Search Results (102)

Cystic fibrosis (CF), the most common hereditary lung disease in Caucasians, is caused by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR). We evaluated CFTR function using a newly developed Ussing chamber system, the Multi Trans Epithelial Current Clamp (MTECC), in an in vitro model of human airway epithelia. Air–liquid interface (ALI) cultures were established from nasal brushings of healthy controls (HC) and CF patients with biallelic CFTR variants. ALI layer thickness was similar between groups (HC: 62 ± 13 µm; CF: 55 ± 9 µm). Immunofluorescence showed apical CFTR expression in HC, but reduced or absent signal in CF cultures. MTECC enabled continuous measurement of transepithelial resistance (Rt), potential difference (PD), and conductance (G_t). G_t was significantly reduced in CF cultures compared to HC (0.825 ± 0.024 vs. −0.054 ± 0.016 mS/cm²), indicating impaired cAMP-inducible ion transport by CFTR. Treatment of CF cultures with elexacaftor, tezacaftor, and ivacaftor (Trikafta^®) increased G_t, reflecting partial restoration of CFTR function. These findings demonstrate the utility of MTECC in detecting functional differences in CFTR activity and support its use as a platform for evaluating CFTR-modulating therapies. Our model may contribute to the development of personalized treatment strategies for CF patients. Full article

This study investigated the effects of potassium magnesium sulfate (PMS) on intestinal dissociation and absorption rate, immune function, and expression of the NOD-like receptor thermal domain-associated protein 3 (NLRP3) inflammasome, aquaporins (AQPs), and potassium and magnesium ion channels in weaned piglets. Experiment 1 involved the assessment of the dissociation rate of PMS in pig digestive fluid and the absorption rate of PMS in the small intestine using an Ussing chamber in vitro. In Experiment 2, 216 healthy 21-day-old weaned piglets were selected and randomly assigned to six groups (0%, 0.15%, 0.30%, 0.45%, 0.60%, and 0.75% PMS), with each group 6 replicates of six piglets per replicate. The in vitro Ussing chamber results indicated that the absorption of K⁺ and Mg²⁺ in the jejunum and ileum was significantly higher than that in the duodenum (p < 0.05). The in vivo study demonstrated that the addition of PMS resulted in a linear increase in serum K⁺, IgG, and interleukin (IL)-2 levels while simultaneously reducing serum IL-1β levels (p < 0.05). Dietary PMS significantly elevated serum IL-10 and Mg²⁺ levels in feces (p < 0.05). Furthermore, supplementation with 0.60% or 0.75% PMS significantly downregulated the mRNA expression of NLRP3 in the jejunum (p < 0.05). Dietary PMS supplementation linearly reduced the mRNA expression levels of cysteine protease 1 (Caspase-1) and IL-1β in both the jejunum and colon as well as the mRNA expression levels of two-pore domain channel subfamily K member 5 (KCNK5) in these regions (p < 0.05). Notably, supplementation with 0.15% PMS significantly decreased the mRNA expression of transient receptor potential channel 6 (TRPM6) in the jejunum and significantly increased the expression of TRPM6 in the colon (p < 0.05). Dietary addition of 0.45% and 0.60% PMS significantly increased the mRNA expression of aquaporin 3 (AQP3) in the colon (p < 0.05), whereas 0.75% PMS significantly increased the mRNA expression of aquaporin 8 (AQP8) in both the jejunum and colon. Moreover, the expression levels of AQP3 and AQP8 were significantly negatively correlated with the diarrhea rate observed between days 29 and 42. In conclusion, dietary PMS supplementation improved immune function, inhibited the activation of intestinal NLRP3, and modulated the expression of water and ion channels in weaned piglets, thereby contributing to the maintenance of intestinal water and ion homeostasis, which could potentially alleviate post-weaning diarrhea in piglets. The recommended supplemental level of PMS in the corn-soybean basal diet for weaned piglets is 0.30%. Full article

Cannabidiol (CBD), a major non-psychoactive cannabinoid of the Cannabis sativa L. plant, has demonstrated anti-inflammatory effects in various studies. However, the therapeutic use of CBD is still limited. Despite its potential, little is known about the molecular mechanisms of CBD on epithelial integrity, particularly concerning effects in native intestinal tissue. To accomplish this, our study aimed to investigate the effects of CBD ex vivo on the follicle-associated epithelium of Peyer’s Patches (PP) and villus epithelium (VE) from porcine intestine. To measure the epithelial barrier, the Ussing chamber technique was employed, followed by immunoblotting and confocal laser-scanning immunofluorescence microscopy of tight junction proteins and specific receptors. The results revealed that CBD significantly strengthens the epithelial barrier of PP by upregulation of sealing tight junction proteins, including occludin, claudin-1, -3, and -7. Additionally, the study showed the potential of CBD to decrease the expression of Tumor necrosis factor alpha (TNFɑ) receptor 1 (TNFR-1) in PP that plays a key role in chronic inflammatory diseases. The study highlights the potential of CBD in the prevention of inflammatory conditions and underlines the important role of PP as a target for bioactive compounds. Full article

Background: This study explores the potential of the Pleurotus eryngii mushroom fermentation supernatant (FS-PEWS) as an intervention for mitigating sodium deoxycholate (SDC)-induced intestinal barrier dysfunction and inflammation. Methods: FS-PEWS was assessed for its protective effects against SDC-induced barrier dysfunction and inflammation using an in vitro Caco-2 cell model and ex vivo colonic biopsies from healthy adult donors, where barrier integrity, permeability, immunomodulation and receptor-mediated pathways were evaluated. Results: In Caco-2 cells, SDC exposure downregulated ZO-1, occludin, and claudin-1 expression, with FS-PEWS restoring ZO-1 and claudin-1 levels while maintaining cell viability. In colonic biopsies from healthy adults, FS-PEWS maintained tissue integrity and selectively mitigated transcellular permeability without affecting paracellular permeability when combined with the stressor. Additionally, FS-PEWS exhibited potent anti-inflammatory effects, reducing pro-inflammatory cytokines, e.g., TNF-α, IL-6, and IL-1β and modulating receptor-mediated pathways, i.e., TLR-4, dectin-1. Conclusions: These results demonstrate the potential of FS-PEWS to sustain intestinal barrier function and modulate immune responses under stress, highlighting its therapeutic potential for managing gut barrier dysfunction and inflammation associated with microbial metabolite-induced disruptions. Full article

Transmucosal drug products, such as aerosols, films, semisolids, suppositories, and tablets, have been developed for the treatment of various human diseases and conditions. Transmucosal drug absorption is highly influenced by the biological structures of the mucosa and the physiological environment specific to the administration route (e.g., nasal, rectal, and vaginal). Over the last few decades, in vitro permeation testing (IVPT) using animal tissues or in vitro cell cultures have been utilized as a cost-effective and efficient tool for evaluating drug release and permeation behavior, assisting in formulation development and quality control of transmucosal drug delivery systems. This review summarizes the key mucosal permeation barriers associated with representative transmucosal administration routes, as well as considerations for IVPT method development. It highlights various IVPT methods, including vertical diffusion cell, flow-through diffusion cell, Ussing chamber, and transwell systems. Additionally, future perspectives are discussed, such as the use of optical methods to study in vitro drug permeation and the development of in vitro–in vivo correlation (IVIVC) for transmucosal drug development. The potential of IVPT as part of in vitro bioequivalence assessment strategies for locally acting transmucosal drug products is also highlighted. Full article

When the protective mechanisms of the gastroesophageal mucosa are overwhelmed by injurious factors, the structural and functional mucosal integrity is compromised, resulting in a wide spectrum of disorders. Poliprotect has recently been shown to be non-inferior to standard-dose omeprazole for the treatment of endoscopy-negative patients with heartburn and/or epigastric pain or burning. Here, we provide preclinical data describing the mechanism of action of the Poliprotect formulation, a 100% natural, biodegradable, and environmental friendly medical device according to EU 2017/745 and containing UVCB (unknown or variable composition, complex-reaction products, or biological materials) substances of botanical and mineral origin, according to the REACH and European Chemical Agency definitions. Different in vitro assays demonstrated the capability of Poliprotect to adhere to mucus-secreting gastric cells and concomitantly deliver a local barrier with buffering and antioxidant activity. In studies conducted in accordance with systems biology principles, we evaluated the effects of this barrier on human gastric cells exposed to acidic stress. Biological functions identified via Ingenuity Pathway Analysis highlighted the product’s ability to create a microenvironment that supports the mucosal structural and functional integrity, promotes healing, and restores a balanced mucosal inflammatory status. Additionally, transepithelial electrical resistance and an Ussing chamber showed the product’s capability of preserving the integrity of the gastric and esophageal epithelial barriers when exposed to an acid solution. Two in vivo models of erosive gastropathy further highlighted its topical protection against ethanol- and drug-induced mucosal injury. Overall, our findings sustain the feasibility of a paradigm shift in therapeutics R&D by depicting a very innovative and desirable mode of interaction with the human body based on the emerging biophysical, rather than the pharmacological properties of these therapeutic agents. Full article

The integrity of esophageal epithelial cells in patients with gastroesophageal reflux disease (GERD) or GERD-like symptoms is the first mechanism of protection to decrease the sensitivity to gastric reflux and heartburn symptoms. We investigated the protective effects of Poliprotect^® (PPRO), a CE-marked medical device, on esophageal epithelial integrity using in vitro and ex vivo models. In vitro, the protective effects of PPRO were tested on Caco-2 cells. PPRO demonstrated safety and protection against oxidative damage induced by hydrogen peroxide. It also preserved epithelial integrity by maintaining transepithelial electrical resistance (TEER) against damage from calcium removal or bile acid exposure (taurodeoxycholic acid, TDCA). Ex vivo, esophageal biopsies from patients subjected to endoscopy were mounted in Ussing chambers and exposed to damaging agents (HCl or HCl + TDCA). Untreated biopsies (control) showed significant loss of epithelial resistance (up to −33%). In contrast, low concentrations of PPRO (50–100 µg/mL) provided strong protection against these damages (p < 0.001), even after 60 min of washing. Histological analysis confirmed the barrier-enhancing effect of PPRO. Overall, PPRO effectively protected the esophageal epithelium from damage in both models, suggesting its potential role in alleviating GERD or GERD-like symptoms by strengthening mucosal barriers and reducing epithelial sensitivity to reflux. Full article

Grana Padano (GP) is an Italian hard cooked cheese characterized by a long ripening process and high protein and Ca contents. After in vitro static simulated gastrointestinal digestion, GP digest contained caseinophosphopeptides that were 6 to 24 amino acids in length, including tri-phosphorylated species incorporating the pSer-pSer-pSer-Glu-Glu cluster. Using rat ileum tissue, the digest was used to assess Ca absorption ex vivo, which showed significantly better results for the GP digest in comparison to the CaCO₃ aqueous solution. An in vitro intestinal model based on Caco-2/HT-29 cell co-culture was able to mimic Ca absorption from GP digest, with Ca-rich water as a control. The metabolite-containing medium was then used to treat osteoblast-like SaOS-2 cells. As a consequence, metabolized GP digest significantly increased the number of osteoblasts, whereas the metabolized water did not exert this effect. Finally, the mice were fed diets containing GP or CaCO₃ and pea isolate and the in vivo outcomes were assessed through fluorescent probe and computed tomography. Mice fed a diet containing GP showed a higher increase in bone remodeling and volume in comparison to those fed a control diet containing CaCO₃ and pea isolate. Overall, the ex vivo, in vitro and in vivo experiments highlighted the effectiveness of GP in improving Ca absorption, osteoblast proliferation and bone remodeling and volume. Full article

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that is dysfunctional in individuals with cystic fibrosis (CF). The permeability of CFTR can be experimentally manipulated though different mechanisms, including activation via inducing the phosphorylation of residues in the regulatory domain as well as altering the gating/open probability of the channel. Phosphorylation/activation of the channel is achieved by exposure to compounds that increase intracellular cAMP, with forskolin and IBMX commonly used for this purpose. C_act-A1 is a unique CFTR activator that does not increase intracellular cAMP, and VX-770 (ivacaftor) is a CFTR potentiator that is used experimentally and therapeutically to increase the open probability of the channel. Using primary human nasal epithelial cell (HNEC) cultures and Fischer rat thyroid (FRT) epithelial cells exogenously expressing functional CFTR, we examined the impact of VX-770, C_act-A1, and forskolin/IBMX on CFTR activity during analysis in an Ussing chamber. Relative contributions of these compounds to maximal CFTR activity were dependent on order of exposure, the presence of chemical and electrical gradients, the level of constitutive CFTR function, and the cell model tested. Increasing intracellular cAMP appeared to change cellular functions outside of CFTR activity that resulted in alterations in the drive for chloride through CFTR. These results demonstrate that one can utilize combinations of small-molecule CFTR activators and potentiators to provide detailed characterization of CFTR-mediated ion transport in primary HNECs and properties of these modulators in both primary HNECs and FRT cells. Future studies using these approaches may assist in the identification of novel defects in CFTR function and the identification of modulators with unique impacts on CFTR-mediated ion transport. Full article

Corneal endothelium cells (CECs) regulate corneal hydration between the leaky barrier of the corneal endothelium and the ionic pumps on the surface of CECs. As CECs do not regenerate, loss of CECs leads to poor vision and corneal blindness. Corneal transplant is the only treatment option; however, there is a severe shortage of donor corneas globally. Cell therapy using propagated primary human CECs is an alternative approach to corneal transplantations, and proof of functionality is crucial for validating such CECs. Expression markers like Na-K-ATPase and ZO-1 are typical but not specific to CECs. Assessing the barrier function of the expanded CECs via electrical resistance (i.e., TEER and Ussing’s chamber) involves difficult techniques and is thus impractical for clinical application. Calcium has been demonstrated to affect the paracellular permeability of the corneal endothelium. Its absence alters morphology and disrupts apical junctions in bovine CECs, underscoring its importance. Calcium signaling patterns such as calcium waves affect the rate of wound healing in bovine CECs. Therefore, observing calcium waves in expanded CECs could provide valuable insights into their health and functional integrity. Mechanical or chemical stimulations, combined with Ca²⁺-sensitive fluorescent dyes and time-lapse imaging, can be used to visualize these waves, which could potentially be used to qualify expanded CECs. Full article

Pyrethroids are pesticides used in agriculture, the textile industry, wood processing, and human and animal medicine. Pyrethroids inhibit voltage-sensitive sodium channels (VSSCs) in insects and mammals. It results in the premature opening and/or delayed closing of the channels, causing a prolonged influx of Na⁺ ions into the cell. Insects absorb pyrethroids throughout the entire body surface, while poisoning in humans most often occurs by inhalation and through the skin. In this study, 52 fragments of human skin taken from the eyelid fold were examined. A modified Ussing chamber was used to measure the active ion transport in epithelial tissue and quantify the tissue viability and integrity. Both permethrin and deltamethrin solutions induced changes in the transport of ions, mainly sodium, but by different mechanisms. Permethrin affected the transepithelial transport of sodium ions in a long-term mechanism, while deltamethrin affected the ability to respond to stimuli in an immediate mechanism. Contact with deltamethrin may cause a delay/slowness of sensation, inflammation, hypersensitivity, and/or allergy. The action of permethrin takes place in the intercellular spaces and is associated with the possibility of faster decomposition/metabolism, while deltamethrin interacts with receptors, channels, and the cell membrane, which translates into slower decomposition and longer action in the tissue. Full article

Extensive evidence indicates that the compromise of airway epithelial barrier function is closely linked to the development of various diseases, posing a significant concern for global mortality and morbidity. Flavonoids, natural bioactive compounds, renowned for their antioxidant and anti-inflammatory properties, have been used for centuries to prevent and treat numerous ailments. Lately, a growing body of evidence suggests that flavonoids can enhance the integrity of the airway epithelial barrier. The objective of this study was to investigate the impact of selected flavonoids representing different subclasses, such as kaempferol (flavonol), luteolin (flavone), and naringenin (flavanone), on transepithelial electrical resistance (TEER), ionic currents, cells migration, and proliferation of a human bronchial epithelial cell line (16HBE14σ). To investigate the effect of selected flavonoids, MTT assay, trypan blue staining, and wound healing were assessed. Additionally, transepithelial resistance and Ussing chamber measurements were applied to investigate the impact of the flavonoids on the electrical properties of the epithelial barrier. This study showed that kaempferol, luteolin, and naringenin at micromolar concentrations were not cytotoxic to 16HBE14σ cells. Indeed, in MTT tests, a statistically significant change in cell metabolic activity for luteolin and naringenin was observed. However, our experiments showed that naringenin did not affect the proliferation of 16HBE14σ cells, while the effect of kaempferol and luteolin was inhibitory. Moreover, transepithelial electrical resistance measurements have shown that all of the flavonoids used in this study improved the epithelial integrity with the slightest effect of kaempferol and the significant impact of naringenin and luteolin. Finally, our observations suggest that luteolin increases the Cl- transport through cystic fibrosis transmembrane conductance regulator (CFTR) channel. Our findings reveal that flavonoids representing different subclasses exert distinct effects in the employed cellular model despite their similar chemical structures. In summary, our study sheds new light on the diverse effects of selected flavonoids on airway epithelial barrier function, underscoring the importance of further exploration into their potential therapeutic applications in respiratory health. Full article

VX-770 is a small-molecule CFTR potentiator that is highly efficacious in individuals with cystic fibrosis caused by mutations in CFTR that result in a defect in channel gating. While studies have reported on the mechanism of action of VX-770, there is still more to learn about the impact that it has on CFTR function in various contexts. The aim of the present study was to examine the longevity and stability of the effect of VX-770 on CFTR function in cultured airway epithelia and to measure the consequences of this interaction. The responses to acute and chronic VX-770 exposure were measured in cultures of expanded and re-differentiated primary human nasal epithelial cells. Acute VX-770 exposure resulted in an increase in CFTR-mediated currents in the absence of exogenous compounds that induce the phosphorylation/activation of CFTR, with acute exposure having the same effect as chronic exposure. The functional impact of VX-770 on CFTR was long-lasting in cultured airway epithelia, as they maintained an electrophysiological profile consistent with the saturation of CFTR with VX-770 over time periods of up to 4 days following a short (0.5 min) or low-dose (100 nM) exposure to VX-770 during an analysis in an Ussing chamber. Rinsing the apical surface prior to VX-770 exposure or exposure during the analysis in the Ussing chamber increased the interaction between VX-770 and the CFTR. Importantly, after short, low-dose exposures to VX-770, the CFTR channels in cultured epithelia appeared to remain saturated with VX-770 for extended periods of time, despite the repetitive rinsing of the apical surface. This finding has implications for patients discontinuing the use of VX-770-containing therapies. Full article

Background: Methionine (Met) is a popular nutritional supplement in humans and animals. It is routinely supplemented to pigs as L-Met, DL-Met, or DL-2-hydroxy-4-(methylthio) butanoic acid (DL-HMTBA). Methods: We investigated the effect of these Met supplements on jejunal amino acid (AA) transport in male castrated Piétrain × Danbred pigs, also including a non-supplemented group. The mucosal-to-serosal flux of ten [¹⁴C]-labeled AAs (L-glutamine, glycine, L-leucine, L-lysine, L-Met, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine) was investigated at two concentrations (50 µM and 5 mM). Inhibition of apical uptake by mucosal L-Met was also measured for these AAs. The intestinal expression of apical AA transporters, angiotensin-converting enzyme II and inflammation-related genes were compared with those of a previous study. Results: Except for tryptophan and lysine at 5 mM, all AA fluxes were Na⁺-dependent (p ≤ 0.05), and the uptake of most AAs, except glycine and lysine, was inhibited by L-Met (p < 0.001). A correlation network existed between Na⁺-dependent fluxes of most AAs (except tryptophan and partly glycine). We observed the upregulation of B⁰AT1 (SLC6A19) (p < 0.001), the downregulation of ATB^0,+ (SLC6A14) (p < 0.001) and a lower expression of CASP1, IL1β, IL8, TGFβ and TNFα in the present vs. the previous study (p < 0.001). Conclusions: The correlating AAs likely share the same Na⁺-dependent transporter(s). A varying effect of the Met supplement type on AA transport in the two studies might be related to a different level of supplementation or a different inflammatory status of the small intestine. Full article

Retinoids are known to improve the condition of the skin. Transepithelial transport of sodium and chloride ions is important for proper skin function. So far, the effect of applying vitamin A preparations to the skin on ion transport has not been evaluated. In the study, electrophysiological parameters, including transepithelial electric potential (PD) and transepithelial resistance (R), of rabbit skin specimens after 24 h exposure to retinol ointment (800 mass units/g) were measured in a modified Ussing chamber. The R of the fragments incubated with retinol was significantly different than that of the control skin samples incubated in iso-osmotic Ringer solution. For the controls, the PD values were negative, whereas the retinol-treated specimens revealed positive PD values. Mechanical–chemical stimulation with the use of inhibitors of the transport of sodium (amiloride) or chloride (bumetanide) ions revealed specific changes in the maximal and minimal PD values measured for the retinol-treated samples. Retinol was shown to slightly modify the transport pathways of sodium and chloride ions. In particular, an intensification of the chloride ion secretion from keratinocytes was observed. The proposed action may contribute to deep hydration and increase skin tightness, limiting the action of other substances on its surface. Full article