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.
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 ...
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...
Sun L, Adams AA, Betancourt A, Stewart JC, Liu C, Horohov DW.Interferon-gamma (IFNγ) plays an important role against viral and intracellular bacterial infections and its production is deficient in foals. Cellular proliferation provides an opportunity for de novo gene expression, though little is known about its role in regulating IFNγ expression in foals. While stimulation of foal peripheral blood mononuclear cells (PBMCs) with concanavalin A (ConA) increased the frequency of IFNγ(+) cells, the overall percentage of IFNγ(+) cells remained below that of adults. By contrast, the proliferative response of foal PBMC was significantly greater than that o...
Hagen J, Hartnett C, Houchins JP, Giguère S, Kalyuzhny AE.Human and mouse immune system cells are the most frequently used specimens in ELISPOT assays. In an effect to expand the application of ELISPOT assay to other species, we developed matched antibody pairs for ready-to-use kits designed for studying the frequency of equine IFNγ- and IL-4-secreting peripheral blood mononuclear cells (PBMCs). Equine PBMCs were stimulated with either concanavalin A (Con A) or calcium ionomycin mixed with phorbol 12-myristate 13-acetate (CaI + PMA). We found that Con A, in general, had a more profound stimulating effect than CaI + PMA on IL-4 secretion, whereas bot...
Liu M, Bordin A, Liu T, Russell K, Cohen N.Focusing on the first 3 weeks of life, this study examined the mRNA transcript development of different Th-type cytokines in foals in response to Rhodococcus equi infection in vitro. Results demonstrated the significant up-regulation in expression of Th1-, Th2-, and Th17-type cytokines (IFN-γ, IL-4, IL-6, IL-8, IL-12p35, IL-12p40, IL-17, IL-23p19, and TNF-α) in R. equi infection of bronchial alveolar lavage (BAL) cells of 10-day-old foals. Consequently, signature cytokines of 3 Th cell types, IFN-γ (Th1), IL-4 (Th2), and IL-17 (Th17), were used to compare temporal response patterns of circu...
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...
Pusterla N, Mapes S, Gebhart C.The cell-mediated immune response to Lawsonia intracellularis, the agent of equine proliferative enteropathy (EPE), was investigated in vaccinated and naturally infected foals. Interferon (IFN)-γ gene expression was determined in peripheral blood mononuclear cells collected from vaccinated (n=6) and control foals (n=6) every 30 days for 180 days following first vaccine administration, and from clinically affected foals (n=16) within 7-10 days of diagnosing EPE. Seroconversion (immunoperoxidase monolayer assay titer ≥60) occurred in 5/6 vaccinated foals between 60 and 90 days following the f...
Hamza E, Steinbach F, Marti E.Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis caused by bites of midges from the genus Culicoides. We have shown previously that peripheral blood mononuclear cells (PBMC) from IBH-affected horses produce higher levels of IL-4 and lower levels of IL-10 and TGF-β1 than those from healthy horses, suggesting that IBH is associated with a reduced regulatory immune response. FoxP3 is a crucial marker of regulatory T cells (Tregs). Here we have determined the proportion of CD4(+)CD25(+)FoxP3(+) T cells by flow cytometry in PBMC directly after isolation or after stimulation with Cu...
Brandt S, Schoster A, Tober R, Kainzbauer C, Burgstaller JP, Haralambus R, Steinborn R, Hinterhofer C, Stanek C.Equine hoof canker is a chronic proliferative pododermatitis of as yet unknown aetiology. Like equine sarcoid disease, canker is a therapy-resistant disorder characterised by hyperkeratosis, acanthosis and a marked tendency to recur. Objective: There is an association of sarcoid-inducing bovine papillomaviruses of types 1 and 2 (BPV-1, BPV-2) with hoof canker disease. Methods: Using PCR-based techniques, we assessed canker tissue, intact skin and/or peripheral blood mononuclear cells (PBMCs) of 25 canker-affected horses for the presence of sarcoid-associated BPV-1 and -2. Results: Conventional...
Zhang L, Franchini M, Wehrli Eser M, Dip R.Accumulation of extracellular adenosine has been closely associated with human asthmatic responses. However, the relevance of adenosine signalling in equine airways has not previously been investigated. Objective: To determine the expression of adenosine receptors (AR) in bronchoalveolar lavage (BAL) cells and assess the reactivity of these cells to AR ligands ex vivo, employing IL-6 as readout of adenosinergic inflammatory signalling. Methods: Eight horses with varying degrees of lower airway inflammation and 10 healthy controls were analysed. Expression of AR-subtypes in each BAL sample was ...
Hurley DJ, Berghaus LJ, Hurley KA, Moore JN.To evaluate effects of black walnut extract (BWE) on equine mononuclear cells and determine whether BWE has direct proinflammatory effects. Methods: Mononuclear cells separated from blood samples from 8 horses. Methods: Aqueous BWE was prepared and processed to eliminate contamination with particulates and microbes. A Limulus amoebocyte lysate assay was used to detect lipopolysaccharide (LPS) contamination in the BWE. Mononuclear cells were incubated in minimal essential medium with or without the addition of 0.6% to 10% (vol/vol) BWE. These mononuclear cells were assessed for viability, activ...
Capomaccio S, Cappelli K, Barrey E, Felicetti M, Silvestrelli M, Verini-Supplizi A.It is known that moderate physical activity may have beneficial effects on health, whereas strenuous effort induces a state resembling inflammation. The molecular mechanisms underlying the cellular response to exercise remain unclear, although it is clear that the immune system plays a key role. It has been hypothesized that the physio-pathological condition that develops in athletes subjected to heavy training is caused by derangement of cellular immune regulation. The purpose of the present study was to obtain information on endurance horse gene transcription under strenuous conditions and t...
Capomaccio S, Verini-Supplizi A, Galla G, Vitulo N, Barcaccia G, Felicetti M, Silvestrelli M, Cappelli K.A large proportion of mammalian genomes is represented by transposable elements (TE), most of them being long interspersed nuclear elements 1 (LINE-1 or L1). An increased expression of LINE-1 elements may play an important role in cellular stress-related conditions exerting drastic effects on the mammalian transcriptome. To understand the impact of TE on the known horse transcriptome, we masked the horse EST database, pointing out that the amount is consistent with other major vertebrates. A previously developed transcript-derived fragments (TDFs) dataset, deriving from exercise-stimulated hor...
Wilsterman S, Soboll-Hussey G, Lunn DP, Ashton LV, Callan RJ, Hussey SB, Rao S, Goehring LS.Infection with equine herpesvirus-1 (EHV-1) causes respiratory disease, late term abortions and equine herpesvirus myeloencephalitis (EHM) and remains an important problem in horses worldwide. Despite increasing outbreaks of EHM in recent years, our understanding of EHM pathogenesis is still limited except for the knowledge that a cell-associated viremia in peripheral blood mononuclear cells (PBMCs) is a critical link between primary respiratory EHV-1 infection and secondary complications such as late-term abortion or EHM. To address this question our objective was to identify which PBMC subpo...
Kabithe E, Hillegas J, Stokol T, Moore J, Wagner B.CD14 is a receptor for the complex of lipopolysaccaride (LPS) and LPS-binding protein. Binding of this complex to CD14 in association with Toll-like receptor 4 provides a major pathway for the initiation of innate immune responses to bacterial pathogens. We used a mammalian expressed extracellular region of equine CD14 (rCD14) derived from an IgG fusion protein to produce monoclonal antibodies (mAbs) to CD14. Eight mAbs were tested by flow cytometric analysis of equine leukocytes and by immunoblotting using rCD14 indicating that the mAbs recognized at least three different epitopes on equine C...
Tong T, Bai Y, Liu G, Wang Q, Zhang W, Xiao Y, Xu S, Liu N, Yang T, Wu D.IL-18 is a cytokine originally discovered as an important modulator of immune responses and subsequently shown to be pleiotropic. In this report, we expressed the recombinant equine mature interleukin-18 (rEMIL-18) in E. coli and purified it by nickel affinity gel column chromatography. Purified rEMIL-18 had biological activity commensurate with recombinant human IL-18, as determined by its synergistic effect with recombinant human IL-12 (rhIL-12) on the induction of IFN-gamma gene expression in equine peripheral blood mononuclear cells (PBMC). Following intraperitoneal (i.p.) immunization of ...
Paillot R, Robinson C, Steward K, Wright N, Jourdan T, Butcher N, Heather Z, Waller AS.Streptococcus equi is the causative agent of strangles, the most frequently diagnosed infectious disease of horses worldwide. The disease is characterized by abscessation and swelling of the lymph nodes of the head and neck, which can literally strangle the horse to death. S. equi produces four recently acquired phage-associated bacterial superantigens (sAgs; SeeH, SeeI, SeeL, and SeeM) that share homology with the mitogenic toxins of Streptococcus pyogenes. The aim of this study was to characterize the contribution of each of these S. equi sAgs to mitogenic activity in vitro and quantify the ...
Hamza E, Torsteinsdottir S, Eydal M, Frey CF, Mirkovitch J, Brcic M, Wagner B, Wilson AD, Jungi TW, Marti E.Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis of horses caused by bites of Culicoides spp. IBH does not occur in Iceland where Culicoides are absent. However, following importation into continental Europe where Culicoides are present, >or=50% of Icelandic horses (1st generation) develop IBH but
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...
Zhang YW, Davis EG, Bai J.Quantitative reverse transcription polymerase chain reaction (RT-PCR) has become a basic, reliable and sensitive modern technique, in both biological research and clinical diagnosis, for investigation of gene expression and validation of cDNA microarray analysis. Accurate mRNA quantification using quantitative RT-PCR commonly requires data normalization through stable housekeeping genes (HKGs). Selection of HKGs for data normalization is critical for accurate mRNA quantification. Our objective was to evaluate a set of candidate HKGs as internal controls for gene expression studies using quanti...
Treonze KM, Alves K, Fischer P, Hagmann WK, Hora D, Kulick A, Vakerich K, Smith ND, Lingham RB, Maniar S, Reger TS, Zunic J, Munoz B, Prasit P....The purpose of this study was to characterize the alpha(4)beta(1) receptor (CD49d/CD29, very late antigen-4, VLA-4) on circulating equine leukocytes and to evaluate the intrinsic potency of an alpha(4)beta(1) receptor antagonist (Compound B) in the horse. Ultimately, these studies would allow us to determine the suitability of treating recurrent airway obstruction (RAO; heaves) affected horses by blocking the cellular recruitment of lymphocytes and neutrophils into the lung. The data demonstrates the alpha(4)beta(1) integrin is present on horse lymphocytes and neutrophils (fluorescence-assiste...
Liu T, Nerren J, Liu M, Martens R, Cohen N.Neonates are thought to be generally deficient in production of Th-1-associated cytokines at birth, and thereby more susceptible to bacterial infections. Using neonatal foals as a model, this study examined the age-dependent maturation of both basal and stimulus-induced immune responses, as reflected by the expression of a panel of Th-1-associated and pro-inflammatory cytokines. Results showed that although the basal production of IFN-gamma and IL-6 was impaired (P<0.05) in PBMCs of neonatal foals at birth, the basal production of IL-8, IL-12(p35/p40) and IL-23(p19/p40) were either in exces...
Lin YZ, Deng XL, Shen N, Lü XL, Zhao LP, Kong XG, Shao YM, Zhou JH.To develop a flow cytometry using (5-carboxyfluorescein diacetate succinmidyl ester, CFSE) to detect the proliferation of specific T lymphocytes from equine infectious anemia virus (EIAV). Methods: Peripheral blood mononuclear cells (PBMC) were isolated, stained with CFSE and incubated with EIAV for 5 days. After interacted with either CD4(+) or CD8(+) antibody, the cells were detected for proliferated population, which contained serially 2-fold reduced CFSE in CD4(+) and CD8(+) T lymphocytes. Results: The concentration of CFSE, and the type, concentration and reaction time of EIAV-specific an...
Wagner B, Freer H.The detection and quantification of equine cytokines has been hampered by the lack of antibodies for many years. With the development of antibody pairs for equine cytokines during the past years, the quantification of these essential regulators of the immune response became possible. After being successfully tested by enzyme-linked immunosorbent assays (ELISA), three of these anti-cytokine reagents were used here to establish the first cytokine multiplex assay for equine IL-4, IL-10 and IFN-alpha. A fluorescent bead-based system was used as matrix for this assay that allows the simultaneous de...
Bailat AS, Soutullo AR, García MI, Veaute CM, Garcia L, Racca AL, Malan Borel IS.Gp90 and gp45 synthetic peptides, which mimic conserved sequences of native viral proteins, are recognized by antibodies to equine infectious anemia virus (EIAV) in asymptomatic carrier horses and generate humoral and cellular responses in immunized mice. Cytokine mRNA levels were evaluated in equine peripheral blood mononuclear cells (PBMCs) after in vitro stimulation with gp90 and gp45 with the aim of determining the cytokine profile associated with the proliferative response. Stimulation index (SI) values indicate that 100 and 60% of EIAV-infected horses recognized gp90 and gp45, respective...
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...
Lin Y, Deng X, Shen N, Zhao L, Meng Q, Max J, Wang J, Shao Y, Zhou J.The attenuated vaccine of equine infectious anemia virus (EIAV) is the first lentiviral vaccine that provides solid protection against the infection of EIAV virulent strains. Study of the immune response induced by EIAV vaccine is an important approach to understand the immunity to other lentiviruses. IFN-gamma expressed by specifically stimulated lymphocytes is an important indicator for the evaluation of T cell-mediated immunity. A flow cytometry based assay was established in this study to accurately and effectively detect IFN-gamma expression in different subtypes of T lymphocytes in EIAV-...
Horohov DW, Breathnach CC, Sturgill TL, Rashid C, Stiltner JL, Strong D, Nieman N, Holland RE.While immune modulators are used routinely in equine medicine, their mechanism of action is not always known. Objective: To determine the effect of a commercial preparation of inactivated parapoxvirus ovis (Orf virus; PPVO) on cytokine gene expression by equine peripheral blood mononuclear cells (PBMC) both in vitro and in vivo. Methods: PBMC were prepared from 6 mixed-breed yearlings and cultured in vitro with PPVO with or without Concanavalin A (Con A) for 24 h. Effects on the expression of IFNalpha, IFNbeta IFNgamma, TNFalpha and IL-18 were analysed by real time quantitative PCR (RT-PCR). I...
Brandt S, Haralambus R, Schoster A, Kirnbauer R, Stanek C.Bovine papillomaviruses of types 1 and 2 (BPV-1 and -2) chiefly contribute to equine sarcoid pathogenesis. However, the mode of virus transmission and the presence of latent infections are largely unknown. This study established a PCR protocol allowing detection of <or=10 copies of the BPV-1/-2 genes E5 and L1. Subsequent screening of peripheral blood mononuclear cell (PBMC) DNA derived from horses with and without BPV-1/2-induced skin lesions demonstrated the exclusive presence of E5, but not L1, in PBMCs of BPV-1/2-infected equines. To validate this result, a blind PCR was performed from ...
Cook RF, Cook SJ, Even DL, Schaffer C, Issel CJ.Horse IL-7 (HIL-7) cDNA was isolated from adult lymph node tissue by reverse transcription polymerase chain reaction (RT-PCR) using oligonucleotide primers based on horse genomic sequences (The Broad Institute). In addition, to the full-length (FL) 531bp reading frame encoding 176 amino acids, shorter open-reading frames of 477, 396 and 264bp were also amplified. Nucleotide sequence analysis of these RT-PCR products demonstrated they were homologous except the shorter species were missing internal sequences consistent with multiple RNA splicing events. Consequently, the shorter open-reading fr...
Pusterla N, Mapes S, Gebhart C.The cell-mediated immune response to Lawsonia intracellularis, the agent of equine proliferative enteropathy (EPE), was investigated in vaccinated and naturally infected foals. Interferon (IFN)-γ gene expression was determined in peripheral blood mononuclear cells collected from vaccinated (n=6) and control foals (n=6) every 30 days for 180 days following first vaccine administration, and from clinically affected foals (n=16) within 7-10 days of diagnosing EPE. Seroconversion (immunoperoxidase monolayer assay titer ≥60) occurred in 5/6 vaccinated foals between 60 and 90 days following the f...
Faber E, Tshilwane SI, Kleef MV, Pretorius A.African horse sickness (AHS) is caused by African horse sickness virus (AHSV), a double stranded RNA (dsRNA) virus of the genus Orbivirus, family Reoviridae. For the development of new generation AHS vaccines or antiviral treatments, it is crucial to understand the host immune response against the virus and the immune evasion strategies the virus employs. To achieve this, the current study used transcriptome analysis of RNA sequences to characterize and compare the innate immune responses activated during the attenuated AHSV serotype 4 (attAHSV4) (in vivo) and the virulent AHSV4 (virAHSV4) (in...
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...
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...
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...
Lonning SM, Zhang W, McGuire TC.Antigen-specific T-helper (Th) lymphocytes are critical for the development of antiviral humoral responses and the expansion of cytotoxic T lymphocytes (CTL). Identification of relevant Th lymphocyte epitopes remains an important step in the development of an efficacious subunit peptide vaccine against equine infectious anemia virus (EIAV), a naturally occurring lentivirus of horses. This study describes Th lymphocyte reactivity in EIAV carrier horses to two proteins, p26 and p15, encoded by the relatively conserved EIAV gag gene. Using partially overlapping peptides, multideterminant and poss...
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...
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: ...
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...
Tschetter JR, Davis WC, Perryman LE, McGuire TC.Eight murine monoclonal antibodies (mAb) were used to identify the equine CD8 alpha or CD8 beta chains and to define the expression of these chains on lymphocytes from various lymphoid tissues. CD8 alpha was a 39 kDa protein and CD8 beta was a 32 kDa protein. Both chains were expressed on most of the CD8+ T lymphocytes in the peripheral blood, spleen, thymus, mesenteric lymph nodes and ileal intraepithelial lymphocytes (IEL), however, in each lymphoid compartment a percentage of lymphocytes expressed only the CD8 alpha chain. The largest percentage of CD8 alpha alpha expressing T lymphocytes w...
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...
Cassart D, Fett T, Sarlet M, Baise E, Coignoul F, Desmecht D.The morphopathological picture of a subset of equine myopathies is compatible with a primary mitochondrial disease, but functional confirmation in vivo is still pending. The cationic dye JC-1 exhibits potential-dependent accumulation in mitochondria that is detectable by a fluorescence shift from green to orange. As a consequence, mitochondrial membrane potential can be optically measured by the orange/green fluorescence intensity ratio. A flow cytometric standardized analytic procedure of the mitochondrial function of equine peripheral blood mononuclear cells is proposed along with a critical...
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...
Gopalakrishnan A, Maji C, Dahiya RK, Suthar A, Kumar R, Gupta AK, Dimri U, Kumar S.The in vitro growth inhibitory efficacies of five drug molecules against Theileria equi were evaluated in in vitro cultured parasites. A continuous microaerophilic stationary-phase culture (MASP) system was established for propagation of T. equi parasites. This in vitro culture system was used to assess the growth inhibitory effect of harmaline hydrochloride dihydrate (HHD), hexadecyltrimethylammonium bromide (HDTAB), hesparidin methyl chalcone (HMC), andrographolide and imidocarb dipropionate against T. equi. The 50% inhibitory concentration value of HHD, HDTAB, HMC, and imidocarb dipropionat...
Singh S, Chopra M, Dilbaghi N, Manuja BK, Kumar S, Kumar R, Rathore NS, Yadav SC, Manuja A.Isometamidium hydrochloride (ISMM) is an effective drug for the treatment of trypanosomosis, but it causes local and systemic toxicity. Isometamidium hydrochloride has limited therapeutic index and exhibit considerable variation in their prophylactic activities. We developed a trypanocidal nanoformulation using ISMM and polymers sodium alginate/gum acacia to enhance the efficacy of the drug at lower doses, while minimizing undesirable side effects. It was characterized by transmission electron microscopy and infrared spectroscopy for evaluation of size, morphology, functional groups, etc. In v...
Zhang L, Franchini M, Wehrli Eser M, Dip R.Accumulation of extracellular adenosine has been closely associated with human asthmatic responses. However, the relevance of adenosine signalling in equine airways has not previously been investigated. Objective: To determine the expression of adenosine receptors (AR) in bronchoalveolar lavage (BAL) cells and assess the reactivity of these cells to AR ligands ex vivo, employing IL-6 as readout of adenosinergic inflammatory signalling. Methods: Eight horses with varying degrees of lower airway inflammation and 10 healthy controls were analysed. Expression of AR-subtypes in each BAL sample was ...
Mißbach S, Aleksic D, Blaschke L, Hassemer T, Lee KJ, Mansfeld M, Hänske J, Handler J, Kammerer R.The CEA gene family is one of the most rapidly evolving gene families in the human genome. The founder gene of the family is thought to be an ancestor of the inhibitory immune checkpoint molecule CEACAM1. Comprehensive analyses of mammalian genomes showed that the CEA gene family is subject to tremendous gene family expansion and contraction events in different mammalian species. While in some species (e.g. rabbits) less than three CEACAM1 related genes exist, were in others (certain microbat species) up to 100 CEACAM1 paralogs identified. We have recently reported that the horse has also an e...
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...
Vandergrifft EV, Swiderski CE, Horohov DW.We have cloned equine interleukin 4 (IL-4) cDNA using the polymerase chain reaction (PCR) and primers based on the human IL-4 sequence. The cDNA was amplified from mitogen-stimulated equine peripheral blood mononuclear cells (PBMC). The cloned PCR product shares extensive homology ith IL-4 sequences from other species.
Carter EI, Valli VE, McSherry BJ.The peripheral blood cells from Standard bred horses were subjected to procedures which will separate equine peripheral blood cells with good precision and efficiency into red cell, leukocyte, and platelet fractions. The separated cells have normal morphology and the differential count of the separated granulocytes and lymphocytes is unchanged from that of the original sample.
Furr M, Pontzer C.The following experiment was performed to test the hypothesis that transforming growth factor beta (TGF-beta) concentration varies in the cerebrospinal fluid and serum of horses with EPM and to determine if cerebrospinal fluid (CSF) alters the interferon-gamma (IFN-gamma) rersponse of equine peripheral blood mononuclear cells (PBMCs). The concentration of transforming growth factor-beta (TGF-beta2) was investigated in the serum and cerebrospinal fluid (CSF) of 18 horses (9 normal, 9 affected with equine protozoal myeloencephalitis [EPM]). The TGF-beta2 assay was validated in a group of 6 norma...
Tiwari M, Sodhi M, Verma P, Vivek P, Kataria RS, Niranjan SK, Bharti VK, Masharing N, Gujar G, Chanda D, Mukesh M.The identification of appropriate references genes is an integral component of any gene expression-based study for getting accuracy and reliability in data interpretation. In this study, we evaluated the expression stability of 10 candidate reference genes (GAPDH, RPL4, EEF1A1, RPS9, HPRT1, UXT, RPS23, B2M, RPS15, ACTB) in peripheral blood mononuclear cells of livestock species that are adapted to high altitude hypoxia conditions of Leh-Ladakh. A total of 37 PBMCs samples from six native livestock species of Leh-Ladakh region such as Ladakhi cattle, Ladakhi yak, Ladakhi donkey, Chanthangi goat...
Bruyninckx WJ, Blancquaert AM.Horse mononuclear cells were separated from whole blood using neutral density gradient centrifugation on Isopaque-Ficoll. The resulting cell suspension was comparable in composition with similarly prepared human and bovine mononuclear cell preparations. The relative concentration of monocytes was increased by the use of a gradient with density lower than that originally proposed by Böyum (Böyum, A. 1968. Scand. J. Clin. Lab. Investig. 21 supple. 97:77-89). Contamination by neutrophils was limited either by using a gradient medium of lower density or by replacing Isopaque-Ficoll by Percoll-0....
Klei TR, Chapman MR, Dennis VA.The adherence of equine leukocytes to Strongylus vulgaris infective larvae (L3) in the presence of normal and immune sera was examined in vitro. Immune sera promoted adherence of buffy coat cells from ponies with S. vulgaris-induced eosinophilia (eosinophilic ponies) to S. vulgaris L3. However, eosinophils in the buffy coat cells were the predominant adherent cell type. Studies using leukocyte populations enriched for eosinophils, neutrophils, and mononuclear cells from eosinophilic ponies support the observations using buffy coat cells that eosinophils were the main effector cells. Adherent e...
Ziegler A, Olzhausen J, Hamza E, Stojiljkovic A, Stoffel MH, Garbani M, Rhyner C, Marti E.Allergen-specific immunotherapy (AIT) constitutes the only curative approach for allergy treatment. There is need for improvement of AIT in veterinary medicine, such as in horses suffering from insect bite hypersensitivity, an IgE-mediated dermatitis to Culicoides. Dendritic cell (DC)-targeting represents an efficient method to increase antigen immunogenicity. It is studied primarily for its use in improvement of cancer therapy and vaccines, but may also be useful for improving AIT efficacy. Immunomodulators, like the Toll-like receptor 4 (TLR-4) agonist monophosphoryl lipid-A (MPLA) has been ...
Lea RG, Bolton AE.Commercial preparations of equine chorionic gonadotrophin (eCG) and extracts of horse placenta taken at 80 days gestation were incorporated into mixed lymphocyte culture and mitogen stimulation assays of horse peripheral blood mononuclear cells. A dose-related inhibition of lymphocyte proliferation, indicative of immunosuppressive activity, was observed in both systems, both with commercial eCG preparations and tissue extracts. Negligible inhibitory activity was observed with an extract of term placenta. The inhibitory activity of the placental samples was not related to their eCG content as m...
Heinz H, Marquardt J, Schuberth HJ, Adolf GR, Leibold W.Peripheral blood mononuclear cells (PBMC) and polymorphonuclear neutrophilic granulocytes (PMN) as well as embryonic equine dermal fibroblasts and the equine fibroblast line E. Derm which were used as controls, were treated with recombinant equine interferon-beta 1 (rEqIFN-beta 1) in vitro which induced the expression of different proteins in these cells. A 74 kDa protein was induced in PBMC and an 82 kDa protein was additionally found in the equine fibroblast E. Derm cell line following treatment with rEqFN-beta 1. Both proteins reacted with anti-mouse and anti-human Mx protein antisera in im...
Herbst AC, Reedy SE, Page AE, Horohov DW, Adams AA.Studies investigating age-related changes in the function of monocytes are currently limited for horses. Thus, the main goal of this study was to determine the effect of aging on monocyte phagocytic capacity and pro-inflammatory cytokine responses to bacterial lipopolysaccharide (LPS). A second goal of this work was to examine the effect of aging on the inflammatory cytokine responses to LPS in a whole blood ex vivo model. Seven healthy young adult (4-6 years of age) and seven healthy senior horses (>20 years of age) were enrolled. Phagocytosis of E. coli, and pro-inflammatory cytokine (TNF...
Rütten S, Schusser GF, Abraham G, Schrödl W.Horses are much predisposed and susceptible to excessive and acute inflammatory responses that cause the recruitment and stimulation of polymorphnuclear granulocytes (PMN) together with peripheral blood mononuclear cells (PBMC) and the release of cytokines. The aim of the study is to develop easy, quick, cheap and reproducible methods for measuring tumor necrosis factor alpha (TNF-α) and interleukin-1 receptor antagonist (IL-1Ra) in the equine whole blood cultures ex-vivo time- and concentration-dependently. Results: Horse whole blood diluted to 10, 20 and 50 % was stimulated with lipopolysa...
Cook RF, Cook SJ, Even DL, Schaffer C, Issel CJ.Horse IL-7 (HIL-7) cDNA was isolated from adult lymph node tissue by reverse transcription polymerase chain reaction (RT-PCR) using oligonucleotide primers based on horse genomic sequences (The Broad Institute). In addition, to the full-length (FL) 531bp reading frame encoding 176 amino acids, shorter open-reading frames of 477, 396 and 264bp were also amplified. Nucleotide sequence analysis of these RT-PCR products demonstrated they were homologous except the shorter species were missing internal sequences consistent with multiple RNA splicing events. Consequently, the shorter open-reading fr...
