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Phenotypical in-situ and in-vitro characterization of canine dorsal root ganglia neurons and satellite glial cells reveal the presence of a unique glial precursor cell population
152:1, 2015
ESVP/ECVP Proceedings 2012
FUNDAMENTAL RESEARCH SOX9 AND CK15 STEM CELL MARKERS IN CANINE SEBACEOUS LESIONS L. Bongiovanni *, M.M. Suter y, A. Inverso *, D. Malatesta *, M. Romanucci *, L. Della Salda * and C. Brachelentez *Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy, y Institute of Animal Pathology (ITPA), Vetsuisse Faculty, University of Bern, Bern, Switzerland and zDepartment of Veterinary Medicine, University of Perugia, Via S. Costanzo 4, Perugia, Italy Introduction: Sebaceous glands are specialized cutaneous adnexal glands, which work under constant hormonal control to produce sebum. They represent a source of adult stem cells that are located in the reserve cell layer. They can give rise to several proliferative lesions and neoplasms (i.e. adenoma, epithelioma and carcinoma). Ck15 and Sox9 represent two stem cell markers, expressed by the adult stem cells of human sebaceous glands, and are expressed in several human sebaceous tumours. Materials and Methods: The aim of the present study was to investigate the immunohistochemical patterns and levels of expression of Ck15 and Sox9 in a set of canine sebaceous gland lesions. Results: Few scattered Ck15-positive cells, but no Sox9 immunolabelling was observed in the reserve cell layer of normal glands. Sox9 was expressed selectively in the nuclei of the innermost cell layer of the outer root sheath of normal canine hair follicles. The spectrum of canine sebaceous tumours, from benign to malignant, contained a subpopulation of cells expressing Ck15, while Sox9 was expressed only in malignant sebaceous tumours. Conclusions: Although further studies are needed, Ck15+ and Sox9+ cells, having some features of stem cells, may play a role in sebaceous tumour development and progression. FELINE T-CELL RECEPTOR SIGNALLING PATHWAY PROTEINS IDENTIFIED BY LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY M. Henrich *, K. Huber y, L. Rydzewski *, S. Kirsten *, ompp y and M. Reinacher* B. Spengler y, A. R€ *Institute for Veterinary Pathology and yInstitute for Inorganic and Analytical Chemistry, Justus-Liebig University Giessen, Giessen, Germany Introduction: The T-cell receptor signalling pathway in man is well described, but in cats only a model of the feline signalling cascade deduced from a public sequence database data exists. This model postulates a strong homology of the feline cascade to the human counterpart. Nevertheless, many of the proteins involved are only predicted from the nucleotide sequence analysis as received from the feline genome project. In this study, members of the T-cell receptor signalling pathway were identified by mass spectrometry in samples of a feline large granular lymphoma cell line. Materials and Methods: Tryptic peptides of homogenates of a feline large granular lymphoma cell line were analyzed by liquid chromatographyeelectrospray ionization tandem mass spectrometry (LCESI-MS/MS). Resulting data were searched against the UniProtKB (Universal Protein Resource) database filtered with Felis catus taxonomy. Results: Seventeen proteins associated with the T-cell receptor pathway were indentified. Fifteen of these were represented by two or more peptides and therefore identification was regarded as valid. Conclusions: Of the 15 identified proteins, two (CD3 epsilon chain and CD8 alpha chain) are already widely recognized by immunohistochemistry, while for the others reports of expression at the protein level are lacking. With this study the existence at the protein level is confirmed. Two proteins were only represented by one peptide and therefore their expression cannot be regarded as proven by this study; however, it remains likely.
73
PHENOTYPICAL IN-SITU AND IN-VITRO CHARACTERIZATION OF CANINE DORSAL ROOT GANGLIA NEURONS AND SATELLITE GLIAL CELLS REVEAL THE PRESENCE OF A UNIQUE GLIAL PRECURSOR CELL POPULATION K. Hahn *,y, A. Lehmbecker *, Y. Wang *,y, A. Habierski *, artner *,y and K. Kegler *,y, K. Schughart z, W. Baumg€ I. Gerhauser* *Department of Pathology, University of Veterinary Medicine Hannover, y Center of Systems Neuroscience Hannover and zHelmholtz Centre for Infection Research, Braunschweig, Germany Introduction: Dorsal root ganglia (DRG) neurons have been used as an in-vitro model in neuroscience in pain, virus and prion protein research. Furthermore, potential stem cell properties have been demonstrated in DRG satellite cells. Recent studies suggested that dog glial cells are a better model for human glia compared with those from rodents, emphasizing the usefulness of dogs as a translational animal model. However, detailed knowledge concerning the structure of canine DRGs and in-vivo and in-vitro expression of different structural and functional proteins by resident cells is lacking. Materials and Methods: Light and transmission electron microscopy, immunohistochemistry and immunofluorescence were used to characterize canine DRG neurons and satellite glial cells (SGCs) in vivo and in vitro. Results: Neuronal class III b-tubulin and non-phosphorylated neurofilaments are reliable markers of canine DRG neurons, which can be maintained over 18 days in vitro. Purification of neurons using Percoll centrifugation caused an enrichment of large over small neurons in DRG cell cultures. Canine SGCs show a co-expression of GFAP, CNPase and vimentin, which are normally found in astrocytes, oligodendrocytes and microglia. The frequent Sox2 expression of SGCs highlights their suggested stem cell properties. Conclusions: The characteristics of canine DRG cell cultures are fundamental for their use in future research in different fields of neuroscience. DRGs may provide a source of cells with stem cell potency, which can easily be obtained and expanded and may be used for new therapeutic concepts in canine and human CNS diseases. IBERIAN PIG PERI-IMPLANTATION PERIOD: UTERINE VEGFeVEGF RECEPTOR SYSTEM EXPRESSION M.A. S anchez, R.A. Garcıa-Fern andez, B. S anchez, P. Garcıa-Palencia, C. Naranjo, A. Palomo and J.M. Flores Departamento de Medicina y Cirugıa Animal, Universidad Complutense de Madrid, Madrid, Spain Introduction: The Iberian pig is an autochthonous breed from the Iberian Peninsula, known worldwide for the production of a curedmeat, Iberian ham, and characterized by a low prolificacy. The aim of this study was to quantify the expression of the vascular endothelial growth factor (VEGF)-system molecules in endometrium during early pregnancy in pregnant Iberian sows. Materials and Methods: Endometrium was collected from eight sows at gestation day (gd) 17 and from 27 sows at gd 22. RTqPCR was performed to detect mRNA expression of VEGF, VEGF receptor 1 (VEGFR1) and VEGF receptor 2 (VEGFR2). A Student’s t-test was conducted to detect differences in VEGF-system expression (statistical significance P !0.05). Results: The mean ( standard error) values for expression of VEGF, VEGFR1 and VEGR2 were 64.80 ( 9.39), 34.66 ( 5.02) and 421.10 (66.27), respectively, in endometrium from sows at gd 17, and 98.13 ( 11.52), 120.60 ( 8.68) and 158.10 (21.23), respectively, in endometrium from sows at gd22. There were statistically significant differences between gd17 and gd22 for all three molecules (P 5 0.0333, P ! 0.0001 and P 5 0.0048 for VEGF, VEGFR1 and VEGFR2, respectively), so that the expression of VEGF and VEGFR1 increased and the expression of VEGFR2 decreased as pregnancy advanced.
ESVP/ECVP Proceedings 2012
FUNDAMENTAL RESEARCH SOX9 AND CK15 STEM CELL MARKERS IN CANINE SEBACEOUS LESIONS L. Bongiovanni *, M.M. Suter y, A. Inverso *, D. Malatesta *, M. Romanucci *, L. Della Salda * and C. Brachelentez *Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy, y Institute of Animal Pathology (ITPA), Vetsuisse Faculty, University of Bern, Bern, Switzerland and zDepartment of Veterinary Medicine, University of Perugia, Via S. Costanzo 4, Perugia, Italy Introduction: Sebaceous glands are specialized cutaneous adnexal glands, which work under constant hormonal control to produce sebum. They represent a source of adult stem cells that are located in the reserve cell layer. They can give rise to several proliferative lesions and neoplasms (i.e. adenoma, epithelioma and carcinoma). Ck15 and Sox9 represent two stem cell markers, expressed by the adult stem cells of human sebaceous glands, and are expressed in several human sebaceous tumours. Materials and Methods: The aim of the present study was to investigate the immunohistochemical patterns and levels of expression of Ck15 and Sox9 in a set of canine sebaceous gland lesions. Results: Few scattered Ck15-positive cells, but no Sox9 immunolabelling was observed in the reserve cell layer of normal glands. Sox9 was expressed selectively in the nuclei of the innermost cell layer of the outer root sheath of normal canine hair follicles. The spectrum of canine sebaceous tumours, from benign to malignant, contained a subpopulation of cells expressing Ck15, while Sox9 was expressed only in malignant sebaceous tumours. Conclusions: Although further studies are needed, Ck15+ and Sox9+ cells, having some features of stem cells, may play a role in sebaceous tumour development and progression. FELINE T-CELL RECEPTOR SIGNALLING PATHWAY PROTEINS IDENTIFIED BY LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY M. Henrich *, K. Huber y, L. Rydzewski *, S. Kirsten *, ompp y and M. Reinacher* B. Spengler y, A. R€ *Institute for Veterinary Pathology and yInstitute for Inorganic and Analytical Chemistry, Justus-Liebig University Giessen, Giessen, Germany Introduction: The T-cell receptor signalling pathway in man is well described, but in cats only a model of the feline signalling cascade deduced from a public sequence database data exists. This model postulates a strong homology of the feline cascade to the human counterpart. Nevertheless, many of the proteins involved are only predicted from the nucleotide sequence analysis as received from the feline genome project. In this study, members of the T-cell receptor signalling pathway were identified by mass spectrometry in samples of a feline large granular lymphoma cell line. Materials and Methods: Tryptic peptides of homogenates of a feline large granular lymphoma cell line were analyzed by liquid chromatographyeelectrospray ionization tandem mass spectrometry (LCESI-MS/MS). Resulting data were searched against the UniProtKB (Universal Protein Resource) database filtered with Felis catus taxonomy. Results: Seventeen proteins associated with the T-cell receptor pathway were indentified. Fifteen of these were represented by two or more peptides and therefore identification was regarded as valid. Conclusions: Of the 15 identified proteins, two (CD3 epsilon chain and CD8 alpha chain) are already widely recognized by immunohistochemistry, while for the others reports of expression at the protein level are lacking. With this study the existence at the protein level is confirmed. Two proteins were only represented by one peptide and therefore their expression cannot be regarded as proven by this study; however, it remains likely.
73
PHENOTYPICAL IN-SITU AND IN-VITRO CHARACTERIZATION OF CANINE DORSAL ROOT GANGLIA NEURONS AND SATELLITE GLIAL CELLS REVEAL THE PRESENCE OF A UNIQUE GLIAL PRECURSOR CELL POPULATION K. Hahn *,y, A. Lehmbecker *, Y. Wang *,y, A. Habierski *, artner *,y and K. Kegler *,y, K. Schughart z, W. Baumg€ I. Gerhauser* *Department of Pathology, University of Veterinary Medicine Hannover, y Center of Systems Neuroscience Hannover and zHelmholtz Centre for Infection Research, Braunschweig, Germany Introduction: Dorsal root ganglia (DRG) neurons have been used as an in-vitro model in neuroscience in pain, virus and prion protein research. Furthermore, potential stem cell properties have been demonstrated in DRG satellite cells. Recent studies suggested that dog glial cells are a better model for human glia compared with those from rodents, emphasizing the usefulness of dogs as a translational animal model. However, detailed knowledge concerning the structure of canine DRGs and in-vivo and in-vitro expression of different structural and functional proteins by resident cells is lacking. Materials and Methods: Light and transmission electron microscopy, immunohistochemistry and immunofluorescence were used to characterize canine DRG neurons and satellite glial cells (SGCs) in vivo and in vitro. Results: Neuronal class III b-tubulin and non-phosphorylated neurofilaments are reliable markers of canine DRG neurons, which can be maintained over 18 days in vitro. Purification of neurons using Percoll centrifugation caused an enrichment of large over small neurons in DRG cell cultures. Canine SGCs show a co-expression of GFAP, CNPase and vimentin, which are normally found in astrocytes, oligodendrocytes and microglia. The frequent Sox2 expression of SGCs highlights their suggested stem cell properties. Conclusions: The characteristics of canine DRG cell cultures are fundamental for their use in future research in different fields of neuroscience. DRGs may provide a source of cells with stem cell potency, which can easily be obtained and expanded and may be used for new therapeutic concepts in canine and human CNS diseases. IBERIAN PIG PERI-IMPLANTATION PERIOD: UTERINE VEGFeVEGF RECEPTOR SYSTEM EXPRESSION M.A. S anchez, R.A. Garcıa-Fern andez, B. S anchez, P. Garcıa-Palencia, C. Naranjo, A. Palomo and J.M. Flores Departamento de Medicina y Cirugıa Animal, Universidad Complutense de Madrid, Madrid, Spain Introduction: The Iberian pig is an autochthonous breed from the Iberian Peninsula, known worldwide for the production of a curedmeat, Iberian ham, and characterized by a low prolificacy. The aim of this study was to quantify the expression of the vascular endothelial growth factor (VEGF)-system molecules in endometrium during early pregnancy in pregnant Iberian sows. Materials and Methods: Endometrium was collected from eight sows at gestation day (gd) 17 and from 27 sows at gd 22. RTqPCR was performed to detect mRNA expression of VEGF, VEGF receptor 1 (VEGFR1) and VEGF receptor 2 (VEGFR2). A Student’s t-test was conducted to detect differences in VEGF-system expression (statistical significance P !0.05). Results: The mean ( standard error) values for expression of VEGF, VEGFR1 and VEGR2 were 64.80 ( 9.39), 34.66 ( 5.02) and 421.10 (66.27), respectively, in endometrium from sows at gd 17, and 98.13 ( 11.52), 120.60 ( 8.68) and 158.10 (21.23), respectively, in endometrium from sows at gd22. There were statistically significant differences between gd17 and gd22 for all three molecules (P 5 0.0333, P ! 0.0001 and P 5 0.0048 for VEGF, VEGFR1 and VEGFR2, respectively), so that the expression of VEGF and VEGFR1 increased and the expression of VEGFR2 decreased as pregnancy advanced.