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Effects of different short-chain fatty acids (SCFA) on gene expression of proteins involved in barrier function in IPEC-J2
Get PDFBACKGROUND: Gut microbial anaerobic fermentation produces short-chain fatty acids (SCFA), which are important substrates for energy metabolism and anabolic processes in mammals. SCFA can regulate the inflammatory response and increase the intestinal barrier integrity by enhancing the tight junction protein (TJp) functions, which prevent the passage of antigens through the paracellular space. The aim of this study was to evaluate the effect of in vitro supplementation with SCFA (acetate, propionate, butyrate, and lactate) at different concentrations on viability, nitric oxide (NO) release (oxidative stress parameter) in cell culture supernatants, and gene expression of TJp (occludin, zonula occludens-1, and claudin-4) and pro-inflammatory pathway-related mediators (β-defensin 1, TNF-α, and NF-κB) in intestinal porcine epithelial cell line J2 (IPEC-J2). RESULTS: The SCFA tested showed significant effects on IPEC-J2, which proved to be dependent on the type and specific concentration of the fatty acid. Acetate stimulated cell viability and NO production in a dose-dependent manner (P < 0.05), and specifically, 5 mM acetate activated the barrier response through claudin-4, and immunity through β-defensin 1 (P < 0.05). The same effect on these parameters was shown by propionate supplementation, especially at 1 mM (P < 0.05). Contrarily, lactate and butyrate showed different effects compared to acetate and propionate, as they did not stimulate an increase of cell viability and regulated barrier integrity through zonula occludens-1 and occludin, especially at 30 mM and 0.5 mM, respectively (P < 0.05). Upon supplementation with SCFA, the increase of NO release at low levels proved not to have detrimental effects on IPEC-J2 proliferation/survival, and in the case of acetate and propionate, such levels were associated with beneficial effects. Furthermore, the results showed that SCFA supplementation induced β-defensin 1 (P < 0.05) that, in turn, may have been involved in the inhibition of TNF-α and NF-κB gene expression (P < 0.05). CONCLUSIONS: The present study demonstrates that the supplementation with specific SCFA in IPEC-J2 can significantly modulate the process of barrier protection, and that particularly acetate and propionate sustain cell viability, low oxidative stress activity and intestinal barrier function
Bioactive Peptides in Dairy Milk: Highlighting the Role of Melatonin
No full text: Melatonin, an endogenous indolamine derived from tryptophan, is primarily synthesized by the pineal gland in mammals and regulated by a complex neural system. Its release follows a circadian rhythm, which is crucial for regulating physiological processes in response to light-dark cycles in both humans and animals. In this review, we report that the presence of this hormone in bovine milk, with significant differences in concentration between daytime and nighttime milking, has increased interest in milk as a natural source of bioactive molecules. Melatonin lowers cortisol levels at night, reduces body temperature and blood pressure, coinciding with decreased alertness and performance, acts as an antioxidant and anti-inflammatory agent, modulates the immune system, offers neuroprotective benefits, and supports gastrointestinal health by scavenging free radicals and reducing oxidative stress in dairy cows. Many factors influence the release of melatonin, such as the intensity of artificial lighting during nighttime milking, the frequency of milkings, milk yield, and genetic differences between animals. Nocturnal milking under low-intensity light boosts melatonin, potentially reducing oxidative damage and mastitis risk. Additionally, ultra-high temperature (UHT) treatment does not significantly affect the melatonin content in milk. However, further research on its stability during milk processing and storage is crucial for ensuring product efficacy. In some countries, nighttime milk with naturally elevated melatonin content is already commercialized as a natural aid for sleep. Thus, naturally melatonin-rich milk may be a promising alternative to synthetic supplements for promoting better sleep and overall well-being
Responsiveness of intestinal epithelial cells to eubiotics: IPEC-J2 as in vitro model in swine
No full textGli effetti degli eubiotici sono di notevole interesse in quanto vengono utilizzati come additivi nell'allevamento suino in risposta alle attuali normative volte ad evitare la resistenza agli antibiotici. Dal 2013, l'Organizzazione mondiale della sanità ha vietato gli antibiotici e lo zinco come promotori della crescita. Nello studio 1, abbiamo caratterizzato un modello di co-coltura composto da cellule epiteliali intestinali (IPEC-J2) e cellule immunitarie del sangue periferico (PBMC). L'arginina è un amminoacido essenziale tra le varie sostanze utilizzate in sostituzione degli antibiotici. Utilizzando questo modello, abbiamo valutato come la privazione di arginina induce un aumento del danno intestinale e la conseguente attivazione della risposta immunitaria intestinale. La privazione porta all'immunoregolazione mediata dall'espressione di pro-infiammatori (fattore di necrosi tumorale (TNF)α, interleuchina (IL)-8, interleuchina (IL)-1α, interleuchina (IL)-6) e antinfiammatori (trasformazione Fattore di crescita (TGF)-β) citochine. Tra le altre molecole alternative, gli acidi grassi a catena corta (SCFA), fonti importanti per il metabolismo energetico e i processi anabolici nei mammiferi, possono regolare la risposta infiammatoria e aumentare l'integrità della barriera intestinale. Nello studio 2, abbiamo valutato gli effetti dell'integrazione di SCFA (acetato, butirrato, propionato e lattato) a diverse concentrazioni in IPEC-J2. Gli SCFA migliorano le funzioni delle proteine della giunzione stretta (TJp), che impediscono il passaggio degli antigeni attraverso la barriera intestinale nello spazio paracellulare. I risultati mostrano impatti su IPEC-J2, che dipendono dal tipo e dalla concentrazione specifica di SCFA. L'acetato e il propionato esercitano un'azione protettiva maggiore rispetto al lattato e al butirrato, alle concentrazioni prescelte. Inoltre, l'integrazione di SCFA stimola l'espressione di TJp e β-defensina 1 (BD-1), che, a sua volta, potrebbe essere stata coinvolta nell'inibizione dell'espressione genica del fattore nucleare (NF)-κB e del TNFα. Infine nello studio 3, basato sullo studio precedente, 5 mM di acetato e 1 mM di propionato sono stati usati come trattamenti per la co-coltura su lipopolisaccaridi (LPS), mimando lo stato infiammatorio acuto. L'acetato e il propionato hanno un effetto protettivo mediante la regolazione del TJp sull'infiammazione acuta.
In sintesi, è stato caratterizzato un modello di co-coltura di IPEC-J2 e PBMC. Il modello potrebbe essere importante per studiare e chiarire il ruolo delle condizioni nutrizionali nella funzionalità e nella reattività delle cellule epiteliali intestinali (IEC) nei suini. La privazione di arginina colpisce le cellule epiteliali intestinali e stimola la loro risposta funzionale; l'amminoacido funge da modulatore della risposta antinfiammatoria locale per adattarsi e reagire a uno stimolo stressante. Inoltre, i dati potrebbero essere tradotti nell'uomo che ci sono somiglianze tra maiali e umani in termini di microbiota intestinale e nei meccanismi dei sistemi di difesa intestinale.
Infine, gli SCFA hanno ruoli protettivi a livello intestinale. Abbiamo evidenziato l'effetto positivo in vitro degli SCFA, anche in presenza di LPS-challenge, sulla vitalità intestinale e sul mantenimento dell'integrità intestinale. Le azioni degli SCFA dipendono dal tipo e dalla concentrazione di essi. Le risposte attivate da arginina e SCFA sono promosse regolando TJp e la secrezione di citochine pro e antinfiammatorie. Pertanto, sarebbe auspicabile che gli SCFA, ampiamente utilizzati nelle diete dei suini, fossero utilizzati in base alle diverse azioni di ciascun SCFA.The effects of eubiotics are of considerable interest as they are used as additives in pig farming in response to current regulations aiming to avoid antibiotic resistance. Since 2013, World Health Organization banned antibiotics and zinc as growth promoters. In study 1, we characterized a co-culture model composed of intestinal epithelial cells (IPEC-J2) and peripheral blood immune cells (PBMC). Arginine is an essential amino acid among the various substances used to replace antibiotics. Using this model, we evaluated how arginine deprivation induced an increase in intestinal damage and the consequent activation of the intestinal immune response. The deprivation leads to the immunoregulation mediated by the expression of pro-inflammatory (Tumor Necrosis factor (TNF)α, Interleukin (IL)-8, Interleukin (IL)-1α, Interleukin (IL)-6) and anti-inflammatory (Transforming Growth Factor (TGF)-β) cytokines. Among other alternative molecules, Short-Chain Fatty Acids (SCFA), important sources for energy metabolism and anabolic processes in mammals, can regulate the inflammatory response and increase the integrity of the intestinal barrier. In Study 2, we evaluated the effects of SCFA supplementation (acetate, butyrate, propionate, and lactate) at different concentrations in IPEC-J2. SCFA enhance the functions of tight junction proteins (TJp), which prevent the passage of antigens across the intestinal barrier into the paracellular space. The results show impacts on IPEC-J2, that depend on the type and specific concentration of SCFA. Acetate and propionate exhibit a greater protective action than lactate and butyrate, at the chosen concentrations. Moreover, SCFA supplementation stimulates the expression of TJp and β-defensin 1 (BD-1), which, in turn, may have been involved in the inhibition of Nuclear factor (NF)-κB and TNFα gene expression. Finally in study 3, based on the previous study, 5 mM acetate and 1 mM propionate were used as treatments for the co-culture upon lipopolysaccharides (LPS) challenge, miming the acute inflammatory state. Acetate and propionate have a protective effect by TJp regulation upon acute inflammation.
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In summary, a co-culture model of IPEC-J2 and PBMC has been characterized. The model could be important to study and elucidate the role of nutritional conditions in the functionality and reactivity of the intestinal epithelial cells (IECs) in pigs. Arginine deprivation affects intestinal epithelial cells and stimulates their functional response; the amino acid acts as a modulator of the local anti-inflammatory response to adapt and react to a stressful stimulus. Moreover, the data could be translated to humans that there are similarities between pigs and humans in terms of intestinal microbiota and in the mechanisms of intestinal defense systems.
Finally, SCFA have protective roles at the level intestinal. We have highlighted the positive in vitro effect of SCFA, even in the presence of LPS-challenge, on intestinal viability and maintaining intestinal integrity. SCFA actions depend on the type and concentration of them. Responses activated by arginine and SCFA are promoted by regulating TJp and the secretion of pro- and anti-inflammatory cytokines. Therefore, it would be desirable for SCFA, widely used in pig diets, to be utilized based on the different actions of each SCFA
Differential Expression of miR-223-3p and miR-26-5p According 2 to Different Stages of Mastitis in Dairy Cows
No full textMastitis is the leading cause of economic losses in dairy farming, significantly impairing 14
animal welfare and the quality and quantity of milk production. MicroRNAs are increasingly gain- 15
ing attention, in both human and veterinary medicine, as biomarkers for various diseases. This 16
study evaluated the diagnostic potential of four circulating microRNAs (miR-26-5p, miR-142-5p, 17
miR-146a, and miR-223-3p) by examining changes in their expression in milk samples from dairy 18
cows at different immune cells subpopulations correlated to different stage of mastitis in validated 19
method. Additionally, the study has analysed the possible source of these circulating microRNA by 20
the measurement of their secretion from activated immune cells (lymphocytes, monocytes, and neu- 21
trophils). miR-223-3p has been significantly expressed in acute stage of mastitis (p<0.01) but not in 22
the chronic or susceptible stages. Conversely, mir-26-5p has been significantly reduced in acute, 23
chronic and susceptible groups of animals. In immune cell cultures miR-26 has been shown to 24
downregulated in Lypopolysaccharide (LPS)-stimulated neutrophils while miR-223 has been 25
shown to be upregulated in Phytohaemagglutinin (PHA)-stimulated lymphocytes. The differential 26
expression of miR-223-3p and miR-26-5p, combined with differential and total somatic cell count, 27
could serve as a useful tool for identifying the evolutionary stage of mastitis-related inflammatory 28
patholog
Hypoxia and platelet lysate sustain differentiation of primary horse articular chondrocytes in xeno-free supplementation culture
Get PDFCurrently, the main limitation for the use of adult differentiated chondrocytes in cell-based therapy and tissue engineering for the repair of articular cartilage is the difficulty of maintaining their state of differentiation during cell expansion. The adult articular cartilage has no direct blood supply, and local oxygen concentrations range from 5%-10% at the surface near the synovial fluid to less than 1% in the deep layer. Low oxygen tension is currently considered an important environmental condition for chondrocytes, and hypoxia has been explored as a signal potentially promoting differentiation and matrix deposition. In the present study, hypoxia and PL supplementation were studied to maintain differentiation in adult articular chondrocytes. Freshly isolated equine articular chondrocytes were grown in monolayer culture at a low seeding density (condition favoring proliferation and dedifferentiation) and in alginate beads (3D culture condition maintaining chondrocyte differentiation) both in normoxic and hypoxic conditions and in various conditions of supplementation or deprivation (fetal bovine serum [FBS]- and PL-free; 10% FBS; 5% PL; 10% PL). Results demonstrated that hypoxia is a micro-environmental condition that reduces chondrocyte dedifferentiation or maintains differentiation during in vitro expansion, as shown by the sustained expression of differentiation markers (COL2, ACAN, SOX9, HIF1a) and the reduction of dedifferentiation marker expression (COL1, RUNX2). In association with hypoxia, PL supplementation demonstrated a positive effect on chondrocyte differentiation in association with hypoxia. This promising result should be confirmed in other conditions of chondrocyte differentiation before proposing PL as a complete alternative to xenogenic serum for the expansion of articular chondrocytes
New knowledge and applied aspects for the study of mucosal immunity in the porcine species: an experimental model
No full textIn 2017, the WHO issued guidelines to ensure the responsible use of antibiotics in farms in order to face the growing phenomenon of antibiotic resistance, including the adoption of strategies aimed at improving animal productivity by modulating the immune response and intestinal function. As a matter of facts the gastrointestinal tract is daily exposed to environmental antigens and it is one of the main sites of infection by external bacterial or viral pathogens. An optimal immune reaction involves coordination between inflammatory response, white line cell activation and cytokine synthesis for which cooperation between immune and non-immune cells is required. The aim of this study was to detect changes in the gene expression of pro-inflammatory, anti-inflammatory cytokines, structural and antimicrobial proteins by porcine intestinal epithelial cells IPEC-J2 when stimulated by activated immune cells and in absence of arginine using Real-Time PCR. While the integration of arginine in animal feed facilitates the activation of immune cells and it increases the ability to fight enteric infections, the presence of activated immune cells was necessary to mimic inflammation. The IPEC-J2 cell line represents an excellent model for studying the defensive mechanisms put in place by the intestinal mucosal epithelium. However further experiments and in-depth studies are needed to better understand how epithelial cells cooperate with inflammatory cells to orchestrate the defensive response in the gut
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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