, mucous metaplasia, and hyperplasia. Upregulation of P2X4 is strongest in MUC5AC-positive goblet cells. Moreover, activation of P2X4 by extracellular ATP augments intracellular Ca2+ signals and mucin secretion, whereas Ca2+ signals and mucin secretion are dampened by inhibition of P2X4 receptors. These data provide new insights into the purinergic regulation of mucin secretion and add to the emerging picture that P2X receptors modulate exocytosis of large secretory organelles and secretion of macromolecular vesicle cargo.
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Señalización del Calcio , Células Caliciformes/metabolismo , Mucinas/metabolismo , Receptores Purinérgicos P2X4/metabolismo , Regulación hacia Arriba , Adenosina Trifosfato/farmacología , Células Caliciformes/patología , Humanos , Inflamación/metabolismo , Inflamación/patologíaRESUMEN
Interleukin-13 (IL-13) drives symptoms in asthma with high levels of T-helper type 2 cells (Th2-cells). Since tight junctions (TJ) constitute the epithelial diffusion barrier, we investigated the effect of IL-13 on TJ in human tracheal epithelial cells. We observed that IL-13 increases paracellular permeability, changes claudin expression pattern and induces intracellular aggregation of the TJ proteins zonlua occludens protein 1, as well as claudins. Furthermore, IL-13 treatment increases expression of ubiquitin conjugating E2 enzyme UBE2Z. Co-localization and proximity ligation assays further showed that ubiquitin and the proteasomal marker PSMA5 co-localize with TJ proteins in IL-13 treated cells, showing that TJ proteins are ubiquitinated following IL-13 exposure. UBE2Z upregulation occurs within the first day after IL-13 exposure. Proteasomal aggregation of ubiquitinated TJ proteins starts three days after IL-13 exposure and transepithelial electrical resistance (TEER) decrease follows the time course of TJ-protein aggregation. Inhibition of JAK/STAT signaling abolishes IL-13 induced effects. Our data suggest that that IL-13 induces ubiquitination and proteasomal aggregation of TJ proteins via JAK/STAT dependent expression of UBE2Z, resulting in opening of TJs. This may contribute to barrier disturbances in pulmonary epithelia and lung damage of patients with inflammatory lung diseases.
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Células Epiteliales/metabolismo , Interleucina-13/farmacología , Uniones Estrechas/metabolismo , Tráquea/metabolismo , Células Cultivadas , Células Epiteliales/efectos de los fármacos , Humanos , Quinasas Janus/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Factores de Transcripción STAT/metabolismo , Uniones Estrechas/efectos de los fármacos , Tráquea/citología , Enzimas Ubiquitina-Conjugadoras/metabolismo , UbiquitinaciónRESUMEN
The apical surface liquid (ASL) layer covers the airways and forms a first line of defense against pathogens. Maintenance of ASL volume by airway epithelia is essential for maintaining lung function. The proteolytic activation of epithelial Na+ channels is believed to be the dominating mechanism to cope with increases in ASL volumes. Alternative mechanisms, in particular increases in epithelial osmotic water permeability (Posm), have so far been regarded as rather less important. However, most studies mainly addressed immediate effects upon apical volume expansion (AVE) and increases in ASL. This study addresses the response of lung epithelia to long-term AVE. NCI-H441 cells and primary human tracheal epithelial cells, both cultivated in air-liquid interface conditions, were used as models for the lung epithelium. AVE was established by adding isotonic solution to the apical surface of differentiated lung epithelia, and time course of ASL volume restoration was assessed by the deuterium oxide dilution method. Concomitant ion transport was investigated in Ussing chambers. We identified a low resorptive state immediately after AVE, which coincided with proteolytic ion transport activation within 10-15 minutes after AVE. The main clearance of excess ASL occurred during a delayed (hours after AVE) high resorptive state, which did not correlate with ion transport activation. Instead, high resorptive state onset coincided with an increase in Posm, which depended on aquaporin up-regulation. In summary, our data demonstrate that, aside from ion transport activation, modulation of Posm is a major mechanism to compensate for long-term AVE in lung epithelia.
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Epitelio/metabolismo , Pulmón/metabolismo , Reología , Agua/metabolismo , Amilorida/farmacología , Acuaporinas/metabolismo , Canales Epiteliales de Sodio/metabolismo , Epitelio/efectos de los fármacos , Humanos , Inmunohistoquímica , Pulmón/efectos de los fármacos , Ósmosis/efectos de los fármacos , Permeabilidad/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Reología/efectos de los fármacos , Propiedades de Superficie , Factores de TiempoRESUMEN
The lung epithelium constitutes a selective barrier that separates the airways from the aqueous interstitial compartment. Regulated barrier function controls water and ion transport across the epithelium and is essential for maintaining lung function. Tight junctions (TJs) seal the epithelial barrier and determine the paracellular transport. The properties of TJs depend especially on their claudin composition. Steroids are potent drugs used to treat a variety of airway diseases. Therefore, we addressed whether steroid hormones directly act on TJ properties in lung epithelia. Primary human tracheal epithelial cells and NCI-H441 cells, both cultivated under air-liquid interface conditions, were used as epithelial cell models. Our results demonstrate that glucocorticoids, but not mineralocorticoids, decreased paracellular permeability and shifted the ion permselectivity of TJs toward Cl(-). Glucocorticoids up-regulated claudin 8 (cldn8) expression via glucocorticoid receptors. Silencing experiments revealed that cldn8 is necessary to recruit occludin at the TJs. Immunohistochemistry on human lung tissue showed that cldn8 is specifically expressed in resorptive epithelia of the conducting and respiratory airways but not in the alveolar epithelium. We conclude that glucocorticoids enhance lung epithelia barrier function and increase paracellular Cl(-) selectivity via modulation of cldn8-dependent recruitment of occludin at the TJs. This mode of glucocorticoid action on lung epithelia might be beneficial to patients who suffer from impaired lung barrier function in various diseased conditions.
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Claudinas/metabolismo , Epitelio/metabolismo , Glucocorticoides/farmacología , Pulmón/metabolismo , Uniones Estrechas/metabolismo , Impedancia Eléctrica , Epitelio/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Silenciador del Gen/efectos de los fármacos , Humanos , Permeabilidad/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Uniones Estrechas/efectos de los fármacos , Factores de Tiempo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: Highly-effective CFTR-modulator therapy with elexa-/teza-/ivacaftor (ETI) has led to improvements in pulmonary outcomes, sweat chloride, body mass index (BMI) and quality of life in people with cystic fibrosis (CF). Improved uptake of fat-soluble vitamins and micronutrients has been reported for CFTR-modulators but data regarding ETI therapy is lacking. METHODS: This single-center retrospective study evaluated forced expiratory volume in one second (FEV-1), sweat chloride, BMI, transaminases (AST, ALT), bilirubin, vitamins A, D, E, zinc and selenium in children and adults eligible for ETI. Parameters were assessed before and up to one year after initiation of ETI. RESULTS: 58 patients (median age m = 28 years, SD ± 11.6 years, 51.7% female14 < 18 years old) were included. FEV-1 and sweat chloride improved significantly after ETI. There were no changes in BMI or AST. ALT was increased significantly after 4 weeks of ETI but returned to normal levels in further course. Bilirubin levels remained elevated after ETI. Vitamin A was significantly higher 12 months after ETI. No changes were found for vitamins D, E, zinc and selenium. CONCLUSIONS: This study adds to the evidence that improvements of some fat-soluble vitamin levels can be found after ETI. No changes regarding micronutrients were noted. Individualized follow-up and supplementation are recommended.
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Cystic fibrosis (CF) is a monogenetic disease caused by an impairment of the cystic fibrosis transmembrane conductance regulator (CFTR). CF affects multiple organs and is associated with acute and chronic inflammation. In 2020, Elexacaftor-Tezacaftor-Ivacaftor (ETI) was approved to enhance and restore the remaining CFTR functionality. This study investigates cellular innate immunity, with a focus on neutrophil activation and phenotype, comparing healthy volunteers with patients with CF before (T1, n = 13) and after six months (T2, n = 11) of ETI treatment. ETI treatment reduced sweat chloride (T1: 95 mmol/l (83|108) vs. T2: 32 mmol/l (25|62), p < 0.01, median, first|third quartile) and significantly improved pulmonal function (FEV1 T1: 2.66 l (1.92|3.04) vs. T2: 3.69 l (3.00|4.03), p < 0.01). Moreover, there was a significant decrease in the biomarker human epididymis protein 4 (T1: 6.2 ng/ml (4.6|6.3) vs. T2: 3.0 ng/ml (2.2|3.7), p < 0.01) and a small but significant decrease in matrix metallopeptidase 9 (T1: 45.5 ng/ml (32.5|140.1) vs. T2: 28.2 ng/ml (18.2|33.6), p < 0.05). Neutrophil phenotype (CD10, CD11b, CD62L, and CD66b) and function (radical oxygen species generation, chemotactic and phagocytic activity) remained largely unaffected by ETI treatment. Likewise, monocyte phenotype and markers of platelet activation were similar at T1 and T2. In summary, the present study confirmed a positive impact on patients with CF after ETI treatment. However, neither beneficial nor harmful effects of ETI treatment on cellular innate immunity could be detected, possibly due to the study population consisting of patients with well-controlled CF.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Humanos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/tratamiento farmacológico , Plaquetas , Monocitos , GranulocitosRESUMEN
Background and Aims: In cystic fibrosis (CF) airways, impaired airway mucociliary clearance and mucus accumulation due to cystic fibrosis transmembrane conductance regulator defects contribute to inflammation, progressive structural lung damage, and decline of lung function. Physiotherapy is essential to promote mucus mobilization and removal in CF and is a key element of rehabilitation measures, but conventional techniques may be suboptimal to mobilize viscous mucus. This study aimed to test the specific effects of a novel bronchial drainage device (BDD) (Simeox®; PhysioAssist) in subjects with CF and evaluate lung function, diaphragm mobility, and sputum properties. Methods: This prospective monocentric clinical cohort study in the setting of outpatient physiotherapy of CF patients (n = 21) with stable CF lung disease collected pulmonary lung function tests (PFT), diaphragm mobility, and sputum properties before and after two physiotherapy sessions using the novel BDD. PFT was assessed using spirometry and diaphragm mobility using m-mode ultrasound analysis. Spontaneous sputum samples were collected before and after using the BDD and analyzed for microstructure and DNA concentrations. Results: PFT parameters (FEV1, FVC, MEF25/50/75) were not affected by the use of the BDD. Ultrasound analysis of diaphragm mobility revealed an increase in maximum diaphragm excursion upon the intervention. Mucus analysis demonstrated altered microstructure and higher DNA concentrations collected after using the BDD compared to samples collected before. Pearson correlation analysis showed significant correlations between changes in mucus properties and DNA levels in respective mucus samples. Conclusion: Our results demonstrate that the novel BDD improves diaphragm mobility and alters sputum properties in subjects with CF. The novel BDD with unique properties may be further studied as a device in CF-specific physiotherapy to facilitate sputum mobilization of CF patients.
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BACKGROUND AND PURPOSE: The comorbidity of psychiatric disorders and cerebrovascular disease appears to be complex with underlying bidirectional influences. Hitherto, research has focused mainly on the evaluation of stroke risk in particular psychiatric disorders; only a few studies have assessed their role in the acute natural history of stroke. The aim of this study was to provide a perspective on psychiatric premorbidity and its impact on stroke severity, psychiatric complications during the initial treatment phase, and the short-term functional outcome of stroke. PATIENTS AND METHODS: We retrospectively studied the impact of a predocumented psychiatric diagnosis (PDPD) on stroke severity, short-term functional outcome, and psychiatric complications in a sample of 798 patients consecutively admitted for acute ischemic or hemorrhagic stroke by performing a chart review. Group comparisons (PDPD vs non-PDPD) with adjustment for covariates were carried out either using multivariate analysis of variance or logistic regression analysis. RESULTS: More severe strokes (ie, mean National Institute of Health Stroke Scale score on admission 10.1±7.9 vs 7.5±7.4; F(10,796)=18.5, p<0.0001) and higher prevalence of poor outcome (73.7 vs 54.9%; OR: 2.6, standard error: 0.5, z=4.82, p<0.0001) was found in patients with a documented psychiatric diagnosis at the time of stroke, as well as a higher rate of psychiatric complications during the initial treatment phase (46.7 vs 28.9%; OR: -0.78, z=4.59, p<0.0001). CONCLUSION: Our data have clinical implications in that they call for identification of psychiatric premorbidity or comorbidity through careful history-taking and particularly close monitoring for psychiatric complications with respect to their potentially negative impact on outcome after stroke.
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MEDICAL HISTORY AND CLINICAL FINDINGS: A 43-year old male patient was admitted because of diffuse abdominal discomfort for two days, which had started in the upper abdomen; medical history, no previous surgical interventions at the abdomen. INVESTIGATIONS: Gastroscopy and abdominal ultrasound revealed only a mild gastritis. Laboratory parameters: showed only a slight leucocytosis (10â610/µL). On the 1st day of the hospital stay, the patient developed a worsening of the abdominal symptoms with distended abdomen, sounding bowel movements and recurrent vomiting, which were interpreted as acute abdomen by the surgeon on call leading to the indication of a surgical intervention. CT scan revealed an intraluminal tumor. DIAGNOSIS: Suspicion of a mechanical ileus by an obstructing tumor of the small intestine. THERAPY: Median laparotomy of the lower abdomen revealed an invagination at the terminal ileum - after devagination, an invaginated Meckel's diverticulum was found. This was resected at its basis and the ileal wall was transversally sutured. COURSE: The postoperative course was uneventful. DISCUSSION (CONCLUSION): An invaginated Meckel's diverticulum belongs rather to the less frequent causes of an ileus of the small intestine and an unclear / acute abdomen in adults.