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Immunology of companion animals
3rd International Veterinary Immunology Symposium - 219 -
Poster Session 20 Immunology of Companion Animals Atsuhiko Hasegawa
PS 20.1
CANINE DISTEMPER VIRUS AND RHEUMATOID ARTHRITIS IN DOGS. Carter, S.D.; May, C.; Bell, S.C. and Bennett, D. Departments of Veterinary Clinical Science & Veterinary Pathology, University of Liverpool, UK
Canine rheumatoid arthritis (CRA) is a chronic inflammatory joint disease with many similarities to the human equivalent, for which it serves as a modell. As in human RA, there is evidence of a number of autoimmune responses in the absence of an identifiable aetiological agent. This study was designed to consider the possible involvement of a number of common dog viruses in CRA. The approach taken was to determine the levels of anti-viral antibodies in sera and synovial fluids and to investigate the presence of viral antigens in immune complexes and in diseased synovial tissues. Dogs with CRA had increased antibodies (by ELISA) to canine distemper virus (CDV) in both sera and, particulaty, synovial fluids. No such increase was seen with any other virus tested. Increased anti-CDV antibodies were also found in precipitated immune complexes. Western blotting of synovial fluid immune complexes and probing with anti-CDV antibodies showed CDV antigens in synovial fluid complexes in CRA but not in normal dogs or dogs with osteoarthritis (OA). In CRA, both circulating immune complexes (IC) and synovial fluid white cell counts correlated with anti-CDV antibodies (pcO.001). No such correlations were seen in dogs with (OA). Preliminary immunocytochemical analyses, using monoclonal antibodies, showed evidence of CDV antigens in rheumatoid synovial tissues and not in Ok The relevance of this study to both canine and human rheumatological disorders is discussed.
3rd International Veterinary Immunology Symposium - 220 PS 20.2
IDENTIFICATION OF GENES ENCODING CANINE T CELL RECEPTOR COMPLEX Goitsuka, R.; Takano, M.; Okuda, M.; Ushiya, S.; Tsujimoto, H. and Hasegawa, A. Department of Veterinary Internal Medicine, University of Tokyo, Tokyo, Japan
To provide the basis for molecular dissection of canine lymphoproliferative diseases, canine genes encoding T cell receptor (TCR) complex including CD3, TCRa and p-chain constant regions (Gz and CjY) were molecularly cloned by the use of PCR with the primers corresponding to the conserved sequences of these genes among species. After PCR-amplification of cDNA prepared from canine lymph node cells, obvious DNA bands of the expected sizes (approximately 220 bp for CD3,320bp for Ca, and 360bp for Cj3) were detected. At the nucleotide and amino acid levels, the DNA 220bp PCR product showed significant homology with human and murine CDM genes rather than with CD3y genes. In the predicted transmembrane region of this clone, 19 out of 27 amino acid residues were identical with human CD%, and a centrally placed negatively charged aspartic acid residue was conserved. Furthermore, two characteristic cysteine residues in the extracellular domain were also found in this clone, indicating that this clone represented a canine homologue of CD3B gene. By using reverse-transcriptional PCR, the transcription of CD3c? gene was observed in CL-l cells which derived from tymic-form malignant lymphoma. The sequence information of Ca and C/l of TCR will be also discussed together with CD3.
3rd International Veterinary Immunology Symposium - 221PS 20.3
IMMUNOSCINTIGRAPHY OF CANINE OSTEOSARCOMAS WITH MONOCLONAL ANTIBODIES TO OSTEOSARCOMAASSOCIATED ANTIGENS Haines, D.M.‘; Bruland, 0.S.2; Matte, G.3; Wilkinson, kk3; Merit, S.M.’ and Fowler, J.D.’ lWestern College of Veterinary Medicine; 3Medica1 Imaging University of Saskatchewan, Saskatoon, Canada S7N OWO and 2Departments of Medical Oncology and Radiotheraphy and of Tumor Biology, Norwegian Radium Hospital, Montebello, Norway
Monoclonal antibody TP-1 selectively binds to human and canine osteosarcoma cells in vitro using immunhistochemical stains. radio-iodinated F(ab’)2 fragments of monoclonal antibody TP-1 was administered intravenously in 4 dogs with primary and/or metastatic spontaneous osteosarcoma. Immunoscintigraphy successfully demonstrated the radiolabeled antibody fragments in 6/6 known primary or metastatic lesions and in addition detected 4 metastatic lesions not diagnosed by conventional radiographs. Concurrent imaging of 99mT~ labeled autologous erythrocytes in two dogs confirmed that the accumulation of radiolabeled antibody fragments was independent of the blood pool. The present study has demonstrated the potential of monoclonal antibody TP-1 F(ab’)2 fragments for early detection of metastatic spread of spontaneous osteosarcoma.
PS 20.4
ENHANCING EFFECTS OF EGG WHITE DERIVATIVE ON PHAGOCYTOSIS AND CHEMOTAXIS OF CANINE PERIPHERAL BLOOD PHAGOCYTES Hirota, Y.t, Yang, M-P.‘, Honjo, K.‘, Ikeda, T.l, Yoshihara, K.‘, Shimizu, S.‘, Araki, S.2 and Onodera, T.l ‘National Institute of Animal Health, 2Eisai Co., Ltd., Tsukuba, Ibaraki 305, Japan
Effects of egg white derivative (EWD) on the functions of peripheral blood leukocytes (PBL) in the dog were examined. PBL from clinically healthy adult beagles were cultured with EWD, and phagocytized FITC-conjugated beats for 1 hr at 37OC. Their phagocytosis was evaluated by flow cytometry. The
3rd International Veterinary immunology Symposium - 222 chemotaxis was measured by a modified Boyden chamber method. PBL stimulated with EWD (200 ,@ml) for 4 hr showed enhanced phagocytosis. The supplement of culture supernatant of mononuclear cells cultured with EWD for 24 hr at 37OC into freshly isolated PBL induced the enhanced phagocytosis. The culture supernatant of EWD-stimulated mononuclear cells also induced significantly enhanced chemotaxis of neutrophils. However, this chemotactic activity was not observed in the culture supernatant from EWD-stimulated neutrophils. These results suggest that EWD has an enhancing effect on phagocytosis and chemotaxis of canine phagocytes and that the events is mediated in part by humoral factors released from EWD-stimulated mononuclear cells but not from EWD-stimulated neutrophils.
PS 20.5
PRODUCTION AND CHARACTERIZATION OF FELINE x MURINE HETEROHYBRIDOMA LINES Leidinger, E.‘; Gemeiner, M.’
Kramberger-Kaplan, E.3; Anrather, J.2 and
‘Institute of Medical Chemistry, 21nternal Clinic for Small Animals, Horses and Poultry, Veterinary Medical University Vienna, Linke Bahng 11, A-1030 Vienna, Austria, 3Vet. Practitioner, Wr. Neustadt, Austria The occurance of myelomas in cats is a rare event and therefore no feline myeloma-lines suitable for hybridizations could be developed so far. In this experiment we produced feline x murine heterohybridomas by fusing feline splenocytes with murine fusion partners, as used for standard murine x murine hybridizations. In order to optimize the fusion efficiency and standardize the following propagation of the heterohybridomas, the effects of different murine fusion lines and media supplements were studied. Up to now several HAT-resistant feline x murine heterohybridomas could be isolated and were characterized by chromosomal analysis and growth properties. Though none of the heterohybridoma lines secreted feline monoclonal antibodies, we hope to succeed by adapting one of them as HAT-resistant fusion line for the production of hybrid-hybridomas (2°-hybridomas).
3rd International Veterinary Immunology Symposium - 223 PS 20.6
DIFFERENT PATTERN OF CANINE ANTINUCLEAR ANTIBODIES ON WESTERN BLOTS Schuberth, H.J.; Kollmer, L. and Leibold, W. Immunology Unit, Veterinary School, Hannover, Germany
Sera from 31 dogs with suspected autoimmune disease were tested in parallel in the standard fluorescent antinuclear antibody test (FANA) on HEp2 and MDCK cells and on nuclear protein preparations of these cells after SDSPAGE and western blotting (ANA-blot: AB). In the FANA assay 18/31 sera reacted with HEp-2 and MDCK showing no differences in their staining pattern. The titre on MDCK was slightly higher. 17/31 sera were positive in the ANA-blot. AB has a r-value of .67 with FANA (3 sera FANA + / AI3 - and 2 sera AI3 + / FANA -). The 17 AB-positive sera showed a total of 12 different significant bands on Western blots occurring separately or in typical combinations. The most promiment reactivity was found with proteins of 46 kDa (lO/lS), 90 + 105 kDa (3/15) and 20 kDa (3/15). The determination of protein- and epitope-specific antibodies in sera of dogs with autoimmune disorders will improve their diagnostic, prognostic and therapeutic values as compared to the standard fluorescent assay.
PS 20.7
DEVELOPMENT OF LYMPHOID CELLS IN NEWBORN BUFFALO CALVES Tewari, D.’ and Gael, M.C.2 ‘Dept. Clinical Vet. Med., Madingley road, Cambridge, UK, 2Veterinary Microbial., H.A.U., Hisar, India.
A study on seven buffalo calves from birth to three months of age was done for identification of lymphoid cells and investigation of their functions. This revealed that the number of T cells, identified as peanut agglutinin binding cells amongst mononuclear cell population remained unaltered throughout this period. While, the number of cells identified as SIg bearing cells increased fourfold by the end of three month period compared to 10 days old calves. T cell subsets were identified as EA rossette forming cells with either IgG or IgM in T cell enriched population. Cells bearing Fc receptors for IgG (Suppressor) was
3’* International Veterinary Immunology Symposium - 224 higher on day of birth and levels decreased by day 60 of age. However, levels of IgM attaching cells (Helper) remained unaltered. The ability of MNC to undergo mitogenisis in response to T cell mitogens was less during first 10 days of life, but the response to B cell mitogen was low throughout the study period. The study indicated functional immaturity of T cells and less numbers of B cells until about three weeks after birth in buffalo calves.
Poster Session 20 Immunology of Companion Animals Atsuhiko Hasegawa
PS 20.1
CANINE DISTEMPER VIRUS AND RHEUMATOID ARTHRITIS IN DOGS. Carter, S.D.; May, C.; Bell, S.C. and Bennett, D. Departments of Veterinary Clinical Science & Veterinary Pathology, University of Liverpool, UK
Canine rheumatoid arthritis (CRA) is a chronic inflammatory joint disease with many similarities to the human equivalent, for which it serves as a modell. As in human RA, there is evidence of a number of autoimmune responses in the absence of an identifiable aetiological agent. This study was designed to consider the possible involvement of a number of common dog viruses in CRA. The approach taken was to determine the levels of anti-viral antibodies in sera and synovial fluids and to investigate the presence of viral antigens in immune complexes and in diseased synovial tissues. Dogs with CRA had increased antibodies (by ELISA) to canine distemper virus (CDV) in both sera and, particulaty, synovial fluids. No such increase was seen with any other virus tested. Increased anti-CDV antibodies were also found in precipitated immune complexes. Western blotting of synovial fluid immune complexes and probing with anti-CDV antibodies showed CDV antigens in synovial fluid complexes in CRA but not in normal dogs or dogs with osteoarthritis (OA). In CRA, both circulating immune complexes (IC) and synovial fluid white cell counts correlated with anti-CDV antibodies (pcO.001). No such correlations were seen in dogs with (OA). Preliminary immunocytochemical analyses, using monoclonal antibodies, showed evidence of CDV antigens in rheumatoid synovial tissues and not in Ok The relevance of this study to both canine and human rheumatological disorders is discussed.
3rd International Veterinary Immunology Symposium - 220 PS 20.2
IDENTIFICATION OF GENES ENCODING CANINE T CELL RECEPTOR COMPLEX Goitsuka, R.; Takano, M.; Okuda, M.; Ushiya, S.; Tsujimoto, H. and Hasegawa, A. Department of Veterinary Internal Medicine, University of Tokyo, Tokyo, Japan
To provide the basis for molecular dissection of canine lymphoproliferative diseases, canine genes encoding T cell receptor (TCR) complex including CD3, TCRa and p-chain constant regions (Gz and CjY) were molecularly cloned by the use of PCR with the primers corresponding to the conserved sequences of these genes among species. After PCR-amplification of cDNA prepared from canine lymph node cells, obvious DNA bands of the expected sizes (approximately 220 bp for CD3,320bp for Ca, and 360bp for Cj3) were detected. At the nucleotide and amino acid levels, the DNA 220bp PCR product showed significant homology with human and murine CDM genes rather than with CD3y genes. In the predicted transmembrane region of this clone, 19 out of 27 amino acid residues were identical with human CD%, and a centrally placed negatively charged aspartic acid residue was conserved. Furthermore, two characteristic cysteine residues in the extracellular domain were also found in this clone, indicating that this clone represented a canine homologue of CD3B gene. By using reverse-transcriptional PCR, the transcription of CD3c? gene was observed in CL-l cells which derived from tymic-form malignant lymphoma. The sequence information of Ca and C/l of TCR will be also discussed together with CD3.
3rd International Veterinary Immunology Symposium - 221PS 20.3
IMMUNOSCINTIGRAPHY OF CANINE OSTEOSARCOMAS WITH MONOCLONAL ANTIBODIES TO OSTEOSARCOMAASSOCIATED ANTIGENS Haines, D.M.‘; Bruland, 0.S.2; Matte, G.3; Wilkinson, kk3; Merit, S.M.’ and Fowler, J.D.’ lWestern College of Veterinary Medicine; 3Medica1 Imaging University of Saskatchewan, Saskatoon, Canada S7N OWO and 2Departments of Medical Oncology and Radiotheraphy and of Tumor Biology, Norwegian Radium Hospital, Montebello, Norway
Monoclonal antibody TP-1 selectively binds to human and canine osteosarcoma cells in vitro using immunhistochemical stains. radio-iodinated F(ab’)2 fragments of monoclonal antibody TP-1 was administered intravenously in 4 dogs with primary and/or metastatic spontaneous osteosarcoma. Immunoscintigraphy successfully demonstrated the radiolabeled antibody fragments in 6/6 known primary or metastatic lesions and in addition detected 4 metastatic lesions not diagnosed by conventional radiographs. Concurrent imaging of 99mT~ labeled autologous erythrocytes in two dogs confirmed that the accumulation of radiolabeled antibody fragments was independent of the blood pool. The present study has demonstrated the potential of monoclonal antibody TP-1 F(ab’)2 fragments for early detection of metastatic spread of spontaneous osteosarcoma.
PS 20.4
ENHANCING EFFECTS OF EGG WHITE DERIVATIVE ON PHAGOCYTOSIS AND CHEMOTAXIS OF CANINE PERIPHERAL BLOOD PHAGOCYTES Hirota, Y.t, Yang, M-P.‘, Honjo, K.‘, Ikeda, T.l, Yoshihara, K.‘, Shimizu, S.‘, Araki, S.2 and Onodera, T.l ‘National Institute of Animal Health, 2Eisai Co., Ltd., Tsukuba, Ibaraki 305, Japan
Effects of egg white derivative (EWD) on the functions of peripheral blood leukocytes (PBL) in the dog were examined. PBL from clinically healthy adult beagles were cultured with EWD, and phagocytized FITC-conjugated beats for 1 hr at 37OC. Their phagocytosis was evaluated by flow cytometry. The
3rd International Veterinary immunology Symposium - 222 chemotaxis was measured by a modified Boyden chamber method. PBL stimulated with EWD (200 ,@ml) for 4 hr showed enhanced phagocytosis. The supplement of culture supernatant of mononuclear cells cultured with EWD for 24 hr at 37OC into freshly isolated PBL induced the enhanced phagocytosis. The culture supernatant of EWD-stimulated mononuclear cells also induced significantly enhanced chemotaxis of neutrophils. However, this chemotactic activity was not observed in the culture supernatant from EWD-stimulated neutrophils. These results suggest that EWD has an enhancing effect on phagocytosis and chemotaxis of canine phagocytes and that the events is mediated in part by humoral factors released from EWD-stimulated mononuclear cells but not from EWD-stimulated neutrophils.
PS 20.5
PRODUCTION AND CHARACTERIZATION OF FELINE x MURINE HETEROHYBRIDOMA LINES Leidinger, E.‘; Gemeiner, M.’
Kramberger-Kaplan, E.3; Anrather, J.2 and
‘Institute of Medical Chemistry, 21nternal Clinic for Small Animals, Horses and Poultry, Veterinary Medical University Vienna, Linke Bahng 11, A-1030 Vienna, Austria, 3Vet. Practitioner, Wr. Neustadt, Austria The occurance of myelomas in cats is a rare event and therefore no feline myeloma-lines suitable for hybridizations could be developed so far. In this experiment we produced feline x murine heterohybridomas by fusing feline splenocytes with murine fusion partners, as used for standard murine x murine hybridizations. In order to optimize the fusion efficiency and standardize the following propagation of the heterohybridomas, the effects of different murine fusion lines and media supplements were studied. Up to now several HAT-resistant feline x murine heterohybridomas could be isolated and were characterized by chromosomal analysis and growth properties. Though none of the heterohybridoma lines secreted feline monoclonal antibodies, we hope to succeed by adapting one of them as HAT-resistant fusion line for the production of hybrid-hybridomas (2°-hybridomas).
3rd International Veterinary Immunology Symposium - 223 PS 20.6
DIFFERENT PATTERN OF CANINE ANTINUCLEAR ANTIBODIES ON WESTERN BLOTS Schuberth, H.J.; Kollmer, L. and Leibold, W. Immunology Unit, Veterinary School, Hannover, Germany
Sera from 31 dogs with suspected autoimmune disease were tested in parallel in the standard fluorescent antinuclear antibody test (FANA) on HEp2 and MDCK cells and on nuclear protein preparations of these cells after SDSPAGE and western blotting (ANA-blot: AB). In the FANA assay 18/31 sera reacted with HEp-2 and MDCK showing no differences in their staining pattern. The titre on MDCK was slightly higher. 17/31 sera were positive in the ANA-blot. AB has a r-value of .67 with FANA (3 sera FANA + / AI3 - and 2 sera AI3 + / FANA -). The 17 AB-positive sera showed a total of 12 different significant bands on Western blots occurring separately or in typical combinations. The most promiment reactivity was found with proteins of 46 kDa (lO/lS), 90 + 105 kDa (3/15) and 20 kDa (3/15). The determination of protein- and epitope-specific antibodies in sera of dogs with autoimmune disorders will improve their diagnostic, prognostic and therapeutic values as compared to the standard fluorescent assay.
PS 20.7
DEVELOPMENT OF LYMPHOID CELLS IN NEWBORN BUFFALO CALVES Tewari, D.’ and Gael, M.C.2 ‘Dept. Clinical Vet. Med., Madingley road, Cambridge, UK, 2Veterinary Microbial., H.A.U., Hisar, India.
A study on seven buffalo calves from birth to three months of age was done for identification of lymphoid cells and investigation of their functions. This revealed that the number of T cells, identified as peanut agglutinin binding cells amongst mononuclear cell population remained unaltered throughout this period. While, the number of cells identified as SIg bearing cells increased fourfold by the end of three month period compared to 10 days old calves. T cell subsets were identified as EA rossette forming cells with either IgG or IgM in T cell enriched population. Cells bearing Fc receptors for IgG (Suppressor) was
3’* International Veterinary Immunology Symposium - 224 higher on day of birth and levels decreased by day 60 of age. However, levels of IgM attaching cells (Helper) remained unaltered. The ability of MNC to undergo mitogenisis in response to T cell mitogens was less during first 10 days of life, but the response to B cell mitogen was low throughout the study period. The study indicated functional immaturity of T cells and less numbers of B cells until about three weeks after birth in buffalo calves.