The Major Histocompatibility Complex (MHC) in horses is a group of genes that play a significant role in the immune system by encoding proteins responsible for antigen presentation. These proteins are essential for the recognition of foreign molecules by the immune system, facilitating the differentiation between self and non-self entities. In horses, the MHC is located on equine chromosome 20 and is divided into several regions, including class I and class II, which present antigens to different subsets of T cells. Variability in the MHC genes is associated with susceptibility or resistance to various diseases, influencing immune responses and vaccine efficacy. This page compiles peer-reviewed research studies and scholarly articles that explore the genetic diversity, expression patterns, and implications of MHC in equine health and disease.
Liu L, Yu W, Cai K, Ma S, Wang Y, Ma Y, Zhao H. () is a zoonotic opportunistic pathogen that can cause life-threatening infections. The rapid evolution of multidrug-resistant and the fact that there is no currently licensed effective vaccine against warrant the need for vaccine development. Reverse vaccinology (RV), which involves screening a pathogen's entire genome and proteome using various web-based prediction tools, is considered one of the most effective approaches for identifying vaccine candidates. Here, we performed a pangenome analysis to determine the core proteins of . We then used the RV approach to examine the subcellular l...
Vasoya D, Tzelos T, Benedictus L, Karagianni AE, Pirie S, Marr C, Oddsdóttir C, Fintl C, Connelley T.The Major Histocompatibility Complex (MHC) genes play a key role in a number of biological processes, most notably in immunological responses. The MHCI and MHCII genes incorporate a complex set of highly polymorphic and polygenic series of genes, which, due to the technical limitations of previously available technologies, have only been partially characterized in non-model but economically important species such as the horse. The advent of high-throughput sequencing platforms has provided new opportunities to develop methods to generate high-resolution sequencing data on a large scale and app...
Plasil M, Oppelt J, Klumplerova M, Bubenikova J, Vychodilova L, Janova E, Stejskalova K, Futas J, Knoll A, Leblond A, Mihalca AD, Horin P.The major histocompatibility complex (MHC) with its class I and II genes plays a crucial role in the immune response to pathogens by presenting oligopeptide antigens to various immune response effector cells. In order to counteract the vast variability of infectious agents, MHC class I and II genes usually retain high levels of SNPs mainly concentrated in the exons encoding the antigen binding sites. The aim of the study was to reveal new variability of selected MHC genes with a special focus on MHC class I physical haplotypes. Long-range NGS to was used to identify exon 2-exon 3 alleles in th...
Heilen LB, Roßgardt J, Dern-Wieloch J, Vogelsberg J, Staszyk C.The lifelong eruption places a great demand on the dental pulp and periodontal ligament (PDL) of horse teeth. Cells within the pulp and PDL seem to play a key role during this remodeling. Unassigned: In this study, we isolated and cultivated MSCs (medicinal signaling cells) from dental pulp, PDL and retrobulbar fat of four horses. Subsequently, we analyzed them by flow cytometry and immunohistochemistry to determine and compare their characteristics. In addition, we localized these cells within the tissue structure via immunohistochemistry of histological sections. For these analyses, several ...
Cequier A, Vázquez FJ, Romero A, Vitoria A, Bernad E, GarcÃa-MartÃnez M, Gascón I, Barrachina L, Rodellar C.The immunomodulatory properties of equine mesenchymal stem cells (MSCs) are important for their therapeutic potential and for their facilitating role in their escape from immune recognition, which may also be influenced by donor-recipient major histocompatibility complex (MHC) matching/mismatching and MHC expression level. Factors such as inflammation can modify the balance between regulatory and immunogenic profiles of equine MSCs, but little is known about how the exposure to the immune system can affect these properties in equine MSCs. In this study, we analyzed the gene expression and secr...
Espinoza-Duarte MR, Ortega-Ochoa C, Baca-Ramirez A, Possani LD, Espino-Solis GP.Horses have played a prominent role in shaping our modern world, with important effects on health. Unfortunately, better characterization of the horse immune system is still needed. In this report, using flow cytometry techniques, four monoclonal antibodies against horse CD11c integrin were characterized and described for their ability to provide a positive recognition signal in peripheral blood mononuclear cells. Further immune cell phenotype experiments were performed using MHC-II, CD14, TLR4 and the specific anti-horse CD11c monoclonal antibody (1C4). With this staining panel, it was possib...
Tang Y, Liu G, Zhao S, Li K, Zhang D, Liu S, Hu D.Major histocompatibility complex (MHC) genes are the most polymorphic in vertebrates and the high variability in many MHC genes is thought to play a crucial role in pathogen recognition. The MHC class II locus DQA polymorphism was analyzed in the endangered Przewalski's horse, , a species that has been extinct in the wild and all the current living individuals descend from 12 founders. We used the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) to detect the polymorphism within the MHC DQA in 31 Przewalski's horses from two reintroduced populations. Consequently, o...
Rosa GDS, Krieck AMT, Padula ET, Stievani FC, Rossi MC, Pfeifer JPH, Basso RM, Braz AMM, Golim MA, Alves ALG.Allogeneic mesenchymal stem cells (MSC) are widely used in clinical routine due to the shorter expansion time and reliability of its quality. However, some recipients can produce alloantibodies that recognize MSCs and activate the immune system, resulting in cell death. Although antibody production was already described after MSC injection, no previous studies described the immune response after intra-articular MSC injection in acute synovitis. This study aimed to evaluate the influence of inflammation on immune response after single and repeated intra-articular injections of synovial membrane...
Cequier A, Romero A, Vázquez FJ, Vitoria A, Bernad E, Fuente S, Zaragoza P, Rodellar C, Barrachina L.Immunomodulation and immunogenicity are pivotal aspects for the therapeutic use of mesenchymal stem cells (MSCs). Since the horse is highly valuable as both a patient and translational model, further knowledge on equine MSC immune properties is required. This study analysed how inflammation, chondrogenic differentiation and compatibility for the major histocompatibility complex (MHC) influence the MSC immunomodulatory-immunogenicity balance. Equine MSCs in basal conditions, pro-inflammatory primed (MSC-primed) or chondrogenically differentiated (MSC-chondro) were co-cultured with either autolo...
Wang M, Li H, Zhang X, Yang L, Liu Y, Liu S, Sun Y, Zhao C.The aim of the present study was to analyze the main factors that have a significant impact on skin thickness, and to further identify the genes and signaling pathways regulating skin growth by RNA-seq in Dezhou donkeys. Skin samples from different body regions of 15 slaughtered donkeys were obtained to study variations in skin thickness over the bodies. Skin thickness data for another 514 donkeys was obtained by minimally invasive skin sampling from the back, and measurements of the donkeys' body size traits and pedigree data were also collected. These data were used to analyze changes in ski...
Murata D, Ishikawa S, Sunaga T, Saito Y, Sogawa T, Nakayama K, Hobo S, Hatazoe T.Medical interventions for subchondral bone cysts in horses have been extensively studied. This study investigated the regeneration of articular cartilage and subchondral bone with scaffold-free three-dimensional (3D) constructs of equine synovial membrane-derived mesenchymal stem cells (SM-MSCs) isolated from three ponies and expanded until over 1.0 × 10 cells at passage 2 (P2). Results: SM-MSCs were strongly positive for CD11a/CD18, CD44, and major histocompatibility complex (MHC) class I; moderately positive for CD90, CD105, and MHC class II; and negative for CD34 and CD45 on flow cytom...
Kamm JL, Riley CB, Parlane NA, Gee EK, McIlwraith CW.Mesenchymal stromal cells (MSCs) are believed to be hypoimmunogeneic with potential use for allogeneic administration. Bone marrow was harvested from Connemara (n = 1), Standardbred (n = 6), and Thoroughbred (n = 3) horses. MSCs were grouped by their level of expression of major histocompatibility factor II (MHC II). MSCs were then sub-grouped by those MSCs derived from universal blood donor horses. MSCs were isolated and cultured using media containing fetal bovine serum until adequate numbers were acquired. The MSCs were cultured in xenogen-free media for 48 h prior to use and d...
Antczak DF, Allen WRT.This chapter focuses on the early stages of placental development in horses and their relatives in the genus Equus and highlights unique features of equid reproductive biology. The equine placenta is classified as a noninvasive, epitheliochorial type. However, equids have evolved a minor component of invasive trophoblast, the chorionic girdle and endometrial cups, which links the equine placenta with the highly invasive hemochorial placentae of rodents and, particularly, with the primate placenta. Two types of fetus-to-mother signaling in equine pregnancy are mediated by the invasive equine tr...
Chen X, Zhao Y, Su L, Wang L, Ma X, Zhang B, Su Y.Strangles, which is caused by Streptococcus equi subspecies equi, is one of the most prevalent equine infectious diseases and poses heavy economic losses worldwide. Although various vaccines have been used for decades, they seemed to be sub-optimal to demonstrate effective protection, and the antigen component of vaccines against S. equi remains to be optimized. In the present study, three target antigens (M-like protein, α2-macroglobulin and IgG-binding protein, and glyceraldehyde-3-phosphate dehydrogenase) were selected and expressed. Mice were immunized and challenged, and their immune res...
Vostry L, Vostra-Vydrova H, Citek J, Gorjanc G, Curik I.Inbreeding depression is the reduction of performance caused by mating of close relatives. In livestock populations, inbreeding depression has been traditionally estimated by regression of phenotypes on pedigree inbreeding coefficients. This estimation can be improved by utilising genomic inbreeding coefficients. Here we estimate inbreeding depression for insect bite hypersensitivity (IBH) prevalence, the most common allergic horse disease worldwide, in Old Kladruber horse. In a deep pedigree with 3214 horses (187 genotyped), we used a generalised linear mixed model with IBH phenotype from 558...
Witkowska-Piłaszewicz O, Pingwara R, Szczepaniak J, Winnicka A.Clenbuterol, the β2-adrenoceptor agonist, is gaining growing popularity because of its effects on weight loss (i.e., chemical liposuction). It is also popular in bodybuilding and professional sports, due to its effects that are similar to anabolic steroids. However, it is prohibited by anti-doping control. On the other hand, it is suggested that clenbuterol can inhibit the inflammatory process. The cells from 14 untrained and 14 well-trained race horses were collected after acute exercise and cultured with clenbuterol. The expressions of CD4, CD8, FoxP3, CD14, MHCII, and CD5 in PBMC, and reac...
Berglund AK, Long JM, Robertson JB, Schnabel LV.Allogeneic mesenchymal stem cells (MSCs) are a promising cell therapy for treating numerous diseases, but major histocompatibility complex (MHC)-mismatched MSCs can be rejected by the recipient's immune system. Pre-treating MSCs with transforming growth factor-β2 (TGF-β2) to downregulate surface expression of MHC molecules may enhance the ability of allogeneic MSCs to evade immune responses. We used lymphocyte proliferation assays and ELISAs to analyze the immunomodulatory potential of TGF-β2-treated equine bone marrow-derived MSCs. T cell activation and cytotoxicity assays were then used t...
Rowland AL, Miller D, Berglund A, Schnabel LV, Levine GJ, Antczak DF, Watts AE.Allogeneic mesenchymal stromal cells (MSCs) have been used clinically for decades, without cross-matching, on the assumption that they are immune-privileged. In the equine model, we demonstrate innate and adaptive immune responses after repeated intra-articular injection with major histocompatibility complex (MHC) mismatched allogeneic MSCs, but not MHC matched allogeneic or autologous MSCs. We document increased peri-articular edema and synovial effusion, increased synovial cytokine and chemokine concentrations, and development of donor-specific antibodies in mismatched recipients compared wi...
Kammerer R, Ballesteros A, Bonsor D, Warren J, Williams JM, Moore T, Dveksler G.In early equine pregnancy, a highly invasive trophoblast cell subpopulation, the chorionic girdle cells, invade the endometrium and form endometrial cups (EC). These cells express classical MHC molecules, thereby stimulating a humoral and cellular immune response, resulting in a massive accumulation of maternal CD4+ and CD8+ T cells around the EC. Nevertheless, no immediate destruction of endometrial cups by maternal lymphoid cells occurs, presumably due to immune tolerance. Although the environment of EC is rich in TGFB and in FOXP3+, CD4+ T cells, the mechanisms leading to tolerance have not...
Klumplerova M, Splichalova P, Oppelt J, Futas J, Kohutova A, Musilova P, Kubickova S, Vodicka R, Orlando L, Horin P.The mammalian Major Histocompatibility Complex (MHC) is a genetic region containing highly polymorphic genes with immunological functions. MHC class I and class II genes encode antigen-presenting molecules expressed on the cell surface. The MHC class II sub-region contains genes expressed in antigen presenting cells. The antigen binding site is encoded by the second exon of genes encoding antigen presenting molecules. The exon 2 sequences of these MHC genes have evolved under the selective pressure of pathogens. Interspecific differences can be observed in the class II sub-region. The family E...
Liu C, Lei H, Ran X, Wang J.The Guizhou pony (GZP) is an indigenous species of equid found in the mountains of the Guizhou province in southwest China. We selected four regions of the equine leukocyte antigen (ELA), including , , and and used them to assess the diversity of the major histocompatibility complex (MHC) class II gene using direct sequencing technology. had the lowest / ratio (0.560) compared with the other three loci, indicating that was conserved and could be conserved after undergoing selective processes. Nine , five , nine and seven codons were under significant positive selection at the antigen b...
Jaworska J, Tobolski D, Janowski T.The failure of the maternal immune system to recognize fetal antigens and vice versa due to MHC similarity between the foal and its dam might result in the lack of placental separation during parturition in mares. The aim of the study was to investigate the influence of MHC similarity between a mare and a foal on the incidence of retained fetal membranes (RFM) in post-partum mares. DNA was sampled from 43 draft mares and their foals. Mares which failed to expel fetal membranes within three hours after foal expulsion were considered the RFM group (n = 14) and mares that expelled fetal membranes...
Futas J, Oppelt J, Janova E, Musilova P, Horin P.Natural killer (NK) cells play important roles in innate and adaptive immunity, as well as in the reproduction of placental mammals. Ly49 (KLRA) molecules represent a lectin-like type of NK cell receptor encoded within a complex genomic region, the NK cell complex. In rodents and horses, an expansion of the genes encoding Ly49 receptors leading to the formation of a gene family was observed. High sequence similarities and frequent high polymorphism of multiple family members represent an obstacle both for their individual identification and for annotation in the reference genomes of their resp...
Dominguez-Medina CC, Rash NL, Robillard S, Robinson C, Efstratiou A, Broughton K, Parkhill J, Holden MTG, Lopez-Alvarez MR, Paillot R, Waller AS.Bacterial superantigens (sAgs) are powerful activators of the immune response that trigger unspecific T cell responses accompanied by the release of proinflammatory cytokines. () and () produce sAgs that play an important role in their ability to cause disease. Strangles, caused by , is one of the most common infectious diseases of horses worldwide. Here, we report the identification of a new sAg of , SpeS, and show that mutation of the putative T cell receptor (TCR)-binding motif (YAY to IAY) abrogated TCR-binding, whilst maintaining interaction with major histocompatibility complex (MHC) c...
Lopez BS, Hurley DJ, Giancola S, Giguère S, Felippe MJB, Hart KA.The impact of culture conditions on equine monocyte-derived dendritic cells (MoDC) generation has not been fully characterized. We hypothesized that 1) MoDC could be cultured in a commercially available serum-free medium (AIM-V); and 2) that differential culture conditions would influence MoDC viability, yield and phenotype. MoDC generated from adult horses were cultured under variable conditions in a series of experiments. Viability was assessed using trypan blue and propidium iodide staining. Yield was determined by manual hemocytometer counting. Phenotype was assessed by flow cytometric ana...
Lange-Consiglio A, Romele P, Magatti M, Silini A, Idda A, Martino NA, Cremonesi F, Parolini O.Equine amniotic mesenchymal stromal cells (AMSCs) and their conditioned medium (CM) were evaluated for their ability to inhibit in vitro proliferation of peripheral blood mononuclear cells (PBMCs) with and without priming. Additionally, AMSC immunogenicity was assessed by expression of MHCI and MHCII and their ability to counteract the in vitro inflammatory process. Horse PBMC proliferation was induced with phytohemagglutinin. AMSC priming was performed with 10 ng/ml of TNF-α, 100 ng/ml of IFN-γ, and a combination of 5 ng/ml of TNF-α and 50 ng/ml of IFN-γ. The CM generated from naÃ...
Barrachina L, Cequier A, Romero A, Vitoria A, Zaragoza P, Vázquez FJ, Rodellar C.Antibody production after allogeneic administration of mesenchymal stem cells (MSCs) could impact their clinical application. Proinflammatory priming of MSCs can potentiate their regulatory ability in vivo but increased expression of major histocompatibility complex (MHC) might augment their immunogenicity, potentially leading to immune memory thus limiting repeated allogeneic administration. This study aimed at evaluating the production of cytotoxic allo-antibodies directed against donor's ELA (equine leukocyte antigen) in mismatched and halfmatched horses receiving repeated intraarticular ad...
Jaworska J, Ropka-Molik K, Wocławek-Potocka I, Siemieniuch M.Polymorphism of major histocompatibility complex (MHC) genes ensures effective immune responses against a wide array of pathogens. However, artificial selection, as performed in the case of domestic animals, may influence MHC diversity. Here, we investigate and compare the MHC diversity of three populations of horses, for which different breeding policies were applied, to evaluate the impact of artificial selection and the environment on MHC polymorphism. Samples of DNA were taken from 100 Polish draft horses, 38 stabled Konik Polski horses and 32 semiferal Konik Polski horses. MHC alleles and...
Jeannerat E, Marti E, Thomas S, Herrera C, Sieme H, Wedekind C, Burger D.The major histocompatibility complex (MHC) influences sexual selection in various vertebrates. Recently, MHC-linked social signaling was also shown to influence female fertility in horses (Equus caballus) diagnosed 17 days after fertilization. However, it remained unclear at which stage the pregnancy was terminated. Here we test if MHC-linked cryptic female choice in horses happens during the first days of pregnancy, i.e., until shortly after embryonic entrance into the uterus and before fixation in the endometrium. We exposed estrous mares to one of several unrelated stallions, instrumentally...
Minato E, Kobayashi A, Aoshima K, Fukushi H, Kimura T.Equine herpesvirus-1 (EHV-1), which causes encephalomyelitis in horses, shows endotheliotropism in the central nervous system of horses, and generally does not infect neurons. However, little is known about the mechanism underlying the resistance of neuron to EHV-1, due to the lack of convenient cell culture systems. In this study, we examined EHV-1 infection in immortalized Rn33B rat neuronal cells, which differentiate into neurons when cultured under nonpermissive conditions. Because murine cell lines are resistant to EHV-1 infections due to the lack of functional entry receptors for EHV-1, ...
Schnabel LV, Pezzanite LM, Antczak DF, Felippe MJ, Fortier LA.The horse is a valuable species to assess the effect of allogeneic mesenchymal stromal cells (MSCs) in regenerative treatments. No studies to date have examined recipient response to major histocompatibility complex (MHC)-mismatched equine MSCs. The purposes of this study were to immunophenotype MSCs from horses of known MHC haplotype and to compare the immunogenicity of MSCs with differing MHC class II expression. Methods: MSCs and peripheral blood leukocytes (PBLs) were obtained from Thoroughbred horses (n=10) of known MHC haplotype (ELA-A2, -A3, and -A9 homozygotes). MSCs were cultured thro...
Pezzanite LM, Fortier LA, Antczak DF, Cassano JM, Brosnahan MM, Miller D, Schnabel LV.This study tested the hypothesis that Major Histocompatibility Complex (MHC) incompatible equine mesenchymal stromal cells (MSCs) would induce cytotoxic antibodies to donor MHC antigens in recipient horses after intradermal injection. No studies to date have explored recipient antibody responses to allogeneic donor MSC transplantation in the horse. This information is critical because the horse is a valuable species for assessing the safety and efficacy of MSC treatment prior to human clinical application. Methods: Six MHC heterozygote horses were identified as non-ELA-A2 haplotype by microsat...
Chambers G, Ellsmore VA, O'Brien PM, Reid SWJ, Love S, Campo MS, Nasir L.The equine sarcoid, a locally aggressive, fibroblastic skin tumour, is the most common dermatological neoplasm reported in horses; there is no consistently effective therapy. It is widely accepted that bovine papillomavirus (BPV) types 1 and 2 are associated with the pathogenesis of sarcoid disease. Most sarcoids appear to contain detectable viral DNA and RNA and are also known to express the BPV types 1 and 2 major transforming protein, E5, but appear not to produce infectious virions. While the mode of transmission of infection has not been elucidated, viral gene expression, in particular of...
Cassano JM, Schnabel LV, Goodale MB, Fortier LA.Inflammatory licensed mesenchymal stem cells (MSCs) have the ability to promote functional tissue repair. This study specifically sought to understand how the recipient tissue environment reciprocally affects MSC function. Inflammatory polarized macrophages, modeling an injured tissue environment, were exposed to licensed MSCs, and the resultant effects of MSC immunomodulation and functionality of the MSC secretome on chondrocyte homeostasis were studied. Inflammatory licensed MSCs were generated through priming with either IFN-γ or polyinosinic:polycytidylic acid (poly I:C). Macrophages were...
Barberini DJ, Freitas NP, Magnoni MS, Maia L, Listoni AJ, Heckler MC, Sudano MJ, Golim MA, da Cruz Landim-Alvarenga F, Amorim RM.Studies with mesenchymal stem cells (MSCs) are increasing due to their immunomodulatory, anti-inflammatory and tissue regenerative properties. However, there is still no agreement about the best source of equine MSCs for a bank for allogeneic therapy. The aim of this study was to evaluate the cell culture and immunophenotypic characteristics and differentiation potential of equine MSCs from bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and umbilical cord (UC-MSCs) under identical in vitro conditions, to compare these sources for research or an allogeneic therapy cell bank. Methods: The BM-MS...
Berglund AK, Schnabel LV.Allogeneic mesenchymal stem cells (MSCs) are a promising cell source for treating musculoskeletal injuries in horses. Controversy exists, however, over whether major histocompatibility complex (MHC)-mismatched MSCs are recognised by the recipient immune system and targeted for death by a cytotoxic antibody response. Objective: To determine if cytotoxic anti-MHC antibodies generated in vivo following MHC-mismatched MSC injections are capable of initiating complement-dependent cytotoxicity of MSCs. Methods: Experimental controlled study. Methods: Antisera previously collected at Days 0, 7, 14 an...
De Schauwer C, Piepers S, Van de Walle GR, Demeyere K, Hoogewijs MK, Govaere JL, Braeckmans K, Van Soom A, Meyer E.During recent years, cell-based therapies using mesenchymal stem cells (MSC) are reported in equine veterinary medicine with increasing frequency. In most cases, the isolation and in vitro differentiation of equine MSC are described, but their proper immunophenotypic characterization is rarely performed. The lack of a single marker specific for MSC and the limited availability of monoclonal antibodies (mAbs) for equine MSC in particular, strongly hamper this research. In this study, 30 commercial mAbs were screened with flow cytometry for recognizing equine epitopes using the appropriate posit...
Lunn DP, Holmes MA, Antczak DF, Agerwal N, Baker J, Bendali-Ahcene S, Blanchard-Channell M, Byrne KM, Cannizzo K, Davis W, Hamilton MJ, Hannant D....The final assignment of antibody clusters for leucocyte antigens and immunoglobulins, as described in detail in Sections 3 and 4, is summarized in Table 4. Together with other mAbs developed outside of ELAW II (Table 9) this pool of reagents represent a powerful array of tools for the study of equine immunity. The Second Equine Leucocyte Antigen Workshop made considerable advances in pursuing the objectives of establishing the specificities of mAbs and achieving consensus on the nomenclature for equine leucocyte and immunoglobulin molecules. Of equal importance, several productive collaboratio...
McGuire TC, Tumas DB, Byrne KM, Hines MT, Leib SR, Brassfield AL, O'Rourke KI, Perryman LE.Cytotoxic T lymphocytes (CTL) can control some viral infections and may be important in the control of lentiviruses, including human immunodeficiency virus type 1. Since there is limited evidence for an in vivo role of CTL in control of lentiviruses, dissection of immune mechanisms in animal lentiviral infections may provide needed information. Horses infected with equine infectious anemia virus (EIAV) a lentivirus, have acute plasma viremia which is terminated in immunocompetent horses. Viremic episodes may recur, but most horses ultimately control infection and become asymptomatic carriers. ...
Takahashi T, Yawata M, Raudsepp T, Lear TL, Chowdhary BP, Antczak DF, Kasahara M.In rodents, the Ly49 family encodes natural killer (NK) receptors interacting with classical MHC class I molecules, whereas the corresponding receptors in primates are members of the killer cell immunoglobulin-like receptor (KIR) family. Recent evidence indicates that the cattle, domestic cat, dog, and pig have a single LY49 and multiple KIR genes, suggesting that predominant NK receptors in most non-rodent mammals might be KIR. Here, we show that the horse has at least six LY49 genes, five with an immunoreceptor tyrosine-based inhibition motif (ITIM) and one with arginine in the transmembrane...
Gustafson AL, Tallmadge RL, Ramlachan N, Miller D, Bird H, Antczak DF, Raudsepp T, Chowdhary BP, Skow LC.A physical map of ordered bacterial artificial chromosome (BAC) clones was constructed to determine the genetic organization of the horse major histocompatibility complex. Human, cattle, pig, mouse, and rat MHC gene sequences were compared to identify highly conserved regions which served as source templates for the design of overgo primers. Thirty-five overgo probes were designed from 24 genes and used for hybridization screening of the equine USDA CHORI 241 BAC library. Two hundred thirty-eight BAC clones were assembled into two contigs spanning the horse MHC region. The first contig contain...
Kydd J, Antczak DF, Allen WR, Barbis D, Butcher G, Davis W, Duffus WP, Edington N, Grünig G, Holmes MA.The First International Workshop on Equine Leucocyte Antigens was organized and convened for the purposes of identifying immunologically relevant cell surface molecules of equine leucocytes and establishing a system of nomenclature for those molecules. Participating members of the workshop represented the majority of laboratories world-wide engaged in the tasks of production and characterization of equine leucocyte and lymphocyte markers using monoclonal antibodies. The workshop confirmed the identification of several equine CD molecules described previously by individual laboratories, and in ...
Paebst F, Piehler D, Brehm W, Heller S, Schroeck C, Tárnok A, Burk J.Horses are an approved large animal model for therapies of the musculoskeletal system. Especially for tendon disease where cell-based therapy is commonly used in equine patients, the translation of achieved results to human medicine would be a great accomplishment. Immunophenotyping of equine mesenchymal stromal cells (MSCs) remains the last obstacle to meet the criteria of the International Society for Cellular Therapy (ISCT) definition of human MSCs. Therefore, the surface antigen expression of CD 29, CD 44, CD 73, CD 90, CD 105, CD 14, CD 34, CD 45, CD 79α, and MHC II in equine MSCs from a...
Böer U, Lohrenz A, Klingenberg M, Pich A, Haverich A, Wilhelmi M.Decellularized equine carotid arteries (dEAC) may represent a reasonable alternative to alloplastic materials in vascular replacement therapy. Acellularity of the matrix is standardly evaluated by DNA quantification what however may not record sufficiently the degree of matrix immunogenicity. Thus, our aim was to analyze dEAC with a low DNA content for residual cellular proteins. A detergent-based decellularization protocol including endonuclease treatment resulted in dEAC with 0.6 ± 0.15 ng DNA/mg dry weight representing 0.33 ± 0.14% of native tissue DNA content. In contrast, when matrices ...
de Mestre A, Noronha L, Wagner B, Antczak DF.Split immunological tolerance refers to states in which an individual is capable of mounting certain types of immune responses to a particular antigenic challenge, but is tolerant of the same antigen in other compartments of the immune system. This concept is applicable to the immunological relationship between mother and fetus, and particularly relevant in equine pregnancy. In pregnant mares, antibody responses to paternal foreign Major Histocompatibility Complex class I antigens are robust, while anti-paternal cytotoxic T cell responses are diminished compared to those mounted by non-pregnan...
Lazary S, Antczak DF, Bailey E, Bell TK, Bernoco D, Byrns G, McClure JJ.Six laboratories participated in the Fifth International Workshop on Lymphocyte Alloantigens of the Horse, testing 132 alloantisera against lymphocytes of 880 horses chosen to represent different families and breeds. Most of the alloantisera were produced by lymphocyte immunization between horses matched at the ELA-A locus. All horses were also tested with antisera contributed to the workshop by participating laboratories which identified ELA specificities A1-A10 and W12-W21. Previously identified workshop specificities ELA-W14, W15 and W19 were accepted as products of the ELA-A locus based on...
Tessier L, Bienzle D, Williams LB, Koch TG.Multipotent mesenchymal stromal cells (MSC) have attracted interest for their cytotherapeutic potential, partly due to their immunomodulatory abilities. The aim of this study was to test the robustness of our equine cord blood (CB) MSC isolation protocol, to characterize the CB-MSC before and after cryopreservation, and to evaluate their immunosuppressive phenotype. We hypothesized that MSC can be consistently isolated from equine CB, have unique and reproducible marker expression and in vitro suppress lymphoproliferation. Preliminary investigation of constitutive cytoplasmic Toll-like recepto...
Tallmadge RL, Campbell JA, Miller DC, Antczak DF.The genomic sequences of 15 horse major histocompatibility complex (MHC) class I genes and a collection of MHC class I homozygous horses of five different haplotypes were used to investigate the genomic structure and polymorphism of the equine MHC. A combination of conserved and locus-specific primers was used to amplify horse MHC class I genes with classical and nonclassical characteristics. Multiple clones from each haplotype identified three to five classical sequences per homozygous animal and two to three nonclassical sequences. Phylogenetic analysis was applied to these sequences, and gr...
Tseng CT, Miller D, Cassano J, Bailey E, Antczak DF.A system for identifying equine major histocompatibility complex (MHC) haplotypes was developed based on five polymorphic microsatellites located within the MHC region on ECA 20. Molecular signatures for 50 microsatellite haplotypes were recognized from typing 353 horses. Of these, 23 microsatellite haplotypes were associated with 12 established equine leucocyte antigen (ELA) haplotypes in Thoroughbreds and Standardbreds. Five ELA serotypes were associated with multiple microsatellite subhaplotypes, expanding the estimates of diversity in the equine MHC. The strong correlations between serolog...
Allen G, Yeargan M, Costa LR, Cross R.An experimental system that permits sensitive and reproducible detection of equine herpesvirus 1 (EHV-1)-specific cytotoxic T-lymphocyte (CTL) activity in the horse was developed. Peripheral blood mononuclear cells (PBMC) collected from immune horses were restimulated in vitro by culture with live EHV-1. Cytotoxic activity against virus-infected, pokeweed mitogen-stimulated lymphoblast targets was assessed in a 4-h 51Cr release assay. The optimal conditions for in vitro stimulation of equine memory CTLs and for preparation of EHV-1-infected target cells expressing viral antigens were systemati...
Tallmadge RL, Lear TL, Antczak DF.The availability of a contig of bacterial artificial chromosome (BAC) clones spanning the equine major histocompatibility complex (MHC) made possible a detailed analysis of horse MHC class I genes. Prior to this study, only a single horse MHC class I gene had been sequenced at the genomic level. Although many ( approximately 60) MHC class I cDNA sequences had been determined and published, from this information, it was not possible to determine how many class I loci are expressed in horses or to assign individual sequences to allelic series. In this study, 15 MHC class I genes were identified ...
Corradin G, Chiller JM.Murine T-lymphocyte specificity was determined in a system of antigen driven in vitro T-cell proliferation using cytochrome c molecules from different species, their derived peptides and reconstituted hybrid proteins. It was observed that primed T cells could discriminate between peptide fragments which differed from each other at a single amino acid residue. These conclusions were substantiated by the pattern of cross-reactivity noted in the response of closely related cytochrome c proteins as well as when artificial hybrid molecules reconstituted by the covalent linkage of peptide fragments ...
Noronha LE, Antczak DF.The horse has proven to be a distinctively informative species in the study of pregnancy immunology for several reasons. First, unique aspects of the anatomy and physiology of the equine conceptus facilitate approaches that are not possible in other model organisms, such as non-surgical recovery of early stage embryos and conceptuses and isolation of pure trophoblast cell populations. Second, pregnant mares make strong cytotoxic antibody responses to paternal major histocompatibility complex class I antigens expressed by the chorionic girdle cells, permitting detailed evaluation of the antigen...
Quintana AM, Landolt GA, Annis KM, Hussey GS.Our understanding of innate immunity within the equine respiratory tract is limited despite growing evidence for its key role in both the immediate defense and the shaping of downstream adaptive immune responses to respiratory disease. As the first interface to undergo pathogen invasion, the respiratory epithelium is a key player in these early events and our goal was to examine the innate immune characteristics of equine respiratory epithelia and compare them to an in vitro equine respiratory epithelial cell model cultured at the air-fluid interface (AFI). Respiratory epithelial tissues, isol...
Ma G, Feineis S, Osterrieder N, Van de Walle GR.Major histocompatibility complex class I (MHC-I) molecules play an important role in host immunity to infection by presenting antigenic peptides to cytotoxic T lymphocytes (CTLs), which recognize and destroy virus-infected cells. Members of the Herpesviridae have developed multiple mechanisms to avoid CTL recognition by virtue of downregulation of MHC-I on the cell surface. We report here on an immunomodulatory protein involved in this process, pUL56, which is encoded by ORF1 of equine herpesvirus type 1 (EHV-1), an alphaherpesvirus. We show that EHV-1 pUL56 is a phosphorylated early protein w...
Ellis SA, Martin AJ, Holmes EC, Morrison WI.Nineteen horse MHC class I specificities have been serologically identified previously at a single locus (ELA-A), and two other specificities appear to be coded at other loci. Biochemical studies indicate that there are at least two expressed loci. In order to establish the number of transcribed horse MHC class I genes, we made a cDNA library from a heterozygous animal (ELA-A3/A7), and screened for positive clones using a bovine class I probe. More than 200 class I clones were isolated in this way, and so far seven unique full length sequences have been identified. All of the sequences are pre...
Murata D, Miyakoshi D, Hatazoe T, Miura N, Tokunaga S, Fujiki M, Nakayama K, Misumi K.Cartilage regeneration with cell therapy following arthroscopic surgery could be used in racehorses with intra-articular fractures (IAF) and osteochondritis dissecans (OCD). The aims of this study were to investigate the origin and multipotency of stromal cells in the synovial fluid (SF) of horses with intra-articular injury and synovitis, and to provide a new strategy for regeneration of lost articular cartilage. Mesenchymal stromal cells were isolated from SF of horses with IAF and OCD. Multipotency was analysed by RT-PCR for specific mRNAs and staining for production of specific extracellul...
Zhang W, Lonning SM, McGuire TC.Most equine infectious anemia virus (EIAV)-infected horses have acute clinical disease, but they eventually control the disease and become lifelong carriers. Cytotoxic T lymphocytes (CTL) are considered an important immune component in the control of infections with lentiviruses including EIAV, but definitive evidence for CTL in the control of disease in carrier horses is lacking. By using retroviral vector-transduced target cells expressing different Gag proteins and overlapping synthetic peptides of 16 to 25 amino acids, peptides containing at least 12 Gag CTL epitopes recognized by virus-st...
