Protective efficacy of antidiarrheal agents in a permeability model of Escherichia coli-infected CacoGoblet® cells.

AIM: To compare the protective efficacy of gelatine tannate/probiotic with other antidiarrheal agents in Escherichia coli-inoculated CacoGoblet® cells. METHODS: Four test compounds - gelatine tannate plus inactivated probiotic, diosmectite, probiotic mixture and Saccharomyces boulardii - were added to E. coli-infected CacoGoblet cells. After 1 and 24 h, transepithelial electrical resistance was measured and a lucifer yellow assay performed. RESULTS: Gelatine tannate/probiotic markedly increased transepithelial electrical resistance by 123.1% (at 1 h) and 149.5% (at 24 h), and produced paracellular flux values of 0.41% (1 h) and 1.34% (24 h), which were considerably less than the E. coli-invasion value (2.41%). CONCLUSION: The protective efficacy of gelatine tannate/probiotic against E. coli-induced reduction of membrane integrity manifests early and is maintained for 24 h.

In vitro method to evaluate the barrier properties of medical devices for cutaneous use.

Barrier creams (BC) are marketed as cosmetic products or locally-applied medical devices to protect skin against damages induced by chemical agents or physical insults. However, the determination of the BC effectiveness is still a matter of discussion at both the clinical and the regulatory level. In this context, this work aimed at the development of a reliable, reproducible and easy-to-perform experimental protocol for the evaluation of BC performances. Preliminarily, an in vivo method based on the measurement of trans-epidermal water loss had been matter of investigation and was discarded: it required too much time and was not robust and sensitive enough. In vitro, reduction of the permeation of caffeine (used as a model of irritant), through an epidermal membrane mounted on a Franz cell or through a reconstructed 3D human epidermis model, was evaluated. Six BC among oil in water (O/W) or water in oil (W/O) creams were investigated with respect to the petrolatum, which is an efficient impermeable barrier against hydrophilic molecules. Despite minor differences, both methods could rate the effectiveness of the tested products in preventing caffeine exposure. Both methods enable to evaluate and quantify the BC effectiveness in a simple and fast manner. Their application may help regulatory agencies to prevent the marketing of ineffective products for the benefit of consumers.

Protective barrier properties of Rhinosectan® spray (containing xyloglucan) on an organotypic 3D airway tissue model (MucilAir): results of an in vitro study.

BACKGROUND: To evaluate barrier protective properties of Rhinosectan® spray, a medical device containing xyloglucan, on nasal epithelial cells (MucilAir). METHODS: MucilAir-Nasal, a three-dimensional organotypic (with different cell types) airway tissue model, was treated with the medical device Rhinosectan® (30 µL) or with controls (Rhinocort-budesonide-or saline solution). The protective barrier effects of Rhinosectan® were evaluated by: TEER (trans-epithelial electrical resistance) (preservation of tight junctions), Lucifer Yellow assay (preservation of paracellular flux) and confocal immunofluorescence microscopy (localization of tight junction proteins). RESULTS: Exposure of MucilAir with Rhinosectan® protected cell tight junctions (increases in TEER of 13.1% vs -6.3% with saline solution after 1 h of exposure), and preserved the paracellular flux, even after exposure with pro-inflammatory compounds (TNF-α and LPS from Pseudomonas aeruginosa 10). Results of confocal immunofluorescence microscopy demonstrated that, after treatment with the pro-inflammatory mixture, Rhinosectan® produced a slight relocation of zona occludens-1 in the cytosol compartment (while Rhinocort induced expression of zona-occludens-1), maintaining the localization of occludin (similarly to negative control). CONCLUSIONS: Results of our study indicates that Rhinosectan® creates a protective physical barrier on nasal epithelial cells in vitro, allowing the avoidance of allergens and triggering factors, thus confirming the utility of this medical device in the management of nasal respiratory diseases, as rhinitis or rhinosinusitis.

Effect of Utipro(®) (containing gelatin-xyloglucan) against Escherichia coli invasion of intestinal epithelial cells: results of an in vitro study.

AIM: To evaluate whether Utipro(®), a natural product approved to prevent urinary tract infections, protects intestinal epithelial cells from Escherichia coli adherence/intracellular invasion in vitro. MATERIALS & METHODS: Caco-2 and CacoGoblet(TM) cells were treated with Utipro (1.5 to 10 mg/ml) or untreated (controls). E. coli adherence/intracellular invasion was evaluated by Trans-Epithelial Electrical Resistance, Lucifer Yellow assay and microbial counts. RESULTS: Utipro was noncytotoxic. Utipro 5 and 10 mg/ml protected cell tight junctions (mean ± SD Trans-Epithelial Electrical Resistance [Ω × cm(2)] 66.83 ± 0.29 and 71.33 ± 0.29, respectively), and protected cells from E. coli intracellular invasion (mean ± SD reductions in total bacteria counts [Log10] 0.9 ± 0.06 and 2.1 ± 0.56, respectively). CONCLUSION: Results indicate that Utipro creates a protective physical barrier on intestinal epithelial cells in vitro which reduces the settling of E. coli reservoirs. These results constitute the first step in the demonstration of the efficacy of Utipro to prevent urinary tract infections. Further research is needed in in vivo models and clinical trials.

Paraquat modulates alternative pre-mRNA splicing by modifying the intracellular distribution of SRPK2.

Paraquat (PQ) is a neurotoxic herbicide that induces superoxide formation. Although it is known that its toxic properties are linked to ROS production, the cellular response to PQ is still poorly understood. We reported previously that treatment with PQ induced genome-wide changes in pre-mRNA splicing. Here, we investigated the molecular mechanism underlying PQ-induced pre-mRNA splicing alterations. We show that PQ treatment leads to the phosphorylation and nuclear accumulation of SRPK2, a member of the family of serine/arginine (SR) protein-specific kinases. Concomitantly, we observed increased phosphorylation of SR proteins. Site-specific mutagenesis identified a single serine residue that is necessary and sufficient for nuclear localization of SRPK2. Transfection of a phosphomimetic mutant modified splice site selection of the E1A minigene splicing reporter similar to PQ-treatment. Finally, we found that PQ induces DNA damage and vice versa that genotoxic treatments are also able to promote SRPK2 phosphorylation and nuclear localization. Consistent with these observations, treatment with PQ, cisplatin or γ-radiation promote changes in the splicing pattern of genes involved in DNA repair, cell cycle control, and apoptosis. Altogether, our findings reveal a novel regulatory mechanism that connects PQ to the DNA damage response and to the modulation of alternative splicing via SRPK2 phosphorylation.