Abstract Introduction: there is a close relationship between obesity, gut health and immune system. A low-grade of inflammation, which could precede obesity, may have implications for the development of metabolic syndrome and insulin resistance. Objective: analyzing the anti-inflammatory capacity of several types of whey (cow, sheep, goat and a mixture of them). Methods: an in vitro model of intestinal inflammation employing a cell co-culture (Caco-2 and RAW 264.7) was performed after an in vitro digestion and fermentation (simulating mouth-to-colon conditions). Inflammatory markers such as IL-8 and TNF-α, as well as the transepithelial electrical resistance (TEER) of Caco-2 monolayer, were determined. Results: digested and fermented whey had a protective effect on cell permeability, being lower in the case of fermented goat whey and mixture. The anti-inflammatory activity of whey was greater the more digestion progressed. Fermented whey showed the greatest anti-inflammatory effect, inhibiting IL-8 and TNF-α secretion, probably due to its composition (protein degradation products such as peptides and amino acids, and SCFA). However, fermented goat whey did not show this degree of inhibition, perhaps due to its low SCFA concentration. Conclusion: milk whey, especially after being fermented in the colon, can be useful nutritional strategy to preserve the intestinal barrier and mitigate the low-grade of inflammation that characterizes metabolic disorders and obesity.

Resumen Introducción: existe una estrecha relación entre obesidad, salud intestinal y sistema inmune. Un bajo grado de inflamación, que precedería a la obesidad, puede tener implicaciones en el desarrollo de síndrome metabólico y resistencia a la insulina. Objetivo: analizar el poder antiinflamatorio de varios tipos de lactosuero (vaca, oveja, cabra y mezcla de los anteriores). Metodología: se utilizó un modelo in vitro de inflamación intestinal, empleando un cocultivo celular (Caco-2 y RAW 264.7). Para ello, se realizó una digestión y fermentación in vitro (simulando las condiciones de boca a colon). Se estudiaron IL-8 y TNF-α como marcadores inflamatorios y la resistencia eléctrica transepitelial celular (RETE) de la monocapa celular Caco-2. Resultados: el suero digerido y fermentado tuvo un efecto protector sobre la permeabilidad celular que fue menor en el caso de lactosuero fermentado de cabra y mezcla. La actividad antiinflamatoria del suero fue mayor cuanto más progresaba la digestión. El lactosuero fermentado mostró el mayor efecto antiinflamatorio, inhibiendo la secreción de IL-8 y TNF-α, probablemente debido a su composición (productos de degradación proteica como péptidos y aminoácidos, y ácidos grasos de cadena corta [AGCC]). Sin embargo, el suero fermentado de cabra no mostró ese grado de inhibición, quizás debido a su baja concentración en AGCC. Conclusión: el lactosuero, sobre todo tras ser fermentado en colon, puede ser una estrategia nutricional útil para preservar la barrera intestinal y mitigar el bajo grado de inflamación que caracteriza a desordenes metabólicos y a la obesidad.

The role of cyclooxygenase (COXs) isoforms in maintaining colonic mucosal integrity is not fully understood. This study aimed to evaluate the role of COX-1 and -2 on colonic mucosal integrity in an experimental colitis model. Colitis was induced in Wistar rats by intracolonic administration of 2,4,6-trinitrobenzenesulfonic acid (20 mg + 50% ethanol). The control group (sham group) received saline only. After 7, 14, or 28 days, colonic samples were removed, and macroscopic lesion scores, wet weight, myeloperoxidase activity, and transepithelial electrical resistance (TER) were determined. In other rat groups, colonic samples from the sham group and a 7th day post-colitis group were mounted in Üssing chambers with the luminal side exposed to a buffer solution (control), acetylsalicylic acid (ASA), SC-560 (COX-1 inhibitor), or celecoxib (COX-2 inhibitor). TER and epithelial permeability to fluorescein were measured. The 7th day colitis group had higher macroscopic damage scores, wet weight, and myeloperoxidase activity and lower basal TER than the sham, 14th day colitis, and 28th day colitis groups. Inhibition of COX-1 but not COX-2 significantly decreased TER and increased permeability to fluorescein in the 7th day post-colitis group compared to the sham group. Additionally, ASA decreased the colonic mucosal integrity on day seven post-colitis compared to the sham group. A decrease in the colonic mucosa integrity in the experimental colitis model can be aggravated only by the inhibition of COX-1, which demonstrated the importance of this enzyme in the maintenance of colonic mucosal integrity. COXs (COXs understood COX1 COX 1 COX- 2 - 2,4,6trinitrobenzenesulfonic 246trinitrobenzenesulfonic trinitrobenzenesulfonic 2,4,6 4 6 20 (2 50 ethanol. ethanol . ethanol) 7 14 days removed scores weight (TER determined groups th postcolitis post control, , (control) ASA, (ASA) SC560 SC 560 SC-56 (COX- inhibitor, inhibitor inhibitor) COX2 inhibitor. measured Additionally COX1, 1, 6trinitrobenzenesulfonic 246 2,4, ( 5 (control (ASA SC56 56 SC-5 (COX 24 2,4 SC5 SC- 2,

Abstract Purpose: The development of an electric impedance meter based on the impedance spectroscopy technique, for in vitro and in situ experimentation, with cellular epithelia submitted to extremely low frequency magnetic fields in a controlled environment. Unlike other reported systems, a strength of the one presented here is that it avoids the influence of external factors on the experiment. Materials and methods: The designed system employs the electrical impedance values obtained by the impedance spectroscopy technique to determine the parameters of the simple equivalent electrical model of a cellular monolayer. The Madin-Darby Canine Kidney (MDCK) cell cultures were used as subjects of study in the experimental protocol. Results: The validation was carried out by comparing the transepithelial electrical impedance data of the cell cultures obtained with the developed system and those of the Cellzscope® commercial system used as the standard. Non-significant differences were obtained. Conclusion: It was confirmed that the developed system provides reliable values of transepithelial electrical impedance to experiment with cell cultures and take advantage of the controlled environment to reduce the effects of experimental management.

RESUMO Introdução: Tem sido sugerido que na doença renal crônica (DRC) a uremia pode causar alterações intestinais, tais como modificações na microbiota e danos à barreira intestinal, e que estas possíveis alterações podem ter uma relação importante com o estado inflamatório e a toxicidade urêmica apresentadas por pacientes com DRC. Objetivos: Avaliar o efeito in vitro do soro urêmico sobre a permeabilidade da monocamada de células epiteliais do intestino, inflamação e apoptose. Métodos: Pools de soro foram preparados a partir de soros de indivíduos saudáveis, pacientes em tratamento conservador e em hemodiálise (Pré e Pós-HD). As células T84 foram incubadas por 24 horas com os diferentes pools. Em seguida a TER foi medida e as células foram submetidas às seguintes análises: apoptose, produção de espécies reativas de oxigênio (EROs) e expressão de receptores toll-like (TLR) por citometria de fluxo e detecção de IL-6 no sobrenadante da cultura por ELISA. Resultados: Não foram encontradas diferenças, entre os grupos, com relação a TER, apoptose, EROs e expressão de TLR-2, TLR-4 e TLR-9. Já a secreção de IL-6 foi maior (p < 0,001) pelas células incubadas com soro pré-HD e pós-HD. Conclusão: Os resultados obtidos a partir deste modelo sugerem que a uremia per se parece não comprometer a integridade das células epiteliais do intestino. O aumento da secreção de IL-6 pelas células incubadas com soro HD (pré e pós) sugere um potencial efeito da uremia sobre a resposta inflamatória intestinal.

ABSTRACT Introduction: In chronic kidney disease (CKD), it has been suggested that alterations within the gut are associated with an inflammatory state and uremic toxicity. Studies suggest that uremia may impair the function of the intestinal barrier via the promotion of increased intestinal permeability. To understand the mechanisms that are involved in intestinal barrier damage in the setting of uremia, we evaluated the in vitro effect of uremic serum on transepithelial electrical resistance (TER), inflammation, and apoptosis in intestinal epithelial cells (T84). Methods: Pools of serum from healthy individuals, patients not on dialysis, and patients on hemodialysis (Pre-HD and Post-HD) were prepared. T84 cells were incubated for 24 h in medium, of which 10% consisted of the pooled serum from each group. After incubation, the TER was measured and the following parameters were determined by flow cytometry: expression of toll-like receptors (TLRs), production of reactive oxygen species (ROS), and apoptosis. The level of IL-6 in the culture supernatant was determined by ELISA. Results: No difference was observed among the groups with respect to TER, apoptosis, and ROS or the expression of TLR-2, TLR-4, and TLR-9. IL-6 secretion was higher (p < 0.001) in cells that were incubated with pre- and post-HD serum. Conclusion: The results that were obtained from this model suggest that uremic serum per se does not seem to impair the integrity of intestinal epithelial cells. The increased IL-6 secretion by cells that were incubated with HD serum suggests a potential effect of uremia in the intestinal inflammatory response.

The epithelium is a highly dynamic system, which plays a crucial role in the homeostasis of the intestinal tract. However, studies on the physiological and pathophysiological functions of intestinal epithelial cells (IECs) have been hampered due to lack of normal epithelial cell models. In the present study, we established a reproducible method for primary culture of mouse IECs, which were isolated from the viable small intestinal crypts of murine fetuses (on embryonic day 19), using type I collagenase and hyaluronidase in a short span of time (≤20 min). With this method, continuously growing mouse IECs, which can be subcultured over a number of passages, were obtained. The obtained cell lines formed a tight cobblestone-like arrangement, displayed long and slender microvilli, expressed characteristic markers (cytokeratin 18 and Notch-1), and generated increasing transepithelial electrical resistance and low paracellular permeability during in vitro culture. The cells also had enzymatic activities of alkaline phosphatase and sucrase-isomaltase, and secreted various cytokines (IL-1β, IL-6, IL-8, and monocyte chemoattractant protein-1), responding to the stimulation of Escherichia coli. These results show that the primary-cultured mouse IECs obtained by the method established here had the morphological and immunological characteristics of IECs. This culture system can be a beneficial in vitro model for studies on mucosal immunology and toxicology.

The objectives of this study were to determine the effect of tumor necrosis factor alpha (TNF-α) on intestinal epithelial cell permeability and the expression of tight junction proteins. Caco-2 cells were plated onto Transwell® microporous filters and treated with TNF-α (10 or 100 ng/mL) for 0, 4, 8, 16, or 24 h. The transepithelial electrical resistance and the mucosal-to-serosal flux rates of the established paracellular marker Lucifer yellow were measured in filter-grown monolayers of Caco-2 intestinal cells. The localization and expression of the tight junction protein occludin were detected by immunofluorescence and Western blot analysis, respectively. SYBR-Green-based real-time PCR was used to measure the expression of occludin mRNA. TNF-α treatment produced concentration- and time-dependent decreases in Caco-2 transepithelial resistance and increases in transepithelial permeability to the paracellular marker Lucifer yellow. Western blot results indicated that TNF-α decreased the expression of phosphorylated occludin in detergent-insoluble fractions but did not affect the expression of non-phosphorylated occludin protein. Real-time RT-PCR data showed that TNF-α did not affect the expression of occludin mRNA. Taken together, our data demonstrate that TNF-α increases Caco-2 monolayer permeability, decreases occludin protein expression and disturbs intercellular junctions.

T84 is an established cell line expressing an enterocyte phenotype whose permeability properties have been widely explored. Osmotic permeability (P OSM), hydraulic permeability (P HYDR) and transport-associated net water fluxes (J W-transp), as well as short-circuit current (I SC), transepithelial resistance (R T), and potential difference (deltaV T) were measured in T84 monolayers with the following results: P OSM 1.3 ± 0.1 cm.s-1 x 10-3; P HYDR 0.27 ± 0.02 cm.s-1; R T 2426 ± 109 omega.cm², and deltaV T 1.31 ± 0.38 mV. The effect of 50 µM 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DCEBIO), a "net Cl<FONT FACE=Symbol>-</FONT> secretory agent", on T84 cells was also studied. We confirm the reported important increase in I SC induced by DCEBIO which was associated here with a modest secretory deltaJ W-transp. The present results were compared with those reported using the same experimental approach applied to established cell lines originating from intestinal and renal epithelial cells (Caco-2, LLC-PK1 and RCCD-1). No clear association between P HYDR and R T could be demonstrated and high P HYDR values were observed in an electrically tight epithelium, supporting the view that a "water leaky" barrier is not necessarily an "electrically leaky" one. Furthermore, the modest secretory deltaJ W-transp was not consistent with previous results obtained with RCCD-1 cells stimulated with vasopressin (absorptive fluxes) or with T84 cells secreting water under the action of Escherichia coli heat stable enterotoxin. We conclude that, while the presence of aquaporins is necessary to dissipate an external osmotic gradient, coupling between water and ion transport cannot be explained by a simple and common underlying mechanism.