Francesca Mercati

Flow cytometric characterization of culture expanded multipotent mesenchymal stromal cells (MSCs) from horse adipose tissue: towards the definition of minimal stemness criteria.

In the last decades, multipotent mesenchymal progenitor cells have been isolated from many adult tissues of different species. The International Society for Cellular Therapy (ISCT) has recently established that multipotent mesenchymal stromal cells (MSCs) is the currently recommended designation. In this study, we used flow cytometry to evaluate the expression of several molecules related to stemness (CD90, CD44, CD73 and STRO-1) in undifferentiated, early-passaged MSCs isolated from adipose tissue of four donor horses (AdMSCs). The four populations unanimously expressed high levels of CD90 and CD44. On the contrary, they were unexpectedly negative to CD73. A small percentage of the cells, finally, showed the expression of STRO-1. This last result might be due to the existence of a small subpopulation of STRO-1+ cells or to a poor cross-reactivity of the antibody. A remarkable donor-to-donor consistency and reproducibility of these findings was demonstrated. The data presented herein support the idea that equine AdMSCs may be easily isolated and selected by adherence to tissue culture plastic and exhibit a surface profile characterized by some peculiar differences in comparison to those described in other species. Continued characterization of these cells will help to clarify several aspects of their biology and may ultimately enable the isolation of specific, purified subpopulations.

Intraluteal regulation of prostaglandin F2α-induced prostaglandin biosynthesis in pseudopregnant rabbits

The objective of the present study was to investigate in rabbit corpora lutea (CL), at both the cellular and molecular level, intraluteal cyclooxygenase (COX)-1, COX-2 and prostaglandin (PG) E2-9-ketoreductase (PGE2-9-K) enzymatic activities as well as in vitro PGE2 and PGF2alpha synthesis following PGF2alpha treatment at either early- (day-4) or mid-luteal (day-9) stage of pseudopregnancy. By immunohistochemistry, positive staining for COX-2 was localized in luteal and endothelial cells of stromal arteries at both the stages. In CL of both stages, basal COX-2 mRNA levels were poorly expressed, but rose (P < 0.01) 4- to 10-fold 1.5-6 h after treatment and then gradually decreased within 24 h. Compared to mid-stage, day-4 CL had lower (P < 0.01) COX-2 and PGE2-9-K basal activities, and PGF2alpha synthesis rate, but higher (P < 0.01) PGE2 production. Independent of luteal stage, PGF2alpha treatment did not affect COX-1 activity. In day-4 CL, PGF2alpha induced an increase (P < 0.01) in both COX-2 activity and PGF2alpha synthesis, whereas that of PGE2 remained unchanged. In day-9 CL, PGF2alpha up-regulated (P < 0.01) both COX-2 and PGE-9-K activities, and PGF2alpha production, but decreased (P < 0.01) PGE2 synthesis. All changes in gene expression and enzymatic activities occurred within 1.5 h after PGF2alpha challenge and were more marked in day-9 CL. Our data suggest that PGF2alpha directs intraluteal PG biosynthesis in mature CL, by affecting the CL biosynthetic machinery to increase the PGF2alpha synthesis in an auto-amplifying manner, with the activation of COX-2 and PGE-9-K; this may partly explain their differentially, age-dependent, luteolytic capacity to exogenous PGF2alpha in rabbits.

Membrane vesicles mediate pro-angiogenic activity of equine adipose-derived mesenchymal stromal cells

Multipotent mesenchymal stromal cells (MSCs) have attracted a great deal of interest, due to several distinctive features, including the ability to migrate to damaged tissue and to participate in tissue regeneration. There is increasing evidence that membrane vesicles (MVs), comprising exosomes and shedding vesicles, represent a key component, responsible for many of the paracrine effects of MSCs. The aim of the present study was to establish whether equine adipose-derived MSCs (E-AdMSCs) produce MVs that are capable of influencing angiogenesis, a key step in tissue regeneration. A morphological study was performed using MSC monolayers, prepared for transmission and scanning electron microscopy and on ultracentrifuged MSC supernatants, to identify production of MVs. The ability of MVs to influence angiogenesis was evaluated by means of the rat aortic ring and scratch assays. The results demonstrated that MVs, constitutively produced by E-AdMSCs, are involved in intercellular communication with endothelial cells, stimulating angiogenesis. Although many questions remain regarding their formation, delivery, content and mechanism of action, the present study supports the concept that MVs released by MSCs have the potential to be exploited as a therapeutic tool for regenerative medicine.

Immunohistochemical identification and localization of orexin A and orexin type 2 receptor in the horse gastrointestinal tract.

The aim of the present study was to investigate the presence and the distribution of cells containing orexin A and orexin type 2 receptor in the horse stomach and gut, by means of immunohistochemical techniques. Orexin A was identified in the stomach fundic and pyloric regions and in the duodenum. In the same stomach regions, a large subset of orexin A-positive cells also showed orexin type 2 receptor-like immunoreactivity. Moreover, in the duodenum, many of them, seemed to store serotonin. Characteristically, enteric neurons or ganglia also displayed orexin A and, sometimes, orexin type 2 receptor immunoreaction. Orexin A and orexin type 2 receptor immunoreactivity was also found in the nerve fibers in the enteric submucosal layer. Our results, together with data present in the literature, could contribute to the understanding of complex mechanisms regulating the horse gut functionality that are depending very likely on the consequence of the co-operation of both a central and a peripheral control.

Identification of orexin A- and orexin type 2 receptor-positive cells in the gastrointestinal tract of neonatal dogs

The presence and distribution of cells positive to orexin A (OXA) and to orexin type 2 receptor (OX2R) were investigated in the gastrointestinal tract of neonatal dogs by means of immunohistochemical techniques. The orexin A-positive cells were identified with some of the endocrine cells in the stomach and in the duodenum; they were both of the open and closed type and were lacking in the large intestine. In the stomach, a large subset of orexin A-positive cells also showed gastrin-like immunoreactivity while, in the duodenum, many of them seemed to store serotonin. The orexin type 2 receptor-positive cells were evidenced all along the gastrointestinal tract examined, also in the large intestine, and they showed the same morphological characteristics as those positive to orexin A. Moreover, the immunohistochemical techniques revealed intense positivity for both orexin A and orexin type 2 receptor in the neurons and fibers of the enteric nervous system. A large subset of orexin A-positive neurons seemed to store substance P.

Immunohistochemical detection of the orexin system in the placenta of cats

The aim of the present study was to investigate the presence and distribution of cells containing orexin A (OXA), and orexin type 1 and 2 receptors (OX1R and OX2R, respectively) in the feline placenta by means of immunohistochemical technique. OXA was identified in several decidual and syncytiotrophoblastic cells present in the lamellar portion of the placenta. In the same placental structures, few decidual and syncytiotrophoblastic cells showed the presence of OX1R-like immunoreactivity. Characteristically, immunopositivity for OX2R, but not for OX1R, was evidenced in the cells of the glandular layer. The orexinic system was not expressed in the uterine structures that were not engaged by the chorion. Our results provide the first evidence of the presence of a placental orexinic system in a mammalian species. Orexin A and both OX1R and OX2R are unequally distributed within the cat placenta. Local OXA production and the presence of specific receptors, differentially expressed in the placental structures of the cat, suggest that the orexinic system may participate in placental growth and development as well as in the regulation of its steroidogenic capacity via endocrine, paracrine and/or autocrine mechanisms.

Differential gene expression and immune localization of the orexin system in the major salivary glands of pigs.

The aim of the present work was to characterize the expression and cell type distribution of the orexinic system in the major swine salivary glands by RT-PCR and immunohistochemistry techniques. Tissue samples of the mandibular (MSG), sub-lingual, and parotid glands were obtained from six pigs. Marked differences in the expression of the orexinergic system were observed among the salivary glands. Prepro-orexin (PPOX) mRNA abundance was approximately 15-fold higher (P<0.05) in MSG than in the others salivary glands. The expression level of OX1R mRNA did not differ among the salivary glands, while that for OX2R transcript was undetectable. The hypothalamus, used as positive control tissues, showed the highest expression level for each component of the orexinic system. Immunoreactivity (IR) for both orexins A and B (OXA and OXB), and cognate receptors (OX1R and OX2R) were identified only in the excretory striated ducts of the MSG while acinar cells were not immunoreactive. Both sub-lingual and parotid glands completely lacked IR for any component of the orexinergic system. In the MSG, parasympathetic neurons and axons of local salivary gland ganglia were IR to both OXA and OXB and also to their receptors.

Neuroendocrine responses in neonatal mother-deprived rabbits.

To study both short- and long-term adaptation mechanisms activated by rabbits which were separated from their mothers (DLS) for 48 h between postnatal days 9 and 11, we examined plasma corticosterone concentrations before, during, and after DLS as well as the expression of glucocorticoid receptor (GR) in the hippocampus and the adrenal axis responses to a mild stress stimuli or exogenous ACTH injection. At postnatal day 10, plasma corticosterone concentration of DLS rabbits was similar to that of controls, but rose two-fold at day 11 (17.7 + or - 1.3 vs. 9.3 + or - 1.2 microg/dl, P<0.01) and then decreased at day 12, when suckling was allowed again, to match those of controls with no difference thereafter. At postnatal day 14, both control and DLS rabbits had similar basal corticosterone concentrations (2.2 + or - 0.4 vs. 2.3 + or - 1.1 microg/dl, respectively) as well as at day 120 (8.8 + or - 3.2 vs. 9.7 + or - 2.8 microg/dl, respectively). After the standardized stress stimulus, plasma corticosterone concentrations were lower in DLS rabbits than controls at postnatal days 14 (P<0.01) and 120 (P<0.05). At day 120, corticosterone levels rose similarly seven-fold (P<0.01) within 30 min after ACTH administration and remained sustained thereafter in both control and DLS rabbits. Positive immunoreactivity for GR was detected in the hippocampus and in the dorsal medial hypothalamic region at postnatal day 14. The present data suggest that 48 h DLS from postnatal days 9 to 11 results in a modified hypothalamic-pituitary-adrenal axis reactivity later in life.

Localization of the orexin system in the gastrointestinal tract of fallow deer

The aim of the present study was to investigate by immunohistochemistry the presence and distribution of the orexin system in the stomach and gut of fallow deer. Abundant orexin A-positive cells were localized in the middle and basal portions of the mucosal glands of the cardial and fundic regions of the stomach. In the same gastric areas, orexin B-positive cells were also found, mainly localized in the basal portion of glands. In the intestinal tract, orexin-containing cells were occasionally found in the duodenal epithelium and in the rectal intestinal glands. Immunoreactivity for orexin receptors, type 1 and 2 (OX1R and OX2R), was not detected in the same stomach regions. OX1R-immunopositivity was observed in the enteric neuron ganglia localized in the submucosal and muscular intestinal layers, while OX2R-immunopositivity was found close in contact with the cytoplasmic membrane of epithelial cells in the small intestine.

Immunohistochemical distribution of leptin receptor in the major salivary glands of horses

The presence of the leptin receptor (ObR) has already been highlighted in the human major salivary glands and it has been hypothesized that leptin may act by regulating the glands growth. No data are reported on domestic animals so, considering the important role that these glands play, not only related to food ingestion and digestion, and the important functional role hypothesized to explain the presence of ObR in humans salivary glands, the aim of the present work was to investigate the presence and the distribution of the leptin receptor in horse parotid and mandibular glands, by immunohistochemical techniques. The presence of ObR was evidenced in parotid and mandibular glands, exclusively localized in duct epithelial cells; their positivity was localized in the cytoplasm and was most evident near its apical portion. Immuno-positivity not only affects the intralobular ducts (intercalated and striated) but also the interlobular ones. Our results indicate that horse major salivary glands, like those of humans, are likely targets of leptin actions, suggesting a functional role of leptin on these glands.