Screening of anti-human leukocyte monoclonal antibodies for reactivity with equine leukocyte

Department of Clinical Sciences, Colorado State University, Fort Collins, Colorado, United States
Veterinary Immunology and Immunopathology (Impact Factor: 1.54). 10/2007; 119(1-2):63-80. DOI: 10.1016/j.vetimm.2007.06.034
Source: PubMed


Three hundred and seventy-nine monoclonal antibodies (mAbs) against various human CD molecules supplied to the HLDA8 animal homologues section (including four isotype controls) were analysed for cross-reactivity with equine leukocytes. First, flow cytometric identification of positively reacting mAbs was performed in one laboratory. Thereafter, a second round of flow cytometric evaluation was performed, involving three laboratories participating in the study. The first test-round indicated 17 mAbs as potentially positive. After the second round of flow cytometric analysis, 14 mAbs remained (directed against CD2, CD11a, CD18, CD44, CD45, CD49d, CD91, CD163 and CD172) where cross-reactivity was anticipated based on similarities between the human and equine staining pattern. Additionally, there was 1 mAb with weak likely positive reactivity, 12 mAbs with positive staining, which likely do not reflect valuable data, 5 mAbs with clear alternate expression pattern from that expected from humans, 5 mAbs with a questionable staining pattern itself, i.e. that was variable between the three labs, 32 mAbs with weak-positive expression and alternate staining pattern, and 279 negative mAbs (including the four isotype controls) were detected. In 31 cases, more appropriate target cells, such as thymocytes or stem cells, were not available for the screening. The results underline the value of this "cross-reactivity" approach for equine immunology. However, as only a few mAbs against leukocyte surface antigens reacted positively (approximately 4% of the mAbs submitted), the analysis of further anti-human mAbs and directed efforts to develop species-specific anti-CD mAb are still required.

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    • "Greater than 90% of the analyzed PBMC were CD172a+, indicating that they were monocytes/macrophages (data not shown). Although the anti-CD172a mAb also identifies equine granulocytes [40], the ficoll hypaque PBMC isolation protocol (which removes the majority of granulocytes) together with the lack of internal complexity in the labeled cells (data not shown), provided further evidence that they were monocytes/macrophages. Overall, these data confirmed the SCID phenotype and indicated that the establishment of T. equi infection in SCID1 and SCID2 did not involve B or T lymphocytes. "
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    ABSTRACT: Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata), the intraleukocyte stage (schizont) of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis, breed susceptibility, and strain virulence.
    PLoS ONE 10/2013; 8(10):e76996. DOI:10.1371/journal.pone.0076996 · 3.23 Impact Factor
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    • "According to Borjesson and Peroni (2011), the capacity to characterize equine MSCs is hampered by the limited availability of commercial antibodies and by variability in cross-reactions . Ibrahim et al. (2007) showed that of 379 human CD antibodies, only 14 (4%) showed cross-reactivity with equine leukocytes. Alternatively, De Schauwer et al. (2011) suggested the use of a panel of cell surface markers for equine MSCs. "
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    ABSTRACT: The aim of this study was to isolate, culture, and characterize mesenchymal stem cells (MSCs) from horse bone marrow (BM) using the techniques of flow cytometry, immunocytochemistry, cytogenetics, and electron microscopy. Immunophenotypic analysis revealed the presence of MSCs with high expression of the CD90 marker, lower expression of the CD44 marker, and absent expression of the CD34 marker. In assays of differentiation, the positive response to osteogenic (OST), chondrogenic (CDG), and adipogenic (ADP) differentiation signals was observed and characterized by deposition of calcium-rich extracellular matrix (OST), proteoglycans and collagen II (CDG) and intracellular deposition of fat drops (ADP). In immunocytochemical characterization, MSCs were immunopositive for CD44, vimentin, and PCNA, and they were negative for CD13. In the ultrastructural analysis of MSCs, the most outstanding characteristic was the presence of rough endoplasmic reticulum with very dilated cisterns filled with a low electrodensity material. Additionally, MSCs had normal karyotypes (2n = 64) as evidenced by cytogenetic analysis, and aneuploidy in metaphase was not observed. The protocols for isolating, culturing, and characterizing equine MSCs used in this study were shown to be appropriate for the production of a cell population with a good potential for differentiation and without aneuploidy that can be used to study future cellular therapies. Microsc. Res. Tech., 2013. © 2013 Wiley Periodicals, Inc.
    Microscopy Research and Technique 03/2013; 5(6). DOI:10.1002/jemt.22208 · 1.15 Impact Factor
    • "Our results thus suggest donor age does not influence the successful isolation of these cells, although we are making this suggestion based on a very small number of samples. Valid immunophenotyping necessitates proper use of isotype controls to exclude non-specific antibody reactions, and positive control cells to confirm cross reactivity in horses, since only some 4% of human antibodies reacts with equivalent equine proteins (Ibrahim et al., 2007). Another important consideration when performing flow cytometry is the possibility that some epitopes can be destroyed by trypsin, resulting in false negative results (Hackett et al., 2011). "
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    ABSTRACT: Although the use of mesenchymal stromal cells (MSCs) for the treatment of orthopaedic injuries in horses has been reported, no official guidelines exist that classify a particular cell as an equine MSC. Given the limited characterisation of peripheral blood (PB)-derived equine MSCs in particular, this study aimed to provide more detailed information in relation to this cell type. Mesenchymal stromal cells were isolated from equine PB samples and colony forming unit (CFU) assays as well as population doubling times (PDTs) (from P(0) to P(10)) were performed. Two types of colonies, 'fingerprint' and dispersed, could be observed based on macroscopic and microscopic features. Moreover, after an initial lag phase (as indicated by a negative PDT at P(0) to P(1)) the MSCs divided rapidly as indicated by a positive PDT at all further passages. Immunophenotyping was carried out with trypsin- as well as with accutase-detached MSC to evaluate potential trypsin-sensitive epitope destruction on particular antigens. Isolated MSC were positive for CD29, CD44, CD90 and CD105, and negative for CD45, CD79α, MHC II and a monocyte/macrophage marker, irrespective of the cell detaching agent used. Trilineage differentiation of the MSCs towards osteoblasts, chondroblasts and adipocytes was confirmed using a range of histochemical stains.
    The Veterinary Journal 06/2012; 195(1). DOI:10.1016/j.tvjl.2012.05.006 · 1.76 Impact Factor
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