Osteoinductive and regenerative potential of premixed calcium-silicate bioceramic sealers on vascular wall mesenchymal stem cells.

AIM: The osteogenic potential of new premixed calcium-silicate-containing bioceramic sealers (Ca-Si sealers) was tested with porcine vascular wall-mesenchymal stem cells (pVW-MSCs). METHODOLOGY: Two Ca-Si-containing sealers: Ceraseal (MetaBiomed, Cheong-si, South Korea) and AH Plus Bioceramic (Maruchi, Wonju-si, South Korea), and an epoxy resin sealer (AH Plus; Dentsply, Konstanz, Germany) as a control, were prepared according to the manufacturers' indications. All samples were allowed to set for 100% of their setting time in a sterile humid cabinet at 37°C and 95% relative humidity. pVW-MSC seeding efficiency and osteogenic differentiation were analysed as marker of gene/protein expression for up to 12 days. Mineralization assay and immunofluorescence staining were performed and evaluated over a period of 21 days. Statistical analyses were conducted using one-way analysis of variance (p < .05). Additional samples were prepared and stored under the same conditions and inspected using an environmental scanning electron microscope equipped with an energy dispersive X-ray spectroscopy system. RESULTS: Significantly higher cell seeding efficiency (p < .05) was observed for both Ca-Si sealers from day 8. pVW-MSCs showed a significant shift towards the osteogenic lineage only when seeded in contact with Ca-Si sealers. Gene expression of osteopontin was upregulated significantly. Collagen I and osteocalcin were clearly expressed by cells in contact with Ca-Si sealers. Mineralization granules were observed in Alizarin red assays and confocal laser scanning microscopy analysis of both Ca-Si sealers. No gene expression or granule mineralization were observed on the epoxy resin sealer. CONCLUSIONS: Premixed Ca-Si sealers displayed a higher potential for osteogenic activity on pVW-MSCs. Epoxy resin sealer was unable to induce any osteogenic activity. The properties of both Ca-Si sealers suggest their potential as osteoinductive platforms for vascular MSCs in periapical bone.

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).

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.

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.

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.

Characterization of metabolic profiles and lipopolysaccharide effects on porcine vascular wall mesenchymal stem cells.

The link between metabolic remodeling and stem cell fate is still unclear. To explore this topic, the metabolic profile of porcine vascular wall mesenchymal stem cells (pVW-MSCs) was investigated. At the first and second cell passages, pVW-MSCs exploit both glycolysis and cellular respiration to synthesize adenosine triphosphate (ATP), but in the subsequent (third to eighth) passages they do not show any mitochondrial ATP turnover. Interestingly, when the first passage pVW-MSCs are exposed to 0.1 or 10 µg/ml lipopolysaccharides (LPSs) for 4 hr, even if ATP synthesis is prevented, the spare respiratory capacity is retained and the glycolytic capacity is unaffected. In contrast, the exposure of pVW-MSCs at the fifth passage to 10 µg/ml LPS stimulates mitochondrial ATP synthesis. Flow cytometry rules out any reactive oxygen species (ROS) involvement in the LPS effects, thus suggesting that the pVW-MSC metabolic pattern is modulated by culture conditions via ROS-independent mechanisms.

Constitutive and LPS-stimulated secretome of porcine Vascular Wall-Mesenchymal Stem Cells exerts effects on in vitro endothelial angiogenesis.

BACKGROUND: MSCs secretome is under investigation as an alternative to whole-cell-based therapies, since it is enriched of bioactive molecules: growth factors, cytokines and chemokines. Taking into account the translational value of the pig model, the leading aim of the present paper was to characterize the secretome of porcine Vascular Wall-Mesenchymal Stem Cells (pVW-MSCs) and its change in presence of LPS stimulation. Moreover, considering the importance of angiogenesis in regenerative mechanisms, we analysed the effect of pVW-MSCs secretome on in vitro angiogenesis. RESULTS: Our results demonstrated that conditioned medium from unstimulated pVW-MSCs contained high levels of IL-8, GM-CSF, IFN-γ and other immunomodulatory proteins: IL-6 IL-18 IL-4 IL-2 IL-10. LPS modulates pVW-MSCs gene expression and secretome composition, in particular a significant increase of IL-6 and IL-8 was observed; conversely, the amount of GM-CSF, IFN-γ, IL-2, IL-4, IL-10 and IL-18 showed a significant transient decrease with the LPS stimulation. Conditioned medium from unstimulated pVW-MSCs induced in vitro endothelial angiogenesis, which is more evident when the conditioned medium was from LPS stimulated pVW-MSCs. CONCLUSIONS: The lines of evidence here presented shed a light on possible future application of secretome derived by pVW-MSCs on research studies in translational regenerative medicine.

Water/ethanol extract of Cucumis sativus L. fruit attenuates lipopolysaccharide-induced inflammatory response in endothelial cells.

BACKGROUND: It is widely accepted the key role of endothelium in the onset of many chronic and acute vascular and cardiovascular diseases. In the last decade, traditional compounds utilized in "folk medicine" were considered with increasing interest to discover new bioactive molecules potentially effective in a wide range of diseases including cardiovascular ones. Since ancient times different parts of the Cucumis sativus L. plant were utilized in Ayurvedic medicine, among these, fruits were traditionally used to alleviate skin problem such as sunburn irritation and inflammation. The main purpose of the present research was, in a well-defined in vitro model of endothelial cells, to investigate whether a water/ethanol extract of Cucumis sativus L. (CSE) fruit can attenuate the damaging effect of pro-inflammatory lipopolysaccharide (LPS). METHODS: Cell viability, gene expression of endothelial cell markers, cytokines secretion and in vitro angiogenesis assay were performed on porcine Aortic Endothelial Cells exposed to increasing doses (0.02; 02; 2 mg/ml) of CSE in the presence of pro-inflammatory lipopolysaccharide (LPS 10 µg/ml). RESULTS: CSE reduced LPS-induced cytotoxicity and decreased the cellular detachment, restoring the expression of tight junction ZO-1. The increase of TLR4 expression induced by LPS was counterbalanced by the presence of CSE, while the protective gene Hemeoxygenase (HO)-1 was increased. Cucumis sativus L. inhibited the early robust secretion of inflammatory IL-8 and GM-CSFs, furthermore inhibition of inflammatory IL-6 and IL-1α occurred late at 7 and 24 h respectively. On the contrary, the secretion of anti-inflammatory IL-10, together with IL-18 and IFN-γ was increased. Moreover, the in vitro angiogenesis induced by inflammatory LPS was prevented by the presence of Cucunis sativus L. extract, at any doses tested. CONCLUSIONS: Our results have clearly demonstrated that Cucumis sativus L. extract has attenuated lipopolysaccharide-induced inflammatory response in endothelial cells.

In vitro differentiation of porcine aortic vascular precursor cells to endothelial and vascular smooth muscle cells.

Recent findings suggest that progenitor and multipotent mesenchymal stromal cells (MSCs) are associated with vascular niches. Cells displaying mesenchymal properties and differentiating to whole components of a functional blood vessel, including endothelial and smooth muscle cells, can be defined as vascular stem cells (VSCs). Recently, we isolated a population of porcine aortic vascular precursor cells (pAVPCs), which have MSC- and pericyte-like properties. The aim of the present work was to investigate whether pAVPCs possess VSC-like properties and assess their differentiation potential toward endothelial and smooth muscle lineages. pAVPCs, maintained in a specific pericyte growth medium, were cultured in high-glucose DMEM + 10% FBS (long-term medium, LTM) or in human endothelial serum-free medium + 5% FBS and 50 ng/ml of hVEGF (endothelial differentiation medium, EDM). After 21 days of culture in LTM, pAVPCs showed an elongated fibroblast-like morphology, and they seem to organize in cord-like structures. qPCR analysis of smooth muscle markers [α-smooth muscle actin (α-SMA), calponin, and smooth muscle myosin (SMM) heavy chain] showed a significant increment of the transcripts, and immunofluorescence analysis confirmed the presence of α-SMA and SMM proteins. After 21 days of culture in EDM, pAVPCs displayed an endothelial cell-like morphology and revealed the upregulation of the expression of endothelial markers (CD31, vascular endothelial-cadherin, von Willebrand factor, and endothelial nitric oxide synthase) showing the CD31-typical pattern. In conclusion, pAVPCs could be defined as a VSC-like population considering that, if they are maintained in a specific pericyte medium, they express MSC markers, and they have, in addition to the classical mesenchymal trilineage differentiation potential, the capacity to differentiate in vitro toward the smooth muscle and the endothelial cell phenotypes.

Cells derived from porcine aorta tunica media show mesenchymal stromal-like cell properties in in vitro culture.

Several studies have already described the presence of specialized niches of precursor cells in vasculature wall, and it has been shown that these populations share several features with mesenchymal stromal cells (MSCs). Considering the relevance of MSCs in the cardiovascular physiopathology and regenerative medicine, and the usefulness of the pig animal model in this field, we reported a new method for MSC-like cell isolation from pig aorta. Filling the vessel with a collagenase solution for 40 min, all endothelial cells were detached and discarded and then collagenase treatment was repeated for 4 h to digest approximately one-third of the tunica media. The ability of our method to select a population of MSC-like cells from tunica media could be ascribed in part to the elimination of contaminant cells from the intimal layer and in part to the overnight culture in the high antibiotic/antimycotic condition and to the starvation step. Aortic-derived cells show an elongated, spindle shape, fibroblast-like morphology, as reported for MSCs, stain positively for CD44, CD56, CD90, and CD105; stain negatively for CD34 and CD45; and express CD73 mRNA. Moreover, these cells show the classical mesenchymal trilineage differentiation potential. Under our in vitro culture conditions, aortic-derived cells share some phenotypical features with pericytes and are able to take part in the formation of network-like structures if cocultured with human umbilical vein endothelial cells. In conclusion, our work reports a simple and highly suitable method for obtaining large numbers of precursor MSC-like cells derived from the porcine aortic wall.

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.

Development of a vessel organ culture system: characterisation of the method and implications for the reduction of animal experiments.

In the field of cardiovascular research, the pig is considered to be an excellent animal model of human diseases. It is well-known that primary cultures of endothelial cells (ECs) are a powerful tool for the study of vascular physiology and pathology, and, according to the principles of the Three Rs, their use results in a substantial reduction in the numbers of experimental animals required. However, a limitation of EC culture is that the cells are not in their physiological context. Here, we describe and characterise a method for the culture of porcine vessels that overcomes the limitation of EC cultures, with the advantage of reducing the number of animals used for research purposes. The organ cultures were set-up by using an aortic cylinder obtained from the arteries of control pigs sacrificed for other experimental purposes. In order to characterise the method, vascular endothelial growth factor (VEGF) secretion, matrix metalloproteinase (MMP) activation and the vessel's structural features were evaluated during organ culture. These analyses confirm that the culture of aortic cylinder lumen, in a medium specific for ECs, results in a stable system in terms of VEGF and MMP secretion. The ECs do not undergo cell division during the organ culture, which is also the case in vivo, if no stimulation occurs. Overall, we show that this novel system closely resembles the in vivo context. Importantly, porcine aortas can be collected from either veterinary surgeries or slaughterhouses, without having to sacrifice animals specifically for the purposes of this type of research.

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.

Detection of Wolbachia DNA in blood for diagnosing filaria-associated syndromes in cats.

A fundamental role for the endosymbiotic bacteria Wolbachia pipientis in the pathogenesis of Dirofilaria immitis infections has emerged in recent years. Diagnostic opportunities arising from this breakthrough have not yet been fully exploited. This study was aimed at developing conventional and real-time PCR assays to carry out a molecular survey in a convenience sample of cats living in an area where D. immitis is endemic and to evaluate the detection of bacterial DNA in blood as a surrogate assay for diagnosing filaria-associated syndromes in cats. COI and FtsZ loci were used as targets for D. immitis and Wolbachia PCR assays, respectively, and real-time TaqMan PCR assays were used only for Wolbachia. A convenience sample of 307 disease-affected or healthy cats examined at a University facility were PCR tested, and their medical records were investigated. Conventional nested PCR for Wolbachia amplified the endosymbionts of both D. immitis and D. repens, while real-time PCR was highly specific only for the former. Observed prevalences of 0.3 and 10.4% were found using conventional nested PCR assays for D. immitis and real-time PCR for Wolbachia, respectively. Similar prevalences were established using the Wolbachia nested PCR (98% concordance with real-time PCR). The group of Wolbachia-positive samples had a significantly higher proportion of subjects with respiratory signs (29.0% versus 9.7%; P = 0.002). The findings of this study indicate that a highly sensitive PCR assay can be used to detect the Wolbachia organism in the peripheral blood of cats with respiratory signs.

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.

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.

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.

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.