Invited review: Dairy proteins and bioactive peptides: Modeling digestion and the intestinal barrier.

Dairy products are one of the most important sources of biologically active proteins and peptides. The health-promoting functions of these peptides are related to their primary structure, which depends on the parent protein composition. A crucial issue in this field is the demonstration of a cause-effect relationship from the ingested protein form to the bioactive form in vivo. Intervention studies represent the gold standard in nutritional research; however, attention has increasingly been focused on the development of sophisticated in vitro models of digestion to elucidate the mechanism of action of dairy nutrients in a mechanistic way and significantly reduce the number of in vivo trials. On the other hand, the epithelial intestinal barrier is the first gate that actively interacts with digestion metabolites, making the intestinal cells the first target tissue of dairy nutrients and respective metabolites. An evolution of the in vitro digestion approach in the study of dairy proteins and derived bioactive compounds is the setup of combined in vitro digestion and cell culture models taking into consideration the endpoint to measure the target organism (e.g., animal, human) and the key concepts of bioaccessibility, bioavailability, and bioactivity. This review discusses the relevance and challenges of modeling digestion and the intestinal barrier, focusing on the implications for the modeling of dairy protein digestion for bioactivity evaluation.

In vitro-digested milk proteins: Evaluation of angiotensin-1-converting enzyme inhibitory and antioxidant activities, peptidomic profile, and mucin gene expression in HT29-MTX cells.

Over the past decades, several studies investigated the health-promoting functions of milk peptides. However, to date many hurdles still exist regarding the widespread use of milk-derived bioactive peptides, as they may be degraded during gastrointestinal digestion. Thus, the aim of our study was to in vitro digest intact whey protein isolate (WPI) and casein proteins (CNP), mimicking in vivo digestion, to investigate their bioactive effects and to identify the potential peptides involved. Whey protein isolate and CNP were digested using a pepsin-pancreatin protocol and ultra-filtered (3-kDa cutoff membrane). A permeate (<3 kDa) and a retentate (>3 kDa) were obtained. Soy protein was included as a control (CTR). Angiotensin-1-converting enzyme inhibitory (ACE1-I) and antioxidant activity (AOX) were assessed and compared with those observed in undigested proteins and CTR. Furthermore, the permeate was characterized by nano-liquid chromatography electrospray ionization tandem mass spectrometry (LC-nano ESI MS/MS) using a shotgun peptidomic approach, and retentate was further digested with trypsin and analyzed by MS using a shotgun proteomic approach to identify potentially bioactive peptides. Further, the effects of WPI, CNP, and CTR retentate on cell metabolic activity and on mucus production (MUC5AC and MUC2 gene expression) were assessed in intestinal goblet HT29-MTX-E12 cells. Results showed that WPI permeate induced a significant ACE1-I inhibitory effect [49.2 ± 0.64% (SEM)] compared with undigested WPI, CNP permeate, and retentate or CTR permeate (10.40 ± 1.07%). A significant increase in AOX (1.58 ± 0.04 and 1.61 ± 0.02 µmol of trolox AOX equivalents per mg of protein, respectively) upon digestion was found in WPI. Potentially bioactive peptides associated with ACE1-I and antihypertensive effects were identified in WPI permeate and CNP retentate. At specific concentrations, WPI, CNP, and CTR retentate were able to stimulate metabolic activity in HT29-MTX-E12 cells. Expression of MUC5AC was increased by CNP retentate and unaltered by WPI retentate; MUC2 expression was significantly increased by 0.33 mg/g of CNP and reduced by 1.33 mg/g of CNP. Our results confirm that milk proteins may be rich sources of bioactive compounds, with the greatest beneficial potential of CNP at the intestinal goblet cell level.

Cytotoxicity, apoptosis, DNA damage and methylation in mammary and kidney epithelial cell lines exposed to ochratoxin A.

This study aimed to investigate the in vitro damage induced by ochratoxin A (OTA) in BME-UV1 and MDCK epithelial cells. Both cells lines were treated with OTA (0 up to 10 µg/mL), and cell viability (MTT assay), membrane stability (lactate dehydrogenase (LDH) release assay) and apoptotic cell rate (Tunel assay) were investigated. Further, the effect of the incubation with OTA has been evaluated at DNA level by the determination of DNA integrity, by the quantification of DNA adduct formation (8-hydroxy-2'-deoxyguanosine (8-OHdG)) and by the assessment of the global DNA methylation status (5-methyl-cytosine (5-mC)). The obtained results showed that after 24 h of OTA treatment, BME-UV1 cell viability was reduced in a dose-dependent way. OTA significantly (P < 0.05) increased LDH release in BME-UV1 cells at all concentrations tested. OTA (1.25 µg/mL) induced 35 % LDH release in MDCK cells (P < 0.05). A significant (P < 0.05) change in percentages of apoptotic BME-UV1 (10 ± 0.86) and MDCK (25 ± 0.88) cells was calculated when the cells were co-incubated with OTA. The level of 8-OHdG adduct formation was significantly (P < 0.05) increased in BME-UV1 cells treated with 1.25 µg/mL of OTA. The results of the present study suggest that a different mechanism of action may occur in these cell lines. Graphical abstract Study results overview.

Effect of growth factors, estradiol 17-ß, and short chain fatty acids on the intestinal HT29-MTX cells : Growth factors and SCFAs effects on intestinal E12 cells.

Peptides growth factors, hormones, and short chain fatty acids (SCFAs) are constantly in contact with the human bowel when secreted by gland or ingested by food, as milk and colostrum, or, as in the case of SCFAs, produced by fermentation processes. This study considers the effect of growth factors, estradiol 17-ß, and SCFAs on the metabolic activity and proliferation of undifferentiated HT29-MTX-E12 (E12) cells. In particular, the aim of the present study was the characterization of the human intestinal cell line E12 for its suitability as an in vitro intestinal model for cell-nutrient interaction studies. The effect of insulin-like growth factors (IGF)-I, epidermal growth factors (EGF), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-ß), estradiol 17-ß and butyrate, propionate, and acetate was assessed on metabolic activity and proliferation of E12 cells using AlamarBlue(TM) assay and PicoGreen® assay, respectively. IGF-I and estradiol 17-ß significantly (P < 0.05; P < 0.001) increased both metabolic activity and proliferation in a concentration-dependent manner, whereas TGF-α, at the concentration of 1 ng/mL, significantly (P < 0.05) reduced the metabolic activity of the cells. Further, a dose-dependent inhibition of cell metabolic activity was detected in the presence of all SCFAs tested. Butyrate showed to be the most active in the inhibition of E12 metabolic activity and its effect was enhanced by the presence of propionate and acetate. E12 cells, in undifferentiated state, showed to be a suitable in vitro model for cell-nutrient interaction studies, providing an opportunity to examine the potential role of growth factors, hormones and SCFAs in the regulation of the intestinal cell viability.

Seed-specific expression of porcine verotoxigenic Escherichia coli antigens in tobacco plants as a potential model of edible vaccines.

Vaccines can reduce the use of antibiotics by preventing specific infective diseases in pigs. Plant-based edible vaccines are particularly attractive because, upon oral ingestion via feed, they can elicit the local immune system against a foreign disease-causing organism. The aim of this study was to engineer two different independent lines of tobacco plants for the seed-specific expression of immunogenic proteins of VTEC as a model of an edible vaccine. For each antigen, fifty Nicotiana tabacum L. cv Xanthi leaf disks were transformed by agroinfection for the seed-specific expression of the structural parts of the fimbrial subunit FedF of F18 and the B-subunit of Vt2e genes. The synthetic genes, optimized by the codon adaptation index for their expression in tobacco, were inserted into expression cassettes under the control of ß-conglycinin promoter. Regenerated tobacco plants (T0) were characterized by molecular and immunoenzymatic techniques. Our results showed that both FedF and Vt2eB genes were integrated into tobacco genome efficiently (> 80%) and they are also maintained in the second generation (T1). Western blotting analyses carried out on the positive producing lines, showed the tissue-specific expression in seeds and the temporal protein accumulation in the mid-late maturation phases. The enzyme-linked immunosorbent assay showed seed expression levels of 0.09 to 0.29% (from 138 to 444 µg/g of seeds) and 0.21 to 0.43% (from 321 to 658 µg/g of seeds) of total soluble protein for the FedF and Vt2eB antigens, respectively. This study confirmed the seed-specific expression of the selected antigens in plant seeds. The expression level is suitable for seed-based edible vaccination systems, which could represent a cost-effective way to prevent VTEC infection. Our findings encourage further in vivo studies focused on the activation of the local immune response.

Antioxidant capacity and peptidomic analysis of in vitro digested Camelina sativa L. Crantz and Cynara cardunculus co-products.

In recent decades, the food system has been faced with the significant problem of increasing food waste. Therefore, the feed industry, supported by scientific research, is attempting to valorise the use of discarded biomass as co-products for the livestock sector, in line with EU objectives. In parallel, the search for functional products that can ensure animal health and performances is a common fundamental goal for both animal husbandry and feeding. In this context, camelina cake (CAMC), cardoon cake (CC) and cardoon meal (CM), due valuable nutritional profile, represent prospective alternatives. Therefore, the aim of this work was to investigate the antioxidant activity of CAMC, CC and CM following in vitro digestion using 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), Ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assays. Total phenolic content (TPC) and angiotensin converting enzyme (ACE) inhibitory activity, actively involved in modulating antioxidant properties, were also studied. Further, a peptidomic analysis was adopted to substantiate the presence of bioactive peptides after in vitro digestion. The results obtained confirmed an interesting nutritional profile of CAMC, CC and CM and relevant antioxidant and ACE inhibitory activities. In particular, considering antioxidant profile, CM and CC revealed a significantly higher (10969.80 ± 18.93 mg TE/100 g and 10451.40 ± 149.17 mg TE/100 g, respectively; p < 0.05) ABTS value than CAMC (9511.18 ± 315.29 mg TE/100 g); a trend also confirmed with the FRAP assay (306.74 ± 5.68 mg FeSO4/100 g; 272.84 ± 11.02 mg FeSO4/100 g; 103.84 ± 3.27 mg FeSO4/100 g, for CC, CM and CAMC, respectively). Similar results were obtained for TPC, demonstrating the involvement of phenols in modulating antioxidant activity. Finally, CAMC was found to have a higher ACE inhibitory activity (40.34 ± 10.11%) than the other matrices. Furthermore, potentially bioactive peptides associated with ACE inhibitory, anti-hypertensive, anti-cancer, antimicrobial, antiviral, antithrombotic, DPP-IV inhibitory and PEP-inhibitory activities were identified in CAMC. This profile was broader than that of CC and CM. The presence of such peptides corroborates the antioxidant and ACE profile of the sample. Although the data obtained report the important antioxidant profile of CAMC, CC, and CM and support their possible use, future investigations, particularly in vivo trials will be critical to evaluate and further investigate their effects on the health and performance of farm animals.

Total Phenolic Content and Antioxidant Activity of In Vitro Digested Hemp-Based Products.

The growth of the world population has prompted research to investigate new food/feed alternatives. Hemp-based products can be considered excellent candidates. Hemp (Cannabis sativa L.) is an environmentally sustainable plant widespread worldwide. Following the reintroduction of its cultivation, hemp is attracting interest, especially in the food/feed industry. To date, scientific research has mainly focused on its nutritional aspect. Therefore, the aim of the work was also to investigate the functional profile (total phenolic content (TPC) and antioxidant activity (Ferric- reducing antioxidant power (FRAP) and 2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS)) of hemp-based products (hempseeds (HSs), flowers, and HS protein extract), following methanol extraction and in vitro digestion, to study the behaviour of the molecules involved. The results show an interesting nutritional value, even when compared to matrices used in the food/feed industry, such as soy and flaxseeds. The functional profile revealed a very interesting TPC following methanol extraction for HSs, flowers, and HS protein extract, respectively, (550.3 ± 28.27; 2982.8 ± 167.78; and 568.9 ± 34.18 mg Tannic Acid Equivalent (TAE)/100 g). This trend was also confirmed for FRAP (50.9 ± 4.30; 123.6 ± 8.08; and 29.73 ± 1.32 mg Ascorbic Acid Equivalent (AAE)/100 g), recording values similar/higher than soy protein extract and flaxseeds (17.4 ± 1.55; and 10.4 ± 0.44 mg AAE/100 g). The results were also maintained following physiological digestion. These results, although promising, need further investigation, confirming what has been observed with different antioxidant activity assays and identifying individual molecules involved in functional pathways. This information will be necessary to gain a better understanding of the functional characteristics of these matrices for use in food/feed formulations.

Comprehensive proteomic analysis reveals omega-3 fatty acids to counteract endotoxin-stimulated metabolic dysregulation in porcine enterocytes.

Omega-3 polyunsaturated fatty acids (n-3 PUFA), such as the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are reported to beneficially affect the intestinal immunity. The biological pathways modulated by n-3 PUFA during an infection, at the level of intestinal epithelial barrier remain elusive. To address this gap, we investigated the proteomic changes induced by n-3 PUFA in porcine enterocyte cell line (IPEC-J2), in the presence and absence of lipopolysaccharide (LPS) stress conditions using shotgun proteomics analysis integrated with RNA-sequencing technology. A total of 33, 85, and 88 differentially abundant proteins (DAPs) were identified in cells exposed to n-3 PUFA (DHA:EPA), LPS, and n-3 PUFA treatment followed by LPS stimulation, respectively. Functional annotation and pathway analysis of DAPs revealed the modulation of central carbon metabolism, including the glycolysis/gluconeogenesis, pentose phosphate pathway, and oxidative phosphorylation processes. Specifically, LPS caused metabolic dysregulation in enterocytes, which was abated upon prior treatment with n-3 PUFA. Besides, n-3 PUFA supplementation facilitated enterocyte development and lipid homeostasis. Altogether, this work for the first time comprehensively described the biological pathways regulated by n-3 PUFA in enterocytes, particularly during endotoxin-stimulated metabolic dysregulation. Additionally, this study may provide nutritional biomarkers in monitoring the intestinal health of human and animals on n-3 PUFA-based diets.

Protective effect of phytogenic plus short and medium-chain fatty acids-based additives in enterotoxigenic Escherichia coli challenged piglets.

Post Weaning Diarrhea (PWD) is the most important multifactorial gastroenteric disease of the weaning in pig livestock. Phytogenic (PHY) natural extracts are largely studied as alternatives to antibiotic treatments in combating the global concern of the antimicrobial resistance. The aim of this study was to evaluate the protective effect of innovative phytogenic premix with or without short and medium chain fatty acids (SCFA and MCFA) in O138 Escherichia coli challenged piglets. Twenty-seven weaned piglets were allotted into four groups fed different diets according to the following dietary treatments: CTRL (n = 13) group fed basal diet, PHY1 (n = 7) fed the basal diet supplemented with 0.2% of phytogenic premix, PHY2 (n = 7) fed the basal diet supplemented with 0.2% of phytogenic premix added with 2000 ppm of SCFA and MCFA. After 6 days of experimental diet feeding, animals were challenged (day 0) with 2 × 109 CFU of E. coli and CTRL group was divided at day 0 into positive (challenged CTRL + ; n = 6) and negative control group (unchallenged CTRL-; n = 7). Body weights were recorded at -14, -6, 0, 4 and 7 days and the feed intake was recorded daily. E. coli shedding was monitored for 4 days post-challenge by plate counting. Fecal consistency was registered daily by a four-point scale (0-3; diarrhea > 1) during the post-challenge period. Tissue samples were obtained for gene expression and histological evaluations at day 7 from four animals per group. Lower average feed intake was observed in CTRL + compared to PHY2 and CTRL during the post-challenge period. Infected groups showed higher E. coli shedding compared to CTRL- during the 4 days post-challenge (p < 0.01). PHY2 showed lower frequency of diarrhea compared to PHY1 and CTRL + from 5 to 7 days post-challenge. No significant alterations among groups were observed in histopathological evaluation. Duodenum expression of occludin tended to be lower in challenged groups compared to CTRL- at 7 days post-challenge (p = 0.066). In conclusion, dietary supplementation of PHY plus SCFA and MCFA revealed encouraging results for diarrhea prevention and growth performance in weaned piglets.

Tobacco Seed-Based Oral Vaccination against Verocytotoxic O138 Escherichia coli as Alternative Approach to Antibiotics in Weaned Piglets.

Post-weaning diarrhoea and enterotoxaemia caused by Escherichia coli are serious threats in the pig (Sus scrofa domesticus) livestock industry and are responsible for economic losses related to mortality, morbidity and stunted growth. The aim of this study was to evaluate the effect of an engineered tobacco seeds-based edible vaccine in O138 Escherichia coli-challenged piglets throughout a multidisciplinary approach. Thirty-six weaned piglets were enrolled and randomly divided into two experimental groups, a control (C; n = 18) group and a tobacco edible vaccination group (T, n = 18), for 29 days of trial. At days 0, 1, 2, 5 and 14, piglets of the T group were fed with 10 g of the engineered tobacco seeds line expressing F18 and VT2eB antigens, while the C group received wild-type tobacco seeds. After 20 days, 6 piglets/group were orally challenged with the Escherichia coli O138 strain (creating four subgroups: UC = unchallenged control, CC = challenged control, UT = unchallenged tobacco, CT = challenged tobacco) and fed with a high protein diet for 3 consecutive days. Zootechnical, clinical, microbiological, histological and immunological parameters were assayed and registered during the 9 days of post-challenge follow up. At 29 days post-challenge, the CT group displayed a lower average of the sum of clinical scores compared to the CC group (p < 0.05), while the CC group showed a higher average sum of the faecal score (diarrhoea) (p < 0.05) than the CT group. A decreased number of days of shedding of the pathogenic strain was observed in the CT compared to the CC group (p < 0.05). Specific anti-F18 IgA molecules were significantly higher in the CT group compared to the CC group's faecal samples during the post-challenge period (p < 0.01). In conclusion, edible vaccination with engineered tobacco seeds showed a protective effect on clinical symptoms and diarrhoea incidence during the post-challenge period, characterized by a limited time of pathogenic strain shedding in faeces.

Changes in the proteomic profile of adipose tissue-derived mesenchymal stem cells during passages.

BACKGROUND: Human mesenchymal stem cells (hMSC) have recently raised the attention because of their therapeutic potential in the novel context of regenerative medicine. However, the safety of these new and promising cellular products should be carefully defined before they can be used in the clinical setting, as. The protein expression profile of these cells might reveal potential hazards associated with senescence and tumoral transformation which may occur during culture. Proteomic is a valuable tool for hMSC characterization and identification of possible changes during expansion. RESULTS: We used Surface Enhanced Laser Desorption/Ionization-Time Of Flight-Mass Spectrometry (SELDI-ToF-MS) to evaluate the presence of stable molecular markers in adipose tissue-derived mesenchymal stem cells (AD-MSC) produced under conditions of good manufacturing practices (GMP). Proteomic patterns of cells prepared were consistent, with 4 up-regulated peaks (mass-to-charge ratio (m/z) 8950, 10087, 10345, and 13058) through subculture steps (P0-P7) with similar trend in three donors. Among the differentially expressed proteins found in the cytoplasmic and nuclear fractions, a cytoplasmic 10.1 kDa protein was upregulated during culture passages and was identified as S100A6 (Calcyclin). CONCLUSIONS: This study suggests for the first time that common variation could occur in AD-MSC from different donors, with the identification of S100A6, a protein prevalently related to cell proliferation and cell culture condition. These results support the hypothesis of common proteomic changes during MSCs expansion and could give important insight in the knowledge of molecular mechanisms intervening during MSC expansion.

Soya protein hydrolysates modify the expression of various pro-inflammatory genes induced by fatty acids in ovine phagocytes.

The objective of the present study was to test the hypothesis that fatty acids are the circulating mediators acting in a pro-inflammatory manner towards activated circulating ovine monocyte/macrophages and neutrophils. Furthermore, whether soya protein hydrolysates (SPH) inhibit the fatty acid-induced increase in the production of pro-inflammatory responses by ovine phagocytes was tested in vitro. All the fatty acids tested (myristic, palmitic, palmitoleic, stearic and oleic) increased (P<0·01; C18>C16>C14) membrane-bound urokinase plasminogen activator (u-PA) and u-PA free binding sites in cell membranes of activated ovine blood monocytes/macrophages, but only the C18 fatty acids (stearic, oleic) were effective towards blood neutrophils. The C18 fatty acids up-regulated (P<0·05) the gene expression of u-PA, u-PA receptor, intercellular adhesion molecule 1 and inducible NO synthase (in monocytes) but not that of cyclo-oxygenase-2, integrin α X and plasminogen activator inhibitor types 1 and 2 by ovine phagocytes. SPH blocked completely or partially all C18 fatty acid-induced changes in the expression of various pro-inflammatory genes. In conclusion, fatty acids selectively 'activate' ovine phagocytes, suggesting that these cells 'sense' metabolic signals derived from adipocytes. Soya protein peptides inhibit all changes in gene expression induced by fatty acids in ovine phagocytes in vitro. This constitutes a novel mechanism of action.

Role of Short Chain Fatty Acids to Counteract Inflammatory Stress and Mucus Production in Human Intestinal HT29-MTX-E12 Cells.

Short chain fatty acids (SCFAs), especially butyrate (BUT), are known to promote intestinal health, but their role in the protection of intestinal barrier integrity is poorly characterized. The aim of the study was to set up an in vitro model of human colon epithelium using HT29-MTX-E12 cells to delineate the potential role of SCFAs under stress conditions. Accordingly, the HT29-MTX-E12 cells were differentiated for 42 days and subsequently exposed to dextran sulphate sodium (DSS). Further, the effects of BUT or its mixture with acetate and propionate (SCFAs-MIX) were tested to study proliferation, epithelial integrity and mucus production. The results showed that the concentration of 10% DSS for 24 h decreased the TEER about 50% compared to the control in HT29-MTX-E12 cells. The pre-treatment on HT29-MTX-E12 cells with BUT or SCFAs-MIX at specific concentrations significantly (p < 0.05) reduced the DSS-induced damage on epithelial cell integrity and permeability. Further, the treatment with specific concentrations of BUT and SCFAs-MIX for 24 h significantly promoted ZO-1, MUC2 and MUC5AC mRNA expression (p < 0.005). The present study demonstrated the suitability of HT29-MTX-E12 cells treated with DSS as an in vitro stress model of inflammatory bowel disease, which enabled us to understand the effect of bioactive SCFAs on the intestinal barrier.

Role of omega-3 polyunsaturated fatty acids, citrus pectin, and milk-derived exosomes on intestinal barrier integrity and immunity in animals.

The gastrointestinal tract of livestock and poultry is prone to challenge by feedborne antigens, pathogens, and other stress factors in the farm environment. Excessive physiological inflammation and oxidative stress that arises firstly disrupts the intestinal epithelial barrier followed by other components of the gastrointestinal tract. In the present review, the interrelationship between intestinal barrier inflammation and oxidative stress that contributes to the pathogenesis of inflammatory bowel disease was described. Further, the role of naturally existing immunomodulatory nutrients such as the omega-3 polyunsaturated fatty acids, citrus pectin, and milk-derived exosomes in preventing intestinal barrier inflammation was discussed. Based on the existing evidence, the possible molecular mechanism of these bioactive nutrients in the intestinal barrier was outlined for application in animal diets.

Effect of growth factors and lactogenic hormones on expression of plasminogen activator-related genes and cell proliferation in a bovine mammary epithelial cell line.

There is conflicting evidence in the literature as to whether up-regulation of urokinase plasminogen activator (u-PA) expression is related to bovine mammary epithelial cell growth. The role of u-PA receptor (u-PAR) and that of the plasminogen activator inhibitors type 1 and type 2 (PAI-1 and PAI-2) in bovine mammary epithelial cell proliferation is not known. The effect of growth factors and various hormones known to affect mammary function on expression of u-PA, u-PAR, PAI-1, PAI-2 and cell proliferation using the BME-UV1 bovine mammary epithelial cell line was examined. Cell proliferation was measured using the MTT assay and direct cell enumeration. Results showed that both IGF-1 and EGF increased cell proliferation but EGF was a more potent mitogen than IGF-1. Furthermore, IGF-1 increased by 2-fold expression of both u-PA and u-PAR while EGF increased by 3·8-fold the expression of only u-PAR. Both growth factors had no effect on expression of PAI-1 and PAI-2. In a manner consistent with changes in gene expression, EGF and to a lesser extent IGF-1 up-regulated total cell associated, membrane-bound and secreted u-PA activity. Thus, a strong correlation exists between u-PAR gene expression along with the activity of u-PA present on cell membranes and cell proliferation. Dexamethasone, prolactin and surprisingly insulin had no effect on cell proliferation. Dexamethasone alone and when combined with insulin or prolactin up-regulated gene expression of both PAI- and PAI-2 but not that of u-PA and u-PAR. Decreased total cell-associated, membrane-bound and secreted u-PA activity was detected in cells cultured in the presence of dexamethasone when combined with insulin or prolactin. However no such effect was observed in the presence of dexamethasone alone. Thus, dexamethasone acting synergistically with prolactin or insulin inhibits the activation of the plasmin-plasminogen system but this inhibition is not correlated with any changes in cell proliferation.

Evaluation of Tannin Extracts, Leonardite and Tributyrin Supplementation on Diarrhoea Incidence and Gut Microbiota of Weaned Piglets.

The effects of the dietary administration of a combination of Quebracho and Chestnut tannins, leonardite and tributyrin were evaluated in weaned piglets. A total of 168 weaned piglets (Landrace × Large White) were randomly allotted to two experimental groups (6 pens/group, 14 piglets/pen). Animals were fed a basal control diet (CTRL) and a treatment diet (MIX) supplemented with 0.75% tannin extracts, 0.25% leonardite and 0.20% tributyrin for 28 days. Individual body weight and feed intake were recorded weekly. Diarrhoea incidence was recorded by a faecal scoring scale (0-3; considering diarrhoea ≥ 2). At 0 and 28 days, faecal samples were obtained from four piglets/pen for microbiological and chemical analyses of faecal microbiota, which were then assessed by V3-V4 region amplification sequencing. At 28 days, blood from two piglets/pen was sampled to evaluate the serum metabolic profile. After 28 days, a reduction in diarrhoea incidence was observed in the MIX compared to CTRL group (p < 0.05). In addition, compared to CTRL, MIX showed a higher lactobacilli:coliform ratio and increased Prevotella and Fibrobacter genera presence (p < 0.01). The serum metabolic profile showed a decreased level of low-density lipoproteins in the treated group (p < 0.05). In conclusion, a combination of tannin extract, leonardite and tributyrin could decrease diarrhoea incidence and modulate the gut microbiota.

Translational Approach to Induce and Evaluate Verocytotoxic E. coli O138 Based Disease in Piglets.

Pig livestock was influenced by several global concerns that imposed a re-thinking of the farming system, which included the reduction in chemical dependency and the development of antimicrobial alternatives. Post-weaning diarrhea and enterotoxaemia caused by Escherichia coli, are serious threats that are responsible for the economic losses related to mortality, morbidity and stunted growth in weaning piglets. The aim of the study was to set up experimental conditions to simulate the simultaneous outbreak of post-weaning diarrhea and enterotoxaemia in weaned piglets, through verocytotoxic O138 Escherichia coli challenge, with a multidisciplinary approach. Eighteen piglets susceptible to F18 VTEC infection were selected by polymerase chain reaction for polymorphism on the fucosyltransferase 1 gene and randomly divided in two experimental groups, non-infected controls (C; n = 6) and infected ones (I; n = 12) and housed into individual pens at the same environmental conditions for 29 days. At day 20, I pigs were orally inoculated with Escherichia coli O138 and fed a high protein ration for 3 days. Zootechnical, clinical, microbiological, histological and immunological parameters were evaluated along the follow up (3 and 9 days). Experimental infection, confirmed by bacteria faecal shedding of the I group, significantly affected the clinical status. The I group showed significantly higher total scores, corresponding to medians of the sum of daily scores from days 1 to 3 (Σ3) and 1 to 9 (Σ9) post infection, epiphora, vitality, hair irregularity, oedema and depression. Histological examination showed evident inflammatory infiltrate of lymphocytes, and follicular hyperplasia in I pigs; in the same group, the immunohistochemical and immunological assays revealed an increase in IgG in the intestinal crypts and CD3-positive T cells in intestinal epithelium. The experimental Escherichia coli infection in controlled conditions is crucial for both the evaluation of innovative compounds and the elucidation of the mechanisms associated with the persistence of antibacterial resistant strains. In conclusion, the adopted infection model, carried out on receptor-mediated susceptible piglets, allowed us to identify a discriminative panel of clinical symptoms related to Escherichia coli O138 infection, and could be used to assess the protective effect of antibiotic alternatives.

Evaluation of Adhesive Characteristics of L. plantarum and L. reuteri Isolated from Weaned Piglets.

Limosilactobacillus reuteri and Lactiplantibacillus plantarum strains, previously isolated from weaned piglets, were considered for the evaluation of their adhesive characteristics. Lactobacilli were treated with LiCl in order to remove the surface protein layer, and probiotic activity was compared with those of untreated strains. The autoaggregation, co-aggregation to E. coli F18+, and adhesive abilities of LiCl-treated Limosilactobacillus reuteri and Lactiplantibacillus plantarum were significantly inhibited (p < 0.05) compared with the respective untreated strain. The hydrophobic and basic phenotypes were observed due to the strong affinity to chloroform and low adherence to ethyl acetate. In particular, L. plantarum showed higher hydrophobicity compared to L. reuteri, which may reflect their different colonizing ability. After treatment with LiCl to remove surface proteins, the adherence capabilities of L. reuteri and L. casei on IPEC-J2 cells decreased significantly (p < 0.001) and L. reuteri adhered more frequently. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that both L. reuteri and L. plantarum had several bands ranging from 20 to 100 kDa. Two-dimensional gel electrophoresis showed an acidic profile of the surface-layer polypeptides for both bacterial strains, and more studies are needed to characterize their profile and functions. The results confirm the pivotal role of surface proteins in the probiotic potential of L. reuteri and L. plantarum.

Omega-3 Polyunsaturated Fatty Acids Counteract Inflammatory and Oxidative Damage of Non-Transformed Porcine Enterocytes.

Marine and plant-based omega-3 polyunsaturated fatty acids (ω-3 PUFAs) are widely added to animal diets to promote growth and immunity. We tested the hypothesis that eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and their 1:2 combination could counteract acute or long-term damage of lipopolysaccharides (LPS), dextran sodium sulphate (DSS) and hydrogen peroxide (H2O2) in Intestinal Porcine Epithelial Cell line-J2 (IPEC-J2). The results showed that 24 h treatment with EPA or DHA exhibited proliferative effects in IPEC-J2 cells at low to moderate concentrations (6.25-50 µM) (p < 0.05). Further, 24 h pretreatment with individual DHA (3.3 µM), EPA (6.7 µM) or as DHA:EPA (1:2; 10 µM) combination increased the mitochondrial activity or cell membrane integrity post-LPS (24 h), DSS (24 h) and H2O2 (1 h) challenge (p < 0.05). Additionally, DHA:EPA (1:2, 10 µM) combination decreased the apoptotic caspase-3/7 activity around twofold after 24 h LPS and DSS challenge (p < 0.05). Our study confirms the proliferative and cytoprotective properties of EPA and DHA in IPEC-J2 cells. Increased intracellular mitochondrial activity and cell membrane integrity by ω-3 PUFAs can play a role in preventing enterocyte apoptosis during acute or chronic inflammatory and oxidative stress.

Potentials and Challenges of Former Food Products (Food Leftover) as Alternative Feed Ingredients.

Former food products (FFPs) are foodstuffs that, even though they are nutritious and safe, have lost their value on the human consumption market for different reasons, such as production errors leading to broken or intermediate foodstuffs, surpluses caused by logistical challenges of daily delivery, or any other reason. The nutritional features of FFPs include carbohydrates, free sugars, and possibly also fats. FFPs tend to have been processed through various technological and heat treatments that impact the nutrients and the kinetics of digestion, as well as animal response and, particularly, gastro-intestinal health. This review integrates some of the most recently published works about the chemical composition, nutritional value, digestibility and glycaemic index of ex-foods. In addition, a view on the relationship between the use of FFPs and safety issues and their effects on pigs' intestinal microbiota are also given.