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Endogenous retroviruses (ERVs) are proviral phases of exogenous retroviruses, which have coevolved with vertebrate genomes for millions of years. The conservation of ERV genes throughout evolution suggests their beneficial effects on their hosts' survival. An example of such positive selection is demonstrated by the syncytin gene, which encodes a protein with affinity for various mammalian placentas that is involved in the formation of syncytiotrophoblasts. Although the horse has an epitheliochorial placenta, in which the fetal trophoblasts are simply apposed to the intact uterine epithelium, we have previously demonstrated that the equine ERV (EqERV) env RNA is unexpectedly expressed in placental tissue. In the present study, we investigated the mRNA expression pattern of the EqERV env gene in different parts of the equine placenta, to gain more insight into its putative role in the fetal–maternal relationship. To this end, we used reverse transcription–quantitative PCR (RT–qPCR) and in situ hybridization assays to analyze different target areas of the equine placenta. The retroviral env gene is expressed in the equine placenta, even though there is no syncytium or erosion of the uterine endometrium. The gene is also expressed in all the sampled areas, although with some quantitative differences. We suggest that these differences are attributable to variations in the density, height, and degree of morphological complexity of the chorionic villi forming the microcotyledons. The involvement of the EqERV env gene in different functional pathways affecting the fetus–mother relationship can be hypothesized.

Background Cashmere goat is known for its precious undercoat. Being photoperiod-dictated, cashmere growth has been studied focusing mainly on hair follicle cycle phases (anagen, catagen and telogen). An accurate molecular knowledge of the goat hair follicle cycle, disentangling gene expression changes during phases and recognizing timing boundaries, could be useful to improve cashmere goat management and ultimately cashmere production. Results To better describe goat’s hair follicle transcriptome we applied RNA-sequencing to isolated hair follicles from five Italian cashmere goats, during the anagen and catagen phase, identifying total of 214 differentially expressed genes (DEGs): 97 were up-regulated while 117 were down-regulated in catagen with respect to anagen. Gene Ontology and pathway analysis were performed. We detected 144 significant pathways spanning from estrogen, pluripotency of stem cells, thermogenesis and fatty acid metabolism that were strongly expressed during the hair follicle phases analysed. Finally, we validated promising DEGs by RT-qPCR in the same set of samples as well as in hair follicles and entire skin biopsies of another cashmere goats cohort accounting for early anagen, anagen, early catagen, and catagen phases. Conclusions As in the isolated hair follicles, some target genes were homogenously modulated during the four hair follicle phases. Ceruloplasmin (CP) and Keratin 4 (K4), confirmed their clear cut expression between growing and resting phase. In fact, K4 was almost absent in catagen phases while CP was barely expressed in anagen phases. In particular, the strong expression of K4 in early anagen makes it an eligible marker to track the beginning of a new hair cycle, and therefore defining the optimum time for cashmere harvesting.

In recent years, extracellular vesicles (EVs), cell-derived micro and nano-sized structures enclosed in a double-layer membrane, have been in the spotlight for their high potential in diagnostic and therapeutic applications. Indeed, they act as signal mediators between cells and/or tissues through different mechanisms involving their complex cargo and exert a number of biological effects depending upon EVs subtype and cell source. Being produced by almost all cell types, they are found in every biological fluid including milk. Milk EVs (MEVs) can enter the intestinal cells by endocytosis and protect their labile cargos against harsh conditions in the intestinal tract. In this study, we performed a metabolomic analysis of MEVs, from three different species (i.e., bovine, goat and donkey) by mass spectroscopy (MS) coupled with Ultrahigh-performance liquid chromatography (UHPLC). Metabolites, both common or specific of a species, were identified and enriched metabolic pathways were investigated, with the final aim to evaluate their anti-inflammatory and immunomodulatory properties in view of prospective applications as a nutraceutical in inflammatory conditions. In particular, metabolites transported by MEVs are involved in common pathways among the three species. These metabolites, such as arginine, asparagine, glutathione and lysine, show immunomodulating effects. Moreover, MEVs in goat milk showed a greater number of enriched metabolic pathways as compared to the other kinds of milk.

Bone marrow (BM) is the major target organ for neuroblastoma (NB) metastasis and its involvement is associated with poor outcome. Yet, the mechanism by which NB cells invade BM is largely unknown. Tumour microenvironment represents a key element in tumour progression and mesenchymal stromal cells (MSCs) have been recognized as a fundamental part of the associated tumour stroma. Here, we show that BM-MSCs isolated from NB patients with BM involvement exhibit a greater osteogenic potential than MSCs from non-infiltrated BM. We show that BM metastasis-derived NB-cell lines secrete higher levels of exosomal miR-375, which promotes osteogenic differentiation in MSCs. Of note, clinical data demonstrate that high level of miR-375 correlates with BM metastasis in NB patients. Our findings suggest, indeed, a potential role for exosomal miR-375 in determining a favourable microenvironment in BM to promote metastatic progression. MiR-375 may, thus, represent a novel biomarker and a potential target for NB patients with BM involvement.

Retinoblastoma (RB) is the most common tumor of the eye in early childhood. Although recent advances in conservative treatment have greatly improved the visual outcome, local tumor control remains difficult in the presence of massive vitreous seeding. Traditional biopsy has long been considered unsafe in RB, due to the risk of extraocular spread. Thus, the identification of new biomarkers is crucial to design safer diagnostic and more effective therapeutic approaches. Exosomes, membrane-derived nanovesicles that are secreted abundantly by aggressive tumor cells and that can be isolated from several biological fluids, represent an interesting alternative for the detection of tumor-associated biomarkers. In this study, we defined the protein signature of exosomes released by RB tumors (RBT) and vitreous seeding (RBVS) primary cell lines by high resolution mass spectrometry. A total of 5666 proteins were identified. Among these, 5223 and 3637 were expressed in exosomes RBT and one RBVS group, respectively. Gene enrichment analysis of exclusively and differentially expressed proteins and network analysis identified in RBVS exosomes upregulated proteins specifically related to invasion and metastasis, such as proteins involved in extracellular matrix (ECM) remodeling and interaction, resistance to anoikis and the metabolism/catabolism of glucose and amino acids. M.C. and V.D.P. are recipients of Fondazione Umberto Veronesi (FUV)-2020 post-doctoral fellowships. We thank Valentina Polcini for proofreading the manuscript. Sí

Similar to the disease affecting humans, osteoarthritis (OA) is a painful musculoskeletal condition affecting 20% of the adult canine population. Several solutions have been proposed, but the results achieved to date are far from being satisfactory. New approaches, such as intra-articular delivery of cells (including mesenchymal stromal cells), have been proposed. Among the many sources, the adipose tissue is considered very promising. We evaluated the safety, feasibility, and efficacy of a single intra-articular injection of autologous and micro-fragmented adipose tissue (MFAT) in 130 dogs with spontaneous OA. MFAT was obtained using a minimally invasive technique in a closed system and injected in the intra- and/or peri-articular space. Clinical outcomes were determined using orthopedic examination and owners’ scores for up to 6 months. In 78% of the dogs, improvement in the orthopedic score was registered 1 month after treatment and continued gradually up to 6 months when 88% of the dogs improved, 11% did not change, and 1% worsened compared with baseline. Considering the owners’ scores at 6 months, 92% of the dogs significantly improved, 6% improved only slightly, and 2% worsened compared with baseline. No local or systemic major adverse effects were recorded. The results of this study suggest that MFAT injection in dogs with OA is safe, feasible, and beneficial. The procedure is time sparing and cost-effective. Post injection cytological investigation, together with the clinical evidence, suggests a long-term pain control role of this treatment. The spontaneous OA dog model has a key role in developing successful treatments for translational medicine.

It has been demonstrated that the biological effector of mesenchymal stem/stromal cells (MSCs) is their secretome, which is composed of a heterogeneous pool of bioactive molecules, partially enclosed in extracellular vesicles (EVs). Therefore, the MSC secretome (including EVs) has been recently proposed as possible alternative to MSC therapy. The secretome can be considered as a protein-based biotechnological product, it is probably safer compared with living/cycling cells, it presents virtually lower tumorigenic risk, and it can be handled, stored and sterilized as an Active Pharmaceutical/Principle Ingredient (API). EVs retain some structural and technological analogies with synthetic drug delivery systems (DDS), even if their potential clinical application is also limited by the absence of reproducible/scalable isolation methods and Good Manufacturing Practice (GMP)-compliant procedures. Notably, EVs secreted by MSCs preserve some of their parental cell features such as homing, immunomodulatory and regenerative potential. This review focuses on MSCs and their EVs as APIs, as well as DDS, considering their ability to reach inflamed and damaged tissues and to prolong the release of encapsulated drugs. Special attention is devoted to the illustration of innovative therapeutic approaches in which nanomedicine is successfully combined with stem cell therapy, thus creating a novel class of “next generation drug delivery systems.”

Aim: Desmoplastic small round cell tumor (DSRCT) is a rare, aggressive mesenchymal tumor, lacking biomarkers for diagnosis, treatment stratification and prognosis. We investigated the exosomal miRNA profile in plasma samples collected from DSRCT patients, evaluating their potential as circulating biomarkers for this tumor. Patients & methods: We isolated exosomes from plasma of three DSRCT adolescents and four age-matched healthy controls; expression of circulating miRNAs was quantified by qPCR. Results: We identified 55 miRNAs significantly modulated compared with healthy controls. Among these miRNAs, 14 were highly dysregulated in at least one patient and 5 were expressed in all patients. Conclusion: To our knowledge, this is the first report describing exosomal miRNAs as promising biomarkers to characterize disease status in DSRCT patients.

Pediatric high-grade gliomas (pHGG) are heterogeneous brain tumors for which new specific diagnostic/prognostic biomarkers are needed. In this study, we aimed to identify new pHGG subgroup specific biomarkers by exploiting exosomes, known vehicles of oncogenic signals. We used plasma from 23 patients (including 6 controls) and conditioned medium from 12 patient-derived cell-lines, representing all locational and molecular subgroups. Upon exosome isolation, total RNA was extracted and miRNAs were assessed using a PCR Panel. Analysis of plasma miRNome showed that tumor exosomal samples were largely clustered together, independently from their locational and/or molecular subgroup. We identified 20 significantly upregulated and 25 downregulated miRNAs compared to controls. Interestingly, 27 miRNAs were expressed only in tumors. Furthermore, the unsupervised clustering showed a clear separation based on locational (hemispheric vs pontine) and mutational (WT vs H3.3G34R or H3.3G34R vs H3K27M) subgroup comparisons, with the identification of distinct miRNomes underlying the key role of location and mutations in defining the pHGG exosomal miRNA profile. This was further confirmed analyzing the miRNOme from cell-line derived exosomes. Moreover, we identified a pool of significantly differentially regulated miRNAs in diagnose vs relapse and biopsy vs autopsy cell-lines. Most importantly, when comparing hemispheric vs pontine and H3.3G34R vs H3.3K27M, we identified respectively four and three miRNas equally dysregulated and in common between plasma and cell-lines. Those were strongly associated mainly to transcriptional regulation and targeting TTC9, linked to cancer invasion and metastasis. Based on this, we suggest exosomal miRNAs as a powerful new pHGG diagnostic/prognostic tool.

Multiple myeloma is an aggressive tumour able to suppress osteoblastogenesis probably mediated by bone marrow mesenchymal stromal cells (BM-MSCs) that can also support plasma cell growth/survival. The use of MSCs for multiple myeloma therapy is a controversial topic because of the contradictory results on the capacity of MSCs to inhibit or to promote cancer growth. Our previous studies demonstrated that MSCs could be loaded with Paclitaxel (PTX) and used to deliver the drug in situ in amount affecting tumour growth (in vitro and in vivo). Therefore, independently on the discussed action of MSCs in myeloma, MSCs could represent a 'trojan horse' to vehicle and deliver anti-tumour agents into bone marrow. This study confirms, by an in vitro 3D dynamic culture system, that PTX loaded BM-MSCs (PTXr-MSCs) are active on the proliferation of RPMI 8226, a human myeloma cell line. Our results demonstrated a dramatic suppression of myeloma cell growth by PTXr-MSCs, suggesting that drug loaded MSCs could be a tool to deliver drug into the bone marrow. Drug releasing MSCs provide a therapeutic approach to potentiate the existing treatments against a very aggressive malignancy as multiple myeloma. Copyright © 2016 John Wiley & Sons, Ltd.

Made available in DSpace on 2018-12-11T17:02:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-04-15 Administration of horse amniotic mesenchymal cells (AMCs) and their conditioned medium (AMC-CM) improves the in vivo recovery of spontaneous equine tendon lesions and inhibits in vitro proliferation of peripheral blood mononuclear cells (PBMC). This process may involve microvesicles (MVs) as an integral component of cell-to-cell communication during tissue regeneration. In this study, the presence and type of MVs secreted by AMCs were investigated and the response of equine tendon cells to MVs was studied using a dose-response curve at different concentrations and times. Moreover, the ability of MVs to counteract in vitro inflammation of tendon cells induced by lipopolysaccharide was studied through the expression of some proinflammatory genes such as metallopeptidase (MPP) 1, 9, and 13 and tumor necrosis factor-α (TNFα), and expression of transforming growth factor-β (TGF-β). Lastly, the immunomodulatory potential of MVs was investigated. Results show that AMCs secrete MVs ranging in size from 100 to 200 nm. An inverse relationship between concentration and time was found in their uptake by tendon cells: the maximal uptake occurred after 72 h at a concentration of 40 × 106 MVs/mL. MVs induced a downregulation of MMP1, MMP9, MMP13, and TNFα expression without affecting PBMC proliferation, contrary to CM and supernatant. Our data suggest that MVs contribute to in vivo healing of tendon lesions, alongside soluble factors in AMC-CM. Large Animal Hospital Reproduction Unit Universita degli Studi di Milano, Via dell'Universita 6 Department of Internal Medicine Molecular Biotechnology Center University of Torino Department of Veterinary Medicine University of Perugia Department of Biochemistry Biology and Genetics Universita Politecnica Delle Marche Department of Animal Reproduction and Radiology FMVZ UNESP Centro di Ricerca E. Menni Fondazione Poliambulanza Istituto Ospedaliero Department of Veterinary Medical Science Universita degli Studi di Milano Department of Animal Reproduction and Radiology FMVZ UNESP

Background aims Pancreatic cancer (pCa) is a tumor characterized by a fibrotic state and associated with a poor prognosis. The observation that mesenchymal stromal cells (MSCs) migrate toward inflammatory micro-environments and engraft into tumor stroma after systemic administration suggested new therapeutic approaches with the use of engineered MSCs to deliver and produce anti-cancer molecules directly within the tumor. Previously, we demonstrated that without any genetic modifications, MSCs are able to deliver anti-cancer drugs. MSCs loaded with paclitaxel by exposure to high concentrations release the drug both in vitro and in vivo , inhibiting tumor proliferation. On the basis of these observations, we evaluated the ability of MSCs (from bone marrow and pancreas) to uptake and release gemcitabine (GCB), a drug widely used in pCa treatment. Methods MSCs were primed by 24-h exposure to 2000 ng/mL of GCB. The anti-tumor potential of primed MSCs was then investigated by in vitro anti-proliferation assays with the use of CFPAC-1, a pancreatic tumor cell line sensitive to GCB. The uptake/release ability was confirmed by means of high-performance liquid chromatography analysis. A cell-cycle study and secretome evaluation were also conducted to better understand the characteristics of primed MSCs. Results GCB-releasing MSCs inhibit the growth of a human pCa cell line in vitro . Conclusions The use of MSCs as a "trojan horse" can open the way to a new pCa therapeutic approach; GCB-loaded MSCs that integrate into the tumor mass could deliver much higher concentrations of the drug in situ than can be achieved by intravenous injection.

Diffuse intrinsic pontine glioma (DIPG) and pediatric glioblastoma (pGBM) are heterogeneous brain tumors characterized by different anatomical and molecular subgroups and the presence of genetically and phenotypically distinct subclonal cell populations. It is recognized that exosomes mediate cross-talk among tumor cells. We hypothesize that there are different exosome-mediated paracrine signaling promoting tumour progression in DIPG and pGBM. Our aim was to determine the specific DIPG and pGBM-derived exosome oncogenic signatures. We used a panel of fifteen patient primary-derived cell lines, which included nine DIPG (seven H3.3 K27M, one H3.3 K27M/ACVR1 and one H3.1 K27M/ACVR1), one diffuse midline glioma H3.3 K27M and three GBM (one H3.3 G34R and two histone WT). Conditioned medium was collected from cells maintained under stem-cell culture condition, adherent on laminin and/or as neurospheres (NS), and exosomes harvested through serial centrifugations. Electron microscopy demonstrated that the isolated microvescicles are exosomes sized between 50–80 nm. DIPG derived-exosomes appeared to have a variable cargo of total protein (µg)/10(6) cells, which was higher than for pGBM-exosomes. Proteomic analysis revealed that proteins associated with vesicle docking, exocytosis and synaptic transmission were exclusively enriched in pontine-derived exosomes, while cell-cell and cell-matrix interaction proteinswere exclusive tohemispheric ones. Proteins in common to the two locations were involved in metabolism and energy pathways. Interestingly, principle component analysis on the different molecular subgroups suggests that ACVR1 may be not implicated in the exosomal proteomic signature. Exosomal miRNA profile appeared to be driven by the two main histone mutated subgroups H3.3 K27M and H3.1 K27M with the latter overexpressing hypoxia and angiogenic-associated miRNAs, leading to distinct oncogenic programs with different specific potential therapeutic targets. This study aimed to development new diagnostic/prognostic tools for DIPG and pGBM patients. Further investigations are aimed to identify new therapeutic strategies to inhibit the cross-talk among glioma subpopulations.

Over the last few years, human microfragmented adipose tissue (MFAT), containing significant levels of mesenchymal stromal cells (MSCs) and obtained from fat lipoaspirate (LP) through a minimal manipulation in a closed system device, has been successfully used in aesthetic medicine as well as in orthopedic and general surgery. Interestingly, in orthopedic diseases, this ready-to-use adipose tissue cell derivative seems to have a prolonged time efficacy even upon a single shot injection into osteoarthritic tissues. Here, we investigated the long-term survival and content of MSCs as well the anti-inflammatory activity of LP and its derived MFAT in vitro, with the aim to better understand a possible in vivo mechanism of action. MFAT and LP specimens from 17 human donors were investigated side by side. During a long-term culture in serum-free medium, we found that the total cell number as well the MSC content in MFAT decreased more slowly if compared to those from LP specimens. The analysis of cytokines and growth factors secreted into the conditioned medium (CM) was similar in MFAT and LP during the first week of culture, but the total amount of cytokines secreted by LP decreased much more rapidly than those produced by MFAT during prolonged culture (up to 28 days). Similarly, the addition of MFAT-CM recovered at early (3-7 days) and late stage (14-28 days) of culture strongly inhibited inflammatory function of U937 monocyte cell line, whereas the anti-inflammatory activity of LP-CM was drastically reduced after only 7 days of culture. We conclude that MFAT is an effective preparation with a long-lasting anti-inflammatory activity probably mediated by a long-term survival of their MSC content that releases a combination of cytokines that affect several mechanisms involved in inflammation processes.

The unit describes protocols for isolating and characterizing extracellular vesicles (EVs) derived from human adipose tissue-derived mesenchymal stromal cells (MSCs). EVs are a mixed population of membrane-surrounded structures with overlapping composition and size. Advances made in recent years have led to a better understanding of the biological role of EVs. In particular, they can be considered key factors responsible for MSC-paracrine activity, mediating their anti-inflammatory effects towards innate immune cells, such as macrophages. The topics comprise description of the MSC-conditioned medium containing vesicles preparation, EV isolation, and characterization mainly by specifically set up flow cytometry and electron microscopy approaches, and in vitro methodologies involved in testing the EV anti-inflammatory capacity. The procedures described here can be easily reproduced and can be employed regardless of the type of progenitor cells used to secrete EVs. © 2018 by John Wiley & Sons, Inc.

The aim of this study was to delineate the pattern of sympathetic innervation in the suprasesamoidean region of the deep digital flexor tendon (DDFT) in horses with tendinopathy by immunohistochemical labelling for tyrosine hydroxylase (TH) and α-1 adrenergic receptor (α1-AR). Twelve forelimbs were obtained from 10 horses with DDFT tendinopathy and six feet obtained from six horses were used as healthy controls. Post-mortem radiographic, ultrasonographic and gross examinations were performed on the suprasesamoidean area of the DDFT to assess the presence of tendinopathy. Longitudinal sections were collected and processed. Lesions were classified as core lesions, dorsal border lesions and parasagittal oblique splits. Immunohistochemistry was performed and the degree of immunoreaction was classified as absent, mild or marked. Seven core lesions, four dorsal border lesions and one parasagittal oblique split were identified. There was no increased expression of sympathetic innervation in samples with a dorsal border lesion of the DDFT compared with healthy samples. In contrast, core lesions showed increased expression of α1-AR and reduced expression of TH, which supports the hypothesis of a compensatory imbalance between the sympathetic mediator and the sympathetic receptors as a cause or effect of structural damage. In addition, adrenergic activation could stimulate cell proliferation and differentiation within these lesions.

The purpose of this study was to delineate the pattern of sympathetic innervation in the suprasesamoidean region of the deep digital flexor tendon (DDFT) in horses using immunohistochemical staining (IHC) for tyrosine hydroxylase (TH) and alpha-1 adrenergic receptor (α1-AR). Fourteen forelimbs were collected from 10 horses. Longitudinal sections of the suprasesamoidean region of healthy DDFTs were harvested. Most of the sympathetic innervation was found to be in the walls of blood vessels. The tendon tissue proper was sparsely innervated, with a lesser degree of innervation within the dorsal fibrocartilage. Increased α1-AR immunostaining was also detected in walls of blood vessels and in spindle cells of fibrocartilage. Both α1-AR and TH immunostaining were detected in tenocytes. These findings support the presence of autocrine/paracrine catecholaminergic signalling in equine tendon tissue.

Mesenchymal Stem Cells (MSCs) are effective therapeutic agents enhancing the repair of injured tissues mostly through their paracrine activity. Increasing evidences show that besides the secretion of soluble molecules, the release of extracellular vesicles (EVs) represents an alternative mechanism adopted by MSCs. Since macrophages are essential contributors toward the resolution of inflammation, which has emerged as a finely orchestrated process, the aim of the present study was to carry out a detailed characterization of EVs released by human adipose derived‐MSCs to investigate their involvement as modulators of MSC anti‐inflammatory effects inducing macrophage polarization. The EV‐isolation method was based on repeated ultracentrifugations of the medium conditioned by MSC exposed to normoxic or hypoxic conditions (EVNormo and EVHypo). Both types of EVs were efficiently internalized by responding bone marrow‐derived macrophages, eliciting their switch from a M1 to a M2 phenotype. In vivo, following cardiotoxin‐induced skeletal muscle damage, EVNormo and EVHypo interacted with macrophages recruited during the initial inflammatory response. In injured and EV‐treated muscles, a downregulation of IL6 and the early marker of innate and classical activation Nos2 were concurrent to a significant upregulation of Arg1 and Ym1, late markers of alternative activation, as well as an increased percentage of infiltrating CD206pos cells. These effects, accompanied by an accelerated expression of the myogenic markers Pax7, MyoD, and eMyhc, were even greater following EVHypo administration. Collectively, these data indicate that MSC‐EVs possess effective anti‐inflammatory properties, making them potential therapeutic agents more handy and safe than MSCs. stem cells translational medicine 2017 Stem Cells Translational Medicine 2017;6:1018–1028

Human amniotic fluid stem cells (hAFS) have shown a distinct secretory profile and significant regenerative potential in several preclinical models of disease. Nevertheless, little is known about the detailed characterization of their secretome. Herein we show for the first time that hAFS actively release extracellular vesicles (EV) endowed with significant paracrine potential and regenerative effect. c-KIT+ hAFS were isolated from leftover samples of amniotic fluid from prenatal screening and stimulated to enhance EV release (24 hours 20% O2 versus 1% O2 preconditioning). The capacity of the c-KIT+ hAFS-derived EV (hAFS-EV) to induce proliferation, survival, immunomodulation, and angiogenesis were investigated in vitro and in vivo. The hAFS-EV regenerative potential was also assessed in a model of skeletal muscle atrophy (HSA-Cre, SmnF7/F7 mice), in which mouse AFS transplantation was previously shown to enhance muscle strength and survival. hAFS secreted EV ranged from 50 up to 1,000 nm in size. In vitro analysis defined their role as biological mediators of regenerative, paracrine effects while their modulatory role in decreasing skeletal muscle inflammation in vivo was shown for the first time. Hypoxic preconditioning significantly induced the enrichment of exosomes endowed with regenerative microRNAs within the hAFS-EV. In conclusion, this is the first study showing that c-KIT+ hAFS dynamically release EV endowed with remarkable paracrine potential, thus representing an appealing tool for future regenerative therapy.

This study investigated whether canine mesenchymal stromal cells (cMSCs) are able to take up and release paclitaxel (PTX) in active form, and therefore whether they have potential as a tool for therapeutic delivery of this drug. cMSCs from bone marrow and adipose tissue were isolated, expanded and characterised phenotypically. cMSCs were loaded with PTX (cMSCs-PTX) and their capacity for release of PTX was determined by their effect on proliferation of cancer cells. cMSCs-PTX derived from bone marrow and adipose tissue were able to take up and then release active PTX. cMSCs-PTC inhibited proliferation of the canine glioma cell line J3T, and the human glioblastoma cell lines T98G and U87MG. The potential of canine cMSCs-PTX for treatment of canine gliomas should be investigated further.

Leptin is a polypeptide secreted by adipocytes which binds to a specific receptor (Ob-R) that is expressed in various tissues. The wide distribution of the Ob-R suggests that leptin might exert diverse biological functions, not only by regulating energy metabolism and appetite, but also by acting as a mitogen in many cell types, including keratinocytes. In this study, the presence and localization of Ob-R was investigated in the skin of the dog using RT-PCR and immunohistochemical techniques. RT-PCR revealed the presence of Ob-R m-RNA in the skin specimens collected from the dorsal region of two smooth coat breed dogs. Through immunohistochemistry performed on the skin of five dogs, the expression of the receptor was observed in the basal layer of the epidermis, in the hair follicles as well as in the apocrine sweat and sebaceous glands. No staining for Ob-R was detected in the suprabasal epidermis layers. Strong positive signals were observed in many cells of the outer root sheath of hair follicles in growing and in regressive phases. The identification of Ob-R in the above targets suggests that leptin may play a role in the regulation of cyclic renewal of the epidermis and skin appendages in dog. This study represents an important contribution to understand the complex mechanisms that are involved in the skin biology in this species.

Background In vitro primary cultures of microvascular endothelial cells from endocrine pancreas are difficult to obtain, but can be a very helpful tool for studies of islet biology, transplantation and regenerative medicine. Methods We applied a protocol recently described for the isolation and culture of brain microvascular endothelial cells (EC) on human pancreatic islets. EC obtained were characterized in terms of morphological (light and transmission electron microscopy), phenotypical (by immunofluorescence and flow cytometry) and functional (cord formation assay and protein secretion by multiplex bead-based assay) characteristics. Results EC were obtained from 25% of islet preparations processed. Two primary endothelial cell lines showed high proliferative potential and were deeply characterized: they presented endothelial cell morphology and expressed CD31, CD49a, CD49e, CD34, von Willebrand Factor (vWF), Vascular Endothelial CAdherin (VE-CAD), Tyrosine Kinase with Ig and EGF Homology Domains-2 (TIE2), Vascular Endothelial Growth Factor Receptor 1 (VEGFR1), Ulex lectin and the endothelium endocrine-specific marker nephrin. Besides, they were able to form cordons in vitro and secreted factors involved in the process of angiogenesis such as Vascular Endothelial Growth Factor (VEGF), Monocyte Chemotactic Protein 1 (MCP-1), interleukin (IL)-8 and Melanoma Growth Stimulatory Activity Alpha (GROα). These cell lines were termed Human Islet Microvascular Endothelial Cells (HIMEC). Discussion This study establishes a simple and effective strategy for isolation and long-term culture of EC derived from human pancreatic islet. HIMEC in culture preserve phenotype and functional properties and are, therefore, a useful tool for future experiments of in vitro pancreas modelling, co-transplantation with pancreatic islets, re-vascularization of scaffold or matrix for regenerative medicine purposes. © 2017 International Society for Cellular Therapy

OBJECTIVE: Gingival tissue is composed of cell types that contribute to the body's defense against many agents in oral environment, wound healing and tissue regeneration. Thanks to their easy and scarcely invasive withdrawal procedure, interdental papilla provide a good source of mesenchymal stromal cells (GinPa-MSCs). We isolated GinPa-MSCs and verified their ability to uptake/release the anticancer agent Paclitaxel (PTX). METHODS: In vitro expanded GinPa-MSCs were characterized for CD markers by FACS, tested for differentiation ability and analyzed by TEM. Their ability to uptake/release PTX was assessed according to a standardized procedure. RESULTS: The CD expression and chondro-adipo-osteo differentiation ability confirmed the mesenchymal feature of GinPa-MSCs. Surprisingly, 28% of GinPa-MSCs expressed CD14 marker and had an impressive pinocytotic activity. GinPa-MSCs were able to take up and release a sufficient amount of PTX to demonstrate effective in vitro activity against pancreatic carcinoma cells, suggesting that the drug was not inactivated. CONCLUSIONS: The procedure to obtain MSCs from interdental papilla is less invasive than that used for both bone marrow and adipose tissue, GinPa-MSCs are easy to expand and can be efficiently loaded with PTX. Taken together these qualities suggest that GinPa-MSCs may prove to be a good tool for cell-mediated drug delivery in cancer, particularly if related to stomatognathic system.

Background Adipose-derived mesenchymal stromal cells (Ad-MSCs) are a promising tool for advanced cell-based therapies. They are routinely obtained enzymatically from fat lipoaspirate (LP) as SVF, and may undergo prolonged ex vivo expansion, with significant senescence and decline in multipotency. Besides, these techniques have complex regulatory issues, thus incurring in the compelling requirements of GMP guidelines. Hence, availability of a minimally manipulated, autologous adipose tissue would have remarkable biomedical and clinical relevance. For this reason, a new device, named Lipogems® (LG), has been developed. This ready-to-use adipose tissue cell derivate has been shown to have in vivo efficacy upon transplantation for ischemic and inflammatory diseases. To broaden our knowledge, we here investigated the angiogenic and anti-inflammatory properties of LG and its derived MSC (LG-MSCs) population. Methods Human LG samples and their LG-MSCs were analyzed by immunohistochemistry for pericyte, endothelial and mesenchymal stromal cell marker expression. Angiogenesis was investigated testing the conditioned media (CM) of LG (LG-CM) and LG-MSCs (LG-MSCs-CM) on cultured endothelial cells (HUVECs), evaluating proliferation, cord formation, and the expression of the adhesion molecules (AM) VCAM-1 and ICAM-1. The macrophage cell line U937 was used to evaluate the anti-inflammatory properties, such as migration, adhesion on HUVECs, and release of RANTES and MCP-1. Results Our results indicate that LG contained a very high number of mesenchymal cells expressing NG2 and CD146 (both pericyte markers) together with an abundant microvascular endothelial cell (mEC) population. Substantially, both LG-CM and LG-MSC-CM increased cord formation, inhibited endothelial ICAM-1 and VCAM-1 expression following TNFα stimulation, and slightly improved HUVEC proliferation. The addition of LG-CM and LG-MSC-CM strongly inhibited U937 migration upon stimulation with the chemokine MCP-1, reduced their adhesion on HUVECs and significantly suppressed the release of RANTES and MCP-1. Conclusions Our data indicate that LG micro-fragmented adipose tissue retains either per se, or in its embedded MSCs content, the capacity to induce vascular stabilization and to inhibit several macrophage functions involved in inflammation.

The dermal sheath (DS) of the hair follicle is comprised by fibroblast-like cells and extends along the follicular epithelium, from the bulb up to the infundibulum. From this structure, cells with stem characteristics were isolated: they have a mesenchymal origin and express CD90 protein, a typical marker of mesenchymal stem cells. It is not yet really clear in which region of hair follicle these cells are located but some experimental evidence suggests that dermal stem cells are localized prevalently in the lower part of the anagen hair follicle. As there are no data available regarding DS stem cells in dog species, we carried out a morphological analysis of the hair follicle DS and performed both an immunohistochemical and an immunocytochemical investigation to identify CD90+ cells. We immunohistochemically evidenced a clear and abundant positivity to CD90 protein in the DS cells located in the lower part of anagen hair follicle. The positive cells showed a typical fibroblast-like morphology. They were flat and elongated and inserted among bundles of collagen fibres. The whole structure formed a close and continuous sleeve around the anagen hair follicle. Our immunocytochemical study allowed us to localize CD90 protein at the cytoplasmic membrane level.

To study the role of endothelial factors in luteal function, the dynamic profiles of genes for endothelin 1 (EDN1), its receptor subtypes, EDNRA and EDNRB, and angiotensin converting enzyme (ACE) were examined in corpora lutea (CL) obtained from rabbits on Days 4 and 9 of pseudopregnancy after prostaglandin (PG) F2alpha analogue (alfaprostol) treatment. The cell type distribution of EDN1 in the ovaries and its mechanisms of actions in vitro and in vivo were also studied. Positive immunostaining for EDN1 was localized in the luteal and endothelial cells, in granulosa cells of the follicles, and in the ovarian epithelium. The basal mRNA levels for EDNRA, EDNRB, and ACE were lower (P

Background Mesenchymal stromal cells (MSCs) are considered an important therapeutic tool in cancer therapy. They possess intrinsic therapeutic potential and can also be in vitro manipulated and engineered to produce therapeutic molecules that can be delivered to the site of diseases, through their capacity to home pathological tissues. We have recently demonstrated that MSCs, upon in vitro priming with anti-cancer drug, become drug-releasing mesenchymal cells (Dr-MCs) able to strongly inhibit cancer cells growth. Methods Murine mesenchymal stromal cells were loaded with Paclitaxel (Dr-MCsPTX) according to a standardized procedure and their ability to inhibit the growth of a murine B16 melanoma was verified by in vitro assays. The anti-metastatic activity of Dr-MCsPTX was then studied in mice injected i.v. with B16 melanoma cells that produced lung metastatic nodules. Lung nodules were counted under a dissecting stereomicroscope and metastasis investigated by histological analysis. Results We found that three i.v. injections of Dr-MCsPTX on day 5, 10 and 15 after tumor injection almost completely abolished B16 lung metastasis. Dr-MCsPTX arrested into lung by interacting with endothelium and migrate toward cancer nodule through a complex mechanism involving primarily mouse lung stromal cells (mL-StCs) and SDF-1/CXCR4/CXCR7 axis. Conclusions Our results show for the first time that Dr-MCsPTX are very effective to inhibit lung metastasis formation. Actually, a cure for lung metastasis in humans is mostly unlikely and we do not know whether a therapy combining engineered MSCs and Dr-MCs may work synergistically. However, we think that our approach using Dr-MCs loaded with PTX may represent a new valid and additive therapeutic tool to fight lung metastases and, perhaps, primary lung cancers in human. Electronic supplementary material The online version of this article (doi:10.1186/s13046-015-0200-3) contains supplementary material, which is available to authorized users.

The immunomodulatory activity of mesenchymal stem cells (MSCs) is largely mediated by paracrine factors. We have recently shown that the immunosuppressive effects of MSCs on B lymphocytes in peripheral blood mononuclear cell (PBMC) culture can be reproduced by extracellular vesicles (EVs) isolated from MSC culture supernatants. Here we investigated the effect of bone marrow-derived MSC-EVs on T cells on PBMC cultures stimulated with anti-CD3/CD28 beads. Stimulation increased the number of proliferating CD3+ cells as well as of regulatory T cells (Tregs). Coculture with MSCs inhibited the proliferation of CD3+ cells, with no significant changes in apoptosis. Addition of MSC-EVs to PBMCs did not affect proliferation of CD3+ cells, but induced the apoptosis of CD3+ cells and of the CD4+ subpopulation and increased the proliferation and the apoptosis of Tregs. Moreover, MSC-EV treatment increased the Treg/Teff ratio and the immunosuppressive cytokine IL-10 concentration in culture medium. The activity of indoleamine 2,3-dioxygenase (IDO), an established mediator of MSC immunosuppressive effects, was increased in supernatants of PBMCs cocultured with MSCs, but was not affected by the presence of MSC-EVs. MSC-EVs demonstrate immunomodulatory effects on T cells in vitro. However, these effects and the underlying mechanisms appear to be different from those exhibited by their cells of origin.

Malignant glial tumors, including glioblastoma multiforme, account for 15 - 20% of pediatric CNS malignancies. They are most resistant to therapy and are associated with a poor prognosis.Given the ability of mesenchymal stem cells (MSCs) to affect glioma growth, we investigated the effects of extracellular vesicles (EVs) derived from MSCs on U87MG glioblastoma cells line.EVs were isolated from culture media of MSCs from different sources, including bone marrow (BM), umbilical cord (UC) and adipose tissue (AT) and added to U87MG culture. The internalization and the effects of BM-, UC- and AT-MSC-EVs on proliferation and apoptosis of tumor cells were evaluated.Both confocal microscopy and FACS analysis showed internalization of EVs into tumor cells. BM- and UC-MSC-EVs decreased cell proliferation, while an opposite effect was observed with AT-MSC-EVs. Moreover, both BM- and UC-MSC-EVs induced apoptosis of glioblastoma cells, while AT-MSC-EVs had no effect. Loading UC-MSC-EVs with Vincristine further increased cytotoxicity when compared both to the free drug and to untreated EVs.Different effects of MSC-EVs on cancer cells were observed depending on their tissue of origin. Moreover, MSC-EVs can deliver antiblastic drugs to glioblastoma cells.

Introduction The goal of cancer chemotherapy is targeting tumor cells and/or tumor-associated microvessels with the lowest systemic toxicity. Mesenchymal stromal cells (MSCs) are promising vehicles for selective drug delivery due to their peculiar ability to home to pathological tissues. We previously showed that MSCs are able to uptake and subsequently to release the chemotherapeutic compound Paclitaxel (PTX) and to impair the growth of subcutaneous glioblastoma multiforme (GBM) xenografts. Here we used an orthotopic GBM model 1) to assess whether PTX-loaded MSCs (PTX-MSCs) retain a tropism towards the tumor cells in the brain context, and 2) to characterize the cytotoxic damage induced by MSCs-driven PTX release in the tumor microenvironment. Methods U87MG GBM cells were fluorescently labeled with the mCherry protein and grafted onto the brain of immunosuppressed rats. In adjacent brain regions, we injected green fluorescent protein-expressing murine MSCs, either loaded with PTX or unloaded. After 1 week survival, the xenografted brain was assessed by confocal microscopy for PTX-induced cell damage. Results Overall, MSCs showed remarkable tropism towards the tumor. In rats grafted with PTX-MSCs, the nuclei of U87MG cells showed changes that are typically induced by PTX, including multi-spindle mitoses, centrosome number alterations, and nuclear fragmentation. Multi-spindle mitoses resulted in multinucleated cells that were significantly higher in tumors co-grafted with PTX-MSCs than in controls. Nuclear changes did not occur in astrocytes and neurons surrounding the tumor. Conclusions MSCs appear particularly suited for anti-neoplastic drug delivery in the brain since PTX-specific damage of GBM cells can be achieved avoiding side effects to the normal tissue.

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 gland’s 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.

Neuroblastoma (NB) is the most common extracranial pediatric solid tumor. Around 70% of patients with metastatic disease at diagnosis present bone-marrow infiltration, which is considered a marker of poor outcome; however, the mechanism underlying this specific tropism has to be elucidated. Tumor-derived exosomes may support metastatic progression in several tumors by interacting with the microenvironment, and may serve as tumor biomarkers. The main objective of this study is to identify an exosomal signature associated with NB metastatic bone-marrow dissemination. Therefore, the proteomic cargo of exosomes isolated from NB cell lines derived from primary tumor and bone-marrow metastasis is characterized. The comparison among exosomal proteins show 15 proteins exclusively present in primary tumor-derived exosomes, mainly involved in neuronal development, and 6 proteins in metastasis-derived exosomes related to cancer progression. Significant proteins obtain with statistical analysis performed between the two groups, reveal that primary tumor exosomes contain a higher level of proteins involved in extra-cellular matrix (ECM) assembly and adhesion, as well as in neuronal development. Exosomes isolated from bone-marrow metastasis exhibit proteins involved in ameboidal cell migration and mitochondrial activity. This work suggests that proteomic profiling of NB-derived exosomes reflects the tumor stage and may be considered as potential tumor biomarker.

The transplantation of multipotent mesenchymal stromal cells (MSCs) is a potentially promising therapy for the treatment of tendon and ligament injuries and some forms of articular pathology in horses. This study investigated the effects of storage conditions on MSCs. Equine adipose tissue-derived MSCs (eAd-MSCs) were stored at 4 °C and at room temperature (RT) for 24 and 48 h, and viability, doubling time, expression of CD44 and CD90 antigens, clonogenic/differentiation potentials, and karyotype were subsequently evaluated. The eAd-MSC viability was significantly affected by the storage conditions, while doubling time was not significantly altered. The findings indicate (1) that storage at 4 °C is preferable to RT as this results in greater numbers of viable, morphologically normal cells, and (2) that cells should be used within 24 h.

Two horses with Rhodococcus equi infection were examined post mortem by an immunohistochemical method (peroxidase-antiperoxidase; PAP) with a monoclonal antibody (Mab 10G5) to the 15-17 kDa antigen of R. equi. One of the horses was also examined bacteriologically, R. equi being isolated in culture. Immunolabelling with this Mab was marked and widespread. On the other hand, the immunohistochemical reactivity of infected macrophages with a polyclonal antibody specific for lysozyme was slight. Thus, Mab 10G5 would appear to be a useful diagnostic reagent in R. equi infection, with or without cultural confirmation.

Summary Current leukaemia therapy focuses on increasing chemotherapy efficacy. Mesenchymal stromal cells (MSCs) have been proposed for carrying and delivery drugs to improve killing of cancer cells. We have shown that MSCs loaded with Paclitaxel (PTX) acquire a potent anti-tumour activity. We investigated the effect of human MSCs (hMSCs) and mouse SR4987 loaded with PTX (hMSCsPTX and SR4987PTX) on MOLT-4 and L1210, two leukaemia cell (LCs) lines of human and mouse origin, respectively. SR4987PTX and hMSCsPTX showed strong anti-LC activity. hMSCsPTX, co-injected with MOLT-4 cells or intra-tumour injected into established subcutaneous MOLT-4 nodules, strongly inhibited growth and angiogenesis. In BDF1-mice-bearing L1210, the intraperitoneal administration of SR4987PTX doubled mouse survival time. In vitro, both hMSCs and hMSCsPTX released chemotactic factors, bound and formed rosettes with LCs. In ultrastructural analysis of rosettes, hMSCsPTX showed no morphological alterations while the attached LCs were apoptotic and necrotic. hMSCs and hMSCsPTX released molecules that reduced LC adhesion to microvascular endothelium (hMECs) and down-modulated ICAM1 and VCAM1 on hMECs. Priming hMSCs with PTX is a simple procedure that does not require any genetic cell manipulation. Once the effectiveness of hMSCsPTX on established cancers in mice is proven, this procedure could be proposed for leukaemia therapy in humans.

The aim of the present study was to demonstrate the presence and the distribution of leptin and its receptor in the pancreas of horses of both sexes by immunohistochemical techniques. The presence and the distribution of leptin receptor were also investigated in the initial portion of the duodenum, near the duodenal ampulla. The immunohistochemical investigation demonstrates the immunolocalization of both leptin and its receptor in the endocrine cells of pancreatic islets, which led us to hypothesize that leptin may possibly exert an autocrine/paracrine action on the endocrine pancreas. Examination of the exocrine pancreas in the same treated sections showed the presence of leptin-positive cells in the wall of the interlobular ducts where, however, the receptor was not found. This observation led us to consider that some cells of the ducts may perform some minimal secretory activity, and that leptin produced by these ductal cells may reach the duodenum in the pancreatic juice. This hypothesis is enhanced by the presence of leptin-receptor in the duodenum of the same animals, where the epithelial cells of the mucosa showed intense immunolocalization in the brush border. Consequently it is possible that the ductular leptin may play a regulatory role on the functionality of the enterocytes.

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.

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.

In veterinary medicine, there is an increasing interest in the study of the endo-cannabinoid system and the possible use of the cannabinoids for the treatment of several diseases. Cannabinoid receptors (CB) are widely distributed in human and laboratory animal tissues, justifying the involvement of the endo-cannabinoid system in a great number of metabolic ways. Since there are no data regarding cannabinoid receptors in hair follicles of domestic animals, we investigated the presence and localization of CB1 receptor in dog hair follicles. By using a goat anti-CB1 polyclonal antibody, we observed CB1 receptor in the proximal part of both primary and secondary hair follicles. Staining was localized in the inner root sheath cells. We suppose that the endo-cannabinoid system is involved in the molecular mechanisms regulating hair follicle activity in dog. The identification of CB1 receptor at the level of the inner root sheath may help in the understanding of hair follicle biology and the possibility that cannabinoid molecules could be considered as suitable therapeutic tools in dog.

CB1 is a member of the G-protein-linked receptor superfamily that is present in the central nervous system as well as in certain peripheral neuronal and non-neuronal tissues. Recently, the presence of CB1 was found in the ductal system of the major salivary glands of laboratory animals, but no data are available for domestic mammals. Thus, in the present study, we examined the presence and distribution of CB1 in the major salivary glands of dogs using immunohistochemical techniques. CB1 was found in the parotid and mandibular glands of adult dogs; positive immunoreaction was localized to the cells of the striated ducts, with a peculiar localization on or near the apical membrane. This particular localization may be explained by the characteristics of this receptor as membrane-associated. The acinar structures were completely negative for CB1. We conclude that CB1 is involved in the control of dog salivary secretion via endogenous substances, likely endocannabinoids. The localization of CB1 highlights that endocannabinoids promote qualitative and/or quantitative changes of the primary saliva in the ductal system.

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.

It is widely documented that a pool of multipotent stem cells located in humans and mice hair follicle outer root sheath (bulge region) is involved in the restoration of the whole follicular unit during each anagen phase. To the authors' knowledge, data regarding the location and characterization of hair follicle stem compartment in dogs have not been reported in the recent relevant literature. In this study, we investigated the haematopoietic stem and progenitor cell antigen CD34 as a marker of putative stem cells located in a bulge-like region of canine hair follicles. The presence of CD34 mRNA and glycoprotein was assessed on formalin-fixed, paraffin-embedded canine skin samples by in situ hybridization technique and by standard immunohistochemistry, respectively. A strong expression of CD34 mRNA and glycoprotein was observed in a well-defined area of the hair follicle isthmic region and appeared uniformly concentrated at the level of the basal layer of the outer root sheath. These findings provide compelling support to the hypothesis that in dogs, a subpopulation of basal keratinocytes located in the hair follicle isthmic region and characterized by the selective expression of CD34 is potentially associated with the stem cell compartment of this skin appendage.

In this study, we have examined the presence and the distribution of receptors for leptin (Ob-R) in the oviduct of rabbits, and the effects of leptin on the release of prostaglandin (PG) F2α and PGE2 and on the activity of nitric oxide (NO) synthase (NOS) by oviducts cultured in vitro. Rabbits were killed during the follicular phase and the oviducts were incubated in vitro with leptin, PGF2α, PGE2, NO donor and inhibitors of NOS and cyclo-oxigenase (COX). Using immunohistochemistry, Ob-R-like positive reaction was observed only in the cytoplasm of secretory cells, having stronger intensity in the infundibulum and ampulla tracts than in the isthmus. Both leptin and NO donor inhibited PGE2 release, whereas they enhanced PGF2α release; NOS inhibitor alone or with leptin increased PGE2 and decreased PGF2α production; NOS activity was enhanced by leptin, while PGs did not affect this enzyme. This study suggests that the oviduct could be a potential target for endocrine regulation by leptin, whose circulating levels may act as a metabolic signal modulating oviductal PG release through mediation of the NOS/NO system.

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.