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.
Bai Y, Tong TG, Zhang WJ, Xu SL, Wang Q, Liu GL, Wu DL.To develop a quantitative ELISA by measuring interferon (IFN-gamma) of equine lymphocytes. Methods: Sandwich ELISA for equine IFN-gamma was developed using mAb A5 as a capture antibody and biotinylated mAb SB10 as a detection antibody. Results: The detection limit of the sandwich ELISA for equine IFN-gamma was 1 microg/L and did not show cross-reactivity with recombinant equine IL-18. Equine IFN-gamma was detected by ELISA in culture medium of the peripheral blood mononuclear cells (PBMCs) stimulated with ConA or PMA/Ionomycin. Conclusions: This method can be used to help understand the role o...
Wagner B, Hillegas JM, Brinker DR, Horohov DW, Antczak DF.Interleukin-10 (IL-10) terminates inflammatory immune responses and inhibits activation and effector functions of T-cells, monocytes, macrophages and dendritic cells. IL-10 has also been found to be a key cytokine expressed by subpopulations of regulatory T-cells. In this report, we describe the generation and characterization of three monoclonal antibodies (mAbs) to equine IL-10. The antibodies were found to be specific for equine IL-10 using different recombinant equine cytokine/IgG fusion proteins. Two of the anti-equine IL-10 mAbs were selected for ELISA to detect secreted IL-10 in superna...
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...
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-...
Xu J, Cai J, Anderson B, Wagner B, Albrecht R, Peek SF, Suresh M, Darien BJ.The recent molecular characterization and sequencing of equine P-selectin (ePsel), and its glycoprotein ligand, P-selectin glycoprotein ligand-1 (PSGL-1), have provided the tools for further investigation into their role in leukocyte trafficking. Here, we report the generation of a genetically engineered chimeric protein (ePsel-IgG) in which the equine P-selectin lectin and epithelial growth factor (EGF) domains were covalently linked to the equine IgG1 heavy chain constant region. The soluble ePsel-IgG was observed to bind to equine monocytes by confocal microscopy and flow cytometry. Further...
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...
Hussey SB, Clark R, Lunn KF, Breathnach C, Soboll G, Whalley JM, Lunn DP.Equine herpesvirus-1 (EHV-1) infection is common in young horses throughout the world, resulting in respiratory disease, epidemic abortion, sporadic myelitis, or latent infections. To improve on conventional diagnostic tests for EHV-1, a real-time polymerase chain reaction (PCR) technique was developed, using primers and probes specific for the EHV-1 gB gene. Amplification efficiencies of 100% +/- 5% were obtained for DNA isolated from a plasmid, infected peripheral blood mononuclear cells (PBMCs), and nasal secretions from infected ponies. The dynamic range of the assay was 8 log10 dilutions,...
Wang Y, Zhang XY, Wei LL, Wu DL, Wang XJ, Xiang WH, Shen RX, Shao YM.To evaluate the relationship between the transcriptional level of IFN-gamma mRNA in peripheral blood mononuclear cells (PBMC) and immune protective response driven by inoculated horses with donkey leukocyte attenuated vaccine of EIAV(DLV), and to elucidate the immune mechanism of DLV. Methods: A real-time PCR method was established for quantitative detection of IFN-gamma mRNA level from horse PBMCs. Twelve horses were divided into vaccination group, healthy control group, challenging control group and EIAV natural infection group. The transcriptional level and distribution of IFN-gamma mRNA in...
Paillot R, Kydd JH, Sindle T, Hannant D, Edlund Toulemonde C, Audonnet JC, Minke JM, Daly JM.In horses, equine influenza virus (EIV) is a leading cause of respiratory disease. Conventional inactivated vaccines induce a short-lived immune response. By comparison, natural infection confers a long-term immunity to re-infection. An aim of new equine influenza vaccines is to more closely mimic natural infection in order to achieve a better quality of immunity. A new live recombinant vaccine derived from the canarypox virus vector and expressing haemagglutinin genes of EIV (subtype H3N8) has been developed. Stimulation of the immune system was studied after immunisation with this canarypox-...
Zhang XY, Li HM, Liang H, Shen T, Ma Y, Xiang WH, Shen RX, Shao YM.To elucidate cellular immune protective mechanism of EIAV. Methods: Four horses were immunized with (DLV) by subcutaneous injection and 2 horses with 0.85% sodium chloride as the negative control. Rectal temperatures and clinical features were recorded daily. Whole blood samples were collected, from which PBMC were separated and used for CTL assay and lymphocyte proliferation assay. Results: The target cells were activated by PWM and treated with DLV or recombinant vaccinia vectors expressing DLV and LN Gag or Env separately. The percentage of EIAV-specific CTL lysis was under 5% in the negati...
Garcia-Tapia D, Loiacono CM, Kleiboeker SB.A cell model of primary monocytes and other mononuclear cells isolated from equine blood was used to study the kinetics of West Nile virus (WNV) replication in a natural host. West Nile virus has emerged on the North American continent as a significant cause of morbidity and mortality in a wide range of avian and mammalian species. While other flaviviruses are known to infect monocytes and lymphocytes, the ability of WNV to productively replicate in specific immune cells of peripheral blood has not been assessed. In this study, enriched populations of monocytes and lymphocytes as well as purif...
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...
Wattrang E, Berg M, Magnusson M.This study aimed to evaluate different nucleic acid preparations as cytokine inducers in equine cells. To induce cytokine production, bacterial plasmid DNA or short synthetic oligodeoxyribonucleotides (ODN), with or without the transfection reagent lipofectin, were added to cultures of purified equine peripheral blood mononuclear cells (PBMC). Cytokine activity was detected with bioassays in cell culture supernatants after 24h of induction and cytokine mRNA expression was detected using RT-PCR at 6h post induction. For IFN-alpha/beta it was found that both plasmid DNA and phosphodiester ODN, c...
Sykes BW, Furr M, Giguère S.Twelve healthy horses were assigned to treatment or control groups. Treated horses received PGG-Glucan[ED-1] (1 mg/kg, IV) 24 hours prior to peripheral blood mononuclear cell (PBMC) isolation. PBMCs were isolated and incubated in the presence of lipopolysachharide (LPS). At 0, 6, 12, 24 and 48 hours messenger RNA (mRNA) was extracted. Reverse transcription polymerase chain reaction (PCR) was performed and cytokine mRNA expression for tumor necrosis factor alpha (TNFalpha), interleukin 1beta (IL-1beta), interleukin 10 (IL-10) and interferon gamma (IFN-gamma) determined using real time PCR. A si...
Jackson KA, Stott JL, Horohov DW, Watson JL.The objectives of this study were to quantify the induction of equine CD23 transcripts in equine peripheral blood mononuclear cells (PBMCs) and pulmonary alveolar macrophages cultured with recombinant equine IL-4 (rEq IL-4). PBMCs were isolated from blood drawn from four healthy horses. Bronchoalveolar lavage (BAL) fluid was collected from three healthy horses and alveolar macrophages were purified using adherence to plastic for 120 min. PBMCs and alveolar macrophages were cultured using four different conditions: rEq IL-4 and LPS, LPS alone, rEq IL-4 alone and a media control. Total RNA was i...
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...
Davis EG, Rush BR, Blecha F.Immunomodulation with Propionibacterium acnes is used for prophylaxis of respiratory disease or in horses suffering from chronic pulmonary inflammation; however, the mechanism for this response is poorly understood. Semiquantitative reverse transcriptase-polymerase chain reaction assays were used to evaluate gene expression of interleukin (IL)-2, IL-10, interferon (IFN)-gamma, and NK-lysin in healthy horses treated with P. acnes. Findings in the study indicated that horses treated with a P. acnes-based immunomodulator exhibited increased IFN-gamma and NK-lysin gene expression in peripheral blo...
van der Meulen KM, Nauwynck HJ, Pensaert MB.Equine herpesvirus-1 (EHV-1) may cause abortion in vaccination- and infection-immune horses. EHV-1-infected peripheral blood mononuclear cells (PBMCs) play an important role in virus immune evasion. The mechanisms by which infected PBMCs can avoid destruction by EHV-1-specific antibody and equine complement were examined. The majority of EHV-1-infected PBMCs (68.6 %) lacked surface expression of viral antigens and these cells were not susceptible to complement-mediated lysis. In infected PBMCs with surface expression of viral antigens, 63 % showed focal surface expression, whereas 37 % showed ...
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...
Vecchione A, Catchpole B, D'Mello F, Kanellos T, Hamblin A.Dendritic cells (DCs) are antigen presenting cells that potently modulate immune responses with varying outcomes depending on the DC sub-population involved. To understand how DC sub-types arise, it is necessary to determine which factors influence their differentiation. At least three major sub-populations of DCs have been described in mice: CD4+/CD8- "myeloid" DCs, CD4-/CD8+ "lymphoid" DCs and Langerhans cell-derived DCs. Whilst somewhat comparable populations have been described in man, in most other species very little is known. The identification of cytokines which stimulate proliferation...
Dunowska M, Meers J, Johnson RD, Wilks CR.Representational difference analysis (RDA) was used to compare gene expression in equine mononuclear cells either infected with equine herpesvirus-2 (EHV-2) or adsorbed with inactivated EHV-2. Seven clones identified in non-infected cells after three rounds of selective subtraction and enrichment for differentially expressed genes contained sequences homologous to equine monocyte chemoattractant protein 1 (MCP-1). This suggested that EHV-2 may down-regulate MCP-1 transcription in infected cells. These findings correlate well with similar findings described for human cytomegalovirus and support...
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...
van der Meulen KM, Nauwynck HJ, Pensaert MB.In the present study, equine herpesvirus-1 (EHV-1)-infected cells were identified in ionomycin/phorbol dibutyrate (IONO/PDB)-stimulated peripheral blood mononuclear cells (PBMC) and the mechanism by which stimulation increases the percentage of infected cells was examined. In the population of viral antigen-positive PBMC, 38.4+/-4.5% were CD5(+) T-lymphocytes (18.1+/-3.2% CD4(+) 13.6+/-1.8% CD8(+)), 18.1+/-5.4% were B-lymphocytes, 8.5+/-3.9% were monocytes and 35% remained unidentified. The role of the cell cycle in the increased susceptibility to EHV-1 upon stimulation was examined by stimula...
Dohmann K, Wagner B, Horohov DW, Leibold W.In the present study, we describe the expression of equine IL2 and IL4. The cDNA of equine IL2 or IL4 was cloned in a mammalian expression vector, containing c-terminal myc- and six histidines His(6)-epitopes for recognition and purification of equine cytokines. The vector constructs were used for transfection of chinese hamster ovary (CHO) cells. Purified equine cytokines were characterised by western blotting. Equine IL2 was secreted with a molecular weight of approximately 17.1kDa, whereas IL4 was expressed in three different sizes of 17.1, 19.6 and 22.1kDa, probably due to different glycos...
Harrold SM, Cook SJ, Cook RF, Rushlow KE, Issel CJ, Montelaro RC.Equine infectious anemia virus (EIAV) infection of horses is characterized by recurring cycles of disease and viremia that typically progress to an inapparent infection in which clinical symptoms are absent as host immune responses maintain control of virus replication indefinitely. The dynamics of EIAV viremia and its association with disease cycles have been well characterized, but there has been to date no comprehensive quantitative analyses of the specific tissue sites of EIAV infection and replication in experimentally infected equids during acute disease episodes and during asymptomatic ...
van Der Meulen KM, Nauwynck HJ, Bí¶®rt W, Pensaert MB.In the present study, the outcome of an inoculation of equine peripheral blood mononuclear cells (PBMC) with equine herpesvirus type 1 (EHV-1) was studied in vitro. Cytoplasmic and plasma membrane expression of viral antigens, intra- and extracellular virus titres, and plaque formation in co-culture were determined. EHV-1 replicated in monocytes, although in a highly restricted way. Viral antigens were found at maximum levels (8.7% of the monocytes) at 12 h post-infection. The infection was productive in 0.16% of the monocytes. The virus yield was 10(0.7) TCID(50) per productive cell. In a pop...
Hammond SA, Horohov D, Montelaro RC.Naive T cells can be activated both in vivo and in vitro by specialized antigen presenting cells, dendritic cells (DC), with potent antigen-specific, immunostimulatory activity. Indeed, DC can provide an extremely powerful and important immunological tool by which to potentiate the immune response for specific recognition of foreign antigens. Until recently, the direct isolation of DC from PBMC required laborious procedures with extremely poor yields (<0.1%). Methods have been developed for the human, lower primate, and murine model systems to propagate large numbers of DC from PBMC or bone...
O'Neill T, Kydd JH, Allen GP, Wattrang E, Mumford JA, Hannant D.The frequency of antigen-specific, genetically restricted cytotoxic T lymphocyte precursors (CTLp) was measured in peripheral blood mononuclear cells (PBMC) of ponies before and after infection with equid herpesvirus 1 (EHV1). Split-well limiting dilution analysis (LDA) was developed to measure CTLp frequency using EHV1-infected 51Cr-labelled lymphoblasts as targets. Extensive characterisation showed that recombinant human interleukin-2, autologous antigen presenting cells and equine serum containing virus neutralising antibody were necessary for maturation of CTLp into effector CTL in vitro. ...
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...
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...
van der Meulen KM, Nauwynck HJ, Pensaert MB.In the present study, equine herpesvirus-1 (EHV-1)-infected cells were identified in ionomycin/phorbol dibutyrate (IONO/PDB)-stimulated peripheral blood mononuclear cells (PBMC) and the mechanism by which stimulation increases the percentage of infected cells was examined. In the population of viral antigen-positive PBMC, 38.4+/-4.5% were CD5(+) T-lymphocytes (18.1+/-3.2% CD4(+) 13.6+/-1.8% CD8(+)), 18.1+/-5.4% were B-lymphocytes, 8.5+/-3.9% were monocytes and 35% remained unidentified. The role of the cell cycle in the increased susceptibility to EHV-1 upon stimulation was examined by stimula...
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...
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...
Suthar A, Maji C, Gopalkrishnan A, Raval SH, Kumar R, Kumar S.Theileria equi and Babesia caballi are the causative agents of equine piroplasmosis (EP). Currently, imidocarb dipropionate (ID) is the only available drug for treating the clinical form of EP. Serious side effects and incomplete clearance of infection is a major drawback of ID. Heat-shock proteins (Hsp) play a vital role in the life cycle of these haemoprotozoans by preventing alteration in protein conformation. These Hsp are activated during transmission of EP sporozoites from the tick vector (poikilotherm) to the natural host (homeotherm) and facilitate parasite survival. In the present stu...
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 ...
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...
Thampi P, Seabaugh KA, Pezzanite LM, Chu CR, Phillips JN, Grieger JC, McIlwraith CW, Samulski RJ, Goodrich LR.Gene therapy approaches using adeno-associated viral vectors have been successfully tested in the equine post-traumatic osteoarthritis (PTOA) model. Owing to differences in the levels of transgene expression and adverse tissue reactions observed in published studies, we sought to identify a safe therapeutic dose of scAAVIL-1ra in an inflamed and injured joint that would result in improved functional outcomes without any adverse events. scAAVIL-1ra was delivered intra-articularly over a 100-fold range, and horses were evaluated throughout and at the end of the 10-week study. A dose-related incr...
Li S, Guo K, Wang X, Lin Y, Wang J, Wang Y, Du C, Hu Z, Wang X.Equine infectious anemia (EIA) has a worldwide distribution and causes severe economic losses to the equine industry. The EIA virus (EIAV) genome sequences from different countries are highly diverse, which poses a great challenge for pathogen identification with PCR. Phylogenetic analysis showed that although gag is the most conserved structural gene, it still has great genome variability. Currently, most existing PCR methods are designed based on the gag gene sequence and therefore do not cover all the viral strains, especially Asian EIAV strains. In this study, we developed a tat-gag-based ...
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...
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...
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...
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...
Tarr MJ, Olsen RG, Krakowka GS, Cockerell GL, Gabel AA.Erythrocyte rosette (ER) formation of equine peripheral blood lymphocytes (PBL) was characterized. Guinea pig and, to a lesser extent, human erythrocytes formed ER; cat, cow, dog, hamster, mouse, rat, and sheep erythrocytes showed negligible rosetting properties. Conditions of the assay were varied to determine which procedure allowed the largest percentage of rosette formation. The PBL from 20 normal horses were then assayed, averaging 38 +/- 2% ER. To characterize the erythrocyte receptor as being on T or B cells, equine thymocytes from 6 foals were assayed; the thymocytes formed an average ...
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-...
Cacciolatti C, Meyer-Ficca ML, Southwood LL, Meyer RG, Bertolotti L, Zarucco L.To evaluate effects of poly(ADP-ribose) polymerase-1 (PARP1) inhibitors on the production of tumor necrosis factor-α (TNF-α) by interferon-γ (IFN-γ)- and lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) of horses as an in vitro model of inflammation in horses. Methods: 1,440 samples of PBMCs from 6 healthy research horses. Methods: From heparinized whole blood samples, PBMC cultures were obtained. An initial dose-response trial on 48 PBMC samples from 2 horses (24 samples each) was used to determine concentrations of IFN-γ and LPS for use as low- and high-lev...
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...
Saini S, Singha H, Shanmugasundaram K, Tripathi BN.Burkholderia mallei causes a highly fatal infectious disease in equines known as glanders. It is one of the OIE listed notifiable diseases, which entails strict control policy measures once B. mallei infection is confirmed in the susceptible hosts. Humans, especially equine handlers, veterinary professionals and laboratory workers are at greater risk to acquire the B. mallei infection directly through prolonged contact with glanderous equines, and indirectly through unprotected handling of B. mallei contaminated materials. Further, natural resistance of B. mallei to multiple antibiotics, aeros...
McKelvie J, Little S, Foster AP, Cunningham FM, Hamblin A.It has been reported that equine peripheral blood mononuclear cells (PBMNs) do not proliferate in response to tetanus toxoid (TT) (Frayne and Stokes 1995, Research in Veterinary Science 59, 79-81). Here we demonstrate that lymphocyte proliferation responses to TT, which are characteristic of a recall antigen, may be achieved under certain culture conditions. Given that TT vaccination is routinely applied to many horses, TT is a suitable antigen for the investigation of cellular immune responses by peripheral blood mononuclear cells in the horse.
