[show abstract][hide abstract] ABSTRACT: A long-term goal of mesenchymal progenitor cell (MPC) research is to identify cell-surface markers to facilitate MPC isolation. One reported MPC feature in humans and other species is lack of CD14 (lipopolysaccharide receptor) expression. The aim of this study was to evaluate CD14 as an MPC sorting marker. Our hypothesis was that cells negatively selected by CD14 expression would enrich MPC colony formation compared with unsorted and CD14-positive fractions. After validation of reagents, bone marrow aspirate was obtained from 12 horses. Fresh and cultured cells were analyzed by flow cytometry and reverse transcription and quantitative polymerase chain reaction to assess dynamic changes in phenotype. In fresh samples, cells did not consistently express protein markers used for lineage classification. Short-term (2-day) culture allowed distinction between hematopoietic and nonhematopoietic populations. Magnetic activated cell sorting was performed on cells from 6 horses to separate adherent CD14(+) from CD14(-) cells. MPC colony formation was assessed at 7 days. Cells positively selected for CD14 expression were significantly more likely to form MPC colonies than both unsorted and negatively selected cells (P ≤ 0.005). MPCs from all fractions maintained low levels of CD14 expression long term, and upregulated CD14 gene and protein expression when stimulated with lipopolysaccharide. The equine CD14 molecule was trypsin-labile, offering a plausible explanation for the discrepancy with MPC phenotypes reported in other species. By definition, MPCs are considered nonhematopoietic because they lack expression of molecules such as CD14. Our results challenge this assumption, as equine MPCs appear to represent a descendant of a CD14-positive cell.
Stem cells and development 04/2011; 20(4):721-35. · 4.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mesenchymal progenitor cells (MPCs) are often characterized using surface markers after expansion and treatment in culture. There are no studies directly comparing gene and protein markers in undifferentiated samples during the very early phases of culture. The goal of this study was to evaluate temporal gene and protein expression changes during establishment of equine MPC cultures. Bone marrow aspirate was obtained from 35 horses and processed by density gradient centrifugation. In freshly isolated bone marrow, mononuclear cells had variable expression of CD44, CD11a/CD18, CD90, and CD45RB cell surface molecules. After 2 h of culture, bone marrow mononuclear cells had a phenotype of CD44(hi), CD29(hi), CD90(lo), CD11a/CD18(hi), and CD45RB(lo). Isolated mononuclear cells were analyzed by flow cytometry and RT-qPCR at 2, 7, 14, 21, and 30 days of culture. At all culture time points, gene expression was in agreement with cell surface protein expression. In established cultures of MPCs, cells remained robustly positive for CD44 and CD29. The proportion of positive cells and the mean fluorescence intensity of positive cells increased in CD90 expression as MPC cultures became more homogeneous. Inversely, the population of cells in culture decreased expression of CD11a/CD18 and CD45RB molecules over time. The decreased expression of the latter molecules makes these useful negative markers of established MPC cultures under normal expansion conditions. The results of this study demonstrate numerous dynamic changes in cell surface molecule expression during early establishment of MPC populations, which may aid to improve MPC isolation methods for research or therapeutic applications.
Stem cells and development 08/2009; 19(2):269-82. · 4.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: Many features of the equine immune system develop during fetal life, yet the naïve or immature immune state of the neonate renders the foal uniquely susceptible to particular pathogens. RT-PCR and immunohistochemical experiments investigated the progressive expression of developmental B cell markers and immunoglobulins in lymphoid tissues from equine fetus, pre-suckle neonate, foal, and adult horses. Serum IgM, IgG isotype, and IgA concentrations were also quantified in pre-suckle foals and adult horses. The expression of essential B cell genes suggests active development and gene recombination during equine gestation, including immunoglobulin isotype switching. The corresponding production of IgM and IgG proteins is detectable in a limited scale at birth. Although the equine neonate humoral response seems competent, B cell activation factors derived from antigen presenting cells and T cells may control critical developmental regulation and immunoglobulin production during the initial months of life.
Developmental and comparative immunology 06/2009; 33(9):1027-38. · 3.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dendritic cells (DCs) are innate immune cells specialized in antigen detection and presentation. They perform an essential role in initiating and guiding the immune response, the direction of which largely depends upon the activation state of the DCs. The objective of this study was to generate mature equine monocyte-derived DCs and, in doing so, to develop a method for measuring the activation state of these cells. Equine DCs were stimulated with UV-inactivated Escherichia coli (E. coli), and the activation status was measured by analyzing cell surface marker expression, cytokine production, and endocytic capacity. Comparisons for each parameter measured were performed between macrophages, non-stimulated DCs and stimulated DCs. Equine monocyte-derived DCs may be distinguished from macrophages based on cell surface expression of MHC class II (p<0.0001) and CD206 (p<0.0001), their capacity for endocytosis of FITC-dextran (p<0.05), and production of TNF-alpha upon stimulation (p<0.001). Furthermore, stimulated DCs can be distinguished from non-stimulated DCs based on increased cell surface expression of MHC class II (p<0.0001) and upregulation of pro-inflammatory cytokine mRNA, particularly IL-12/IL-23p40 (p<0.05) and IL-23p19 (p<0.05). The ability to measure DC activation state will facilitate future investigations of equine DC function.
Veterinary Immunology and Immunopathology 05/2009; 131(3-4):259-67. · 1.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: Susceptibility of foals to Rhodococcus equi pneumonia is exclusive to the first few months of life. The objective of this study was to investigate the immediate immunologic response of foal and adult horse antigen-presenting cells (APCs) upon infection with R. equi. We measured the activation of the antigen-presenting major histocompatibility complex (MHC) class II molecule, costimulatory molecules CD40 and CD86, the cytokine interleukin-12 (IL-12), and the transcriptional factor interferon regulatory factor 1 (IRF-1) in monocyte-derived macrophages (mMOs) and dendritic cells (mDCs) of adult horses and foals of different ages (from birth to 3 months of age) infected with virulent R. equi or its avirulent, plasmid-cured derivative. Infection with virulent or avirulent R. equi induced (P <or= 0.01) the expression of IL-12p35 and IL-12p40 mRNAs in foal mMOs and mDCs at different ages. This response was likely mediated by the higher (P=0.008) expression of IRF-1 in foal mDCs at birth than in adult horse mDCs. R. equi infection promoted comparable expression of costimulatory molecules CD86 and CD40 in foal and adult horse cells. The cytokine and costimulatory response by foal mDCs was not accompanied by robust MHC class II molecule expression. These data suggest that foal APCs detect the presence of R. equi and respond with the expression of the Th1-inducing cytokine IL-12. Nevertheless, there seems to be a limitation to MHC class II molecule expression which we hypothesize may compromise the efficient priming of naïve effector cells in early life.
[show abstract][hide abstract] ABSTRACT: INTRODUCTION: Common variable immunodeficiency (CVID) in horse patients is characterized by late-onset B cell lymphopenia or depletion, hypo- or agammaglobulinemia, impaired humoral response to tetanus toxoid vaccination, and recurrent fevers and bacterial infections. DISCUSSION: This study describes the clinical and immunologic findings of 14 affected horses (average age 10.7 +/- 4.4 years) of both genders (six females, eight males) and different breeds (eight Thoroughbreds, four Quarter Horses, one Warmblood, one Pony). Serial immunological testing in peripheral blood revealed persistent, severe B cell lymphopenia (mean 1.3 +/- 2.3% positive cells) in all patients. Serum IgG (range <200 to 800 mg/dL) and IgM (<or=25.0 mg/dL) deficiency was common to all horses. Serum IgA concentrations declined with time. Histopathology and immunohistochemistry revealed absence of lymphoid follicles and B cells in primary and secondary lymphoid tissues. CVID is a cause of recurrent pneumonia, septicemia, and meningitis in adult horses and has a grave prognosis for clinical management and survival.
Journal of Clinical Immunology 08/2008; 29(1):107-16. · 3.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: Interferon-alpha (IFN-alpha) is a type I interferon that is secreted during the early stages of the innate immune response and is often induced upon infection with viral pathogens. IFN-alpha production affects multiple downstream events influencing both innate and adaptive immune responses. Here, we describe the expression of an equine rIFN-alpha/IgG4 fusion protein in mammalian cells. The anti-viral activity of rIFN-alpha/IgG4 was found to be 70-fold higher than that of a previously described IFN-gamma/IgG1 as tested by bioassay. The purified rIFN-alpha was subsequently used for the generation of six monoclonal antibodies (mAbs) to equine IFN-alpha. Four of these mAbs inhibited the protective anti-viral effect of equine leukocyte IFN in bioassays. One mAb (clone 240-2) showed a high-neutralizing capacity. An ELISA was established using two anti-equine IFN-alpha mAbs (clones 29B and 240-2) and its analytical sensitivity for was found to be around 800 pg/ml and 3 U/ml for rIFN-alpha and equine leukocyte IFN, respectively. When analyzing samples with a likely dominance of IFN-alpha among type I IFNs, such as supernatants from equine peripheral blood mononuclear cells stimulated with CpG-oligodeoxyribonucleotides, the results obtained by ELISA and IFN bioassay showed a high agreement (r(2)(sp)=0.98). When analyzing samples likely containing a mixture of type I IFNs, such as serum and nasal secretions from virally infected horses, the ELISA only detected some of the IFN-activity recorded in the bioassay. Overall, the data showed that the new anti-equine IFN-alpha mAbs are valuable tools to detect native IFN-alpha for further characterization of the early innate immune response and anti-viral immunity in horses.
Veterinary Immunology and Immunopathology 06/2008; 125(3-4):315-25. · 1.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have reported on the reactivity of anti-human CD molecules with equine leukocytes by single-colour flow cytometry (this issue). The objectives of this additional study were to test for the reliability of the results obtained, and to obtain further information on the positive populations of lymphocytes. Two-colour flow cytometry and immunohistochemistry were performed, using many of the positive mAbs and a few questionable ones from the first part of the study. All mAbs analysed by two-colour flow cytometry could be confirmed to their previous designation as "positive" or "questionable". Most of the mAbs tested were effective in immunohistochemistry, supporting previous results. Examples of positive results will be presented and limitations of the study will be discussed briefly.
Veterinary Immunology and Immunopathology 10/2007; 119(1-2):92-9. · 1.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cytosine-phosphate-guanosine oligodeoxynucleotide (CpG-ODN) has been used successfully to induce immune responses against viral and intracellular organisms in mammals. The main objective of this study was to test the effect of CpG-ODN on antigen presenting cells of young foals.
Peripheral blood monocytes of foals (n = 7) were isolated in the first day of life and monthly thereafter up to 3 months of life. Adult horse (n = 7) monocytes were isolated and tested once for comparison. Isolated monocytes were stimulated with IL-4 and GM-CSF (to obtain dendritic cells, DC) or not stimulated (to obtain macrophages). Macrophages and DCs were stimulated for 14-16 hours with either CpG-ODN, LPS or not stimulated. The stimulated and non-stimulated cells were tested for cell surface markers (CD86 and MHC class II) using flow cytometry, mRNA expression of cytokines (IL-12, IFNalpha, IL-10) and TLR-9 using real time quantitative RT-PCR, and for the activation of the transcription factor NF-kappaB p65 using a chemiluminescence assay.
The median fluorescence of the MHC class II molecule in non-stimulated foal macrophages and DCs at birth were 12.5 times and 11.2 times inferior, respectively, than adult horse cells (p = 0.009). That difference subsided at 3 months of life (p = 0.3). The expression of the CD86 co-stimulatory molecule was comparable in adult horse and foal macrophages and DCs, independent of treatment. CpG-ODN stimulation induced IL-12p40 (53 times) and IFNalpha (23 times) mRNA expression in CpG-ODN-treated adult horse DCs (p = 0.078), but not macrophages, in comparison to non-stimulated cells. In contrast, foal APCs did not respond to CpG-ODN stimulation with increased cytokine mRNA expression up to 3 months of age. TLR-9 mRNA expression and NF-kB activation (NF-kB p65) in foal DCs and macrophages were comparable (p > 0.05) to adult horse cells.
CpG-ODN treatment did not induce specific maturation and cytokine expression in foal macrophages and DCs. Nevertheless, adult horse DCs, but not macrophages, increased their expression of IL-12 and IFNalpha cytokines upon CpG-ODN stimulation. Importantly, foals presented an age-dependent limitation in the expression of MHC class II in macrophages and DCs, independent of treatment.
Journal of Immune Based Therapies and Vaccines 02/2007; 5:1.
[show abstract][hide abstract] ABSTRACT: Phagocytic activity of neonatal foals has been reported to be similar to that of adult horses, but serum opsonization capacity develops with age and may be further altered when opsonins are consumed during infection.
Phagocytosis, oxidative burst activity, and serum opsonization capacity in neonatal foals admitted to an intensive care unit are reduced in comparison with control foals.
Blood samples were collected from hospitalized neonatal foals and from control foals. Hospitalized foals were characterized as sick or septic on the basis of a sepsis score and received intravenous plasma transfusion.
Phagocytosis, oxidative burst activity, and serum opsonization capacity were tested with flow cytometric analysis. Serum immunoglobulin and complement component 3 concentrations were determined with radial immunodiffusion. Serum amyloid A concentration was assayed with a commercially available solid-phase Sandwich ELISA Kit. Data were analyzed with nonparametric and regression methods. Alpha was set at P = .05.
Phagocytic functions of septic and sick foals were lower than control foals in the initial phase of the study (P = .01). Opsonization capacity was significantly higher when bacteria were opsonized with serum from septic (P = .029) and sick (P = .006) foals than from control foals on day 1. Opsonization capacity in septic foals was comparable with control foals on days 2 and 5. This effect was not accompanied by an increase in serum complement C3 or immunoglobulin G concentrations independently.
Our results suggest that phagocytic function could be decreased in hospitalized foals. The synergistic effect of opsonic elements provided by plasma transfusion may sustain opsonization capacity during sepsis.
Journal of Veterinary Internal Medicine 01/2007; 21(4):797-805. · 2.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: The mannose-binding lectin (MBL), a pattern recognition serum protein, participates in the innate immune system of mammals as an opsonin. In humans, MBL plays a key role in first-line host defense against infection during the lag period prior to the development of a specific immune response. MBL also activates complement via the lectin pathway that requires a MBL-associated serine protease-2 (MASP-2). Homologues of human MBL (hMBL) have been identified in a variety of mammals, fish, and primitive animals such as ascidians. In this study, we report that equine MBL (eMBL) has properties that are similar to hMBL. In addition, we found low levels of MBL:MASP activity in sick horses compared to healthy horses. These results suggest that eMBL is involved in the immune response of the horse and that low MBL:MASP activity could be used to monitor immune function and clinical outcome.
Biochemical and Biophysical Research Communications 06/2006; 343(3):928-36. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Three adult horses were evaluated for signs of musculoskeletal pain, dullness, ataxia, and seizures. A diagnosis of bacterial meningitis was made on the basis of results of CSF analysis. Because primary bacterial meningitis is so rare in adult horses without any history of generalized sepsis or trauma, immune function testing was pursued. Flow cytometric phenotyping of peripheral blood lymphocytes was performed, and proliferation of peripheral blood lymphocytes in response to concanavalin A, phytohemagglutinin, pokeweed mitogen, and lipopolysaccharide was determined. Serum IgA, IgM, and IgG concentrations were measured by means of radial immunodiffusion, and serum concentrations of IgG isotypes were assessed with a capture antibody ELISA. Serum tetanus antibody concentrations were measured before and 1 month after tetanus toxoid administration. Phagocytosis and oxidative burst activity of isolated peripheral blood phagocytes were evaluated by means of simultaneous flow cytometric analysis. Persistent B-cell lymphopenia, hypogammaglobulinemia, and abnormal in vitro responses to mitogens were detected in all 3 horses, and a diagnosis of common variable immunodeficiency was made.
Journal of the American Veterinary Medical Association 08/2005; 227(1):114-22, 87. · 1.72 Impact Factor
[show abstract][hide abstract] ABSTRACT: Observations in early equine pregnancy clearly reveal maternal immune recognition of and response to the presence of the conceptus. Nevertheless, both maternal cellular and humoral responses appear ineffective in destroying the developing placenta and fetus in early pregnancy. Our previous studies had shown that the pre-conditioned medium generated from the culture of equine invasive trophoblast inhibited mitogen-induced lymphocyte proliferation and the expression of cytokine messenger RNA in vitro. Those findings also suggested that lymphocytes might have been halted in the G0/G1 phase of the cell cycle. To characterize the cell cycle and the intracellular mechanisms involved in the inhibition of lymphocyte proliferation, equine peripheral blood lymphocytes were cultured in the presence or absence of pokeweed mitogen (PWM) in fresh medium, or in medium pre-conditioned through cell culture of invasive trophoblast cells or fetal fibroblasts. Two-color flow cytometric analysis for bromodeoxyuridine (BrdU) incorporation by stimulated lymphocytes, and concomitant DNA staining with 7-amino-actinomycin D (7-AAD), indicated that a greater proportion of lymphocytes were found in the G0/G1 phase of the cell cycle when cultured in the invasive trophoblast cell pre-conditioned medium compared to controls. Analysis using carboxyfluorescein diacetate succinimidyl ester (CFSE) fluorescence intensity demonstrated that lymphocytes cultured in the presence of invasive trophoblast cell pre-conditioned medium had fewer cells going through division, but that those fewer cells sustained similar numbers of cell divisions as in control cultures. Hypophosphorylated retinoblastoma (Rb) protein expression was increased and p27Kip1 expression was maintained at higher levels in lymphocytes cultured in invasive trophoblast pre-conditioned medium compared to fresh medium. In agreement with these data, flow cytometric measurement of the Ki-67 protein expression in lymphocytes cultured in invasive trophoblast pre-conditioned medium was lower in comparison to controls. These findings suggest that the equine lymphocyte proliferation is at least partially regulated by the expression of proliferation inhibitory proteins such as p27Kip1 and hypophosphorylated Rb. These proteins seem to be important regulators of cell cycle transition between G1 and S phase in equine lymphocytes.
Veterinary Immunology and Immunopathology 01/2005; 102(4):363-77. · 1.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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 commencement of a 9-week conditioning program and 24h after the completion of a maximum exercise test conducted in week 12. Samples were obtained simultaneously from control horses. Differential counts were performed on the bronchoalveolar lavage cells. Real-time PCR was performed on the pulmonary and PBMCs to quantitate cytokine expression using equine-specific primers and Taqman probes. Target gene expression was normalized to 18s rRNA expression. With the exception of IL-4 in the BALF cells, mRNA for the three cytokines was detected in the mononuclear cells from all horses at both sampling times. There were no significant differences in the cytokine mRNA concentrations between the two groups of horses at either of the sampling times. These findings demonstrate that strenuous treadmill exercise does not exert a deleterious effect on gene expression for IL-12p35, IFN-gamma or IL-4 when assessed in horses 24h following the intense physical activity.
Veterinary Immunology and Immunopathology 02/2003; 91(1):61-71. · 1.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: A 12-year-old Quarter Horse mare that was nonresponsive to medical treatment was evaluated for chronic respiratory disease and hepatobiliary disease. Serum immunoglobulin concentrations were measured by use of radial immunodiffusion that revealed trace to nondetectable concentrations of IgG, IgG(T), IgM, and IgA. Use of serum protein electrophoresis confirmed agammaglobulinemia by the absence of the expected peak in the gamma region. In addition, vaccination with tetanus toxoid did not result in specific immunoglobulin production. Flow cytometric analysis of blood lymphocyte subpopulations revealed the absence of B cells in blood. Immunohistochemical analysis of tissue sections revealed the absence of B lymphocytes in bone marrow and spleen, with occasional B cells in the peripheral lymph nodes. Blood lymphocyte proliferation assays revealed weak responses to pokeweed mitogen and no response to stimulation with lipopolysaccharide. Considering the age and sex of the horse, results of the immunologic tests suggested a diagnosis of common variable immunodeficiency.
Journal of the American Veterinary Medical Association 12/2002; 221(9):1296-302, 1267. · 1.72 Impact Factor
[show abstract][hide abstract] ABSTRACT: Studies in infants and foals indicate an age-dependent maturation of peripheral lymphocyte subsets. The age-dependent relationship for maturation of cellular immune responses, such as phagocytosis and lymphocyte responses of the peripheral and pulmonary-derived leukocytes, has not been characterized in foals. Lymphocyte subpopulations, mitogen stimulation response of lymphocytes, lymphokine-activated killing cell activity, phagocytosis and oxidative burst activity, and serum immunoglobulin (Ig) classes G and M concentrations were determined in developing foals. This study illustrates age-dependent changes in immunoglobulin class concentrations, lymphocyte subsets, and EqMHC Class II expression in cells of the peripheral blood and lungs of developing neonatal-to-weanling foals. The increase in peripheral blood and BAL B-lymphocytes and serum immunoglobulins in developing foals suggests expansion of immune cell populations during a time in which environmental pathogen exposure is great. General immune function, mitogenic responses, LAK cell activity, opsonized phagocytosis, and oxidative burst activity of newborns was similar to the adult horse. Total immune-cell numbers, rather than function, seemed to be the limiting factor in the development of the equine neonatal immune system. There was an age-related percent increase in the appearance of pulmonary lymphocytes, but a percent decrease in macrophages. Although development of the respiratory immune system follows changes in the peripheral blood, cellular expansion, activation, and migration may occur at a slower pace, making the respiratory environment susceptible to pathogens prior to optimal immune system maturity.
Veterinary Immunology and Immunopathology 04/2000; · 1.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: We describe a method for creating differentiated equine bronchial epithelial cell cultures that can be used for in vitro studies
including airway disease mechanisms and pathogen–host interactions. Our method is based on the culturing of human tracheobronchial
epithelial cells at an air–liquid interface (ALI) in specific serum-free, hormone-supplemented medium. Bronchial epithelial
cells are isolated and grown on T-Clear® insert membranes. Within 2 to 3wk, cells differentiate into ciliated and mucus producing
cells as demonstrated by confocal and electron microscopy. Furthermore, the demonstration of the two major gel-forming mucin
species, Muc5ac and Muc5b, in our bronchial epithelial cell culture system validates this method for studies of respiratory
tract disease of the horse.
KeywordsBronchial epithelial cells-Horse-Cilia-Mucins