Basic Three-Dimensional (3D) Intestinal Model System with an Immune Component.

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the pharmacological screening of potentially beneficial substances, and for toxicity studies on potentially harmful food components. Of relevance, there is a current demand for the development of cell-based in vitro models to substitute animal models. Here, a protocol for a basic, "healthy tissue" three-dimensional (3D) intestinal equivalent model using cell lines is presented with the dual benefit of providing both experimental simplicity (standardized and easily repeatable system) and physiological complexity (Caco-2 enterocytes with a supporting immune component of U937 monocytes and L929 fibroblasts). The protocol also includes paraffin embedding for light microscopic evaluation of fixed intestinal equivalents, thereby providing the advantage of analyzing multiple visual parameters from a single experiment. Hematoxylin and eosin (H&E) stained sections showing the Caco-2 columnar cells forming a tight and regular monolayer in control treatments are used to verify the efficacy of the model as an experimental system. Using gluten as a pro-inflammatory food component, parameters analyzed from sections include reduced monolayer thickness, as well as disruption and detachment from the underlying matrix (H&E), decreased tight junction protein expression as shown from occludin staining (quantifiable statistically), and immune-activation of migrating U937 cells as evidenced from the cluster of differentiation 14 (CD14) staining and CD11b-related differentiation into macrophages. As shown by using lipopolysaccharide to simulate intestinal inflammation, additional parameters that can be measured are increased mucus staining and cytokine expression (such as midkine) that can be extracted from the medium prior to fixation. The basic three-dimensional (3D) intestinal mucosa model and fixed sections can be recommended for inflammatory status and barrier integrity studies with the possibility of analyzing multiple visual quantifiable parameters.

Spermidine-Eugenol Supplement Preserved Inflammation-Challenged Intestinal Cells by Stimulating Autophagy.

Increases in non-communicable and auto-immune diseases, with a shared etiology of defective autophagy and chronic inflammation, have motivated research both on natural products in drug discovery fields and on the interrelationship between autophagy and inflammation. Within this framework, the tolerability and protective effects of a wheat-germ spermidine (SPD) and clove eugenol (EUG) combination supplement (SUPPL) were investigated on inflammation status (after the administration of lipopolysaccharide (LPS)) and on autophagy using human Caco-2 and NCM460 cell lines. In comparison to the LPS treatment alone, the SUPPL + LPS significantly attenuated ROS levels and midkine expression in monocultures, as well as occludin expression and mucus production in reconstituted intestinal equivalents. Over a timeline of 2-4 h, the SUPPL and SUPPL + LPS treatments stimulated autophagy LC3-11 steady state expression and turnover, as well as P62 turnover. After completely blocking autophagy with dorsomorphin, inflammatory midkine was significantly reduced in the SUPPL + LPS treatment in a non-autophagy-dependent manner. After a 24 h timeline, preliminary results showed that mitophagy receptor BNIP3L expression was significantly downregulated in the SUPPL + LPS treatment compared to the LPS alone, whereas conventional autophagy protein expression was significantly higher. The SUPPL shows promise in reducing inflammation and increasing autophagy to improve intestinal health.

Wheat Germ Spermidine and Clove Eugenol in Combination Stimulate Autophagy In Vitro Showing Potential in Supporting the Immune System against Viral Infections.

Impaired autophagy, responsible for increased inflammation, constitutes a risk factor for the more severe COVID-19 outcomes. Spermidine (SPD) is a known autophagy modulator and supplementation for COVID-19 risk groups (including the elderly) is recommended. However, information on the modulatory effects of eugenol (EUG) is scarce. Therefore, the effects of SPD and EUG, both singularly and in combination, on autophagy were investigated using different cell lines (HBEpiC, SHSY5Y, HUVEC, Caco-2, L929 and U937). SPD (0.3 mM), EUG (0.2 mM) and 0.3 mM SPD + 0.2 mM EUG, significantly increased autophagy using the hallmark measure of LC3-II protein accumulation in the cell lines without cytotoxic effects. Using Caco-2 cells as a model, several crucial autophagy proteins were upregulated at all stages of autophagic flux in response to the treatments. This effect was verified by the activation/differentiation and migration of U937 monocytes in a three-dimensional reconstituted intestinal model (Caco-2, L929 and U937 cells). Comparable benefits of SPD, EUG and SPD + EUG in inducing autophagy were shown by the protection of Caco-2 and L929 cells against lipopolysaccharide-induced inflammation. SPD + EUG is an innovative dual therapy capable of stimulating autophagy and reducing inflammation in vitro and could show promise for COVID-19 risk groups.

An Overview on Dietary Polyphenols and Their Biopharmaceutical Classification System (BCS).

Polyphenols are natural organic compounds produced by plants, acting as antioxidants by reacting with ROS. These compounds are widely consumed in daily diet and many studies report several benefits to human health thanks to their bioavailability in humans. However, the digestion process of phenolic compounds is still not completely clear. Moreover, bioavailability is dependent on the metabolic phase of these compounds. The LogP value can be managed as a simplified measure of the lipophilicity of a substance ingested within the human body, which affects resultant absorption. The biopharmaceutical classification system (BCS), a method used to classify drugs intended for gastrointestinal absorption, correlates the solubility and permeability of the drug with both the rate and extent of oral absorption. BCS may be helpful to measure the bioactive constituents of foods, such as polyphenols, in order to understand their nutraceutical potential. There are many literature studies that focus on permeability, absorption, and bioavailability of polyphenols and their resultant metabolic byproducts, but there is still confusion about their respective LogP values and BCS classification. This review will provide an overview of the information regarding 10 dietarypolyphenols (ferulic acid, chlorogenic acid, rutin, quercetin, apigenin, cirsimaritin, daidzein, resveratrol, ellagic acid, and curcumin) and their association with the BCS classification.

Durable Self-Cleaning Coatings for Architectural Surfaces by Incorporation of TiO2 Nano-Particles into Hydroxyapatite Films.

To prevent soiling of marble exposed outdoors, the use of TiO2 nano-particles has been proposed in the literature by two main routes, both raising durability issues: (i) direct application to marble surface, with the risk of particle leaching by rainfall; (ii) particle incorporation into inorganic or organic coatings, with the risk of organic coating degradation catalyzed by TiO2 photoactivity. Here, we investigated the combination of nano-TiO2 and hydroxyapatite (HAP), previously developed for marble protection against dissolution in rain and mechanical consolidation. HAP-TiO2 combination was investigated by two routes: (i) sequential application of HAP followed by nano-TiO2 ("H+T"); (ii) simultaneous application by introducing nano-TiO2 into the phosphate solution used to form HAP ("HT"). The self-cleaning ability was evaluated before and after prolonged exposure to simulated rain. "H+T" and "HT" coatings exhibited much better resistance to nano-TiO2 leaching by rain, compared to TiO2 alone. In "H+T" samples, TiO2 nano-particles adhere better to HAP (having flower-like morphology and high specific surface area) than to marble. In "HT" samples, thanks to chemical bonds between nano-TiO2 and HAP, the particles are firmly incorporated in the HAP coating, which protects them from leaching by rain, without diminishing their photoactivity and without being degraded by them.

A quest for supramolecular gelators: silver(I) complexes with quinoline-urea derivatives.

The quinoline urea derivatives 1,3-di(quinolin-5-yl)urea (DQ5U), 1-phenyl-3-(quinolin-6-yl)urea (PQ6U), 1-(isoquinolin-5-yl)-3-phenylurea (PiQ5U) and 1-phenyl-3-(3,5-bis(pyrid-2-yl)-1,2,4-triazol-4-yl)urea (PPT4U) have been synthesised and structurally characterized by powder and single crystal X-ray diffraction. Their gelator behaviour in the formation of Ag-complexes has been explored. Compound DQ5U proved capable of gelating the mixed solvent EtOH-DMF 1 : 2 (v/v) when mixed with 1 equivalent of AgNO3. In the case of PQ6U, two polymorphic forms of the complex [Ag(PQ6U)2]NO3, plus the solvated form [Ag(PQ6U)2]NO3·CH3CN, were crystallized. Photophysical characterization of the ligands has been conducted in solution, while fluorescence microscopy has been used to examine the microstructure and photophysical properties of the gels formed by PQ5U and DQ5U with AgNO3.

Polymorphs from supramolecular gels: four crystal forms of the same silver(I) supergelator crystallized directly from its gels.

A new silver(I) complex with 1-phenyl-3-(quinolin-5-yl)urea has been prepared, acting as a supramolecular supergelator in the presence of polar solvents. In open vials temperature dependent, reversible gel-to-xerogel transition is observed, while in sealed vials direct crystallization of four polymorphs of the same gelling compound is achieved, depending on the gelling solvent.