Mononuclear cells in horses are a type of immune cell that includes lymphocytes and monocytes. These cells are integral components of the equine immune system, participating in the body's defense mechanisms against pathogens. Lymphocytes are involved in the adaptive immune response, while monocytes are part of the innate immune system and can differentiate into macrophages or dendritic cells. Mononuclear cells circulate in the bloodstream and migrate to sites of infection or inflammation, where they perform various functions such as phagocytosis, antigen presentation, and cytokine production. This page compiles peer-reviewed research studies and scholarly articles that explore the characteristics, functions, and clinical relevance of mononuclear cells in equine health.
Pretorius A, Faber FE, van Kleef M.Development of African horsesickness (AHS) subunit vaccines will have to include a rational approach that uses knowledge of how the virus interacts with the host immune system. The global in vivo immune response induced by attenuated AHSV serotype 4 in horses was characterised using transcriptome sequencing. PBMC were collected with 24h intervals for four days after inoculation and four days after a second boost, 21 days later. Transcriptome data were normalised to the day 0 naïve transcriptome and up- or down-regulated immune genes identified using the CLC workbench. Peak expression was obse...
Pacholewska A, Jagannathan V, Drögemüller M, Klukowska-Rötzler J, Lanz S, Hamza E, Dermitzakis ET, Marti E, Leeb T, Gerber V.Recurrent airway obstruction (RAO) is a common and potentially debilitating lower airway disease in horses, which shares many similarities with human asthma. In susceptible horses RAO exacerbation is caused by environmental allergens and irritants present in hay dust. The objective of this study was the identification of genes and pathways involved in the pathology of RAO by global transcriptome analyses in stimulated peripheral blood mononuclear cells (PBMCs). We performed RNA-seq on PBMCs derived from 40 RAO affected and 45 control horses belonging to three cohorts of Warmblood horses: two h...
Douglas HF, Southwood LL, Meyer-Ficca ML, Hart SK, Meyer RG.To evaluate the poly (ADP-ribose) polymerase-1 (PARP1) enzyme and its inhibition in horses and explore its potential as a novel therapeutic target for equine intestinal ischemia-reperfusion injury by (1) identifying poly (ADP-ribose) (PAR) as an indication of PARP1 activation in equine cells using available immunoblot analytical techniques, (2) inducing PARP1 activation in an in vitro oxidative DNA damage model, (3) and demonstrating the inhibition of PARP1 in equine cells using commercially available PARP1 inhibitors. Methods: Experimental study. Methods: Blood samples were collected from sys...
Remacha AR, Barrachina L, Álvarez-Arguedas S, Ranera B, Romero A, Vázquez FJ, Zaragoza P, Yañez R, Martín-Burriel I, Rodellar C.The immunomodulatory capacities of mesenchymal stem cells (MSCs) have made them the subject of increased clinical interest for tissue regeneration and repair. We have studied the immunomodulatory capacity of equine MSCs derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) in cocultures with allogeneic peripheral blood mononuclear cells (PBMCs). Different isoforms and concentrations of phytohaemaglutinin (PHA) were tested to determine the best stimulation conditions for PBMC proliferation and a proliferation assay was performed for 7 days to determine the optimal day of stimulation o...
Pacholewska A, Drögemüller M, Klukowska-Rötzler J, Lanz S, Hamza E, Dermitzakis ET, Marti E, Gerber V, Leeb T, Jagannathan V.Complete transcriptomic data at high resolution are available only for a few model organisms with medical importance. The gene structures of non-model organisms are mostly computationally predicted based on comparative genomics with other species. As a result, more than half of the horse gene models are known only by projection. Experimental data supporting these gene models are scarce. Moreover, most of the annotated equine genes are single-transcript genes. Utilizing RNA sequencing (RNA-seq) the experimental validation of predicted transcriptomes has become accessible at reasonable costs. To...
Ranera B, Antczak D, Miller D, Doroshenkova T, Ryan A, McIlwraith CW, Barry F.Recently, it has been shown that mesenchymal stem cells (MSCs) do not express the major histocompatibility complex (MHC) II antigen and are able to inhibit proliferation of MHC-mismatched stimulated lymphocytes, enabling their use as in vivo allogeneic transplants. However, prior to clinical application of allo-MSCs, in vitro tests are required to confirm the safety of treatment protocols. Objective: To evaluate the immunosuppressive capabilities of equine bone-marrow-derived MSCs (BM-MSCs) on MHC-mismatched lymphocytes. Methods: In vitro experiment. Methods: Phytohaemagglutinin-stimulated per...
Espino-Solis GP, Quintero-Hernandez V, Olvera-Rodriguez A, Calderon-Amador J, Pedraza-Escalona M, Licea-Navarro A, Flores-Romo L, Possani LD.The αX I-domain of the horse integrin CD11c was successfully expressed in Escherichia coli, purified, biochemically characterized and used as immunogen to generate murine monoclonal antibodies against horse CD11c, which are not yet commercially available. One monoclonal antibody mAb-1C4 against the αX I-domain, is an IgG2a able to interact with the recombinant I-domain, showing an EC50=2.4ng according to ELISA assays. By western blot with horse PBMCs lysates the mAb-1C4 recognized a protein of 150kDa which corresponds well with the CD11c molecule. Using immunohistochemistry in horse lymph no...
Burton AG, Clark KC, Borjesson DL, Carrade DD, Burges J, Owens SD.Volume reduction and RBC depletion of equine bone marrow specimens are necessary processing steps for the immediate therapeutic use of bone marrow (BM)-derived mesenchymal stem cells (MSC), and for MSC expansion in culture. Objective: The purpose of the study was to evaluate the ability of the PrepaCyte-CB processing system to reduce volume, deplete RBC, and recover mononuclear cells (MNC) from equine BM specimens. Methods: One hundred and twenty mL of heparinized BM were obtained from each of 90 horses. A CBC was performed on the BM pre- and post-PrepaCyte-CB processing. Volume and RBC reduct...
Hatzel JN, Bouma GJ, Cleys ER, Bemis LT, Ehrhart EJ, McCue PM.Early pregnancy factor has been identified as a 10-kDa extracellular homolog of heat shock protein 10 (Hsp10). Hsp10 has been detected during early pregnancy in serum of mice, sheep, pigs, horses, cows, and humans by the rosette inhibition test. Hsp10 has also been associated with several neoplastic and autoimmune diseases. The goal of the present study was to determine if Hsp10 could be detected in the early equine embryo through the use of immunohistochemistry and quantitative real-time PCR. Additionally, analysis of systemically harvested peripheral blood mononuclear cells (PBMCs) from both...
Dossa RG, Alperin DC, Garzon D, Mealey RH, Brown WC, Jervis PJ, Besra GS, Cox LR, Hines SA.α-GalCer is a potent immunomodulatory molecule that is presented to NKT cells via the CD1 antigen-presenting system. We hypothesized that when used as an adjuvant α-GalCer would induce protective immune responses against Rhodococcus equi, an important pathogen of young horses. Here we demonstrate that the equine CD1d molecule shares most features found in CD1d from other species and has a suitable lipid-binding groove for presenting glycolipids to NKT cells. However, equine CTL stimulated with α-GalCer failed to kill cells infected with R. equi, and α-GalCer did not increase killing by C...
Manuja A, Kumar P, Kumar R, Kumar B, Singha H, Sharma RK, Yadav SC.Trypanosoma evansi is the causative agent of surra, which is the most common and widespread trypansomal disease. The infection is mainly restricted to animals, but it has also been documented in human. Trypanosomes possess the thick immunogenic surface coat known as variant surface glycoprotein (VSG). The parasite modifies the VSG constantly resulting in continuous antigenic variations and thus evades the host immune response. Due to antigenic variations, vaccination against trypanosomosis is not useful. Therefore, alternate strategies to augment the immune response are required. CpG-ODN class...
Hansen S, Baptiste KE, Fjeldborg J, Betancourt A, Horohov DW.An imbalance in various cytokine mRNA expression in bronchoalveolar lavage (BAL) and peripheral blood mononuclear cells (PBMC) in horses suffering from recurrent airway obstruction (RAO) has been demonstrated; however, the natural variation of cytokine expression in healthy horses has yet to be described. The objectives of this study were to: (1) identify age-related differences in the expression of pro-inflammatory cytokines in BAL and PBMC, (2) compare the expression of pro-inflammatory cytokines between BAL and PBMC, and (3) evaluate the relationship between BAL pro-inflammatory cytokine ex...
Schnabel CL, Wagner S, Wagner B, Durán MC, Babasyan S, Nolte I, Pfarrer C, Feige K, Murua Escobar H, Cavalleri JM.Research on equine cytokines is often performed by analyses of mRNA. For many equine cytokines an analysis on the actual protein level is limited by the availability of antibodies against the targeted cytokines. Generation of new antibodies is ongoing but time consuming. Thus, testing the reactivity of commercially available antibodies for cross-reactivity with equine cytokines is of particular interest. Fifteen monoclonal antibodies against IL-1β, IL-6, IL-8, IL-12, IL-18 and Granulocyte Macrophage Colony stimulating factor (GM-CSF) of different species were evaluated for reactivity with the...
Silva A, Wagner B, McKenzie HC, Desrochers AM, Furr MO.The objectives of this study were to (1) evaluate the effects of equine soluble CD14 (sCD14) and monoclonal antibodies (mAb) to equine CD14 on lipopolysaccharide-induced tumor necrosis factor α (TNF-α) secretion from equine peripheral blood mononuclear cells (PBMC); and to (2) determine serum concentrations of sCD14 in a population of horses with gastrointestinal diseases or other illnesses likely to result in endotoxemia. Equine PBMC isolated from 10 healthy horses were incubated with Escherichia coli LPS plus CD14 mAb or sCD14 and assayed for TNF-α activity. Pre-incubation with CD14 mAb d...
Lanz S, Gerber V, Marti E, Rettmer H, Klukowska-Rötzler J, Gottstein B, Matthews JB, Pirie S, Hamza E.Equine recurrent airway obstruction (RAO) is an inflammatory, obstructive airway disease induced by exposure of susceptible horses to inhaled organic dust particles. The immunological process underlying RAO is still unclear. Previous studies have shown that RAO is linked to the Interleukin-4 receptor (IL-4R) gene in one Warmblood family (F1), but not in another (F2). It has also been shown that in F1, but not in F2, RAO is associated with resistance against parasites, suggesting that this association may have an immuno-genetic basis. Therefore, we hypothesized that the T helper (h)1/Th2/regula...
Vendrig JC, Coffeng LE, Fink-Gremmels J.Dietary supplementation with oligosaccharides has been proven to be beneficial for health in several mammalian species. Next to prebiotic effects resulting in a modulation of gut micro biota, immunomodulatory effects of oligosaccharides have been documented in vivo. Supplementation with defined oligosaccharide fractions has been shown to attenuate allergic responses and enhance defensive immune responses. Despite the accumulating evidence for immunomodulatory effects, very limited information is available regarding the direct mechanism of action of oligosaccharides. This study aims to elucidat...
Vendrig JC, Coffeng LE, Fink-Gremmels J.Deficient innate and adaptive immune responses cause newborn mammals to be more susceptible to bacterial infections than adult individuals. Toll-like receptors (TLRs) are known to play a pivotal role in bacterial recognition and subsequent immune responses. Several studies have indicated that activation of certain TLRs, in particular TLR-2, can result in suppression of inflammatory pathology. In this study, we isolated peripheral blood mononuclear cells (PBMCs) from adult and newborn horses to investigate the influence of TLR-2 activation on the inflammatory response mediated by TLR-4. Data we...
Duran MC, Willenbrock S, Müller JM, Nolte I, Feige K, Murua Escobar H.Interleukin-12 (IL-12) and interferon gamma (IFN-γ) are key cytokines in immunemediated equine melanoma therapy. Currently, a method for accurate simultaneous quantification of these equine cytokines is lacking. Therefore, we sought to establish an assay that allows for accurate and simultaneous quantification of equine IL-12 (eIL-12) and IFN-γ (eIFN-γ). Methods: Several antibodies were evaluated for cross-reactivity to eIL-12 and eIFN-γ and were used to establish a bead-based Luminex assay, which was subsequently applied to quantify cytokine concentrations in biological samples. Results: ...
Vendrig JC, Coffeng LE, Fink-Gremmels J.Increasing evidence suggests that reactions to lipopolysaccharide (LPS), particularly in the gut, can be partly or completely mitigated by colostrum- and milk-derived oligosaccharides. Confirmation of this hypothesis could lead to the development of new therapeutic concepts. Objective: To demonstrate the influence of equine colostral carbohydrates on the inflammatory response in an in vitro model with equine peripheral blood mononuclear cells (PBMCs). Methods: Carbohydrates were extracted from mare colostrum, and then evaluated for their influence on LPS-induced inflammatory responses in PBMCs...
Keggan A, Freer H, Rollins A, Wagner B.Horses have 11 immunoglobulin isotypes: IgM, IgD, IgA, IgE, and seven IgG subclasses designated as IgG1-IgG7, each of which are distinguished by separate genes encoding the constant heavy chain regions. Immunoglobulin (Ig) isotypes have different functions during the immune response and pathogen-specific isotypes can be used as indicators for immunity and protection from disease. In addition to existing monoclonal antibodies to various equine Igs, quantification of the individual isotypes requires pure isotype standards. In this report, we describe a fusion between X63-Ag8.653 mouse myeloma ce...
Bonin CP, Baccarin RY, Nostell K, Nahum LA, Fossum C, de Camargo MM.Engagement of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) is a master trigger of the deleterious effects of septic shock. Horses and humans are considered the most sensitive species to septic shock, but the mechanisms explaining these phenomena remain elusive. Analysis of tlr4 promoters revealed high similarity among LPS-sensitive species (human, chimpanzee, and horse) and low similarity with LPS-resistant species (mouse and rat). Four conserved nuclear factor kappa B (NFκB) binding sites were found in the tlr4 promoter and two in the md2 promoter sequences that are likely to be t...
De Schauwer C, van de Walle GR, Piepers S, Hoogewijs MK, Govaere JL, Meyer E, van Soom A.The therapeutic potential of mesenchymal stromal cells for cellular therapy has generated increasing interest in human as well as veterinary medicine. Considerable research has been performed on the cryopreservation of expanded mesenchymal stromal cells, but little information is available on the cryopreservation of the original mononuclear cell fraction. Objective: The present study describes a protocol to expand equine mesenchymal stromal cells after cryopreserving the mononuclear cells of umbilical cord blood. Methods: To this end, mononuclear cells were isolated from 7 umbilical cord blood...
Kappmeyer LS, Thiagarajan M, Herndon DR, Ramsay JD, Caler E, Djikeng A, Gillespie JJ, Lau AO, Roalson EH, Silva JC, Silva MG, Suarez CE, Ueti MW....Transmission of arthropod-borne apicomplexan parasites that cause disease and result in death or persistent infection represents a major challenge to global human and animal health. First described in 1901 as Piroplasma equi, this re-emergent apicomplexan parasite was renamed Babesia equi and subsequently Theileria equi, reflecting an uncertain taxonomy. Understanding mechanisms by which apicomplexan parasites evade immune or chemotherapeutic elimination is required for development of effective vaccines or chemotherapeutics. The continued risk of transmission of T. equi from clinically silent,...
Durán MC, Willenbrock S, Carlson R, Feige K, Nolte I, Murua Escobar H.CD14 positive (CD14+) cells are the precursor cells of monocyte-derived dendritic cells (DCs). In horses their potent antigen-presenting capacity and ability to induce an effective immune response classify these cells suitable for several therapeutic approaches such as for equine sarcoid. However, in horses, the generation efficiency of DCs from adherent peripheral blood mononuclear cells (PBMCs) is currently still poor. Objective: Establishment of a simple short protocol to enhance DC generation in horses by using a human CD14 monoclonal antibody (mAb) and an automated magnetic activated cell...
Binley K, Widdowson PS, Kelleher M, de Belin J, Loader J, Ferrige G, Carlucci M, Esapa M, Chipchase D, Angell-Manning D, Ellis S, Mitrophanous K....RetinoStat(®) is an equine infectious anemia virus-based lentiviral gene therapy vector that expresses the angiostatic proteins endostatin and angiostatin that is delivered via a subretinal injection for the treatment of the wet form of age-related macular degeneration. We initiated 6-month safety and biodistribution studies in two species; rhesus macaques and Dutch belted rabbits. After subretinal administration of RetinoStat the level of human endostatin and angiostatin proteins in the vitreous of treated rabbit eyes peaked at ∼1 month after dosing and remained elevated for the duration o...
Delling U, Lindner K, Ribitsch I, Jülke H, Brehm W.The objective of this study was to compare bone marrow (BM) aspirates from the sternum and the tuber coxae of middle-aged horses. Bone marrow was obtained from the sternum and both tubera coxae of 12 healthy, 13-year-old geldings. Two different puncture techniques were used for the tuber coxae. The 2 syringes used for sternal sampling were evaluated separately. The mononuclear cell (MNC) fraction of the BM was isolated and the mesenchymal stem cells (MSCs) were culture-expanded. At the sternum, BM aspiration was always possible. Bone marrow aspiration at the tuber coxae required straight and d...
Liu C, Cook FR, Cook SJ, Craigo JK, Even DL, Issel CJ, Montelaro RC, Horohov DW.Distinct from human lentivirus infection, equine infectious anemia virus (EIAV)-infected horses will eventually enter an inapparent carrier state in which virus replication is apparently controlled by adaptive immune responses. Although recrudescence of disease can occur after immune suppression, the actual immune correlate associated with protection has yet to be determined. Therefore, EIAV provides a model for investigating immune-mediated protective mechanisms against lentivirus infection. Here, we have developed a method to monitor EIAV-envelope specific cellular immunity in vivo. An EIA c...
Spaas JH, De Schauwer C, Cornillie P, Meyer E, Van Soom A, Van de Walle GR.Although the use of mesenchymal stromal cells (MSCs) for the treatment of orthopaedic injuries in horses has been reported, no official guidelines exist that classify a particular cell as an equine MSC. Given the limited characterisation of peripheral blood (PB)-derived equine MSCs in particular, this study aimed to provide more detailed information in relation to this cell type. Mesenchymal stromal cells were isolated from equine PB samples and colony forming unit (CFU) assays as well as population doubling times (PDTs) (from P(0) to P(10)) were performed. Two types of colonies, 'fingerprint'...
Frellstedt L, McKenzie HC, Barrett JG, Furr MO.Endotoxemia is responsible for severe illness in horses. Individuals can become clinically unresponsive to the endotoxin molecule after an initial exposure; a phenomenon referred to as 'endotoxin tolerance' (ET). ET has been observed clinically in horses in vivo; however, cytokine expression associated with ET has not been investigated. The purpose of this study was to develop and validate a method for inducing ET in equine peripheral blood mononuclear cells (PBMC) in vitro, and to describe selected cytokine responses which are associated with ET. ET was induced by culturing cells with three c...
Longworth KE.Pulmonary intravascular macrophages are an important part of the mononuclear phagocyte system in some species of mammals, mainly sheep and other ruminants, pigs, and horses. These cells phagocytize foreign particles, cell debris and pathogens that pass through the pulmonary circulation. Species with intravascular macrophages localize intravenously injected tracer particles and bacteria predominantly in the lung rather than the liver, and exhibit pulmonary hypertension when these cells are activated. Both in vivo and in vitro studies show that pulmonary intravascular macrophages have distinct s...
The Journal of nutritionJuly 31, 2004
Volume 134, Issue 8 Suppl 2133S-2140S doi: 10.1093/jn/134.8.2133S
Marlin DJ, Johnson L, Kingston DA, Smith NC, Deaton CM, Mann S, Heaton P, Van Vugt F, Saunders K, Kydd J, Harris PA.Oxidative stress occurs when antioxidant defense mechanisms are overwhelmed by free radicals and may lead to DNA damage, which has been implicated in processes such as aging and diseases such as cancer. The two main techniques presently used to quantify DNA damage are measurement of 8-hydroxydeoxyguanosine and the Comet assay (also known as single-cell gel electrophoresis). The aim of this study was to apply the comet assay to equine peripheral blood mononuclear cells (PBMCs) and identify two conditions in which we hypothesized that oxidative DNA damage would be increased in PBMCs: aging and e...
BMC research notesJuly 19, 2016
Volume 9 353 doi: 10.1186/s13104-016-2161-9
Tarlinton RE, Alder L, Moreton J, Maboni G, Emes RD, Tötemeyer S.Toll like receptors are one of the major innate immune system pathogen recognition systems. There is little data on the expression of the TLR10 member of this family in the horse. Results: This paper describes the genetic structure of the Equine TLR10 gene and its RNA expression in a range of horse tissues. It describes the phylogenetic analysis of the Equine TLR1,6,10,2 annotations in the horse genome, firmly identifying them in their corresponding gene clades compared to other species and firmly placing the horse gene with other TLR10 genes from odd-toed ungulates. Additional 3' transcript e...
Mealey RH, Littke MH, Leib SR, Davis WC, McGuire TC.Although CTL are important for control of lentiviruses, including equine infectious anemia virus (EIAV), it is not known if CTL can limit lentiviral replication in the absence of CD4 help and neutralizing antibody. Adoptive transfer of EIAV-specific CTL clones into severe combined immunodeficient (SCID) foals could resolve this issue, but it is not known whether exogenous IL-2 administration is sufficient to support the engraftment and proliferation of CTL clones infused into immunodeficient horses. To address this question we adoptively transferred EIAV Rev-specific CTL clones into four EIAV-...
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...
Hellman S, Hjertner B, Morein B, Fossum C.The immunomodulatory effect of a new particulate adjuvant, G3, alone or in combination with agonists to TLR2/1 or TLR5 was evaluated in cultures of equine PBMC. Exposure to the G3 adjuvant up-regulated genes encoding IFN-γ, IL-1β, IL-6, IL-8, IL-12p40 and IL-23p19 in the majority of the horses tested, indicating that the G3 adjuvant induced a pro-inflammatory and Th1 dominated profile. In accordance, genes encoding IL-13, IL-4, IL-10 and TGF-β remained unaffected and genes encoding IFN-α, IL-17A and TNF-α were only occasionally and weakly induced. The two TLR agonists Pam3CSK4 (TLR2/1) an...
Cappelli K, Felicetti M, Capomaccio S, Pieramati C, Silvestrelli M, Verini-Supplizi A.The stress response is a critical factor in the training of equine athletes; it is important for performance and for protection of the animal against physio-pathological disorders.In this study, the molecular mechanisms involved in the response to acute and strenuous exercise were investigated using peripheral blood mononuclear cells (PBMCs). Results: Quantitative real-time PCR (qRT-PCR) was used to detect modifications in transcription levels of the genes for matrix metalloproteinase-1 (MMP-1) and interleukin 8 (IL-8), which were derived from previous genome-wide expression analysis. Signific...
Murphy BA, Vick MM, Sessions DR, Cook RF, Fitzgerald BP.Peripheral clocks receive timing signals from the master mammalian pacemaker in the suprachiasmatic nucleus (SCN) and function to adaptively anticipate daily changes that influence local physiology. Evidence suggests that peripheral immune activation may act as a resetting signal for circadian clocks in peripheral tissues. We wished to investigate whether acute systemic inflammation could synchronize clock gene expression in equine peripheral blood, a tissue that does not normally oscillate in this species. We report that in vivo administration of lipopolysaccharide (LPS) results in significan...
Fraser DG, Leib SR, Zhang BS, Mealey RH, Brown WC, McGuire TC.The effect of immunization with five lipopeptides, three containing T-helper (Th) epitopes and two with both Th and cytotoxic T-lymphocyte (CTL) epitopes, on equine infectious anemia virus (EIAV) challenge was evaluated. Peripheral blood mononuclear cells from EIAV lipopeptide-immunized horses had significant proliferative responses to Th peptides compared with those preimmunization, and the responses were attributed to significant responses to peptides Gag from positions 221 to 245 (Gag 221-245), Gag 250-269, and Pol 326-347; however, there were no consistent CTL responses. The significant pr...
Tallmadge RL, Wang M, Sun Q, Felippe MJB.During the neonatal period, the ability to generate immune effector and memory responses to vaccines or pathogens is often questioned. This study was undertaken to obtain a global view of the natural differences in the expression of immune genes early in life. Our hypothesis was that transcriptome analyses of peripheral blood mononuclear cells (PBMCs) of foals (on day 1 and day 42 after birth) and adult horses would show differential gene expression profiles that characterize natural immune processes. Gene ontology enrichment analysis provided assessment of biological processes affected by age...
Hansen S, Otten ND, Fjeldborg J, Baptiste KE, Horohov DW.The objectives of this study were to evaluate the natural age-related variation and compare the level of pro-inflammatory cytokines in the peripheral blood and lower airways of horses. The mRNA expression of IL-1β, IL-6, IL-8 TNF-α TLR-4 in bronchoalveolar lavage (BAL) fluid and peripheral blood mononuclear cells (PBMC) were studied by quantitative real time polymerase chain reaction (PCR) and differential cell count cytology from 44 horses of different ages. A significant age-related increase was found for the mRNA expression of IL-6, IL-8, TLR-4 and TNF-α in stimulated BAL cells and for T...
Bruschetta G, D'Ascola A, Medica P, Ferlazzo AM.Serotonin (5-hydroxytryptamine [5-HT]) may induce metabolic effects in different cell types, including leukocytes. In horses, 5-HT is involved in physiological and behavioral functions. Physical exercise is known to increase the amounts of 5-HT both in brain and periphery, but so far, the signal mechanism in response to exercise is not known. The aim of the study was to investigate the effect of a racehorse intensive training session on plasma 5-HT levels, serotonin transporter (SERT), 5HT, 5-HT, 5-HT, 5-HT receptor, interleukin-1 beta, and tumor necrosis factor-alpha expression in horse perip...
Ainsworth DM, Appleton JA, Eicker SW, Luce R, Julia Flaminio M, Antczak DF.The effect of strenuous exercise on the mRNA concentrations of interleukin-12p35 subunit (IL-12p35), interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in equine pulmonary and peripheral blood mononuclear cells (PBMCs) was investigated. We hypothesized that strenuous exercise would suppress the expression of IL-12p35, IFN-gamma and augment the expression of IL-4. Eleven horses were randomly divided into two groups, a stall-confined control group (n=5) and an exercise-conditioned treatment group (n=6). Bronchoalveolar and PBMCs were obtained from horses in the treatment group prior to the co...
Hannant D, Mumford JA.Cytotoxic cell precursors and/or cytotoxic memory cells were demonstrated in the peripheral blood of ponies after aerosol infection with influenza A/equine/Newmarket/79 (H3N8). In order to reveal their cytotoxic potential, peripheral blood mononuclear cells required a secondary antigenic stimulation. In vitro induced cytotoxic cells showed activity against influenza infected target cells in a 3-4 h 51Cr-release assay. The reactivity of cytotoxic cells was markedly influenced by the conditions of the secondary induction culture. If high concentrations of exogenous crude equine IL-2 were used, v...
Wattrang E, Palm AK, Wagner B.Synthetic oligodeoxyribonucleotides (ODN) may prove useful immune modulators in equine medicine. It is however important to assess the effects of each specific ODN in the species it is intended to be used in. The present study therefore aimed to evaluate some ODN for induction of cytokine production; i.e. type I interferons (IFN), IFN-γ, tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β), and proliferation of equine peripheral blood mononuclear cells (PBMC). A panel of four ODN containing unmethylated cytosine-guanosine sequences (CpG) was used: ODN 1 and ODN 8 repre...
Frellstedt L, McKenzie HC, Barrett JG, Furr MO.Endotoxemia is responsible for severe illness in horses. Individuals can become clinically unresponsive to the endotoxin molecule after an initial exposure; a phenomenon referred to as 'endotoxin tolerance' (ET). ET has been observed clinically in horses in vivo; however, cytokine expression associated with ET has not been investigated. The purpose of this study was to develop and validate a method for inducing ET in equine peripheral blood mononuclear cells (PBMC) in vitro, and to describe selected cytokine responses which are associated with ET. ET was induced by culturing cells with three c...
Schulz AK, Kersten S, Dänicke S, Coenen M, Vervuert I.The present study examined the short-term effects of deoxynivalenol (DON), administered at two different concentrations via a feed preparation using naturally contaminated wheat, on feed intake, liver and kidney metabolism and immunomodulatory properties in horses. Twelve geldings were randomly assigned to one of three dietary treatments for 21 days. DON was provided via naturally contaminated wheat (14.6 ± 6.5 mg DON/kg dry matter). The daily feed intake was adjusted to 4 kg of wheat and 1.7 kg of silage per 100 kg of body weight (BW). Horses were fed one of the following diets: control whea...
Ziegler A, Gerber V, Marti E.Insect bite hypersensitivity (IBH) is an equine allergic dermatitis to Culicoides spp. antigens. Attempts at using allergen-specific immunotherapy (AIT) as a treatment for IBH have so far proven unsuccessful. Toll-like receptor (TLR) agonists can promote a shift in the immune response from the allergy-promoting T helper cell 2 (Th2) response towards a Th1 and/or regulatory response. The aim of this study was to evaluate two immunomodulatory TLR agonists in vitro as potential vaccine adjuvants for a more efficacious AIT in IBH. Peripheral blood mononuclear cells (PBMCs) from healthy and IBH-aff...
Schnabel CL, Wagner S, Wagner B, Durán MC, Babasyan S, Nolte I, Pfarrer C, Feige K, Murua Escobar H, Cavalleri JM.Research on equine cytokines is often performed by analyses of mRNA. For many equine cytokines an analysis on the actual protein level is limited by the availability of antibodies against the targeted cytokines. Generation of new antibodies is ongoing but time consuming. Thus, testing the reactivity of commercially available antibodies for cross-reactivity with equine cytokines is of particular interest. Fifteen monoclonal antibodies against IL-1β, IL-6, IL-8, IL-12, IL-18 and Granulocyte Macrophage Colony stimulating factor (GM-CSF) of different species were evaluated for reactivity with the...
Vendrig JC, Coffeng LE, Fink-Gremmels J.Deficient innate and adaptive immune responses cause newborn mammals to be more susceptible to bacterial infections than adult individuals. Toll-like receptors (TLRs) are known to play a pivotal role in bacterial recognition and subsequent immune responses. Several studies have indicated that activation of certain TLRs, in particular TLR-2, can result in suppression of inflammatory pathology. In this study, we isolated peripheral blood mononuclear cells (PBMCs) from adult and newborn horses to investigate the influence of TLR-2 activation on the inflammatory response mediated by TLR-4. Data we...
Fedorka CE, Ball BA, Walker OF, Conley AJ, Corbin CJ, Lu KG, Hanneman JM, Troedsson MHT, Adams AA.Progestins are immunomodulatory in a variety of species. In the horse, the most commonly administered synthetic progestin is altrenogest (ALT), but its effect on the immune system of the non-pregnant mare is unknown. Peripheral blood mononuclear cells (PBMCs) from diestrous mares were incubated with varying concentrations of progesterone (P4) or ALT to assess intracellular production of IFNγ and the expression of select cytokines. Additionally, ten mares received either ALT or VEH daily utilizing a switchback design beginning on the day of ovulation and continuing for 7 days. Circulating PBM...
Anzai T, Sheoran AS, Kuwamoto Y, Kondo T, Wada R, Inoue T, Timoney JF.Streptococcus equi causes equine strangles. The acute disease has many of the hallmarks of an acute response including high fever, elevated plasma fibrinogen and neutrophilia, affects known to be mediated by proinflammatory cytokines. The objective of this study was to screen-culture supernatants from equine clinical isolates of S. equi and S. zooepidemicus for stimulation of mitogenic responses by horse peripheral blood mononuclear cells. Mitogenicity comparable to that of concanavalin A was detected in culture supernatants of S. equi strains but not in those of S. zooepidemicus. Mitogenicity...
Dietze B, Cierpka E, Schäfer M, Schill W, Lutz MB.Equine dendritic cells (eqDC) can be generated from peripheral blood monocytes by propagation in GM-CSF and IL-4. Despite similarities with the generation of human DC, we found significant improvements for eqDC generation and functional influences on eqDC maturation. The fractionation of peripheral blood mononuclear cells (PBMC) by two subsequent gradients at densities of 1.090 and 1.077 as well as an adherence step in AIM V((R)) medium on dishes coated with extracellular matrix components (Primaria) improved the purity and yield of DC. After 3 days, eqDC cultures with GM-CSF alone developed i...
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...
Parkinson NJ, Buechner-Maxwell VA, Witonsky SG, Pleasant RS, Werre SR, Ahmed SA.The innate immune response to lipopolysaccharide contributes substantially to the morbidity and mortality of gram-negative sepsis. Horses and humans share an exquisite sensitivity to lipopolysaccharide and thus the horse may provide valuable comparative insights into this aspect of the inflammatory response. MicroRNAs, small non-coding RNA molecules acting as post-transcriptional regulators of gene expression, have key roles in toll-like receptor signaling regulation but have not been studied in this context in horses. The central hypothesis of this study was that lipopolysaccharide induces di...
Schumann B, Winkler J, Mickenautsch N, Warnken T, Dänicke S.Both deoxynivalenol (DON), zearalenone (ZEN), and their metabolites are known to modulate immune cells in various species whereby viability and proliferation are influenced. Such effects were rarely examined in horses. Therefore, one aim of the present study was to titrate the inhibitory concentrations of DON, 3-acetyl-DON (3AcDON), de-epoxy-DON (DOM-1), ZEN, and α- and β-zearalenol (ZEL) at which viability and proliferation of equine PBMC were reduced by 50 % (IC50) and 10 % (IC10) in vitro. For evaluation of practical relevance of the in vitro findings, a further aim was to screen horses...
Hatzel JN, Bouma GJ, Cleys ER, Bemis LT, Ehrhart EJ, McCue PM.Early pregnancy factor has been identified as a 10-kDa extracellular homolog of heat shock protein 10 (Hsp10). Hsp10 has been detected during early pregnancy in serum of mice, sheep, pigs, horses, cows, and humans by the rosette inhibition test. Hsp10 has also been associated with several neoplastic and autoimmune diseases. The goal of the present study was to determine if Hsp10 could be detected in the early equine embryo through the use of immunohistochemistry and quantitative real-time PCR. Additionally, analysis of systemically harvested peripheral blood mononuclear cells (PBMCs) from both...
Schnabel CL, Babasyan S, Freer H, Wagner B.C-X-C motif ligand 10 (CXCL10) is a pro-inflammatory chemokine and has been extensively evaluated in people and mice. In horses, CXCL10 and its involvement in host immunity has rarely been analyzed due to the lack of specific antibodies. We generated a mAb specific for the equine chemokine CXCL10 using hybridoma technology. Antibody specificity was confirmed by intracellular staining and flow cytometric analysis of Chinese Hamster Ovary (CHO) cells expressing equine rCXCL10, while CHO cells expressing equine rCXCL9 were not detected. Native CXCL10 expression in PBMC from horses of different ag...
Dahlgren AR, Knych HK, Arthur RM, Durbin-Johnson BP, Finno CJ.Recombinant human erythropoietin (rHuEPO) is a well-known performance enhancing drug in human athletes, and there is anecdotal evidence of it being used in horse racing for the same purpose. rHuEPO, like endogenous EPO, increases arterial oxygen content and thus aerobic power. Micro-doping, or injecting smaller doses over a longer period of time, has become an important concern in both human and equine athletics since it is more difficult to detect. Horses offer an additional challenge of a contractile spleen, thus large changes in the red blood cell mass occur naturally. To address the challe...
Hansen S, Baptiste KE, Fjeldborg J, Betancourt A, Horohov DW.An imbalance in various cytokine mRNA expression in bronchoalveolar lavage (BAL) and peripheral blood mononuclear cells (PBMC) in horses suffering from recurrent airway obstruction (RAO) has been demonstrated; however, the natural variation of cytokine expression in healthy horses has yet to be described. The objectives of this study were to: (1) identify age-related differences in the expression of pro-inflammatory cytokines in BAL and PBMC, (2) compare the expression of pro-inflammatory cytokines between BAL and PBMC, and (3) evaluate the relationship between BAL pro-inflammatory cytokine ex...
