Flow cytometry is a technology used to analyze the physical and chemical characteristics of cells or particles in horses. This technique involves suspending cells in a fluid stream and passing them through a laser beam, where they are individually measured for various parameters such as size, complexity, and fluorescence intensity. In equine research, flow cytometry is applied to study immune cell populations, assess cellular responses to pathogens, and evaluate hematological parameters. It is a valuable tool for veterinary diagnostics, allowing for detailed analysis of blood and tissue samples. This page compiles peer-reviewed research studies and scholarly articles that explore the applications, methodologies, and findings related to flow cytometry in equine health and disease.
Schwab UE, Tallmadge RL, Matychak MB, Felippe MJB.OBJECTIVE To develop an in vitro system for differentiation of equine B cells from bone marrow hematopoietic progenitor cells on the basis of protocols for other species. SAMPLE Bone marrow aspirates aseptically obtained from 12 research horses. PROCEDURES Equine bone marrow CD34 cells were sorted by use of magnetic beads and cultured in medium supplemented with cytokines (recombinant human interleukin-7, equine interleukin-7, stem cell factor, and Fms-like tyrosine kinase-3), murine OP9 stromal cell preconditioned medium, and equine fetal bone marrow mesenchymal stromal cell preconditioned me...
Esteves CL, Donadeu FX.The discovery that pericytes are in vivo counterparts of Mesenchymal Stem/Stromal Cells (MSCs) has placed these perivascular cells in the research spotlight, bringing up hope for a well-characterized cell source for clinical applications, alternative to poorly defined, heterogeneous MSCs preparations currently in use. Native pericytes express typical MSC markers and, after isolation by fluorescence-activated cell sorting, display an MSC phenotype in culture. These features have been demonstrated in different species, including humans and horses, the main targets of regenerative treatments. Sig...
Schubert S, Brehm W, Hillmann A, Burk J.For clinical applications of multipotent mesenchymal stromal cells (MSCs), serum-free culture is preferable to standardize cell products and prevent contamination with pathogens. In contrast to human MSCs, knowledge on serum-free culture of large animal MSCs is limited, despite its relevance for preclinical studies and development of veterinary cellular therapeutics. This study aimed to evaluate the suitability of a commercially available serum-free human MSC medium for culturing equine adipose-derived MSCs in comparison with human adipose MSCs. Enzyme-free isolation by explant technique and e...
Michler JK, Hillmann A, Savkovic V, Mülling CKW.The easily accessible niche represented by skin and its appendages may serve as a promising source to complement modern regenerative medicine for horses. In humans and in animal models for human medicine, the hair follicle and its stem cell niches are well characterized. Since literature in this field of equine research is scarce, we sought to analyze cells of the dermal stem cell niche of the equine hair follicle morphologically and for a subset of markers useful for cell characterization via immunolabeling. We cultured equine forelock skin explants to obtain cultures with cells migrating fro...
Oldenhof H, Bigalk J, Hettel C, de Oliveira Barros L, Sydykov B, Bajcsy ÁC, Sieme H, Wolkers WF.In this study, modeling and experimental approaches were used to investigate the interplay between cooling rate and protectant concentration for cryopreservation of stallion sperm. Glycerol (GLY), ethylene glycol (EG), dimethylformamide (DMF), propylene glycol (PG), and dimethyl sulfoxide (DMSO) were tested as cryoprotective agents (CPAs), using concentrations up to 1500 mM and cooling rates ranging from 5°C to 55°C min. Modeling of the extent of sperm dehydration during freezing was done using previously determined values of the sperm membrane permeability to water to predict optimal cool...
Freer H, Hillegas JM, Wimer C, Baldwin C, LaBresh J, Wagner B.Interleukin-2 (IL-2) is a T cell growth factor and major modulator of T helper (Th) cell differentiation. Here, we have developed and characterized a monoclonal antibody to equine IL-2 (anti-IL-2 mAb, clone 158-1). The IL-2 mAb detected rIL-2 by ELISA, intracellular staining and flow cytometry analysis and Western blotting. The IL-2 mAb was also paired with a polyclonal IL-2 detection antibody in both ELISA and a fluorescent bead-based assay. When these two assays were compared using identical reagents there was an improved analytical sensitivity (46pg/ml) and wider linear quantification range...
Ness SL, Frye AH, Divers TJ, Rishniw M, Erb HN, Brooks MB.OBJECTIVE To determine effects of oral administration of Yunnan Baiyao on platelet activation, coagulation, and fibrinolysis in healthy horses. ANIMALS 12 healthy adult horses. PROCEDURES In a randomized blinded crossover study that included a 4-week washout period between treatments, horses were orally administered a paste containing Yunnan Baiyao (15 mg/kg) or placebo at 12-hour intervals for 3 days. Blood samples were collected before start of treatment (time 0) and at 24 and 72 hours for a CBC, measurement of fibrinogen concentration, coagulation screening tests, and a panel of assays to a...
Rink BE, Amilon KR, Esteves CL, French HM, Watson E, Aurich C, Donadeu FX.Equine mesenchymal stromal/stem cells (MSCs) are most commonly harvested from bone marrow (BM) or adipose tissue, requiring the use of surgical procedures. By contrast, the uterus can be accessed nonsurgically, and may provide a more readily available cell source. While human endometrium is known to harbor mesenchymal precursor cells, MSCs have not been identified in equine endometrium. This study reports the isolation, culture, and characterization of MSCs from equine endometrium. The presence of MSC and pericyte markers in endometrial sections was determined using immunohistochemistry. Strom...
Ortiz-Rodriguez JM, Anel-Lopez L, Martín-Muñoz P, Álvarez M, Gaitskell-Phillips G, Anel L, Rodríguez-Medina P, Peña FJ, Ortega Ferrusola C.Testicular function is particularly susceptible to vascular insult, resulting in a negative impact on sperm production and quality of the ejaculate. A prompt diagnosis of testicular dysfunction enables implementation of appropriate treatment, hence improving fertility forecasts for stallions. The present research aims to: (1) assess if Doppler ultrasonography is a good tool to diagnose stallions with testicular dysfunction; (2) to study the relationship between Doppler parameters of the testicular artery and those of sperm quality assessed by flow cytometry and (3) to establish cut off values ...
Maia L, de Moraes CN, Dias MC, Martinez JB, Caballol AO, Testoni G, de Queiroz CM, Peña RD, Landim-Alvarenga FC, de Oliveira E.To the best of our knowledge, this is the first study describing the proteome of equine umbilical cord intervascular matrix mesenchymal stem cells (UCIM-MSCs) in a global and functional manner. The aim of this work was to analyze the proteome of previously characterized UCIM-MSCs to determine protein abundance and classify the identified proteins according to Gene Ontology (GO) terms. Protein classification analysis according to biological process, molecular function and cellular component was performed using the PANTHER (Protein ANalysis THrough Evolutionary Relationships) Classification Syst...
Esteves CL, Sheldrake TA, Dawson L, Menghini T, Rink BE, Amilon K, Khan N, Péault B, Donadeu FX.Mesenchymal stem/stromal cells (MSCs) have been used in human and equine regenerative medicine, and interest in exploiting their potential has increased dramatically over the years. Despite significant effort to characterize equine MSCs, the actual origin of these cells and how much of their native phenotype is maintained in culture have not been determined. In this study, we investigated the relationship between MSCs, derived from adipose tissue (AT) and bone marrow (BM), and pericytes in the horse. Both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD90, and CD73) markers were detected in ...
Battut IB, Kempfer A, Lemasson N, Chevrier L, Camugli S.Spermatozoa from some stallions do not maintain an acceptable fertility after freezing and thawing. The selection of frozen ejaculates that would be suitable for insemination is mainly based on post-thaw motility, but the prediction of fertility remains limited. A recent study in our laboratory has enabled the determination of a new protocol for the evaluation of fresh stallion semen, combining microscopical observation, computer-assisted motility analysis and flow cytometry, and providing a high level of fertility prediction. The purpose of the present experiment was to perform similar invest...
Esteves CL, Sheldrake TA, Mesquita SP, Pesántez JJ, Menghini T, Dawson L, Péault B, Donadeu FX.In contrast to humans in which mesenchymal stem/stromal cell (MSC) therapies are still largely in the clinical trial phase, MSCs have been used therapeutically in horses for over 15 years, thus constituting a valuable preclinical model for humans. In human tissues, MSCs have been shown to originate from perivascular cells, namely pericytes and adventitial cells, which are identified by the presence of the cell surface markers CD146 and CD34, respectively. In contrast, the origin of MSCs in equine tissues has not been established, preventing the isolation and culture of defined cell population...
Lang HM, Schnabel LV, Cassano JM, Fortier LA.Mesenchymal stem cells (MSCs) are frequently delivered via needle injection for treatment of musculoskeletal injuries. The purpose of this study was to evaluate the effect of needle diameter on the viability of MSCs. Methods: Equine bone marrow-derived MSCs from 5 horses were suspended in PBS, and held at room temperature for 7 hours to mimic shipping conditions. Two replicate samples for each needle size (20, 22, 23, or 25-gauge [ga]) were aspirated into a 3 mL syringe and re-injected into the holding vial 3 times, to reproduce the resuspension of cells prior to injection in clinical cases. C...
Pfaender S, Walter S, Grabski E, Todt D, Bruening J, Romero-Brey I, Gather T, Brown RJ, Hahn K, Puff C, Pfankuche VM, Hansmann F, Postel A, Becher P....Hepatitis C virus (HCV) displays a restricted host species tropism and only humans and chimpanzees are susceptible to infection. A robust immunocompetent animal model is still lacking, hampering mechanistic analysis of virus pathogenesis, immune control, and prophylactic vaccine development. The closest homolog of HCV is the equine nonprimate hepacivirus (NPHV), which shares similar features with HCV and thus represents an animal model to study hepacivirus infections in their natural hosts. We aimed to dissect equine immune responses after experimental NPHV infection and conducted challenge ex...
Peña FJ, Ball BA, Squires EL.Multiparametric assessment of stallion sperm quality using flow cytometry can be a useful adjunct in semen evaluation; however, the availability of flow cytometers in veterinary practice is limited. The ability to preserve and transport sperm samples for later flow cytometric analysis using fixable probes would potentially facilitate this process. In the current study, we validated the combination of live/dead Zombie Green (a fixable dye used to assess live and dead sperm) and MitoTracker Deep Red (used to assess mitochondrial membrane potential). The assay was validated against classic, non-f...
Elzinga SE, Rohleder B, Schanbacher B, McQuerry K, Barker VD, Adams AA.Extracts derived from the leaves of the stevia plant (stevioside) are commonly used as sweeteners for humans and horses. Stevioside appears to be safe for human consumption, including for individuals with insulin dysregulation. In the horse, the safety or metabolic effects of stevioside on normal animals or on those with metabolic dysfunction are unknown. Furthermore, the inflammatory response to a glycemic challenge or to stevioside in horses is not well defined. Therefore, the objective of this study was to measure the effects of stevioside and a glycemic challenge on insulin, glucose, and i...
Mancini F, Nannarone S, Buratta S, Ferrara G, Stabile AM, Vuerich M, Santinelli I, Pistilli A, Chiaradia E.To assess the effects of xylazine and dexmedetomidine on equine chondrocytes, in vitro. Methods: Prospective, experimental study. Methods: Equine articular chondrocytes from five male horses. Methods: Chondrocytes were isolated from healthy equine articular cartilage of the metacarpo/metatarsophalangeal joints. Cell viability was assessed using the WST-8 assay by exposing chondrocytes to xylazine (0.5, 1, 2, 4, 8, 16.6, 25, 50 mg mL) or dexmedetomidine (0.001, 0.005, 0.01, 0.05, 0.175, 0.25 mg mL) for 15, 30 and 60 minutes. Based on the results of these tests, cells were treated with xyla...
Prieto JMB, Tallmadge RL, Felippe MJB.Identification and classification of B cell subpopulations has been shown to be challenging and inconsistent among different species. Our study tested aspects of ontogeny, phenotype, tissue distribution, and function of equine CD5 B cells, which represented a greater proportion of B cells early in development and in the peritoneal cavity. CD5 and CD5 B cells differentially expressed B cell markers (CD2, CD21, IgM) measured using flow cytometry, but similar mRNA expression of signature genes (DGKA, FGL2, PAX5, IGHM, IL10) measured using quantitative RT-PCR. Sequencing lambda light chain segment...
Hernandez D, Yeo WM, Brooks MB, Ness SL, Divers TJ, Stokol T.OBJECTIVE To evaluate the effects of treatment of horses with standard platelet inhibitors on ex vivo inhibition of platelet activation by equine herpesvirus type I (EHV-I). ANIMALS II healthy adult horses. PROCEDURES In a double-blinded, placebo-controlled crossover study, horses were treated orally for 5 days with theophylline (5 mg/kg, q 12 h), pentoxifylline (10 mg/kg, q 12 h), clopidogrel bisulfate (4 mg/kg, q 24 h), acetylsalicylic acid (20 mg/kg, q 24 h), or placebo. Horses received all treatments, each separated by a 3-week washout period. Platelet-rich plasma was prepared from citrate...
Stokol T, Serpa PBS, Zahid MN, Brooks MB.Equid herpes virus type-1 (EHV-1) is a major pathogen of horses, causing abortion storms and outbreaks of herpes virus myeloencephalopathy. These clinical syndromes are partly attributed to ischemic injury from thrombosis in placental and spinal vessels. The mechanism of thrombosis in affected horses is unknown. We have previously shown that EHV-1 activates platelets through virus-associated tissue factor-initiated thrombin generation. Activated platelets participate in thrombus formation by providing a surface to localize coagulation factor complexes that amplify and propagate thrombin genera...
Ziegler A, Everett H, Hamza E, Garbani M, Gerber V, Marti E, Steinbach F.Dendritic cells are professional antigen-presenting cells that play an essential role in the initiation and modulation of T cell responses. They have been studied widely for their potential clinical applications, but for clinical use to be successful, alternatives to xenogeneic substances like fetal bovine serum (FBS) in cell culture need to be found. Protocols for the generation of dendritic cells ex vivo from monocytes are well established for several species, including horses. Currently, the gold standard protocol for generating dendritic cells from monocytes across various species relies u...
Sharpe AN, Seeto WJ, Winter RL, Zhong Q, Lipke EA, Wooldridge AA.OBJECTIVE To evaluate optimal isolation of endothelial colony-forming cells (ECFCs) from peripheral blood of horses. SAMPLE Jugular and cephalic venous blood samples from 17 adult horses. PROCEDURES Each blood sample was divided; isolation was performed with whole blood adherence (WBA) and density gradient centrifugation (DGC). Isolated cells were characterized by uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI-Ac-LDL), vascular tubule formation, and expression of endothelial (CD34, CD105, vascular endothelial growth ...
Owens SD, Kol A, Walker NJ, Borjesson DL.Background. It is unknown whether horses that receive allogeneic mesenchymal stem cells (MSCs) injections develop specific humoral immune response. Our goal was to develop and validate a flow cytometric MSC crossmatch procedure and to determine if horses that received allogeneic MSCs in a clinical setting developed measurable antibodies following MSC administration. Methods. Serum was collected from a total of 19 horses enrolled in 3 different research projects. Horses in the 3 studies all received unmatched allogeneic MSCs. Bone marrow (BM) or adipose tissue derived MSCs (ad-MSCs) were admini...
Ziegler A, Marti E, Summerfield A, Baumann A.Dendritic cells (DC) are antigen-presenting cells that can be classified into three major cell subsets: conventional DC1 (cDC1), cDC2 and plasmacytoid DCs (pDC), none of which have been identified in horses. Therefore, the objective of this study was to identify and characterize DC subsets in equine peripheral blood, emphasizing on pDC. Surface marker analysis allowed distinction of putative DC subsets, according to their differential expression of CADM-1 and MHC class II. Equine pDC were found to be Flt3(+) CD4(low) CD13(-) CD14(-) CD172a(-) CADM-1(-) MHCII(low). The weak expression of CD4 on...
Burk J, Berner D, Brehm W, Hillmann A, Horstmeier C, Josten C, Paebst F, Rossi G, Schubert S, Ahrberg AB.Tendon disease has been treated with multipotent mesenchymal stromal cells (MSCs) in the equine large-animal model with promising success. The aim of this study was to gain more insight into the fate and biodistribution of MSCs after local application into tendon lesions by long-term cell tracking in this large-animal model. Superficial digital flexor tendon lesions were induced in all limbs in six horses and injected with 10106 Molday ION Rhodamine B-labeled MSCs suspended in serum or serum alone. Follow-up was performed using low-field magnetic resonance imaging (MRI), flow cytometry, and hi...
Murata D, Yamasaki A, Matsuzaki S, Sunaga T, Fujiki M, Tokunaga S, Misumi K.Dedifferentiated fat (DFAT) cells have been shown to be multipotent, similar to mesenchymal stem cells (MSCs). In this study, we aimed to establish and characterize equine DFAT cells. Equine adipocytes were ceiling cultured, and then dedifferentiated into DFAT cells by the seventh day of culture. The number of DFAT cells was increased to over 10 million by the fourth passage. Flow cytometry of DFAT cells showed that the cells were strongly positive for CD44, CD90, and major histocompatibility complex (MHC) class I; moderately positive for CD11a/18, CD105, and MHC class II; and negative for CD3...
Anderson SL, Singh B.Horses that develop colitis invariably exhibit signs of a systemic inflammatory response syndrome (SIRS). A significant contributor to the development of SIRS in human subjects is delayed neutrophil apoptosis, but this has not been specifically studied in horses. Objective: To determine the occurrence of ex vivo neutrophil apoptosis and its contribution to the development of SIRS in an equine colitis model. Methods: Experiment using a colitis model. Methods: Neutrophils were isolated before and after the induction of colitis using an oligofructose overdose model, placed into culture for 12 h o...
Gibb Z, Grupen CG, Maxwell WM, Morris LH.The fertility of sex-sorted, cryopreserved stallion sperm must be improved for the sex-sorting technology to be applied commercially. Objective: To optimise the conditions used to liquid store stallion sperm prior to sex-sorting and assess the fertility of sperm following sex-sorting and cryopreservation. Methods: Both in vitro experiment and randomised controlled trial in healthy, client-owned mares. Methods: Stallion ejaculates (n = 9) were diluted in either a skimmed milk (KMT) or BSA (I-BSA) based media to 25 × 106 sperm/ml directly (+SP25) or washed to remove seminal plasma and diluted t...
Murcia RY, Vargas A, Lavoie JP.Glucocorticoids (GCs) are the most effective drugs for the treatment of human asthma. However, a subgroup of asthmatic patients with neutrophilic airway inflammation is insensitive to GCs. Interleukin-17 (IL-17), a cytokine upregulated in the airways of a subset of human asthmatic patients, contributes to the recruitment of neutrophils and induces a glucocorticoid resistance in human airway epithelial cells. We hypothesized that IL-17 similarly activates neutrophils and contributes to their persistence in the asthmatic airways in spite of glucocorticoid therapy. To determine whether IL-17 dire...
Macieira S, Rivard GE, Champagne J, Lavoie JP, Bédard C.An 18-month-old Oldenbourg filly was presented with a bleeding diathesis. Laboratory testing included platelet count, gingival bleeding time, prothrombin time (PT), activated partial thromboplastin time (aPTT), von Willebrand factor (vWf) antigen, clottable fibrinogen, clot retraction time, PFA-100 closure time, platelet aggregometry (on platelet-rich plasma), and thrombelastography (TEG). TEG was performed by using kaolin and tissue factor as coagulation activators. Expression of the platelet receptor for fibrinogen was assessed by flow cytometry by using anti CD41 (alpha(IIb) or glycoprotein...
McTaggart C, Yovich JV, Penhale J, Raidal SL.Flow cytometric assays were used to compare phagocytic and oxidative burst activity of neutrophils from healthy foals less than 7 days of age with the activity of cells from healthy adult horses. The phagocytosis of Staphylococcus aureus by foal neutrophils was less than that observed for adult neutrophils when autologous serum was used as the source of opsonins in the assay. The use of adult serum did not significantly improve the ability of foal neutrophils to attach bacteria. The oxidative burst activity of foal neutrophils was equivalent to that of adult cells. However, when serum or plasm...
Lavoie-Lamoureux A, Beauchamp G, Quessy S, Martin JG, Lavoie JP.To compare innate immune responses of peripheral blood leukocytes from healthy and asymptomatic heaves-affected horses. Methods: Heaves-affected horses (n=5-6) and healthy controls (n=4-5) kept under low dust environments (pasture or shavings and pellets). Methods: Blood neutrophil and neutrophil-depleted cell populations were isolated using MACS system. Cells were incubated with or without bacterial products (lipopolysaccharide (LPS), 100 ng/mL and fMLP, 5 ng/mL, 5 h). Cytokine (IL-1β, IL-8, TNF, IL-4, INFγ and IL-10) and receptor (TLR4) mRNA expression was assessed by qPCR. TNF concentrati...
Silva MG, Bastos RG, Stone Doggett J, Riscoe MK, Pou S, Winter R, Dodean RA, Nilsen A, Suarez CE.The most common apicomplexan parasites causing bovine babesiosis are Babesia bovis and B. bigemina, while B. caballi and Theileria equi are responsible for equine piroplasmosis. Treatment and control of these diseases are usually achieved using potentially toxic chemotherapeutics, such as imidocarb diproprionate, but drug-resistant parasites are emerging, and alternative effective and safer drugs are needed. The endochin-like quinolones (ELQ)-300 and ELQ-316 have been proven to be safe and efficacious against related apicomplexans, such as Plasmodium spp., with ELQ-316 also being effective aga...
Warner S, Hartley CA, Stevenson RA, Ficorilli N, Varrasso A, Studdert MJ, Crabb BS.Equine rhinitis A virus (ERAV) is a respiratory pathogen of horses and is classified as an Aphthovirus, the only non-Foot-and-mouth disease virus (FMDV) member of this genus. In FMDV, virion protein 1 (VP1) is a major target of protective antibodies and is responsible for viral attachment to permissive cells via an RGD motif located in a distal surface loop. Although both viruses share considerable sequence identity, ERAV VP1 does not contain an RGD motif. To investigate antibody and receptor-binding properties of ERAV VP1, we have expressed full-length ERAV VP1 in Escherichia coli as a glutat...
Aguiar C, Theoret C, Smith O, Segura M, Lemire P, Smith LC.Induced pluripotent stem cells (iPSC) have brought immense hope to cellular therapy and regenerative medicine. However, the antigenicity of iPSC has not been well documented and remains a hurdle for clinical applications. Expression of major histocompatibility complex (MHC) molecules by human and murine iPSC is downregulated, making these cells potentially safe for transplantation. No such data are available for any large animal model. Objective: To measure expression of MHC molecules on equine iPSC (eiPSC) and describe their antigenicity using intradermal testing. The hypothesis was that allo...
Hamza E, Akdis CA, Wagner B, Steinbach F, Marti E.Insect bite hypersensitivity (IBH) is a recurrent allergic dermatitis of horses with similarities to human atopic eczema, caused by bites of insects of the genus Culicoides. Previous studies suggested a dysregulated T cell tolerance to Culicoides allergen in IBH-affected horses. Objective: We have investigated whether the suppressive function of CD4(+) CD25(high) cells is impaired in IBH-affected horses and possible ways to restore it. Methods: CD4(+) CD25(-) cells sorted from peripheral blood mononuclear cells (PBMC) were stimulated with irradiated autologous PBMC pulsed with Culicoides or te...
Bullido R, Doménech N, Alvarez B, Alonso F, Babín M, Ezquerra A, Ortuño E, Domínguez J.A set of five monoclonal antibodies (mAb) against porcine major histocompatibility complex (MHC), or swine leukocyte antigens (SLA), class II molecules has been characterized. These mAbs appear to recognize monomorphic determinants on SLA-DR (2F4, 1F12 and 2E9/13) and SLA-DQ (BL2H5 and BL4H2) molecules, as assessed by flow cytometry and immunoprecipitation. By Western blot, the 2F4, 1F12, BL2H5 and BL4H2 epitopes were located on the beta-chains of these molecules. mAbs 2F4 and 1F12 crossreact with leucocytes of dog, cattle, horse and human; mAbs 2E9/13, BL2H5 and BL4H2 bind leucocytes of cattl...
Ibrahim S, Steinbach F.Earlier studies investigating the cross-reactivity of antibodies submitted to the HLDA8 had used flow cytometry as a method of choice to screen mAbs for reactivity with equine leukocytes, including two-color flow-cytometry to characterize the lymphocyte population they detect. In addition, immuno-histochemistry (IHC) was used to detect distribution of positive cells in lymphoid tissue sections. In this study we performed immunoprecipitation (IP) to complement the previous results and add valuable information regarding the molecules detected by the cross-reacting antibodies. Surface molecules f...
Steinbach F, Stark R, Ibrahim S, Gawad EA, Ludwig H, Walter J, Commandeur U, Mauel S.The myeloid cell system comprises of monocytes, macrophages (MPhi), dendritic cells (DC), Kupffer cells, osteoclasts or microglia and is also known as the mononuclear phagocytic system (MPS). Essential cytokines to differentiate or activate these cells include GM-CSF or IL-4. Important markers for characterization include CD1, CD14, CD68, CD163 and CD206. All these markers, however, were not cloned or further characterized in equids by use of monoclonal antibodies earlier. To overcome this problem with the present study, two approaches were used. First, we cloned equine cytokines and markers, ...
Peña FJ, Ortega Ferrusola C, Martín Muñoz P.Flow cytometry is currently recognized as a robust tool for the evaluation of sperm quality and function. However, within equine reproduction, this technique has not reached the sophistication of other areas of biology and medicine. In recent years, more sophisticated flow cytometers have been introduced in andrology laboratories, and the number of tests that can be potentially used in the evaluation of sperm physiology has increased accordingly. In this review, recent advances in the evaluation of stallion spermatozoa will be discussed. These new techniques in flow cytometry are able to simul...
Gressler AE, Lübke S, Wagner B, Arnold C, Lohmann KL, Schnabel CL.Equine asthma (EA) is a highly relevant disease, estimated to affect up to 20% of all horses, and compares to human asthma. The pathogenesis of EA is most likely immune-mediated, yet incompletely understood. To study the immune response in the affected lower airways, mixed leukocytes were acquired through bronchoalveolar lavage (BAL) and the cell populations were analyzed on a single-cell basis by flow cytometry (FC). Samples of 38 horses grouped as respiratory healthy or affected by mild to moderate (mEA) or severe EA (sEA) according to their history, clinical signs, and BAL cytology were ana...
Swegen A, Smith ND, Gibb Z, Curry BJ, Aitken RJ.Serine proteases are emerging as important players in the spermatozoon's acquisition of functional competence. This study aimed to characterize the serine protease testisin (PRSS21) in stallion spermatozoa, examining its surface expression, possible origins in the testis and epididymis, and changes in response to capacitation and acrosome reaction, as well as its capacity to form high molecular weight complexes and interact with other proteins. The role of serine proteases in spontaneous capacitation and acrosome reaction of stallion spermatozoa was established using the serine protease inhibi...
Aziz DM, Ahlswede L, Enbergs H.The assay of MTT reduction depends on the ability of metabolically active cells to reduce the tetrazolium salt (3[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide) to formazan. This study was conducted to examine and validate of a simple and less costly MTT test in determining equine sperm viability and compare the efficiency of this test with a flow cytometer. Fresh ejaculates from 11 stallions (warm blood) were included in this study. Semen was diluted to 100 million cells/ml in a Hepes 0.1% BSA. The rates of MTT reduction were measured in microtiter plates after incubation for 1 an...
O'Shea CM, Werre SR, Dahlgren LA.(1) To compare the performance of 4 platelet counting technologies in equine platelet concentrates and (2) to evaluate the ability of the Magellan platelet rich plasma (PRP) system to concentrate equine platelets. Methods: Experimental study to assess method agreement. Methods: Adult mixed breed horses (n = 32). Methods: Acid citrate dextrose-A anti-coagulated whole blood was collected and PRP produced using the Magellan system according to the manufacturer's instructions. Platelets were quantified using 4 counting methods: optical scatter (Advia 2120), impedance (CellDyn 3700), hand counting,...
Katila T.The article reviews methods used for in vitro evaluation of sperm, with particular emphasis on frozen-thawed stallion sperm. The techniques, limitations of the methods and correlations with fertility results are discussed. Very few studies have tried to find correlation between fertility of frozen stallion semen and laboratory tests. It is difficult and expensive to inseminate an adequate number of mares to achieve statistically significant differences. Significant, but low correlations have been demonstrated between the foaling rate and subjective motility of sperm incubated for 2 h and 4 h a...
Braune S, Walter M, Schulze F, Lendlein A, Jung F.For in vitro studies assessing the interaction of platelets with implant materials, common and standardized protocols for the preparation of platelet rich plasma (PRP) are lacking, which may lead to non-matching results due to the diversity of applied protocols. Particularly, the aging of platelets during prolonged preparation and storage times is discussed to lead to an underestimation of the material thrombogenicity. Here, we study the influence of whole blood- and PRP-storage times on changes in platelet morphology and function. Blood from apparently healthy subjects was collected according...
Norris JW, Pratt SM, Auh JH, Wilson SJ, Clutter D, Magdesian KG, Ferraro GL, Tablin F.Bleeding in racing horses associated with exercise appears to be multifactorial, and clinical investigation into severe cases rarely occurs. Previously, we reported a severe bleeding diathesis in a Thoroughbred mare. Herein, we describe the cellular physiology of this defect, provide a diagnostic tool for identifying it, and demonstrate that the dysfunction is heritable. Objective: The subject has a heritable defect in platelet secretion that reduces thrombin generation in the absence of additional plasma factors and delays the onset of thrombin production even in the presence of these factors...
Ziegler A, Marti E, Summerfield A, Baumann A.Dendritic cells (DC) are antigen-presenting cells that can be classified into three major cell subsets: conventional DC1 (cDC1), cDC2 and plasmacytoid DCs (pDC), none of which have been identified in horses. Therefore, the objective of this study was to identify and characterize DC subsets in equine peripheral blood, emphasizing on pDC. Surface marker analysis allowed distinction of putative DC subsets, according to their differential expression of CADM-1 and MHC class II. Equine pDC were found to be Flt3(+) CD4(low) CD13(-) CD14(-) CD172a(-) CADM-1(-) MHCII(low). The weak expression of CD4 on...
Lawler DF, Hopkins J, Watson ED.Recent evidence indicates that the cells of the immune system and their large network of secretory products, or cytokines, play an active role in the ovary throughout the oestrous cycle. In the present study, immune cell populations (T and B lymphocytes, macrophages, granulocytes and eosinophils) and expression of major histocompatibility complex (MHC) class II were investigated in corpora lutea from mares in early (days 2-4), mid- (days 7-10) and late (days 12-14) dioestrus, the post-luteolytic phase (days 16-17) and early pregnancy. The number of T lymphocytes within the corpus luteum increa...
Petersen GF, Hilbert B, Trope G, Kalle W, Strappe P.Equine adipose-derived mesenchymal stem cells (EADMSC) provide a unique cell-based approach for treatment of a variety of equine musculoskeletal injuries, via regeneration of diseased or damaged tissue, or the secretion of immunomodulatory molecules. These capabilities can be further enhanced by genetic modification using lentiviral vectors, which provide a safe and efficient method of gene delivery. We investigated the suitability of lentiviral vector technology for gene delivery into EADMSC, using GFP expressing lentiviral vectors pseudotyped with the G glycoprotein from the vesicular stomat...
Flaminio MJ, LaCombe V, Kohn CW, Antczak DF.A 12-year-old Quarter Horse mare that was nonresponsive to medical treatment was evaluated for chronic respiratory disease and hepatobiliary disease. Serum immunoglobulin concentrations were measured by use of radial immunodiffusion that revealed trace to nondetectable concentrations of IgG, IgG(T), IgM, and IgA. Use of serum protein electrophoresis confirmed agammaglobulinemia by the absence of the expected peak in the gamma region. In addition, vaccination with tetanus toxoid did not result in specific immunoglobulin production. Flow cytometric analysis of blood lymphocyte subpopulations rev...
Chapman HS, Gale AL, Dodson ME, Linardi RL, Ortved KF.Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are being investigated for their potential in the treatment of musculoskeletal injuries, including tendon and ligament lesions, and cartilage lesions. Culture expansion of cells has traditionally been performed in medium supplemented with fetal bovine serum (FBS), however, concerns regarding the antigenicity and potential viral or prion contamination of FBS have prompted interest in alternative medium supplements. Platelet lysate (PL) contains elevated concentrations of growth factors, including transforming growth factor-β (TGF-β), pla...
Furusawa Y, Yamada S, Itai S, Sano M, Nakamura T, Yanaka M, Handa S, Mizuno T, Maeda K, Fukui M, Harada H, Kaneko MK, Kato Y.Podoplanin (PDPN), a type I transmembrane glycoprotein, is expressed in several body tissues, including podocytes of renal glomerulus, type I alveolar cells of lung, and lymphatic endothelial cells. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) presented on platelets. Monoclonal antibodies (mAbs) against human-, mouse-, rat-, rabbit-, dog-, bovine-, and cat-PDPN have already been established. However, anti-horse PDPN mAbs have not yet been developed. In this study, we immunized mice with synthetic horse PDPN peptides and developed anti-horse PDPN mAbs...
Hansen S, Sun L, Baptiste KE, Fjeldborg J, Horohov DW.Diseases of the lower airways represent some of the most common conditions affecting horses of all ages, but the type and severity tends to follow the horses' age. The age-related dysregulation of pro-inflammatory cytokines may, in part contribute to the development of the diseases. Therefore, we hypothesize that the expression of pro-inflammatory cytokines increases with age. Peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage (BAL) cells from clinically healthy horses of different ages were used for the investigation. The cells were stimulated and the production of IFN-γ an...
Ibrahim S, Steinbach F.In addition to the 379 monoclonal antibodies (mAbs) tested in the animal homologues section of HLDA8, another 155 mAbs were screened at the Institute for Zoo and Wildlife Research, Berlin for cross-reactivity with equine leukocytes. For this purpose, one colour flow-cytometric analysis was performed as screening test. This additional screening indicated further 16 mAbs as positive with staining homologous to human pattern, 1 mAb with weak (positive) reactivity, 11 mAbs with positive, but likely not valuable staining, 12 mAbs with alternate expression pattern from that expected from human immun...
Noronha LE, Harman RM, Wagner B, Antczak DF.The immunoreceptor NKp46 is considered to be the most consistent marker of NK cells across mammalian species. Here, we use a recombinant NKp46 protein to generate a panel of monoclonal antibodies that recognize equine NKp46. The extracellular region of equine NKp46 was expressed with equine IL-4 as a recombinant fusion protein (rIL-4/NKp46) and used as an immunogen to generate mouse monoclonal antibodies (mAbs). MAbs were first screened by ELISA for an ability to recognize NKp46, but not IL-4, or the structurally related immunoreceptor CD16. Nine mAbs were selected and were shown to recognize ...
Ghosh S, Serafini R, Love CC, Teague SR, Hernández-Avilés C, LaCaze KA, Varner DD.Effects of different media and promoters on lipid peroxidation (LPO) in viable stallion sperm have not been reported. Aims of this study were to determine effects of three media (INRA-96™, Equipro CoolGuard™, and Biggers, Whitten and Whittingham [BWW]), and promoters (iron sulfate-Fe; ultraviolet light-UV; or control-no exposure to promoters) on viable sperm LPO using four different flow cytometric assays (i.e., BODIPY, Liperfluo, 4-hydroxylnonenal [4HNE], malonaldehyde [MDA]). Significant media x promoter interactions were detected using the Liperfluo, 4HNE, and MDA assays (P < 0.05)...
Love CC.Sperm quality assays have increased in number in the last 10 years. Most of these assays are flow cytometry based in application and are modified from assays that have been developed to measure somatic cell function. The goal of any sperm quality assay should be to advance the clinicians/researchers understanding of sperm cell function and the relationship to fertility. While these assays appear to measure somatic cell-like functions in sperm there tends to be little understanding how the results of these assays relate to fertility.
Viveiros MM, Antczak DF.Natural killer (NK) cells are an important component of the innate immune system. Though intensively studied in humans and rodents. NK cells remain less well characterized in other species. Studies are often limited by the lack of specific cell markers; however, the mAb NK-5C6 has been suggested to recognize an evolutionarily conserved molecule on NK cells and reacts with cells from several species. This mAb was used in the current investigation to identify and characterize equine NK cells, and was found to label approximately 10% of peripheral blood lymphocytes (PBL). Two-color flow cytometry...
