Cell culture in horses involves the in vitro cultivation of equine cells under controlled conditions. This technique is employed to study various cellular processes, including growth, differentiation, and response to external stimuli, in an isolated environment. Equine cell cultures can be derived from various tissues, such as skin, muscle, or bone, and are used in a range of research applications, including genetic studies, drug testing, and disease modeling. These cultures provide a valuable platform for understanding cellular mechanisms and developing therapeutic strategies. This page compiles peer-reviewed research studies and scholarly articles that explore the methodologies, applications, and findings related to cell culture in equine research.
Dobrinski I, Jacob JR, Tennant BC, Ball BA.Equine oviductal epithelial cells (OEC) were transformed with simian virus 40 large T antigen (SV 40 T-ag) to create a cell line for the study of the interaction of equine spermatozoa with oviductal epithelium. One cell line was established based on the expression of the SV 40 T-ag and extended lifespan in culture. Immortalized equine OEC retained the characteristics of differentiated OEC such as the formation of monolayers with characteristic epithelial morphology and cell polarization as well as expression of cytokeratin and equine major histocompatibility complex I. Monolayers of immortaliz...
The Journal of parasitologyMarch 4, 2000
Volume 86, Issue 1 164-166 doi: 10.1645/0022-3395(2000)086[0164:DOTAOD]2.0.CO;2
Lindsay DS, Dubey JP.Diclazuril is a benzeneacetonitril anticoccidial that has excellent activity against the extraintestinal stages of Toxoplasma gondii and Neospora caninum. It also is highly active against intestinal coccidia of poultry. The present study examined the efficacy of diclazuril in inhibiting merozoite production of Sarcocystis neurona and Sarcocystis falcatula in bovine turbinate cell cultures. Diclazuril inhibited merozoite production by more than 80% in cultures of S. neurona or S. falcatula treated with 0.1 ng/ml diclazuril and greater than 95% inhibition of merozoite production was observed whe...
Pusterla N, Madigan JE, Chae JS, DeRock E, Johnson E, Pusterla JB.We report successful helminthic transmission of Ehrlichia risticii, the causative agent of Potomac horse fever, using trematode stages collected from Juga yrekaensis snails. The ehrlichial agent was isolated from the blood of experimentally infected horses by culture in murine monocytic cells and identified as E. risticii ultrastructurally and by characterization of three different genes.
Miller CB, Wilson DA, Keegan KG, Kreeger JM, Adelstein EH, Ganjam VK.To determine if there is a difference in in vitro growth of fibroblasts isolated from the trunk and distal aspect of the limb of horses and ponies. To determine the effects of a corticosteroid and monokine on in vitro growth of fibroblasts isolated from the trunk and distal aspect of the limb of horses and ponies. Methods: Growth of fibroblasts from tissues harvested from the trunk and limb were compared from horse and pony samples grown in control media and control media with triamcinolone or monokine added. Methods: Dermal and subcutaneous tissue from 22 horses and 17 ponies of various ages ...
Reedy SE, Powell DM, Williams NM, Dodson MV, Fitzgerald BP.This paper describes attempts to initiate equine adipocyte cultures from necropsy cases with varying intervals from time of death to isolation and culture. Equine adipocytes were isolated from 21 necropsy cases, regardless of the interval from time after death to establishment in primary ceiling cultures. However, while all cultures produced adipocytes, only 2 attempts to produce long-term equine adipocyte cultures from the subcutaneous rump fat depots were successful and not contaminated. Findings from these experiments indicate that it is possible to collect and culture equine adipocytes fro...
Netolitzky DJ, Schmaltz FL, Parker MD, Rayner GA, Fisher GR, Trent DW, Bader DE, Nagata LP.The complete nucleotide sequence of the 71V-1658 strain of western equine encephalitis virus (WEE) was determined (minus 25 nucleotides from the 5' end). A 5' RACE reaction was used to sequence the 5' terminus from WEE strain CBA87. The deduced WEE genome was 11508 nucleotides in length, excluding the 5' cap nucleotide and 3' poly(A) tail. The nucleotide composition was 28% A, 25% C, 25% G and 22% U. Comparison with partial WEE sequences of strain 5614 (nsP2-nsP3 of the nonstructural region) and strain BFS1703 (26S structural region) revealed comparatively little variation; a total of 149 nucl...
MacDonald GH, Johnston RE.The initial steps of Venezuelan equine encephalitis virus (VEE) spread from inoculation in the skin to the draining lymph node have been characterized. By using green fluorescent protein and immunocytochemistry, dendritic cells in the draining lymph node were determined to be the primary target of VEE infection in the first 48 h following inoculation. VEE viral replicon particles, which can undergo only one round of infection, identified Langerhans cells to be the initial set of cells infected by VEE directly following inoculation. These cells are resident dendritic cells in the skin, which mi...
Sternberg S, Johannisson A, Magnusson U, Jensen-Waern M.After exposure of equine granulocytes from both foals and adult horses to culture supernatants from clinical isolates of Actinobacillus equuli, phagocytic capacity and respiratory burst was examined by flow-cytometry and a chemiluminescence assay, respectively. One haemolytic isolate of an equine Actinobacillus was also included in the study. An average decrease of 22% in total number of granulocytes, in the flow cytometric assay (P < 0.01), and an average decrease of 26% in light emission, in the chemiluminescence assay (P < 0.001), was seen after exposure to bacterial culture supernata...
Hammond SA, Horohov D, Montelaro RC.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...
Murphy AJ, Mansfield LS.Sarcocystis neurona is a protozoan parasite that causes a neurological disease in horses called equine protozoal myeloencephalitis. The route of transmission is speculated to be by fecal-oral transfer of sporocysts shed from opossums. Controversy exists regarding both the natural life cycle for this parasite as well as the species identity of opossum Sarcocystis. To provide stage-specific material for species comparison, 27 opossums from southern Michigan were screened for Sarcocystis spp. sporocysts. Seven opossums were positive for Sarcocystis sporocysts by fecal flotation. A simplified, eff...
Walter I, Nowotny N.Effects of infection with two different strains of equine herpes virus type 1 (EHV-1; Piber 178/83, Kentucky D) on the cytoskeleton of Vero cells were investigated immunohistochemically, and evaluated by confocal laser scanning microscopy. Twenty four hours post EHV-1 infection the assembly of the microtubulus system of Vero cells was heavily disturbed. The Golgi region was dispersed into vesicles spread throughout the cytoplasm as demonstrated by WGA lectin binding. Other cytoskeletal elements such as cytokeratin, vimentin, and filamentous actin (F-actin) were not affected by EHV-1 infection....
Dubey JP, Mattson DE, Speer CA, Baker RJ, Mulrooney DM, Tornquist SJ, Hamir AN, Gerros TC.An isolate of Sarcocystis neurona (SN6) was obtained from the spinal cord of a horse from Oregon with neurologic signs. The parasite was isolated in cultures of bovine monocytes and equine spleen cells. The parasite divided by endopolygeny and completed at least one asexual cycle in cell cultures in three days. Two gamma interferon knockout mice inoculated with cell culture-derived merozoites became ill 35 d later and S. neurona schizonts and merozoites were found in encephalitic lesions. The parasite in tissue sections of mice reacted with S. neurona-specific antibodies and S. neurona was rei...
Huang Y, Ko WH, Chung YW, Wong PY.The patch-clamp recording technique was used to examine the properties of the K+ channels in cultured equine sweat gland epithelial cells. With symmetric K+ solutions (140 mM), a single population of K+ channels was identified with a slope conductance of 187 pS and a reversal potential of around 0 mV. The channel was selective for K+ over Na+. Channel activity was increased by membrane depolarization. A 10-fold increase in [Ca2+]i produced an approximate 60 mV negative shift in the open state probability (Popen)-voltage curve. Externally applied tetraethylammonium ions (TEA+) caused a rapid an...
Frean SP, Abraham LA, Lees P.The effects of hyaluronan and carprofen (both racemic mixture and separate R and S enantiomers) on proteoglycan (PG) synthesis by equine cultured chondrocytes and cartilage explants were examined. Hyaluronan stimulated PG synthesis in both cell and explant cultures. The concentration-response curve of the latter was bell-shaped. Racemic carprofen and R and S enantiomers also stimulated PG synthesis, although concentration-response relationships varied for each preparation and high concentrations inhibited synthesis. It was concluded that (a) hyaluronan exerts a stimulatory effect on PG synthes...
Loubière L, Tiraby M, Cazaux C, Brisson E, Grisoni M, Zhao-Emonet J, Tiraby G, Klatzmann D.The herpes simplex virus type 1 thymidine kinase suicide gene (HSV1tk) together with ganciclovir (GCV) have been successfully used for in vivo treatment of various experimental tumors, and many clinical trials using this system have been launched. With the aim to improve this therapeutic system, we compared the potential efficacy of different herpes virus derived thymidine kinases (HSV1, varicella-zoster virus, equine herpes virus type-4 and Epstein-Barr virus) as suicide genes in association with the nucleoside analogs acyclovir, ganciclovir and bromovinyldeoxyur- idine. Using various murine ...
Carver SE, Heath CA.Equine articular chondrocytes, embedded within a polyglycolic acid nonwoven mesh, were cultured with various combinations of intermittent pressure, fluid flow, and mixing to examine the effects of different physical stimuli on neochondrogenesis from young cells. The cell/polymer constructs were cultured first in 125 ml spinner flasks for 1, 2, or 4 weeks and then in a perfusion system with intermittent pressure for a total of up to 6 weeks. Additional constructs were either cultured for all 6 weeks in the spinner flasks or for 1 week in spinners followed by 5 weeks in the perfusion system with...
Wilson SM, Gallagher M, Rakhit S, Remsbury AL, Ko WH.Uridine triphosphate (UTP) evoked inhibition of adrenaline-evoked cAMP accumulation in cultured equine epithelial cells (EC50, 1.8 +/- 0.2 microM) and this effect was mimicked by 5-Br-UTP (EC50, 6.6 +/- 1.8 microM) and uridine diphosphate (UDP; EC50, 96 +/- 26 microM). This inhibitory action of UTP was abolished by pre-treating cells with pertussis toxin (10 ng ml-1, 24 h). UTP (EC50, 2.3 +/- 0.3 microM) and 5-Br-UTP (EC50, 29.4 +/- 9.4 microM) also increased intracellular free calcium ([Ca2+]i) whilst UDP did not; the two effects are thus differentially sensitive to these pyrimidine nucleotid...
Julliand V, de Vaux A, Millet L, Fonty G.Detection and quantification of cellulolytic bacteria with oligonucleotide probes showed that Ruminococcus flavefaciens was the predominant species in the pony and donkey cecum. Fibrobacter succinogenes and Ruminococcus albus were present at low levels. Four isolates, morphologically resembling R. flavefaciens, differed from ruminal strains by their carbohydrate utilization and their end products of cellobiose fermentation.
Ellington JE, Samper JC, Jones AE, Oliver SA, Burnett KM, Wright RW.Formation of a spermatozoa ('sperm') reservoir in the mare is thought to occur through lectin-mediated sperm attachment to the oviductal epithelium. Once attached, prefertilization sperm survival is supported by oviductal factors. Cryopreservation of stallion sperm decreases the number of sperm attaching to oviduct epithelial cells (OEC) and the length of time these sperm survive. Quantification of in vitro interactions between sperm and OEC in a co-culture system may provide an assay for functional integrity of cryopreserved or fresh sperm samples. Additionally, superior additives for in vitr...
Lindsay DS, Dubey JP.Equine protozoal myeloencephalitis (EPM) is a neurologic syndrome in horses from the Americas and is usually caused by infection with the apicomplexan parasite, Sarcocystis neurona. The activities of pyrimethamine, trimethoprim, sulfachloropyridazine, sulfadiazine, sulfadimethoxine, sulfamethoxazole, sulfamethazine, and sulfathiazole were examined against developing S. neurona merozoites in bovine turbinate cell cultures. A microtiter plate host cell lesion based assay was used to determine the effects of agents on developing merozoites. A cell culture flask assay was used to determine if sele...
Hess S, Gulati R, Peluso JJ.The present studies showed that sequential treatment with equine CG (eCG) and hCG not only induced an increase in ovarian weight, but also caused an estimated 4.6-fold increase in the number of ovarian surface epithelial cells. In addition, eCG-hCG treatment increased ovarian hepatocyte growth factor (HGF) messenger RNA levels. These studies also demonstrated that rat primary ovarian surface epithelial cells as well as a cell line derived from rat ovarian surface epithelium (i.e. ROSE-179 cells) do not express the LH (hCG) receptor. Both of these cells express c-Met, the receptor for HGF. To a...
Aggarwal N, Holmes MA.The aim of this study was to characterise CD4+T-cells in equines, as these cells are pivotal in establishing immune responses or regulating established ones. Peripheral blood mononuclear cells from a pony immunised with ovalbumin were cultured in vitro in the presence of the specific antigen and autologous antigen presenting cells. During the antigen starvation phase, cells were maintained on recombinant equine IL-2. After 35 days of culture, most of the cells were CD4+, CD8-and sIg-. Cells proliferated specifically in the presence of antigen, as tested on day 42 of culture. These cells were a...
Ellington JE, Samper J, Jones A, Oliver SA, Burnett K, Wright RW.To compare function of cultured cryopreserved stallion spermatozoa in a modified Tyrode's medium (TM), with or without bovine serum albumin (BSA), or in uterine tube (oviduct) epithelial cell (OEC) coculture in TM, with or without BSA. Methods: Cryopreserved spermatozoa from 6 proven stallions and OEC from bovine reproductive tracts in follicular phase. Methods: Thawed spermatozoa were cultured in TM, with or without BSA, or cocultured with OEC monolayers in TM, with or without BSA. Percentages of capacitated and acrosome-reacted spermatozoa were measured at 5 hours for TM cultures. Spermatozo...
Bramucci M, Miano A, Quassinti L, Maccari E, Murri O, Amici D.This study was conducted to determine the presence of the angiotensin converting enzyme in commercial sera used in cell culture medium. The aim of the research was to bring the presence of proteinases (angiotensin converting enzyme) to cell culture users' knowledge and to give some data for solving problems about the development of peptides as useful drugs. The enzymes, purified from foetal bovine, adult bovine, foetal equine, adult equine, and human sera, showed molecular weights of about 170 kDa. Captopril and lisinopril inhibited enzyme activities at nanomolar concentrations. The enzymes we...
Pollitt CC, Pass MA, Pollitt S.A method for culturing explants of lamellar hoof was developed to investigate the process of lamellar separation that occurs in laminitis. Explants, consisting of hoof wall, dermal and epidermal lamellae and the adjacent sub-lamellar connective tissue remained intact when cultured in tissue culture medium for 2 days. However, when cultured in the presence of the matrix metalloproteinase (MMP) activator aminophenylmercuric acetate (APMA), the lamellae separated when tension was applied by pulling the hoof wall in an opposite direction to the connective tissue. The separation occurred between th...
Wunn D, Wardrop KJ, Meyers K, Kramer J, Ragle C.To develop methods to isolate, culture, and characterize equine hoof endothelial cells (EC) and keratinocytes. Methods: Cells harvested from the forelimbs of 8 horses. Methods: EC were obtained via catheters placed in the palmar digital arteries of the disarticulated lower portion of the forelimbs from 4 horses that had been heparinized prior to euthanasia. Phosphate-buffered saline solution was used to remove and discard RBC from blood vessels, and collagenase was used to loosen and flush EC from the vasculature. Hoof keratinocytes were obtained from 4 recently euthanatized horses by use of d...
Gray AW, Davies ME, Jeffcott LB.We report on preliminary results of a novel in vitro culture system designed to generate equine osteoclasts in large numbers. Osteoclast generation, as determined by the expression of tartrate resistant acid phosphatase (TRAP) and ability to resorb bone, was enhanced in equine bone marrow cultures supplemented with fibroblastic cell (L929) conditioned medium (L929-CM). Bone marrow was collected from a total of 12 horses and ponies and TRAP-positive cells with bone resorbing ability were generated in significant numbers in the last seven. TRAP-positive mononuclear cells appeared after three day...
Raidal SL, Bailey GD, Love DN.Flow cytometry was used to assess the phagocytosis of fluorescent-labelled bacteria by equine peripheral blood neutrophils and pulmonary alveolar macrophages. Cell populations were prepared from venous blood following ammonium chloride lysis and from washed bronchoalveolar lavage derived samples. Discrete clusters of cells, corresponding to different leucocyte groups, were readily identified on the basis of differing light scattering properties and could thus be discriminated, negating the need for prior cell separation. Cells able to associate with fluorescent-labelled bacteria (by attachment...
Clegg PD, Coughlan AR, Carter SD.Matrix metalloproteinases (MMPs) are the main enzymes involved in connective tissue turnover. Regulation of MMPs is achieved by controlling production, activation of the pro-enzymes together with the presence of inhibitors, such as, tissue inhibitors of metalloproteinases (TIMPS). The presence of TIMPs in equine synovial fluid was assessed by the ability of the fluid to inhibit equine MMP-9 activity using a gelatin degradation ELISA. The cellular source of the TIMPs was determined using culture supernatants of resident articular cells (chondrocytes and synovial fibroblasts) and invading inflam...
Fortier LA, Nixon AJ, Williams J, Cable CS.To isolate mesenchymal stem cells from adult horses and determine specific monolayer culture conditions required to enhance biochemically and phenotypically defined chondrocytic differentiation. Methods: 2 adult horse bone marrow donors without skeletal or hematologic abnormalities. Methods: Bone marrow was aspirated from the sternebra, and mesenchymal stem cells were isolated by centrifugation and cultured in monolayers. Subcultures were established in 24-well plates on day 13. Culture medium was harvested every 2 days, and culture of 12 of the 24 wells was terminated on day 6 and of the rema...
Ząbek T, Witarski W, Semik-Gurgul E, Szmatoła T, Kowalska K, Bugno-Poniewierska M.We investigated the activity of chondrogenic markers and variation of methylation patterns in equine cartilaginous cells cultivated in monolayer. The transcriptional and epigenetic effect of the long-term culture of chondrocytes has been evaluated using several passages of chondrocyte cell-lines derived from equine articular cartilage. Using 3 genes as endogenous control we tested the expression of 7 genes important for different stages of chondrocyte differentiation and maturation. CpG islands in RUNX3 locus were inspected for the evaluation of differential methylation state of passaged cell-...
Mok CH, MacLeod JN.Within developing synovial joints, interzone and anlagen cells progress through divergent chondrogenic pathways to generate stable articular cartilage and transient hypertrophic anlagen cartilage, respectively. Understanding the comparative cell biology between interzone and anlagen cells may provide novel insights into emergent cell-based therapies to support articular cartilage regeneration. The aim of this study was to assess the kinetics of gene expression profiles in these skeletal cell lines after inducing chondrogenesis in culture. Interzone and anlagen cells from seven equine fetuses w...
Del Campo MR, Donoso X, Parrish JJ, Ginther OJ.Equine oocytes (n = 537) were collected from slaughterhouse ovaries (n = 118 mares) by scraping the internal follicular wall. Preculture record was made of the appearance of oocyte investments (no cumulus, corona radiata only, compact cumulus, expanded cumulus), appearance of cytoplasm (homogeneous, condensed heterogeneous/fragmented), and nuclear maturation stages (germinal vesicle, germinal-vesicle breakdown, metaphase I, metaphase II, degenerated). There was no difference between follicles > 30 mm and follicles < or = 30 mm in the preculture frequency distribution among the 5 nuclear stages...
Choi YH, Chung YG, Seidel GE, Squires EL.The objective was to compare culture media for in vitro maturation of equine oocytes and for in vitro culture of zygotes produced from IVF of partially zona-removed oocytes. Cumulus-oocyte complexes from slaughterhouse-derived ovaries were washed in m-Dulbecco's PBS and cultured in TCM-199, F10-DMEM or c-F10-DMEM (50% F10-DMEM + 50% F10-DMEM conditioned medium from culture of an equine trophoblast monolayer for 3 or 4 days). All media included FSH, LH, E2, and 10% FCS. After 28 to 30 h maturation, cumulus expansion was scored from 0 (no expansion) to 4 (fully expanded). Oocytes with a 1st pola...
Morier L, Cantelar N, Soler M.Eastern Equine Encephalitis (EEE) was in Cuba before the 1940s; the virus has been isolated from horses, birds, and rodents during epizootic as well as interepizootic periods. The only isolation of Western Equine Encephalitis (WEE) virus was from a sick pigeon found in the vicinity of Havana University. Both viruses can cause human disease; the isolation of WEE virus from the centre of an urban area emphasises the need for the prompt isolation and rapid identification of these agents. The object of this work was to compare the sensitivity of a continuous cell line (XL-2) from the toad, Xenopus...
Schumacher J, Chambers M, Hanselka DV, Morton LD.Eighteen stored split thickness meshed skin grafts were applied to surgically created lesions on the metacarpal and metatarsal regions of six horses. Donor skin was harvested from the sternal region, meshed and stored at 4 degrees C in a cell culture medium containing 10% serum. Stored grafts were applied to the wounds at 1, 2, and 3 week intervals. Acceptance of the grafts stored for 1 week was generally poor (1 of 6 grafts), whereas that of the 2 and 3 week old grafts was generally excellent (10 of 12 grafts). Poor acceptance of the 1 week old grafts was attributed to streptococcal infection...
O'Niell FD, Issel CJ.Growth kinetics of equine influenza virus-A1, equine herpesvirus-1, and equine rhinovirus-1 were determined in susceptible cell monolayers and in organ cultures of equine fetal tracheal and nasal turbinate epithelium. Equine influenza virus-A1 was replicated in cell and organ cultures and was released more readily and for longer periods from nasal turbinate epithelium than from tracheal epithelium. Equine herpesvirus-1 was also replicated in cell and organ cultures. During the first 24 hours after inoculation, equine herpesvirus-1 was released more readily from tracheal epithelium than from na...
Ericson KK, Yang TJ.During our studies of cytostatic cytokines in the mixed leukocyte culture, we found that horse serum in the medium control contained a tumor cell growth-inhibitory factor. The fraction isolated by molecular sieving and ion exchange chromatography inhibited the growth and DNA synthesis of the primary culture and passaged cell line of the canine transmissible venereal sarcoma, murine T (L5178Y) and B (P3-X63-Ag8.653) lymphoid tumor cells, murine mammary tumor cells (RIII), bovine lymphoid tumor cells (BL3), and the nontransformed cell line of baby hamster kidney cells. Nontransformed cell lines ...
Silva GB, De La Côrte FD, Brass KE, Palma HE, Gallio M, Cantarelli C, Bertolin K, Krause A, Wergutz J, Fontanari Krause LM, Antoniazzi AQ.Chondrocyte health is altered when exposed to local anesthetics, raising concerns as to the long-term effects of local anesthetics intra-articularly for diagnosis and analgesia. To investigate the drug with the lowest toxic potential, the effect of ropivacaine and mepivacaine on chondrocytes was evaluated. Articular cartilage from normal metacarpophalangeal joints of five equine cadaver specimens was used to establish chondrocyte cultures. Following seven days, chondrocytes were exposed to standard culture medium (DMEM), ropivacaine 7.5 mg/ml (ROP7.5), ropivacaine 10 mg/ml (ROP10), mepivacaine...
Yang Q, Lopez MJ.The equine hoof dermal-epidermal interface requires progenitor cells with distinct characteristics. This study was designed to provide accurate ultrastructural depictions of progenitor cells isolated from inflamed tissue and normal tissue before and after cryopreservation and following selection of cells expressing both keratin (K) 14 (ectodermal) and cluster of differentiation (CD) 105 (mesodermal). Passage 3 cell ultrastructure was assessed following 2D culture and after 3D culture on decellularized hoof tissue scaffolds. Outcome measures included light, transmission electron, and scanning e...
Zweygarth E, Ahmed JS, Rehbein G, Voigt WP.The capacity of equine peripheral blood lymphocytes (PBL) to proliferate in the presence of Babesia equi-transformed lymphoblastoid stimulator cells was tested in an autologous as well as in an allogenic one way mixed lymphocyte reaction (MLR). It was found that both autologous and allogeneic responder lymphocytes incorporated high amounts of 3H-thymidine. The incorporation of 3H-thymidine was lower in MLR using as stimulator cells lymphocytes from which the cell line had previously been established, than when using parasitized culture cells as stimulator. Proliferation of PBL was achieved onl...
Oliveira Spila D, Maranhão RPA, Ocarino NM, de Lima JTB, Melo FG, Boeloni JN, Serakides R.The effects of two concentrations of triiodothyronine (T3; 0.01 and 1,000 nM) on the osteogenic and chondrogenic differentiation abilities of equine adipose-derived mesenchymal stem cells (AD-MSCs) were evaluated. The osteogenic study evaluated the effect of T3 using alkaline phosphatase activity (ALP) assay; cell viability and density; and formation of mineralized nodules at Days 7, 14, and 21 in culture. The chondrogenic study tested the effect of T3 through ALP assay, mitochondrial metabolism, cell density, and periodic acid-Schiff-positive (PAS+) matrix percentage at Days 7 and 14. In bot...
Maia L, Dias MC, de Moraes CN, de Paula Freitas-Dell'Aqua C, da Mota LS, Santiloni V, da Cruz Landim-Alvarenga F.Cryopreservation is a feasible alternative to maintaining several cell lines, particularly for immediate therapeutic use, transportation of samples, and implementation of new in vitro studies. This work parts from the hypothesis that the medium of cryopreservation composed by 90% of conditioned medium (CM) supports cryopreservation of equine umbilical cord intervascular matrix mesenchymal stem cells (UCIM-MSCs), allowing the maintenance of the biological properties for the establishment of cell banks intended for therapeutic use and in vitro studies. Thus, we evaluated the viability, apoptosis...
Storch C, Fuhrmann H, Schoeniger A.Osteoarthritis the quality and span of life in horses. Previous studies focused on nasal cartilage as a possible source for autologous chondrocyte implantation (ACI) in cartilage defects in humans. "HOX gene-negative" nasal chondrocytes adapted articular HOX patterns after implantation into caprine joint defects and produced cartilage matrix proteins. We compared the HOX gene profile of equine chondrocytes of nasal septum, anterior and posterior fetlock to identify nasal cartilage as a potential source for ACI in horses. Cartilage was harvested from seven horses after death and derived chondro...
Wise LM, Stuart GS, Sriutaisuk K, Adams BR, Riley CB, Theoret CL.Fibroproliferative disorders occur in both humans and horses following skin injury. In horses, wound healing on the limb is often complicated by the formation of fibroproliferative exuberant granulation tissue, characterized by persistent expression of pro-fibrotic transforming growth factor-beta1 (TGF-β1) and deficient expression of anti-inflammatory interleukin-10 (IL-10). IL-10 has been shown to directly modulate fibrotic gene expression in human fibroblasts, so we hypothesized that equine IL-10 (eIL-10) may exert similar anti-fibrotic effects on equine dermal fibroblasts. Cell-lines were ...
Thomas PG, Ball BA, Ignotz GG, Dobrinski I, Parks JE, Currie WB.Before fertilization, equine spermatozoa adhere to oviduct epithelial cells (OEC) of the mare. The biochemical basis for this adhesion has not been determined. Our objective was to produce an antiserum to block this interaction. Ejaculated spermatozoa were subjected to nitrogen cavitation and spermatozoal plasma membranes enriched by sucrose density gradient centrifugation; membrane enrichment was confirmed by comparative alkaline phosphatase analysis, electron microscopy, and one- and two-dimensional PAGE. Periacrosomal plasma membrane was used as an immunogen for the production of an antiser...
Zhou H, Liu C, Wang W.This study was conducted to reconstruct heterogeneous embryos using equine skin fibroblast cells as donor karyoplasts and the bovine oocytes as recipient cytoplast for investigating the reprogramming of equine somatic cell nuclear in bovine oocyte cytoplasm and the developmental potential of the reconstructed embryos. Adult horse skin fibroblast cells serum-starved were used as donor somatic cells. Bovine oocytes matured in vitro were employed as recipient cytoplasts. The fusion of fibroblast cells into recipient cytoplasm was induced by electofusion. The fused eggs were activated by inomycin ...
Fernandez-Fuente M, Terracciano CM, Martin-Duque P, Brown SC, Vassaux G, Piercy RJ.Dysfunctional skeletal muscle calcium homeostasis plays a central role in the pathophysiology of several human and animal skeletal muscle disorders, in particular, genetic disorders associated with ryanodine receptor 1 (RYR1) mutations, such as malignant hyperthermia, central core disease, multiminicore disease and certain centronuclear myopathies. In addition, aberrant skeletal muscle calcium handling is believed to play a pivotal role in the highly prevalent disorder of Thoroughbred racehorses, known as Recurrent Exertional Rhabdomyolysis. Traditionally, such defects were studied in human an...
Grünig G, Hulliger C, Hermann M, Winder C, von Fellenberg R.Bronchopulmonary lavage was performed in 10 healthy horses and in 39 horses with chronic pulmonary disease. The predominant cell types were macrophages in healthy horses and neutrophils in severely diseased horses. Procoagulant activity (PCA) was detected in all 32 cell-free supernatants examined and in all 49 unpurified cell suspensions. Cells were separated by centrifugation on discontinuous gradients prepared either with Percoll or with Metrizamide. Macrophages were enriched in subpopulations of low density. Neutrophils could not be purified by density gradient centrifugation using either g...
Choi YH, Love LB, Varner DD, Hinrichs K.The effect of medium-to-embryo ratio on blastocyst development of equine embryos from oocytes with compact cumuli was evaluated in the present experiment. In addition, two methods for holding oocytes before in vitro maturation were compared. In Experiment 1, oocytes cultured with roscovitine for 16-18h before maturation were fertilized by intracytoplasmic sperm injection and cultured individually in 2.5, 5, 10 or 50microl droplets. In Experiment 2, oocytes were either cultured with roscovitine or held in a modified M199 with 20% serum at room temperature (EH treatment) for 16-18h, then matured...
Baldwin DM, Roser JF, Muyan M, Lasley B, Dybdal N.Enzymatically dispersed anterior pituitary cells from donor mares were cultured for 48 h in alpha-modified Eagles' medium containing 10% steroid-free horse serum. The cells were then incubated for 24 h in fresh medium oestrogen followed by a 4-h incubation with or without GnRH. Media and cell extracts were analyzed for LH by radioimmunoassay. In the first series of experiments, pituitary cells from Day-3 dioestrous mares were preincubated with ethanol (control) or different concentrations of E2 (10(-11) to 10(-7) M) for 24 h prior to a 4-h incubation without (basal) or with 1.0 nM GnRH. E2 inc...
Riley CB, Archer FJ, Bailey JV.To compare the effects of different commercial nutrient media and sera on protein synthesis and maintenance of cellular density in cultures of the equine superficial digital flexor tendon (SDFT). Methods: 8 healthy 2- to 4-year-old horses. Methods: First Dulbecco's modified Eagle's medium, Ham's F12 nutrient mixture, RPMI 1640 medium, minimum essential medium with Earle's salts, minimum essential medium with Hanks' salts, and a Dulbecco's modified Eagle's medium/Ham's F12 nutrient mixture with 10% fetal bovine serum (FBS) were compared. Then FBS, fetal equine serum, and donor horse serum, each...
Belloli C, Re G, Arioli F, Badino P, Carcano R, Odore R, Girardi C, Beretta C.To identify beta-adrenoceptor subtypes involved in motility inhibition of circular and longitudinal smooth muscle layers of equine ileum. Methods: Isolated strips of equine ileum circular smooth muscle and membrane preparations from circular and longitudinal muscle layers. Methods: Functional assays of circular muscle preparations and radioligand binding assays and measurements of cAMP production in smooth muscle membranes from circular and longitudinal layers. Results: Selective beta-adrenergic agonists exerted inhibitory effects on circular muscle preparations. Binding studies of cell membra...
Wijnker JJ, Bull S, Van Dijk P, Veenman JN, Rutten VP, Klein WR, Fink-Gremmels J.The detrimental effects of the pro-inflammatory cytokine TNF-alpha during equine acute abdominal disease are well known. Its pivotal role in many human diseases has led to various in-depth studies regarding its release mechanism, in particular by TNF-alpha converting enzyme (TACE). In this study we investigated the inhibitory effect of a TACE-inhibitor on cytokine release (TNF-alpha, IL-1alpha and IL-6) in three different cell models, including U937 cells, a recently established equine macrophage cell line, known as eCAS, and primary equine PBMC. The aim was to show the similarity of TNF-alpha...
Mitchell SM, Zajac AM, Davis WL, Lindsay DS.Toxoplasma gondii is an important apicomplexan parasite of humans and other warm-blooded animals. Ponazuril is a triazine anticoccidial recently approved for use in horses in the United States. We investigated the mode of action of ponazuril against developing RH strain T. gondii tachyzoites in African green monkey kidney cells. Host cells were infected with 2.0 x 10(5) tachyzoites and treated with 5 microg/ml ponazuril. Cultures were fixed and examined by transmission electron microscopy 3 days after treatment. Ponazuril interfered with normal parasite division. This led to the presence of mu...
Santos VH, Pfeifer JPH, de Souza JB, Milani BHG, de Oliveira RA, Assis MG, Deffune E, Moroz A, Alves ALG.The original article [1] contained a minor error regarding the mean diameter of the alginate microcapsules described in relation to Fig. 4 in the Results section. The microcapsules had an actual mean diameter of 3000 μm instead of 1000 μm as mistakenly mentioned in the original article.
Boorman S, Hanson RR, Velloso Alvarez A, Zhong K, Hofmeister E, Boone LH.To determine the effect of concurrent versus delayed treatment with corticosteroid on equine articular tissues also treated with local anesthetic in vitro in the presence of inflammatory mediators. Methods: Controlled laboratory study. Methods: Five geldings, one mare (aged 3-18 years). Methods: From each horse, 24 synovial and 12 osteochondral explants were cultured in a 12-well plate (2 wells/group, 2 synovial and 1 osteochondral explant/well, total 216 explants in the study). Explants were stimulated in culture medium with 10 μg/ml recombinant equine interleukin-1β and 10 μg/ml tumor...
Nino-Fong R, Esparza Gonzalez BP, Rodriguez-Lecompte JC, Montelpare W, McD￮ L.Bone repair in horses implies invasive surgeries and increased cost. Research on musculoskeletal disorders therapy in horses includes cell-based therapy with mesenchymal stromal cells (MSCs). Mesenchymal stromal cells can be obtained from bone marrow (BMMSCs). Unfortunately, BMMSCs have limited cell replication . The objective of this study was to develop a biologically immortalized equine stem cell line derived from bone marrow, with unlimited proliferation and the ability to differentiate into bone cells. Equine BMMSCs were transfected and immortalized with human telomerase reverse transcri...
Stewart F, Tuffnell PP.A 514 bp cDNA transcript coding for 78% of horse (Equus caballus.) GH has been cloned and sequenced. The deduced amino acid sequence corresponded precisely to that previously obtained by protein sequencing and, in addition, provided new sequence information for the signal peptide. The missing 3' fragment of the cDNA was reconstructed using synthetic oligonucleotides and site-specific directed mutagenesis. The complete cDNA sequence was then inserted into an expression vector (PIN-III-lppp-5) which utilizes a bacterial signal peptide to secrete the expressed product into the periplasmic space o...
Singh B, Minhas KJ, Atwal OS.The pulmonary intravascular macrophages (PIMs) of the ponies possess all the characteristics of a fully differentiated resident macrophage, which forms adhesive plaques with the capillary-endothelial cells. In addition, it has a unique surface coat which does not conform to the concept of a carbohydrate rich membrane bound glycocalyx generally associated with macrophages. We studied the responses of these cells especially in the context of its globular surface coat to multiple doses of MB intravenously: 0.2 ml/kg body copper tracer substance. The ponies were treated with three doses of MB intr...
