Isolation and characterization of mammary epithelial cells derived from Göttingen Minipigs: A comparative study versus hybrid pig cells from the IMI-ConcePTION Project.

The value of pig as "large animal model" is a well-known tool for translational medicine, but it can also be beneficial in studying animal health in a one-health vision. The ConcePTION Project aims to provide new information about the risks associated with medication use during breastfeeding, as this information is not available for most commonly used drugs. In the IMI-Conception context, Göttingen Minipigs have been preferred to hybrid pigs for their genetic stability and microbiological control. For the first time, in the present research, three primary cell cultures of mammary epithelial cells were isolated and characterized from Göttingen Minipigs (mpMECs), including their ability to create the epithelial barrier. In addition, a comparative analysis between Göttingen Minipigs and commercial hybrid pig mammary epithelial cells (pMECs) was conducted. Epithelial markers: CKs, CK18, E-CAD, ZO-1 and OCL, were expressed in both mpMECs and pMECs. RT2 Profiler PCR Array Pig Drug Transporters showed a similar profile in mRNA drug transporters. No difference in energy production under basal metabolic condition was evidenced, while under stressed state, a different metabolic behaviour was shown between mpMECs vs pMECs. TEER measurement and sodium fluorescein transport, indicated that mpMECs were able to create an epithelial barrier, although, this turned out to be less compact than pMECs. By comparing mpMECs with mammary epithelial cells isolated from Hybrid pigs (pMECs), although both cell lines have morphological and phenotypic characteristics that make them both useful in barrier studies, some specific differences exist and must be considered in a translational perspective.

Isolation of Vascular Wall Mesenchymal Stem Cells from the Thoracic Aorta of Adult Göttingen Minipigs: A New Protocol for the Simultaneous Endothelial Cell Collection.

Two main classes of perivascular multipotent populations have been described: the microvascular pericytes and the vascular wall mesenchymal stem cells (VW-MSCs). VW-MSCs are isolated from large vessels in many species and they participate in vascular remodeling together with other cellular components such as endothelial cells. Considering that the Göttingen Minipigs are widely used in Europe as a translational model in the field of cardiovascular diseases, the aim of the present research was to isolate VW-MSCs from the adult aorta of Göttingen Minipigs while preserving and also collecting endothelial cells. The results obtained in the present research demonstrated that this new protocol allows us to obtain a pure population of VW-MSCs and endothelial cells. VW-MSCs from Göttingen Minipigs responded fully to the MSC minima international criteria, being positive to CD105, CD90, and CD44 and negative to CD45 and CD34. Moreover, VW-MSCs presented a differentiative potential towards osteogenic, chondrogenic, and adipogenic lineages. Overall, the present protocol, preserving the viability and phenotypic features of the two isolated populations, opens future possibilities of using minipig VW-MSCs and endothelial cells in in vitro vascular remodeling studies.

Expression of cannabinoid (CB1 and CB2) and cannabinoid-related receptors (TRPV1, GPR55, and PPARα) in the synovial membrane of the horse metacarpophalangeal joint.

Background: The metacarpophalangeal joint undergoes enormous loading during locomotion and can therefore often become inflamed, potentially resulting in osteoarthritis (OA). There are studies indicating that the endocannabinoid system (ECS) modulates synovium homeostasis, and could be a promising target for OA therapy. Some cannabinoid receptors, which modulate proliferative and secretory responses in joint inflammation, have been functionally identified in human and animal synovial cells. Objective: To characterize the cellular distribution of the cannabinoid receptors 1 (CB1R) and 2 (CB2R), and the cannabinoid-related receptors transient receptor potential vanilloid type 1 (TRPV1), G protein-related receptor 55 (GPR55) and peroxisome proliferator-activated receptor alpha (PPARα) in the synovial membrane of the metacarpophalangeal joint of the horse. Animals: The dorsal synovial membranes of 14 equine metacarpophalangeal joints were collected post-mortem from an abattoir. Materials and methods: The dorsal synovial membranes of 14 equine metacarpophalangeal joints were collected post-mortem from an abattoir. The expression of the CB1R, CB2R, TRPV1, GPR55, and PPARα in synovial tissues was studied using qualitative and quantitative immunofluorescence, and quantitative real-time reverse transcriptase PCR (qRT-PCR). Macrophage-like (MLS) and fibroblast-like (FLS) synoviocytes were identified by means of antibodies directed against IBA1 and vimentin, respectively. Results: Both the mRNA and protein expression of the CB2R, TRPV1, GPR55, and PPARα were found in the synoviocytes and blood vessels of the metacarpophalangeal joints. The synoviocytes expressed the mRNA and protein of the CB1R in some of the horses investigated, but not in all. Conclusions and clinical importance: Given the expression of the CB1R, CB2R, TRPV1, GPR55, and PPARα in the synovial elements of the metacarpophalangeal joint, these findings encouraged the development of new studies supporting the use of molecules acting on these receptors to reduce the inflammation during joint inflammation in the horse.

Maternal amoxicillin affects piglets colon microbiota: microbial ecology and metabolomics in a gut model.

The first weeks of life represent a crucial stage for microbial colonization of the piglets' gastrointestinal tract. Newborns' microbiota is unstable and easily subject to changes under stimuli or insults. Nonetheless, the administration of antibiotics to the sow is still considered as common practice in intensive farming for pathological conditions in the postpartum. Therefore, transfer of antibiotic residues through milk may occurs, affecting the piglets' colon microbiota. In this study, we aimed to extend the knowledge on antibiotic transfer through milk, employing an in vitro dedicated piglet colon model (MICODE-Multi Unit In vitro Colon Model). The authors' focus was set on the shifts of the piglets' microbiota composition microbiomics (16S r-DNA MiSeq and qPCR-quantitative polymerase chain reaction) and on the production of microbial metabolites (SPME GC/MS-solid phase micro-extraction gas chromatography/mass spectrometry) in response to milk with different concentrations of amoxicillin. The results showed an effective influence of amoxicillin in piglets' microbiota and metabolites production; however, without altering the overall biodiversity. The scenario is that of a limitation of pathogens and opportunistic taxa, e.g., Staphylococcaceae and Enterobacteriaceae, but also a limitation of commensal dominant Lactobacillaceae, a reduction in commensal Ruminococcaceae and a depletion in beneficial Bifidobactericeae. Lastly, an incremental growth of resistant species, such as Enterococcaceae or Clostridiaceae, was observed. To the authors' knowledge, this study is the first evaluating the impact of antibiotic residues towards the piglets' colon microbiota in an in vitro model, opening the way to include such approach in a pipeline of experiments where a reduced number of animals for testing is employed. KEY POINTS: • Piglet colon model to study antibiotic transfer through milk. • MICODE resulted a robust and versatile in vitro gut model. • Towards the "3Rs" Principles to replace, reduce and refine the use of animals used for scientific purposes (Directive 2010/63/UE).

Anticancer activity of an Artemisia annua L. hydroalcoholic extract on canine osteosarcoma cell lines.

Since ancient times, Artemisia annua (A. annua) has been used as a medicinal plant in Traditional Chinese Medicine. In addition, recent studies have investigated the cytotoxic effects of A. annua extracts towards cancer cells. The leading aim of the present research is to evaluate the cytotoxic effects of an hydroalcoholic extract of A. annua on two canine osteosarcoma (OSA) cell lines, OSCA-8 and OSCA-40, focusing on the possible involvement of ferroptosis. The quantitative determination of artemisinin concentration in the extract, culture medium and OSA cells was carried out through the use of an instrumental analytical method based on liquid chromatography coupled with spectrophotometric detection and tandem mass spectrometry (LC-DAD-MS/MS). OSCA-8 and OSCA-40 were exposed to different dilutions of the extract for the EC50 calculation then the uptake of artemisinin by the cells, the effects on the cell cycle, the intracellular iron level, the cellular morphology and the lipid oxidation state were evaluated. A concentration of artemisinin of 63.8 ± 3.4 µg/mL was detected in the extract. A dose-dependent cytotoxic effect was evidenced. In OSCA-40 alterations of the cell cycle and a significantly higher intracellular iron content were observed. In both cell lines the treatment with the extract was associated with lipid peroxidation and with the appearance of a "ballooning" phenotype suggesting the activation of ferroptosis. In conclusion the A. annua idroalcoholic extract utilized in this study showed anticancer activity on canine OSA cell lines that could be useful in treating drug resistant canine OSAs.

Cellular Metabolism and Bioenergetic Function in Human Fibroblasts and Preadipocytes of Type 2 Familial Partial Lipodystrophy.

LMNA mutation is associated with type-2 familial partial lipodystrophy (FPLD2). The disease causes a disorder characterized by anomalous accumulation of body fat in humans. The dysfunction at the molecular level is triggered by a lamin A/C mutation, impairing the cell metabolism. In human fibroblasts and preadipocytes, a trend for ATP production, mainly supported by mitochondrial oxidative metabolism, is detected. Moreover, primary cell lines with FPLD2 mutation decrease the mitochondrial ATP production if compared with the control, even if no differences are observed in the oxygen consumption rate of bioenergetic parameters (i.e., basal and maximal respiration, spare respiratory capacity, and ATP turnover). Conversely, glycolysis is only inhibited in FPLD2 fibroblast cell lines. We notice that the amount of ATP produced in the fibroblasts is higher than in the preadipocytes, and likewise in the control, with respect to FPLD2, due to a more active oxidative phosphorylation (OXPHOS) and glycolysis. Moreover, the proton leak parameter, which characterizes the transformation of white adipose tissue to brown/beige adipose tissue, is unaffected by FPLD2 mutation. The metabolic profile of fibroblasts and preadipocytes is confirmed by the ability of these cell lines to increase the metabolic potential of both OXPHOS and glycolysis under energy required independently by the FPLD2 mutation.

Influence of Dietary Supplementation with Boswellia serrata and Salix alba on Performance and Blood Biochemistry in Free-Range Leghorn Laying Hens.

This study was conducted to evaluate the safety and the beneficial effects of dietary supplementation with Boswellia serrata (Bs) and Salix alba (Sa) in Leghorn hens during the critical pre-laying and laying phases. A total of 120 pullets, 17 weeks of age, were assigned to two groups (Control­C; Treated­T, n = 60 each). For 12 weeks, the T group received a diet supplemented with 0.3% of dry extracts of Bs (5%) and Sa (5%). The study lasted 19 weeks. Productive performance, serum analytes, H/L ratio, IgA and anti-IBV antibodies were investigated. Water intake was significantly higher, while body and egg weight was significantly lower for the T group (p < 0.05). No other differences were detected in performance parameters, serum analytes, IgA and H/L ratio excluding t0, with a significantly (p < 0.05) higher H/R ratio and higher titers of anti-IBV antibody for the T group. Overall, the data obtained in this study show that the supplementation with Bs and Sa was safe and resulted in an increase in water consumption, a decrease in egg weight, and a sedative effect in the hens. In the future, it would be interesting to test this supplement in hens reared on intensive farms.

Efficacy of Stem Cell Therapy in Large Animal Models of Ischemic Cardiomyopathies: A Systematic Review and Meta-Analysis.

Stem-cell therapy provides a promising strategy for patients with ischemic heart disease. In recent years, numerous studies related to this therapeutic approach were performed; however, the results were often heterogeneous and contradictory. For this reason, we conducted a systematic review and meta-analysis of trials, reporting the use of stem-cell treatment against acute or chronic ischemic cardiomyopathies in large animal models with regard to Left Ventricular Ejection Fraction (LVEF). The defined research strategy was applied to the PubMed database to identify relevant studies published from January 2011 to July 2021. A random-effect meta-analysis was performed on LVEF mean data at follow-up between control and stem-cell-treated animals. In order to improve the definition of the effect measure and to analyze the factors that could influence the outcomes, a subgroup comparison was conducted. Sixty-six studies (n = 1183 animals) satisfied our inclusion criteria. Ischemia/reperfusion infarction was performed in 37 studies, and chronic occlusion in 29 studies; moreover, 58 studies were on a pig animal model. The meta-analysis showed that cell therapy increased LVEF by 7.41% (95% Confidence Interval 6.23−8.59%; p < 0.001) at follow-up, with significative heterogeneity and high inconsistency (I2 = 82%, p < 0.001). By subgroup comparison, the follow-up after 31−60 days (p = 0.025), the late cell injection (>7 days, p = 0.005) and the route of cellular delivery by surgical treatment (p < 0.001) were significant predictors of LVEF improvement. This meta-analysis showed that stem-cell therapy may improve heart function in large animal models and that the swine specie is confirmed as a relevant animal model in the cardiovascular field. Due to the significative heterogeneity and high inconsistency, future translational studies should be designed to take into account the evidenced predictors to allow for the reduction of the number of animals used.

Development of a Pig Mammary Epithelial Cell Culture Model as a Non-Clinical Tool for Studying Epithelial Barrier-A Contribution from the IMI-ConcePTION Project.

The ConcePTION project aims at generating further knowledge about the risks related to the use of medication during breastfeeding, as this information is lacking for most commonly used drugs. Taking into consideration multiple aspects, the pig model has been considered by the consortium as the most appropriate choice. The present research was planned to develop an efficient method for the isolation and culture of porcine Mammary Epithelial Cells (pMECs) to study the mammary epithelial barrier in vitro. Mammary gland tissues were collected at a local slaughterhouse, dissociated and the selected cellular population was cultured, expanded and characterized by morphology, cell cycle analysis and immunophenotyping. Their ability to create a barrier was tested by TEER measurement and sodium fluorescein transport activity. Expression of 84 genes related to drug transporters was evaluated by a PCR array. Our results show that primary cells express epithelial cell markers: CKs, CK18, E-Cad and tight junctions molecules ZO-1 and OCL. All the three pMEC cellular lines were able to create a tight barrier, although with different strengths and kinetics, and express the main ABC and SLC drug transporters. In conclusion, in the present paper we have reported an efficient method to obtain primary pMEC lines to study epithelial barrier function in the pig model.

Seaweed Supplementation Failed to Affect Fecal Microbiota and Metabolome as Well as Fecal IgA and Apparent Nutrient Digestibility in Adult Dogs.

The present study investigated in dogs the dietary effects of intact seaweeds on some fecal bacterial populations and metabolites, fecal IgA and apparent total tract digestibility (ATTD). Ten healthy adult dogs were enrolled in a 5 × 5 replicated Latin square design to evaluate five dietary treatments: control diet (CD); CD + Ascophyllum nodosum; CD + Undaria pinnatifida; CD + Saccharina japonica; CD + Palmaria palmata (n replicates per treatment = 10). Seaweeds were added to food at a daily dose of 15 g/kg. The CD contained silica as a digestion marker. Each feeding period lasted 28 d, with a 7 d wash-out in between. Feces were collected at days 21 and 28 of each period for chemical and microbiological analyses. Fecal samples were collected during the last five days of each period for ATTD assessment. Dogs showed good health conditions throughout the study. The fecal chemical parameters, fecal IgA and nutrient ATTD were not influenced by algal supplementation. Similarly, microbiological analyses did not reveal any effect by seaweed ingestion. In conclusion, algal supplementation at a dose of 15 g/kg of diet failed to exert noticeable effects on the canine fecal parameters evaluated in the present study.

Testicular Melatonin and Its Pathway in Roe Deer Bucks (Capreolus capreolus) during Pre- and Post-Rut Periods: Correlation with Testicular Involution.

Roe deer are seasonal breeders with a complete yearly testicular cycle. The peak in reproductive activity is recorded during summer, the rutting period, with the highest levels of androgens and testicular weight. Melatonin plays a pivotal role in seasonal breeders by stimulating the hypothalamus-pituitary-gonads axis and acting locally; in different species, its synthesis within testes has been reported. The aim of this study was to evaluate the physiological melatonin pattern within roe deer testes by comparing data obtained from animals sampled during pre- and post-rut periods. Melatonin was quantified in testicular parenchyma, along with the genetic expression of enzymes involved in its local synthesis (AANAT and ASMT) and function (UCP1). Melatonin receptors, MT1-2, were quantified both at protein and gene expression levels. Finally, to assess changes in reproductive hormonal profiles, testicular dehydroepiandrosterone (DHEA) was quantified and used for a correlation analysis. Melatonin and AANAT were detected in all samples, without significant differences between pre- and post-rut periods. Despite DHEA levels confirming testicular involution during the post-rut period, no correlations appeared between such involution and melatonin pathways. This study represents the first report regarding melatonin synthesis in roe deer testes, opening the way for future prospective studies in the physiology of this species.

Relationship between serum concentration, functional parameters and cell bioenergetics in IPEC-J2 cell line.

The foetal bovine serum (FBS) concentration could influence functional parameters of IPEC-J2 cells. IPEC-J2 is a non-transformed continuous epithelial cell line that represents an established in vitro model to study porcine gut inflammation and alterations of intestinal integrity. This cell line also represents a good translational model thanks to the high similitudes between pig and human gastrointestinal tract. With the aim to assess if the FBS-dependent functional variations are linked to the bioenergetic aspects, the addition of 5% and 10% FBS in the IPEC-J2 culture medium were tested. Doubling time and TEER measurement indicated that cells cultured at higher FBS dose grow faster and as a more compact monolayer. 10% FBS increases ATP production and mitochondrial oxidative phosphorylation (OxPhos) and does not affect glycolysis. Both at 5% and 10% FBS ATP production mainly comes from OxPhos and FBS concentration does not affect the cell respiration bioenergetic parameters. Noteworthy, IPEC-J2 treated with 5% and 10% FBS have a metabolic potential since both OxPhos and glycolysis increase by > 100% and < 50%, respectively in comparison with baseline metabolism. Moreover, glucose, fatty acids and glutamine constitute the preferred metabolic fuel for mitochondrial respiration at both FBS conditions tested. Accordingly, the cells flexibility to oxidize these substrates shows that IPEC-J2 mitochondria cannot maintain the basal ATP production without oxidizing all the substrates available irrespective of FBS concentration. To sum up, in IPEC-J2 cells OxPhos increases with the FBS-stimulated functional physiological parameters to fulfil ATP requirements.

Doxorubicin treatment modulates chemoresistance and affects the cell cycle in two canine mammary tumour cell lines.

BACKGROUND: Doxorubicin (DOX) is widely used in both human and veterinary oncology although the onset of multidrug resistance (MDR) in neoplastic cells often leads to chemotherapy failure. Better understanding of the cellular mechanisms that circumvent chemotherapy efficacy is paramount. The aim of this study was to investigate the response of two canine mammary tumour cell lines, CIPp from a primary tumour and CIPm, from its lymph node metastasis, to exposure to EC50(20h) DOX at 12, 24 and 48 h of treatment. We assessed the uptake and subcellular distribution of DOX, the expression and function of P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP), two important MDR mediators. To better understand this phenomenon the effects of DOX on the cell cycle and Ki67 cell proliferation index and the expression of p53 and telomerase reverse transcriptase (TERT) were also evaluated by immunocytochemistry (ICC). RESULTS: Both cell lines were able to uptake DOX within the nucleus at 3 h treatment while at 48 h DOX was absent from the intracellular compartment (assessed by fluorescence microscope) in all the surviving cells. CIPm, originated from the metastatic tumour, were more efficient in extruding P-gp substrates. By ICC and qRT-PCR an overall increase in both P-gp and BCRP were observed at 48 h of EC50(20h) DOX treatment in both cell lines and were associated with a striking increase in the percentage of p53 and TERT expressing cells by ICC. The cell proliferation fraction was decreased at 48 h in both cell lines and cell cycle analysis showed a DOX-induced arrest in the S phase for CIPp, while CIPm had an increase in cellular death without arrest. Both cells lines were therefore composed by a fraction of cells sensible to DOX that underwent apoptosis/necrosis. CONCLUSIONS: DOX administration results in interlinked modifications in the cellular population including a substantial effect on the cell cycle, in particular arrest in the S phase for CIPp and the selection of a subpopulation of neoplastic cells bearing MDR phenotype characterized by P-gp and BCRP expression, TERT activation, p53 accumulation and decrease in the proliferating fraction. Important information is given for understanding the dynamic and mechanisms of the onset of drug resistance in a neoplastic cell population.

Expression of Proteinase-Activated Receptor 2 During Colon Volvulus in the Horse.

Large colon volvulus in horses is associated with a poor prognosis, especially when ischemic-reperfusion injury of the affected intestinal tract develops. Proteinase-activated receptor 2 (PAR2) plays an important role in the pathogenesis of inflammation in the gastrointestinal tract. The aim of this study was to evaluate the distribution and expression of PAR2 in colonic pelvic flexure of horses spontaneously affected by large colon volvulus (CVH group). Eight horses admitted for severe abdominal colon volvolus and which underwent surgery were included. Colon samples were collected after enterotomy. Data previously obtained from healthy horses were used as a control group. Histologic evaluation was carried out to grade the severity of the colon lesions. Immunofluorescence, western blot and quantitative polymerase chain reaction (RT-qPCR) were carried out on colon samples to evaluate PAR2 expression. In addition, the transcriptional profile of cytokines and chemokines was evaluated using RT2 Profiler™ PCR Array Horse Cytokines & Chemokines. Three out of the eight patients were euthanised due to clinical deterioration. Immunostaining for PAR2 was observed in the enterocytes, intestinal glands and neurons of the submucosal and myenteric plexi. In the CVH horses, the expression of PAR2 mesenger RNA (mRNA) did not differ significantly from that of the healthy animals; western blots of the mucosa of the colon tracts showed a clear band of the expected molecular weight for PAR2 (~44 kDa) and a band smaller than the expected molecular weight for PAR2 (25kDa), suggesting its activation. The gene expressions for C-X-C motif ligand 1 (CXCL1); interleukin 8 (IL8), macrophage inflammatory protein 2 beta (MIP-2BETA) were upregulated in the colic horses as compared with the colons of the healthy horses. Therefore, in the present study, the expression and activation of PAR2 in the colons of horses in the presence of an inflammatory reaction like that occurring in those with spontaneous colon volvulus was confirmed.

Barrier Effect of a New Topical Agent on Damaged Esophageal Mucosa: Experimental Study on an ex vivo Swine Model.

PURPOSE: AL2106 is a new medical device based on a mixture of chondroitin sulphate in a xyloglucan and glycerol solution made to maximize its bioadhesive capability to the esophageal mucosa. The aim of the present study was twofold to evaluate the AL2106 protective effect on the esophageal mucosa when exposed to an acidic solution mimicking gastric reflux and to assess the resilience of this effect to saline washing. MATERIALS AND METHODS: A porcine ex vivo model was used and the effects of the new medical device were compared to a sodium alginate suspension (SAS) already present on the market which was assumed as reference. Mucosal damage was induced in 19 porcine esophagi by perfusion with an acidic solution added with pepsin, and Evans blue dye (EBD) tissue uptake was used as an indicator of mucosal permeability. The EBD penetration, expressed as EBD µg/g of dry tissue, was assessed in specimens of untreated damaged mucosa and in specimens treated with AL2106 or SAS. The same evaluation was carried out after washing with normal saline. RESULTS: Both topical agents tested significantly reduced the EBD uptake by more than 60% (AL2106 8.4±4.5, SAS 3.6±2.7 vs control 23.2±13.1, p<0.01). The saline washing did not cause any significant reduction in the protective effect of AL2106 (8.6±5.9), while it significantly reduced that of SAS (5.9±4.3, p<0.05). CONCLUSION: The new AL2106 medical device showed a good barrier effect against a reflux-like damaging solution and preserved this effect after the mucosal washing test, thus suggesting its possible relevance for the treatment of gastroesophageal reflux disease.

Clinopodium tomentosum (Kunth) Govaerts Leaf Extract Influences in vitro Cell Proliferation and Angiogenesis on Primary Cultures of Porcine Aortic Endothelial Cells.

Clinopodium tomentosum (Kunth) Govaerts is an endemic species in Ecuador, where it is used as an anti-inflammatory plant to treat respiratory and digestive affections. In this work, effects of a Clinopodium tomentosum ethanolic extract (CTEE), prepared from aerial parts of the plant, were investigated on vascular endothelium functions. In particularly, angiogenesis activity was evaluated, using primary cultures of porcine aortic endothelial cells (pAECs). Cells were cultured for 24 h in the presence of CTEE different concentrations (10, 25, 50, and 100 µg/ml); no viability alterations were found in the 10-50 µg/ml range, while a slight, but significant, proliferative effect was observed at the highest dose. In addition, treatment with CTEE was able to rescue LPS-induced injury in terms of cell viability. The CTEE ability to affect angiogenesis was evaluated by scratch test analysis and by an in vitro capillary-like network assay. Treatment with 25-50 µg/ml of extract caused a significant increase in pAEC's migration and tube formation capabilities compared to untreated cells, as results from the increased master junctions' number. On the other hand, CTEE at 100 µg/ml did not induce the same effects. Quantitative PCR data demonstrated that FLK-1 mRNA expression significantly increased at a CTEE dose of 25 µg/ml. The CTEE phytochemical composition was assessed through HPLC-DAD; rosmarinic acid among phenolic acids and hesperidin among flavonoids were found as major phenolic components. Total phenolic content and total flavonoid content assays showed that flavonoids are the most abundant class of polyphenols. The CTEE antioxidant activity was also showed by means of the DPPH and ORAC assays. Results indicate that CTEE possesses an angiogenic capacity in a dose-dependent manner; this represents an initial step in elucidating the mechanism of the therapeutic use of the plant.

Effects of Hydrogen Sulfide Donor NaHS on Porcine Vascular Wall-Mesenchymal Stem Cells.

Hydrogen sulfide (H2S) is now considered not only for its toxicity, but also as an endogenously produced gas transmitter with multiple physiological roles, also in maintaining and regulating stem cell physiology. In the present work, we evaluated the effect of a common H2S donor, NaHS, on porcine vascular wall-mesenchymal stem cells (pVW-MSCs). pVW-MSCs were treated for 24 h with increasing doses of NaHS, and the cell viability, cell cycle, and reactive oxygen species (ROS) production were evaluated. Moreover, the long-term effects of NaHS administration on the noteworthy characteristics of pVW-MSCs were analyzed. The MTT test revealed no alteration in cell viability, however, the cell cycle analysis demonstrated that the highest NaHS dose tested (300 µM) determined a block in S phase, which did not depend on the ROS production. Moreover, NaHS (10 µM), continuously administered in culture for 21 days, was able to significantly reduce NG2, Nestin and PDGFR-ß expression. The pro-angiogenic attitude of pVW-MSCs was partially reduced by NaHS: the cells maintained the ability to grow in spheroid and sprouting from that, but endothelial markers (Factor VIII and CD31) were reduced. In conclusion, NaHS can be toxic for pVW-MSCs in high doses, while in low doses, it influences cellular physiology, by affecting the gene expression with a slowing down of the endothelial lineage.

Non-invasive Assessment of Fecal Stress Biomarkers in Hunting Dogs During Exercise and at Rest.

Intense exercise causes to organisms to have oxidative stress and inflammation at the gastrointestinal (GI) level. The reduction in intestinal blood flow and the exercise-linked thermal damage to the intestinal mucosa can cause intestinal barrier disruption, followed by an inflammatory response. Furthermore, the adaptation to exercise may affect the gut microbiota and the metabolome of the biofluids. The aim of the present research was to evaluate the presence of a GI derangement in hunting dogs through a non-invasive sampling as a consequence of a period of intense exercise in comparison with samples collected at rest. The study included nine dogs that underwent the same training regime for hunting wild boar. In order to counterbalance physiological variations, multiple-day replicates were collected and pooled at each experimental point for each dog. The samples were collected immediately at rest before the training (T0), after 60 days of training (T1), after 60 days of hunting wild boar (T2), and finally, at 60 days of rest after hunting (T3). A number of potential stress markers were evaluated: fecal cortisol metabolites (FCMs) as a major indicator of altered physiological states, immunoglobulin A (IgA) as an indicator of intestinal immune protection, and total antioxidant activity [total antioxidant capacity (TAC)]. Since stool samples contain exfoliated cells, we investigated also the presence of some transcripts involved in GI permeability [occludin (OCLN), protease-activated receptor-2 (PAR-2)] and in the inflammatory mechanism [interleukin (IL)-8, IL-6, IL-1b, tumor necrosis factor alpha (TNFα), calprotectin (CALP), heme oxygenase-1 (HO-1)]. Finally, the metabolome and the microbiota profiles were analyzed. No variation in FCM and IgA content and no differences in OCLN and CALP gene expression between rest and training were observed. On the contrary, an increase in PAR-2 and HO-1 transcripts, a reduction in total antioxidant activity, and a different profile of microbiota and metabolomics data were observed. Collectively, the data in the present study indicated that physical exercise in our model could be considered a mild stressor stimulus.

Proteinase Activated Receptor 4 in the Jejunum of Healthy Horses and of Horses With Epiploic Hernia.

Proteinase activated receptor 4 (PAR4) in the gastrointestinal tract is involved in the regulation of inflammation and pain pathways. The aim of the present study was to evaluate the distribution and expression of PAR4 in the jejunum of healthy horses and in the pathologic tracts from horses undergoing surgery for herniation of the small intestine through the epiploic foramen. Eight healthy horses (Group H) and eight horses with epiploic hernia (Group EH) were included; the jejunum samples were collected at the slaughter or intraoperatively after enterectomy, respectively. To evaluate PAR4 expression in sections of the jejunum, immunofluorescence, western blot and quantitative polymerase chain reaction (qRT-PCR) were performed. Immunohistochemistry of PAR4 in the jejunum of the healthy horses showed that receptors are predominantly expressed in the immune cell population scattered throughout the lamina propria of the mucosa and in the submucosa. Quantitative PCR data demonstrated that PAR4 mRNA was detectable in all of the samples analyzed without any difference between the H and the EH groups, however the PAR4 protein level was significantly lower in the jejunums of the EH horses. In the Group EH horses, PAR4 immunoreactivity was mainly expressed in the mast cells and was extensively distributed in the sierosa. In the lamina propria of mucosa of Group EH, leukocytes were less abundant than in Group H. In this study, the distribution and expression of PAR4 in the jejunums of the healthy horses and in those with spontaneous occurring epiploic hernia was demonstrated.

Influence of Lactobacillus kefiri on Intestinal Microbiota and Fecal IgA Content of Healthy Dogs.

The increasing incidence of gastrointestinal tract pathologies in dogs and the worrisome topic of antibiotic resistance have raised the need to look for new therapeutic frontiers. Of these, the use of probiotics represents a potential therapeutic alternative. Lactobacillus kefiri (Lk) is a species of Lactobacillus isolated from kefir. Previous studies have demonstrated that its administration in mice downregulates the expression of proinflammatory mediators and increases anti-inflammatory molecules in the gut immune system. It also regulates intestinal homeostasis, incrementing immunoglobulin A (IgA) secretion. Since Lk has never been studied as a single probiotic in dogs, the aim of this study was to evaluate the safety of Lk in dogs, and its effect on IgA secretion and on intestinal microbiota composition. Ten healthy dogs without a history of gastrointestinal diseases were included. The dogs received Lk at a dose of 107 live microorganisms orally, once daily for 30 days. The fecal samples were tested before administration, in the middle, at the end, and 30 days after discontinuation. The IgA secretion concentration and the microbiota composition were evaluated on the fecal samples. The results in this study suggested that Lk did not influence the concentration of IgA, nor significant changes of the intestinal microbiota were observed during and after the treatment. Therefore, additional studies are needed to investigate if a higher daily dosage of Lk can influence the intestinal homeostasis of dogs.