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1.
OBJECTIVE: To isolate and characterize bone marrow-derived equine mesenchymal stem cells (MSCs) for possible future therapeutic applications in horses. SAMPLE POPULATION: Equine MSCs were isolated from bone marrow aspirates obtained from the sternum of 30 donor horses. PROCEDURES: Cells were cultured in medium (alpha-minimum essential medium) with a fetal calf serum content of 20%. Equine MSC features were analyzed to determine selfrenewing and differentiation capacity. For potential therapeutic applications, the migratory potential of equine MSCs was determined. An adenoviral vector was used to determine the transduction rate of equine MSCs. RESULTS: Equine MSCs can be culture-expanded. Equine MSCs undergo cryopreservation in liquid nitrogen without altering morphologic characteristics. Furthermore, equine MSCs maintain their ability to proliferate and differentiate after thawing. Immunocytochemically, the expression of the stem cell marker CD90 can be detected on equine MSCs. The multilineage differentiation potential of equine MSCs was revealed by their ability to undergo adipogenic, osteogenic, and chondrogenic differentiation. CONCLUSIONS AND CLINICAL RELEVANCE: Our data indicate that bone marrow-derived stromal cells of horses can be characterized as MSCs. Equine MSCs have a high transduction rate and migratory potential and adapt to scaffold material in culture. As an autologous cell population, equine MSCs can be regarded as a promising cell population for tissue engineering in lesions of the musculoskeletal system in horses.  相似文献   

2.
Dendritic cells (DCs) are innate immune cells specialized in antigen detection and presentation. They perform an essential role in initiating and guiding the immune response, the direction of which largely depends upon the activation state of the DCs. The objective of this study was to generate mature equine monocyte-derived DCs and, in doing so, to develop a method for measuring the activation state of these cells. Equine DCs were stimulated with UV-inactivated Escherichia coli (E. coli), and the activation status was measured by analyzing cell surface marker expression, cytokine production, and endocytic capacity. Comparisons for each parameter measured were performed between macrophages, non-stimulated DCs and stimulated DCs. Equine monocyte-derived DCs may be distinguished from macrophages based on cell surface expression of MHC class II (p < 0.0001) and CD206 (p < 0.0001), their capacity for endocytosis of FITC-dextran (p < 0.05), and production of TNF-α upon stimulation (p < 0.001). Furthermore, stimulated DCs can be distinguished from non-stimulated DCs based on increased cell surface expression of MHC class II (p < 0.0001) and upregulation of pro-inflammatory cytokine mRNA, particularly IL-12/IL-23p40 (p < 0.05) and IL-23p19 (p < 0.05). The ability to measure DC activation state will facilitate future investigations of equine DC function.  相似文献   

3.
Objective  To establish a reproducible method for the culture of primary equine corneal epithelial cells, keratocytes, and endothelial cells and to describe each cell's morphologic characteristics, immunocytochemical staining properties and conditions required for cryopreservation.
Procedures  Corneas from eight horses recently euthanized for reasons unrelated to this study were collected aseptically and enzymatically separated into three individual layers for cell isolation. The cells were plated, grown in culture, and continued for several passages. Each cell type was characterized by morphology and immunocytochemical staining.
Results  All three equine corneal cell types were successfully grown in culture. Cultured corneal endothelial cells were large, hexagonal cells with a moderate growth rate. Keratocytes were small, spindloid cells that grew rapidly. Epithelial cells had heterogenous morphology and grew slowly. The endothelial cells and keratocytes stained positive for vimentin and were morphologically distinguishable from one another. The epithelial cells stained positive for cytokeratin. Keratocytes and endothelial cells were able to be cryopreserved and recovered. The cryopreserved cells maintained their morphological and immunocytochemical features after cryopreservation and recovery.
Discussion  This work establishes reproducible methods for isolation and culture of equine corneal keratocytes and endothelial cells. Cell morphology and cytoskeletal element expression for equine corneal epithelial cells, keratocytes, and endothelial cells are also described. This has not previously been reported for equine corneal cells. This report also demonstrates the ability to preserve equine keratocytes and endothelial cells for extended periods of time and utilize them long after the primary-cell collection, a feature that has not been reported for veterinary corneal cell culture.  相似文献   

4.
Cyathostomins (Cyathostominae) are regarded as the most pathogenic equine nematode worldwide. These nematodes are difficult to control in equine populations due to emerging anthelmintic resistance and evasion of encysted larval cyathostomins to regular modern anthelmintics. Mast cells and their proteinases have been shown to play a role in the mammalian immune response to nematode infections. Involvement of mast cells and mast cell proteinases in the equine immune response to cyathostomin infection is proposed. A technique was established to perform immunohistochemical staining using polyclonal rabbit anti-equine mast cell proteinase-1 (eqMCP-1) and anti-equine tryptase on formalin-fixed large intestinal sections, from horses classified as cyathostomin positive and negative at the time of death based upon larval enumeration. Quantitative analysis of antibody labelled mast cells was used to detect mast cell proteinases in equine large intestinal sections positive and negative for cyathostomin larvae. This demonstrated an increase in equine tryptase labelled mucosal and submucosal mast cells in cyathostomin positive horses. This study has established an immunohistochemical technique to demonstrate mast cell proteinases in formalin-fixed large intestinal sections. This technique may be used to determine possible involvement of mast cells and their proteinases in the equine immune response to cyathostomin larvae. Further studies are required to define a specific role.  相似文献   

5.
Stem cell therapy and cell-based therapies using other progenitor cells are becoming the treatment of choice for many equine orthopedic lesions. Important criteria for obtaining autogenous equine progenitor cells in vitro for use in clinical cell-based therapy include the ability to isolate and expand cells repeatedly to high numbers (millions) required for therapy, in a clinically relevant time frame. Cells must also maintain their ability to differentiate into the tissue type of choice. The objective of this study was to compare isolation and expansion techniques for preparation of periosteal-derived osteogenic progenitor cells for use in commercial autogenous cell-based therapy. Cells were allowed to migrate spontaneously from periosteal tissue or were enzymatically released. Isolated cells were expanded using enzymatic detachment of cells and subsequent monolayer or dynamic culture techniques. Viable osteogenic progenitor cells from each group were counted at 2 weeks, and osteogenic potential determined. Cells isolated or expanded using the explant or bioreactor technique yielded cells at a much lower number per gram of tissue compared with that of enzyme digestion and monolayer expansion, but all cells were able to differentiate into the ostoblast phenotype. Osteogenic progenitor cells isolated by enzymatic release and expanded using monolayer culture reached the highest number of viable cells per gram of donor periosteal tissue while maintaining the ability to differentiate into bone forming cells in vitro. This technique would be an easy, consistent method of preparation of equine osteogenic cells for clinical cell based therapy for orthopedic conditions.  相似文献   

6.
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7.
Accumulation of equine eosinophils at sites of parasite infestation or allergic inflammation depends upon their adherence to vascular endothelial cells and subsequent migration through the endothelium and extracellular matrix. This study has examined whether cytokines, which cause endothelial cell-dependent eosinophil adherence in other species, and histamine and substance P, which increase adherence of equine eosinophils to protein coated plastic, induce equine eosinophil adherence to cultured equine digital vein endothelial cell (EDVEC) monolayers. The EDVEC monolayers were stimulated with recombinant human (rh) interleukin (IL)-1beta, rhTNFalpha, substance P or histamine for different times and with a range of concentrations of mediators and the adherence of blood eosinophils from normal horses examined. All four mediators caused time- and concentration-dependent increases in adherence. However, neither the response to substance P, nor that to histamine, reached a maximum at the highest concentration tested (10-3 M: 10.6 +/- 2.6% and 4.5 +/- 0.6% adherent cells vs. background adherence of 1.9 +/- 0.4% and 1.1 +/- 0.2%; values for substance P and histamine, respectively, expressed as a percentage of total cells added initially; n=4). These data suggest that, as in other species, cytokines induce endothelial cell-dependent eosinophil adherence and mediators released during allergic inflammation may play a role in eosinophil recruitment by this mechanism.  相似文献   

8.
Increases in procoagulant activities (PCA) in equine lung macrophages were induced by non-adherent blood lymphocytes which were prestimulated with phytohaemagglutinin for 48 to 72 hours or by supernatants harvested from prestimulated blood lymphocyte cultures. However, prestimulated lymphocyte suspensions themselves expressed PCA which was most probably derived from contaminating monocytes. Because non-adherent cells from lymphocyte suspensions may have attached to adherent macrophages, cells within lymphocyte suspensions might have contributed to the PCAs expressed by lymphocyte-stimulated lung macrophages. Stimulation of lung macrophages for 24 hours by supernatants of phytohaemagglutinin-prestimulated blood lymphocytes induced a significantly greater PCA increase than stimulation by phytohaemagglutin alone. Thus, cytokines from lymphocyte cultures might have triggered or enhanced PCA induction. Direct stimulation of lung cell preparations with phytohaemagglutinin for 48 hours resulted in a progressive increase of PCA in only two of five specimens tested. The failure to induce PCA in three specimens could be due to the absence of sufficient numbers of T cells within the adherent lung cell preparations. In conclusion, PCA response of equine lung macrophages might be lymphocyte-stimulated in which case PCA might be a useful tool for monitoring the processes of cell-mediated immunity in horses.  相似文献   

9.
A study was performed to determine the effect of proadifen hydrochloride on prostacyclin (prostaglandin I2 [PGI2]) and thromboxane A2 (TxA2) synthesis by equine peritoneal macrophages and the effect of proadifen on endotoxin-induced synthesis of PGI2 and TxA2 by equine macrophages. Peritoneal macrophages (2.5 x 10(6)/ml) were incubated for 6 hours in tissue culture media containing 1) nothing (nontreated control), 2) proadifen hydrochloride (20, 100, 250, and 500 mumol/L, 3) endotoxin (5 ng/ml), or 4) the calcium ionophore A23187 (0.95 mumol/L). In a second series of experiments, peritoneal macrophages were incubated with endotoxin (5 ng/ml) and proadifen (250 umol/L), for 6 hours. Concentrations of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and thromboxane B2, the stable metabolites of PGI2 and TxA2, were determined in the incubation media by radioimmunoassay. Proadifen caused increased synthesis of PGI2 by equine macrophages, without affecting TxA2 production. The increased PGI2 production was similar to that induced by endotoxin and calcium ionophore; however, the latter 2 agents significantly stimulated TxA2 production as well (P less than 0.05). There were no significant differences among mean concentrations of 6-keto-PGF1 alpha in media from macrophages treated with 100, 250, or 500 mumol/L proadifen, but there was a significant curvilinear regression between their concentrations. The ratio of thromboxane B2 to 6-keto-PGF1 alpha was significantly lower than baseline in incubation media from macrophages exposed to proadifen, endotoxin, and calcium ionophore. Proadifen hydrochloride did not significantly change equine peritoneal macrophage production of PGI2 or TxA2 in response to endotoxin.  相似文献   

10.
This study evaluated the effect of dexamethasone on endotoxin-induced production of tumor necrosis factor (TNF) activity in vitro by equine peritoneal macrophages. Peritoneal macrophages from adult horses were cultured in the presence of dexamethasone (1-100 microM) for various time periods (2 hour, 0.5 hour, 0 hour) prior to the addition of endotoxin (5 ng/ml), then the secretion of TNF activity was evaluated. Macrophage supernatant concentrations of TNF activity were estimated by a modified in vitro cytotoxicity bioassay using the murine fibrosarcoma cell line, WEHI 164 clone 13. An experiment was performed to determine whether dexamethasone interfered with the cytolytic bioassay's ability to detect TNF activity. The endotoxin-induced TNF activity production by equine peritoneal macrophages was significantly reduced by co-incubation with 100 microM dexamethasone, but not by tested concentrations of dexamethasone less than 100 microM. This concentration of dexamethasone greatly exceeds those generally attained by therapeutic use of dexamethasone in horses. Preincubation time did not affect the ability of 100 microM dexamethasone to reduce TNF production by equine macrophages. The quantitation of equine TNF activity by its cytolytic bioassay was not altered by dexamethasone.  相似文献   

11.
Prostaglandin E2 (PGE2) and stromelysin are produced by equine chondrocytes and synovial cells in vitro in response to recombinant human (rh) interleukin-1 (IL-1) alpha and beta, and equine mononuclear cell supernatants (MCS) containing IL-1. However, culture conditions are important. PGE2 concentrations increase in proportion to the concentration of fetal calf serum (FCS) in the culture medium, whereas stromelysin concentrations are inversely proportional to the concentration of FCS. Equine MCS, containing a lower concentration of IL-1 than the concentration of rhIL-1 used in these experiments, stimulated production of much higher levels of PGE2 than rhIL-1. In addition, equine MCS induced the production of broadly similar levels of PGE2 by both chondrocytes and synovial cells, whereas rhIL-1 was more active on equine synovial cells than equine chondrocytes. Although equine MCS induced both stromelysin and PGE2 production by equine articular cells, on the whole rhIL-1 failed to induce stromelysin production. This supports previous observations of species restrictions in the activity of human IL-1 on equine cells. Therefore, experiments using mammalian cells and heterologous IL-1 should be interpreted with caution.  相似文献   

12.
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13.
Equine satellite cells are responsible for muscle healing and regeneration in the mature horse. We describe the in vitro cell culture conditions required for clonal populations of equine satellite cells to undergo both proliferation and differentiation. Our hypothesis is that these in vitro conditions model regeneration of muscle and can be used to evaluate potential therapeutics. In this study, 2 areas of satellite cell response were tested: proliferation of clones induced by growth factors, and fusion induced by culture conditions. Equine satellite cell clones showed differences in their response to growth factors as well as accumulation of cellular protein concentrations. Equine satellite cells proliferate in response to both human and bovine FGF. IGF-1, a powerful mitogen of other satellite cell culture systems, was not as effective for inducing equine satellite cell proliferation. Protein concentrations were also measured in satellite cell cultures. Clones differed in cellular protein produced depending on growth conditions. Conditions inducing differentiation into myotubes was also determined for a 96 well assay and can be used to study the final stage of functioning muscle production. This in vitro model is the first step in identifying potential therapeutics to speed wound healing and promote muscle regeneration in horses.  相似文献   

14.
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15.
OBJECTIVE: To determine the amount of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) enzymes induced in vitro in equine alveolar macrophages in response to lipopolysaccharide (LPS). Sample Population-Alveolar macrophages obtained from 12 horses. PROCEDURE: Alveolar macrophages were collected by bronchoalveolar lavage from 12 horses and incubated for 6 hours with LPS (0.001 to 10 microg/ml) or vehicle. Total RNA was extracted and purified. After first-strand cDNA synthesis, mRNA induction was measured, using a polymerase chain reaction (PCR) technique for COX-2, iNOS, and glyceraldehyde 3-phosphate dehydrogenase. In a second study, cells were incubated with LPS or vehicle for 24 hours. Culture medium was assayed for COX-2 and iNOS activity by determining prostaglandin E2 (PGE2) and total nitrite concentrations, respectively. RESULTS: Lipopolysaccharide induces COX-2 and iNOS mRNA in equine alveolar macrophages. Sequencing revealed that PCR products for COX-2 and iNOS had a high degree of nucleotide homology with the human sequences (91% COX-2, 93% iNOS). Production of mRNA for COX-2 and iNOS was accompanied by induction of enzyme activity. Comparing PCR fragment production, expression of mRNA for iNOS appeared to be less than that for COX-2. Induction of COX-2, but not iNOS, was LPS-concentration dependent. Conclusion-Lipopolysaccharide induces COX-2 and iNOS in equine macrophages. CLINICAL RELEVANCE: The induction of iNOS and COX-2 by LPS in equine macrophages suggests these enzymes may be important in the pathophysiology of sepsis. Pharmacologic modulation of iNOS and COX-2 activity may represent a novel therapeutic target in the management of endotoxemia in horses.  相似文献   

16.
REASONS FOR PERFORMING STUDY: The equine oviduct is the site of fertilisation and location of embryonic development during the first 5 or 6 days. It therefore has an important influence on mare fertility. Although histopathological changes have been described previously, there is limited information regarding lymphocyte subtypes present in the mucosa of the normal equine oviduct. OBJECTIVES: To characterise the distribution of CD3+, CD4+, CD8+ and B lymphocytes in the equine oviduct from inseminated mares during oestrus and dioestrus, and from noninseminated mares during the immediate post ovulatory period. METHODS: Oviductal tissues were collected from noninseminated mares at oestrus (> 30 mm follicle, n = 4), at Day 1 post ovulation (n = 3) and at dioestrus (Day 7 post ovulation; n = 4). Oviducts were also collected from inseminated mares at Days 1, 2, and 3 post ovulation (n = 4 for each period). Cross-sections of tissues from the ampullar-isthmic junction from each oviduct were snap frozen and cryostat sections stained by the immunoperoxidase technique with monoclonal antibodies directed against equine lymphocyte surface markers for B cells as well as CD3+, CD4+ and CD8+ cells. RESULTS: In all oviductal sections examined, B cells were rare whereas T cells were relatively abundant. The predominant cell type found was the CD8+ phenotype, with a lesser number of CD4+ cells. Among mares, individual variation was large; therefore, although breeding status and stage of oestrous cycle appeared to alter lymphocyte populations, these differences were not significant. CONCLUSIONS AND POTENTIAL RELEVANCE: A population of CD3+, CD4+ and CD8+ cells exists within the mucosal region of the equine oviduct. The density of these cells is similar to that described in the human oviduct. Their function is not currently known, but they may be involved with modulation of the maternal response to the presence of spermatozoa or the early conceptus within the equine oviduct. As our capacity to differentiate these cell types improves, along with the ability to identify the specific cytokines they produce, their functional significance will become more apparent.  相似文献   

17.
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18.
Interleukin-10 (IL-10) terminates inflammatory immune responses and inhibits activation and effector functions of T-cells, monocytes, macrophages and dendritic cells. IL-10 has also been found to be a key cytokine expressed by subpopulations of regulatory T-cells. In this report, we describe the generation and characterization of three monoclonal antibodies (mAbs) to equine IL-10. The antibodies were found to be specific for equine IL-10 using different recombinant equine cytokine/IgG fusion proteins. Two of the anti-equine IL-10 mAbs were selected for ELISA to detect secreted IL-10 in supernatants of mitogen stimulated equine peripheral blood mononuclear cells (PBMC). The sensitivity of the ELISA for detecting secreted IL-10 was found to be around 200pg/ml. The production of intracellular IL-10 was measured in equine PBMC by flow cytometry. PBMC were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of the secretion blocker Brefeldin A. All three anti-IL-10 mAbs detected a positive population in PMA stimulated lymphocytes which was absent in the medium controls. Around 80% of the IL-10(+) cells were CD4(+). Another 15% were CD8(+) cells. Double staining with IL-4 or interferon-gamma (IFN-gamma) indicated that PMA and ionomycin stimulation induced 80% IL-10(+)/IFN-gamma(+) lymphocytes, while only 5% IL-10(+)/IL-4(+) cells were observed. By calculation, at least 60% of the IL-10(+)/IFN-gamma(+) cells were CD4(+) lymphocytes. This expression profile corresponds to the recently described T regulatory 1 (T(R)1) cell phenotype. In summary, the new mAbs to equine IL-10 detected native equine IL-10 by ELISA and flow cytometry and can be used for further characterization of this important regulatory cytokine in horses.  相似文献   

19.
Naive T cells can be activated both in vivo and in vitro by specialized antigen presenting cells, dendritic cells (DC), with potent antigen-specific, immunostimulatory activity. Indeed, DC can provide an extremely powerful and important immunological tool by which to potentiate the immune response for specific recognition of foreign antigens. Until recently, the direct isolation of DC from PBMC required laborious procedures with extremely poor yields (<0.1%). Methods have been developed for the human, lower primate, and murine model systems to propagate large numbers of DC from PBMC or bone marrow ex vivo with various cytokines. However, all other model systems, including equine, still require the laborious isolation procedures to obtain DC. In this study, we have adapted the methods developed for the human system to generate large numbers of equine DC from PBMC precursors using recombinant human GM-CSF and recombinant equine IL-4. Our report is the first documentation of ex vivo generated DC from PBMC in a domesticated animal model system. Equine DC derived from PBMC were rigorously characterized by analyzing morphological, phenotypic, and functional properties and were determined to have similar attributes as DC generated from human PBMC. Equine DC appeared stellate with large projectiles and veils and had cell surface antigens at similar levels as those defined on human and murine DC. Furthermore, functional attributes of the DC included rapidly capturing antigens by pinocytosis, receptor-mediated endocytosis, and phagocytosis, activating naive T cells in a mixed leukocyte reaction to a much greater extent than macrophage or lymphoblasts, presenting soluble and particulate antigen 10-100 fold more effectively to T cells on a per cell basis than macrophage or lymphoblasts, and presenting soluble and particulate antigen to both CD4+ and CD8+ T cells. Taken together, our study provides a framework by which equine DC can now be readily produced from PBMC precursors and presents an impetus for and model by which DC can be simply generated in other animal model systems.  相似文献   

20.
Although the use of mesenchymal stromal cells (MSCs) for the treatment of orthopaedic injuries in horses has been reported, no official guidelines exist that classify a particular cell as an equine MSC. Given the limited characterisation of peripheral blood (PB)-derived equine MSCs in particular, this study aimed to provide more detailed information in relation to this cell type. Mesenchymal stromal cells were isolated from equine PB samples and colony forming unit (CFU) assays as well as population doubling times (PDTs) (from P0 to P10) were performed.Two types of colonies, ‘fingerprint’ and dispersed, could be observed based on macroscopic and microscopic features. Moreover, after an initial lag phase (as indicated by a negative PDT at P0 to P1) the MSCs divided rapidly as indicated by a positive PDT at all further passages. Immunophenotyping was carried out with trypsin- as well as with accutase-detached MSC to evaluate potential trypsin-sensitive epitope destruction on particular antigens. Isolated MSC were positive for CD29, CD44, CD90 and CD105, and negative for CD45, CD79α, MHC II and a monocyte/macrophage marker, irrespective of the cell detaching agent used. Trilineage differentiation of the MSCs towards osteoblasts, chondroblasts and adipocytes was confirmed using a range of histochemical stains.  相似文献   

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