Effects of the Myokine Irisin on Stromal Cells from Swine Adipose Tissue.

Irisin is a hormone able to reproduce some of the positive effects of physical activity and diet. Recently, we demonstrated the presence of Irisin at the ovarian level as a potential physiological regulator of follicular function. Adipose tissue is crucial for reproductive function through its metabolic activity and the production of adipokines. At present, the exact nature of adipocyte precursors is still under debate, but an important role has been assigned to the population of adipose tissue mesenchymal stromal cells (ASCs) of perivascular origin. It should be noted that, when appropriately stimulated, ASCs can differentiate into preadipocytes and, subsequently, adipocytes. Therefore, this present study was undertaken to explore the potential effect of Irisin on ASCs, known for their high differentiative potential. Since Irisin expression in ASCs was confirmed by PCR, we tested its potential effects on the main functional activities of these cells, including proliferation (BrdU uptake); metabolic activity (ATP production); redox status, evaluated as the generation of free molecules such as superoxide anion and nitric oxide; and scavenger activities, assessed as both enzymatic (superoxide dismutase) and non-enzymatic antioxidant power. Moreover, we tested the effect of Irisin on ASCs adipogenic differentiation. BrdU uptake was significantly (p < 0.001) inhibited by Irisin, while ATP production was significantly (p < 0.05) increased. Both superoxide anion and nitric oxide generation were significantly increased (p < 0.001) by Irisin, while scavenger activity was significantly reduced (p < 0.05). Irisin was found to significantly (p < 0.05) inhibit ASCs adipogenic differentiation. Taken together, the present results suggest a potential local role of Irisin in the regulation of adipose tissue function.

Nipah Virus Disease: Epidemiological, Clinical, Diagnostic and Legislative Aspects of This Unpredictable Emerging Zoonosis.

Nipah virus (NiV) infection is a viral disease caused by a Henipavirus, belonging to the Paramyxoviridae family, responsible for a zoonosis. The course of the disease can be very serious and lead to death. NiV natural hosts are fruit bats (also known as megabats) belonging to the Pteropodidae family, especially those of the Pteropus genus. Natural infection in domestic animals has been described in farming pigs, horses, domestic and feral dogs and cats. Natural NiV transmission is possible intra-species (pig-to-pig, human-to-human) and inter-species (flying bat-to-human, pig-to-human, horse-to-human). The infection can be spread by humans or animals in different ways. It is peculiar how the viral transmission modes among different hosts also change depending on the geographical area for different reasons, including different breeding methods, eating habits and the recently identified genetic traits/molecular features of main virus proteins related to virulence. Outbreaks have been described in Malaysia, Singapore, Bangladesh, India and the Philippines with, in some cases, severe respiratory and neurological disease and high mortality in both humans and pigs. Diagnosis can be made using different methods including serological, molecular, virological and immunohistochemical methods. The cornerstones for control of the disease are biosecurity (via the correct management of reservoir and intermediate/amplifying hosts) and potential vaccines which are still under development. However, the evaluation of the potential influence of climate and anthropogenic changes on the NiV reservoir bats and their habitat as well as on disease spread and inter-specific infections is of great importance. Bats, as natural reservoirs of the virus, are responsible for the viral spread and, therefore, for the outbreaks of the disease in humans and animals. Due to the worldwide distribution of bats, potential new reports and spillovers are not to be dismissed in the future.

Effects of Orexin B on Swine Granulosa and Endothelial Cells.

In addition to the well-known central modulatory role of orexins, we recently demonstrated a peripheral involvement in swine granulosa cells for orexin A and in adipose tissue for orexin B (OXB). The aim of present research was to verify immunolocalization of OXB and its potential role in modulating the main features of swine granulosa cells. In particular, we explored the effects on granulosa cell proliferation (through the incorporation of bromodeoxyuridine), cell metabolic activity (as indirect evaluation by the assessment of ATP), steroidogenic activity (by immunoenzymatic examination) and redox status (evaluating the production of superoxide anion by means of the WST test, production of nitric oxide through the use of the Griess test and the non-enzymatic reducing power by FRAP test). Our data point out that OXB does not modify granulosa cell growth, steroidogenesis and superoxide anion generation. On the contrary, the peptide stimulates (p < 0.05) nitric oxide output and non-enzymatic reducing power. Since new vessel growth is crucial for ovarian follicle development, a further aim of this study was to explore the expression of prepro-orexin and the effects of OXB on swine aortic endothelial cells. We found that the peptide is ineffective in modulating cell growth, while it inhibits redox status parameters. In addition, we demonstrated a stimulatory effect on angiogenesis evaluated in fibrin gel angiogenesis assay. Taken together, OXB appears to be potentially involved in the modulation of redox status in granulosa and endothelial cells and we could argue an involvement of the peptide in the follicular angiogenic events.

A pathogenic HEXA missense variant in wild boars with Tay-Sachs disease.

Gangliosidoses are inherited lysosomal storage disorders caused by reduced or absent activity of either a lysosomal enzyme involved in ganglioside catabolism, or an activator protein required for the proper activity of a ganglioside hydrolase, which results in the intra-lysosomal accumulation of undegraded metabolites. We hereby describe morphological, ultrastructural, biochemical and genetic features of GM2 gangliosidosis in three captive bred wild boar littermates. The piglets were kept in a partially-free range farm and presented progressive neurological signs, starting at 6 months of age. Animals were euthanized at approximately one year of age due to their poor conditions. Neuropathogens were excluded as a possible cause of the signs. Gross examination showed a reduction of cerebral and cerebellar consistency. Central (CNS) and peripheral (PNS) nervous system neurons were enlarged and foamy, with severe and diffuse cytoplasmic vacuolization. Transmission electron microscopy (TEM) of CNS neurons demonstrated numerous lysosomes, filled by parallel or concentric layers of membranous electron-dense material, defined as membranous cytoplasmic bodies (MCB). Biochemical composition of gangliosides analysis from CNS revealed accumulation of GM2 ganglioside; furthermore, Hex A enzyme activity was less than 1% compared to control animals. These data confirmed the diagnosis of GM2 gangliosidosis. Genetic analysis identified, at a homozygous level, the presence of a missense nucleotide variant c.1495C > T (p Arg499Cys) in the hexosaminidase subunit alpha gene (HEXA), located within the GH20 hexosaminidase superfamily domain of the encoded protein. This specific HEXA variant is known to be pathogenic and associated with Tay-Sachs disease in humans, but has never been identified in other animal species. This is the first report of a HEXA gene associated Tay-Sachs disease in wild boars and provides a comprehensive description of a novel spontaneous animal model for this lysosomal storage disease.

Melatonin modulates swine luteal and adipose stromal cell functions.

Based on our previous study in follicles, the first aim of this work was to evaluate the effect of melatonin in the swine corpus luteum (CL). Luteal cells were exposed to 10 and 20pg mL-1 melatonin. We evaluated the effect on proliferation (bromo-deoxy-uridine uptake), steroidogenesis (progesterone) and redox status by means of Griess test (nitric oxide production), WST-1 test (superoxide anion generation) and FRAP test (non-enzymatic antioxidant power). The results showed a significant increase in antioxidant power, as well as a reduction in the other parameters analysed. These data and the expression of MT2 observed in luteal cells allow us to hypothesise a physiological role of melatonin in the regulation of CL functionality. The reproductive function is dependent on energy reserves stored in adipose tissue. Therefore, we sought to verify the effect of melatonin on adipose stromal cells (ASCs). MT2 receptor expression was detected in ASCs and the presence of gene markers (PPARγ and leptin) before and after adipogenic differentiation was verified. The differentiation was significantly inhibited by melatonin, as well as cell viability. In conclusion, present results suggest that melatonin exerts a potential inhibitory action on luteal function and adipogenesis, possibly mediated by MT2.

Xenobiotic-Free Medium Guarantees Expansion of Adipose Tissue-Derived Canine Mesenchymal Stem Cells Both in 3D Fibrin-Based Matrices and in 2D Plastic Surface Cultures.

Mesenchymal stem cells (MSCs) have been recently introduced in veterinary medicine as a potential therapeutic tool for several pathologies. The large-scale in vitro expansion needed to ensure the preparation of a suitable number of MSCs for clinical application usually requires the use of xenogeneic supplements like the fetal bovine serum (FBS). The substitution of FBS with species-specific supplements would improve the safety of implanted cells, reducing the risk of undesired immune responses following cell therapy. We have evaluated the effectiveness of canine adipose tissue-derived stromal vascular fraction (SVF) and MSCs (ADMSCs) expansion in the presence of canine blood-derived supplements. Cells were cultured on traditional plastic surface and inside a 3D environment derived from the jellification of different blood-derived products, i.e., platelet-poor plasma (PPP), platelet-rich plasma (PRP), or platelet lysate (PL). PPP, PRP, and PL can contribute to canine ADMSCs in vitro expansion. Both allogeneic and autologous PPP and PL can replace FBS for ADMSCs culture on a plastic surface, exhibiting either a similar (PPP) or a more effective (PL) stimulus to cell replication. Furthermore, the 3D environment based on homospecific blood-derived products polymerization provides a strong stimulus to ADMSCs replication, producing a higher number of cells in comparison to the plastic surface environment. Allogeneic or autologous blood products behave similarly. The work suggests that canine ADMSCs can be expanded in the absence of xenogeneic supplements, thus increasing the safety of cellular preparations. Furthermore, the 3D fibrin-based matrices could represent a simple, readily available environments for effective in vitro expansion of ADMSCs using allogeneic or autologous blood-products.

Endocanalicular transendothelial crossing (ETC): A novel intravasation mode used by HEK-EBNA293-VEGF-D cells during the metastatic process in a xenograft model.

In cancer metastasis, intravasation of the invasive tumor cell (TCi) represents one of the most relevant events. During the last years, models regarding cancer cell intravasation have been proposed, such as the "endocanalicular transendothelial crossing" (ETC) theory. This theory describes the interplay between two adjacent endothelial cells and the TCi or a leukocyte during intravasation. Two endothelial cells create a channel with their cell membranes, in which the cell fits in without involving endothelial cell intercellular junctions, reaching the lumen through a transendothelial passage. In the present study, ten SCID mice were subcutaneously xenotransplanted with the HEK-EBNA293-VEGF-D cell line and euthanized after 35 days. Post-mortem examinations were performed and proper specimens from tumors were collected. Routine histology and immunohistochemistry for Ki-67, pAKT, pERK, ZEB-1, TWIST-1, F-actin, E-cadherin and LYVE-1 were performed followed by ultrastructural serial sections analysis. A novel experimental approach involving Computed Tomography (CT) combined with 3D digital model reconstruction was employed. The analysis of activated transcription factors supports that tumor cells at the periphery potentially underwent an epithelial-to-mesenchymal transition (EMT)-like process. Topographical analysis of LYVE-1 immunolabeled lymphatics revealed a peritumoral localisation. TEM investigations of the lymphatic vessels combined with 3D digital modelling enhanced the understanding of the endotheliocytes behavior during TCi intravasation, clarifying the ETC theory. Serial ultrastructural analysis performed within tumor periphery revealed numerous cells during the ETC process. Furthermore, this study demonstrates that ETC is an intravasation mode more frequently used by the TCi than by leukocytes during intravasation in the HEK-EBNA293-VEGF-D xenograft model and lays down the potential basis for promising future studies regarding intravasation blocking therapy.

Evaluation of the Anti-Tumor Activity of Small Molecules Targeting Eph/Ephrins in APC min/J Mice.

The Eph receptors are the largest receptors tyrosine kinases (RTKs) family in humans and together with ephrin ligands constitute a complex cellular communication system often dysregulated in many tumors. The role of the Eph-ephrin system in colorectal cancer (CRC) has been investigated and different expression of Eph receptors have been associated with tumor development and progression. In light of this evidence, we investigated if a pharmacological approach aimed at inhibiting Eph/ephrin interaction through small molecules could prevent tumor growth in APC min/J mice. The 8-week treatment with the Eph-ephrin antagonist UniPR129 significantly reduced the number of adenomas in the ileum and decreased the diameter of adenomas in the same region. Overall our data suggested as UniPR129 could be able to slow down the tumor development in APC min/J mice. These results further confirm literature data about Eph kinases as a new valuable target in the intestinal cancer and for the first time showed the feasibility of the Eph-ephrin inhibition as a useful pharmacological approach against the intestinal tumorigenesis. In conclusion this work paves the way for further studies with Eph-ephrin inhibitors in order to confirm the Eph antagonism as innovative pharmacological approach with preventive benefit in the intestinal tumor development.

Multifocal cutaneous non-epitheliotropic B-cell lymphoma in a cat.

CASE SUMMARY: Skin tumours are the second-most common form of feline cancer after haematopoietic neoplasms and are often malignant. Cutaneous lymphoma is uncommon in cats and can be classified as epitheliotropic (typically of T-cell origin) or non-epitheliotropic (either of T-cell or B-cell origin). The present study describes a case of multifocal cutaneous non-epitheliotropic B-cell lymphoma. The skin nodules were multiple and variable in size; showed rapid progression; were alopecic and erythematous in appearance and pruritic and ulcerated; and were mostly located on the trunk. Nodule biopsies revealed the presence of uniform medium-to-large round neoplastic cells that infiltrated the dermis and subcutis. The neoplasias were consistent with a round cell cutaneous tumour and did not show evidence of epitheliotropism. Furthermore, immunohistochemical assessments indicated an immunophenotype characterised by round cells with a strong membrane and cytoplasmic positivity for the CD20 antigen, consistent with a lymphocyte of B-cell origin. RELEVANCE AND NOVEL INFORMATION: Cutaneous non-epitheliotropic B-cell lymphoma in cats is rare and was previously reported to appear as single dermal and subcutaneous masses that are variable in size and generally develop in the tarsal region. To our knowledge, this is the first report to describe multifocal cutaneous non-epitheliotropic B-cell lymphoma in a cat.

The effect of pathogen-associated molecular patterns on the swine granulosa cells.

Recent studies have demonstrated the surprising ability of reproductive endocrine cells to express receptors of innate immunity useful to sense danger in order to avoid disruption of tissue homeostasis. Present research demonstrates the presence of pattern recognition receptors, i.e. toll like receptors (TLR) TLR2, TLR4 and TLR 5 and NOD like receptors (NLR) NOD1 and NOD2 in swine granulosa cells from ovarian follicles> 5 mm. Therefore, our second goal was to expose granulosa cells to different concentrations (1000, 100 and 10 ng/ml) of lipopolysaccharide (LPS) and N-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)- propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]- lysine (Pam3CSK4), two substances associated with pathogen molecular patterns. Their potential effects on the main functional parameters were monitored: proliferation (through the incorporation of Bromo-deoxy-Uridine), cell viability (by testing the metabolization of MTT salt), steroidogenic activity (by immunoenzymatic examination) and redox status (evaluating the production of superoxide anion by means of the WST test, production of nitric oxide through the use of the Griess test, and the non-enzymatic reducing power, by FRAP test). The data collected show a significant inhibition (p < 0.01) of cell proliferation after treatment with both LPS and Pam3CSK4, while cell viability has not been modified. As for steroidogenesis, treatment with both LPS and Pam3CSK4 significantly inhibited (p < 0.05) the production of 17ß-estradiol and progesterone. LPS and Pam3CSK4 stimulated (p < 0.05) the production of superoxide anion and nitric oxide, while inhibited (p < 0.05) the antioxidant power. In conclusion, the study shows that the functionality of granulosa cells is compromised by the exposure to molecular profiles associated with pathogens; the knowledge gathered could lay the theoretical basis for the definition of therapeutic treatments related to diseases that can affect normal reproductive processes.

Lead and copper in hunted wild boars and radiographic evaluation of bullet fragmentation between ammunitions.

The purpose of the present study was to evaluate the content of lead in carcasses of wild boars shot with lead bullets, in comparison with that of copper caused by lead-free ammunitions. Radiographic images of hunted boars were obtained in order to assess the degree of bullet fragmentation in the carcasses. Samples of meat were collected from different body areas at increasing distance from bullet trajectory, to be analysed by ICP-MS for lead and copper levels. In wild boars shot with lead ammunitions, a massive dispersion of bullet fragments and very high lead levels were detected. By contrast, in wild boars killed with copper ammunitions no radiographic signs of bullet fragmentation were observed. Copper ammunitions seem therefore a safer alternative to standard lead-core ones, due to their minimal fragmentation and the relatively low toxicity of this metal.

Rifaximin anti-inflammatory activity on bovine endometrium primary cell cultures: a preliminary study.

Rifaximin is an unabsorbed oral antibiotic showing anti-inflammatory properties in human pathologies like irritable bowel syndrome and inflammatory bowel disease. In veterinary medicine, rifaximin is primarily used in the treatment of dermatological diseases in all animal species, in therapy and prophylaxis of mastitis in cows and in the treatment of endometritis in cattle and horses. The aim of this preliminary study was to evaluate the anti-inflammatory properties of rifaximin on primary cell cultures from bovine endometrium in which inflammatory response was induced by Lipopolysaccaride (LPS) treatment. Epithelial and stromal cells were isolated from bovine endometrium and separately incubated for 24 h with 1 µg mL-1 LPS after rifaximin (10, 50 and 100 µmol L-1 ) or dexamethasone (10 µmol L-1 ) pre-treatment for 24 h. Supernatants were collected 24 h after LPS treatment and interleukin (IL)-6 and IL-8 accumulation was measured by ELISA. Rifaximin (10, 50 and 100 µmol L-1 ) dose dependently inhibited the LPS-induced increase in IL-6 and IL-8 in stromal cells, whereas in epithelial cells it was not possible to detect any accumulation of these interleukins. Rifaximin reduced IL-6 and IL-8 production, showing a potential anti-inflammatory effect that opens up to new possibilities for the use of this drug in uterine inflammatory diseases.

Immunolocalization of Orexin A and its receptors in the different structures of the porcine ovary.

Orexins are neuropeptides with pleiotropic functions, involved in the coordination of multiple versatile physiological processes, in particular related to food intake and several aspects of the reproductive process. Their actions are carried out through the bond with the related Orexin 1 (OXR1) and Orexin 2 (OXR2) G-protein-coupled receptors. Studies on the expression of the orexinergic system in the female genital organs are scarce and limited to preovulatory gametogenic follicles and corpora lutea isolated from the rest of the ovary. As the description of only these structures is insufficient to provide a complete picture of the organ, the present study is aimed to give a panoramic view of all the ovarian structures and cells expressing Orexin A (OXA) and its receptors in their original localization. Double labeling immunofluorescent methods, applied on frozen sections of the whole organ in both follicular and luteal phase, were used to highlight the particular distribution and colocalization of the proteins. For a better recognition of cellular morphology and a better distinction between gametogenic (healthy) and atretic follicles, also a single labeling immunolocalization of OXA on formalin fixed paraffin embedded tissues and a TUNEL staining were performed. The results indicate that OXA and its two receptors subtypes are expressed in all the different structures composing the swine ovary, albeit in different ways, in both phases of the ovarian cycle. In general, OXA and OXR2 appear diffusely distributed within "health", proliferating and steroid producing cells, while has granular appearance, being presumably associated to cytoplasmic vesicles, in degenerating cells, independently if apoptotic or not. The immunoreactivity for OXR1, instead, is often associated with the nuclear envelope but it is also detectable, to a lesser extent, diffusely distributed in the cytoplasm of growing or steroid producing cells. When cells undertake the path leading to degeneration, also OXR1 immunoreactivity assumes a granular appearance in the cytoplasm and is colocalized with OXA and OXR2. Different roles for the two receptors in the same cell and a different regulation of their expression remain to be investigated. Their comprehension could help studies of follicle development in pig, as part of in vitro oocyte maturation and fertilization programs in livestock.

Swine Granulosa Cells Show Typical Endothelial Cell Characteristics.

Granulosa cells, which belong to the somatic compartment of the ovarian follicle, are actively involved as endocrine cells in follicle growth. Recently, it has been proposed that these cells are not terminally differentiated and possess multipotency. Therefore, we cultured swine granulosa cells in specific endothelial cell culture medium (EBM-2), and phenotypic and functional characteristics of endothelial cells were assessed. The collected data suggest that these endocrine cells can also behave as endothelial cells, therefore potentially contributing to follicular angiogenesis, a crucial process in follicle growth and selection.

Immunoregulatory signal FoxP3, cytokine gene expression and IFN-γ cell responsiveness upon porcine reproductive and respiratory syndrome virus (PRRSV) natural infection.

The study aims at evaluating gene expression of pro-inflammatory (IL-1ß, IL-8, TNF-α), pro-immune (IFN-γ), anti-inflammatory (IL-10) cytokines and of the immunoregulatory signal FoxP3 in association with PRRSV-specific IFN-γ secreting cell (SC) responsiveness upon PRRSV natural infection. Forty PRRSV-negative pigs were assigned to two groups: 20 pigs were vaccinated at 3 weeks of age (weaning) against PRRSV (V-PRRSV) with a modified live virus vaccine (MLV) and 20 pigs were kept non-vaccinated (NV) as controls. Blood samples were collected at 3 (vaccination), 6, 8, 10, 12, 14, and 16 weeks of age. Natural infection occurred from 8 weeks of age onward in both groups and viremia lasted 8 weeks. In the early phase of infection, pro-inflammatory cytokines (IL-1ß, IL-8, TNF-α) showed a delayed increase concomitant with the peak of viremia in both groups. In both groups, IL-10 peaked at 12 weeks in association with the increase of pro-inflammatory cytokines. Conversely, in vaccinated pigs (V-PRRSV), IFN-γ showed higher gene expression during the early phase of infection and a more intense secreting cell (SC) response in the late phase. Differently, gene expression of the transcription factor FoxP3, expressed by T regulatory lymphocytes (Tregs), increased significantly in controls only and was associated with the rise of the viral load. Moreover, FoxP3 levels remained significantly higher during the late phase of infection and paralleled with lower levels of IFN-γ SC detected by ELISPOT. The expression/production of immunoregulatory signals involved in Treg activation could be a promising marker to study the immunobiology of PRRSV infection.

Preparation and application of an innovative thrombocyte/leukocyte-enriched plasma to promote tissue repair in chelonians.

Platelet concentrates are widely used in mammalian regenerative medicine to improve tissue healing. Chelonians (Testudines) would benefit from the application of thrombocyte preparations to regenerate damaged tissues, since traumatic injuries are leading causes of morbidity and mortality for both wild-living and domesticated animals. The aim of this study was to establish a protocol that optimized the recovery of the thrombocytes from blood samples and to show the efficacy of thrombocyte-enriched plasma in chelonians. Peripheral blood samples were obtained from Testudo spp. (n = 12) and Trachemys scripta elegans (n = 10). Blood cells were fractionated by sodium diatrizoate-sodium polysucrose density gradient using a two-step centrifugation protocol. Thrombocytes and leukocytes were isolated and resuspended to obtain thrombocyte-leucocyte rich plasma (TLRP). The mean recovery of leukocytes and thrombocytes was 48.9% (±4.0 SEM, n = 22) of the whole blood cell content. No statistically significant difference was observed between blood samples collected from different turtle species. The ability of TLRP to form a gel was evaluated by adding variable concentrations of calcium gluconate at room temperature and at 37°C. A reliable and consistent clotting of the TLRP was obtained in glass tubes and dishes by adding 5-20% v/v of a 100 mg/ml solution of calcium gluconate. Furthermore, in order to test the clinical efficacy of TLRP, a preliminary evaluation was performed on four turtles (Testudo spp.) with traumatic injuries. In all the four animals, a successful clinical outcome was observed. The results demonstrated that a thrombocyte-enriched plasma, comparable to mammalian platelet rich plasma, can be prepared from chelonian blood samples. Furthermore, although the low number of cases presented does not allow definitive conclusions from a clinical point of view, their outcome suggests that TLRP application could be further investigated to improve the healing process of both soft and hard tissue injuries in chelonians.