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Molecular cloning of bovine thymopoietin gene and its expression in different calf tissues
798
Abstracts
t74.
Molecular cloning of Bovine thymopoietin gene and its expression in different calf tissues O. Zevin-Sonkin, El llan, D. Gutman, J. Riss, L. Theodor and J. Shoham. Departmentof Life Sciences, Bar-llan University, Ramat-Gan 52900, Israel Thymopoietin (TP), a 49 amino acid peptide isolated from bovine thymus in the form of two closely related isoforms, is regarded as a thymic hormone, secreted specifically by some thymic epithelial cells and exerting multitude effects on T lineage cells exclusively. We used synthetic oligonucleotides to isolate bovine TPgene and a partial TP cDNAclone. The cDNAclone is 1.1 kb long, containing 147 b.p. coding for TP, at its 5' end. The deduced amino acid sequence is identical to TP-II isoform, with the following exceptions: amino acid 43 is as in TPI and amino acids 47 and 48 d i f f e r from either. A cDNA subclone containing 126 b.p. coding for aminoacids 1-42 of TP-II was used to assay TP mRNAby $I nuclease protection method in following calf tissues: thymus, thymocytes, thymocyte, depleted thymus, spleen, lymph nodes, tonsils, testes, kidney, l i v e r , adrenal glands, small intestine, lung, heart, striated muscle, cerebrum, cerebellum and hypothalamus, peripheral blood mononuclearcells and cultured thymic stroma cells. Our results indicate that in contrast to reported studies using anti TP antibodies, TP gene is expressed to a varying degree by all lymphatic tissues with predominant, strong expression in thymocytes (and not in thymic epithelial cells as thought before). Most other tissues, except cerebellum, displayed a weak, or barely detectable TP mRNA. These results suggest that TP may have an important role in the immune system, in a way different from that thought about before.
175.
Analysis of Cytokines Secreted by HumanThymic Epithelial Cells in Culture Aviram Meilin, Yedida Sharabi and Jacob Shoham. Department o f L i f e Sciences, B a r - I l a n U n i v e r s i t y , Ramat-Gan 52900, I s r a e l The culture supernatant (CS) of unstimulated human thymic epithelial (HTE) cells, grown on extracellular matrix in defined medium, was found to have a strong immunomodulatoryactivity on thymocytes (see our other Abstract in this Conference). We demonstrated that HTE cell culture supernatant contained interleukin-6 (IL-6) biologic activity, as determined by the a b i l i t y to support proliferation of Bg and by inhibition of its activity by anti human IL-6 antibodies. HTE-CS contained also two CSF activities, G-CSF and M-CSF, as determined by proliferation of the murine cell lines 32D and 14MI.4 which are dependent on the respective factors and by specific inhibition of their activity with anti G-CSF and anti M-CSF antibodies. We did not find activities on cell lines dependent on IL-I or l l - 2 (DIO and CTLL, respectively). No antiviral, interferon activity was found either. Thus, using biological assays, we demonstrated that cultured human epithelial cells produce both immunoregulatoryand hematopoietic cytokines. Our findings suggest that thymic stromal cells play an important role in proliferation and differentiation of hematopoietic precursor cell which should conceivably be linked to T cell maturation in the thymus.
176.
Imunomodulatory Activities in Culture Supernatant (CS) of Human Thymic Epithelial Cells Grown Under Defined Conditions Licita Schreiber, llana Eshel, Avlram Meilin, Yedida Sharabi and Jacob Shoham. Department Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel
of
We previously reported on the use of extracellular matrix and defined medium for the selective cultivation of murine epithelial (MTE) and mesenchymal thymic stromal cells - (J. Immunol. 144, 1554, 1990) and on the analysis of cytokines secreted by these cells (J. Immunol. 144, 1563, 1990). We applied the same approach for the cultivation of human thymic epithelial (HTE) cells and tested its CS. HTE-CS strongly enhanced the responsiveness of both human and murine thymocytes to mitogens (PHA, Con A), as measured by 3H-thymidine incorporation and by lymphokine (IL-2 and IL-3) production, with a higher effect in the homologouscombination. The responsive thymocyte population was the more mature, PNA-negative, hydrocortisone resistant population. Comparison of HTE-CS activity with that of 3 known thymic factors (thymulin, thymopoietin and thymostimulin) indicated that all the thymic factors had an enhancing effect in the assays, but without the species preference, clearly observed with HTE-CS. The results indicate a strong immunomodulatoryactivity of the CS tested, and calls for further analysis of the factors or cytokines involved.
Abstracts
t74.
Molecular cloning of Bovine thymopoietin gene and its expression in different calf tissues O. Zevin-Sonkin, El llan, D. Gutman, J. Riss, L. Theodor and J. Shoham. Departmentof Life Sciences, Bar-llan University, Ramat-Gan 52900, Israel Thymopoietin (TP), a 49 amino acid peptide isolated from bovine thymus in the form of two closely related isoforms, is regarded as a thymic hormone, secreted specifically by some thymic epithelial cells and exerting multitude effects on T lineage cells exclusively. We used synthetic oligonucleotides to isolate bovine TPgene and a partial TP cDNAclone. The cDNAclone is 1.1 kb long, containing 147 b.p. coding for TP, at its 5' end. The deduced amino acid sequence is identical to TP-II isoform, with the following exceptions: amino acid 43 is as in TPI and amino acids 47 and 48 d i f f e r from either. A cDNA subclone containing 126 b.p. coding for aminoacids 1-42 of TP-II was used to assay TP mRNAby $I nuclease protection method in following calf tissues: thymus, thymocytes, thymocyte, depleted thymus, spleen, lymph nodes, tonsils, testes, kidney, l i v e r , adrenal glands, small intestine, lung, heart, striated muscle, cerebrum, cerebellum and hypothalamus, peripheral blood mononuclearcells and cultured thymic stroma cells. Our results indicate that in contrast to reported studies using anti TP antibodies, TP gene is expressed to a varying degree by all lymphatic tissues with predominant, strong expression in thymocytes (and not in thymic epithelial cells as thought before). Most other tissues, except cerebellum, displayed a weak, or barely detectable TP mRNA. These results suggest that TP may have an important role in the immune system, in a way different from that thought about before.
175.
Analysis of Cytokines Secreted by HumanThymic Epithelial Cells in Culture Aviram Meilin, Yedida Sharabi and Jacob Shoham. Department o f L i f e Sciences, B a r - I l a n U n i v e r s i t y , Ramat-Gan 52900, I s r a e l The culture supernatant (CS) of unstimulated human thymic epithelial (HTE) cells, grown on extracellular matrix in defined medium, was found to have a strong immunomodulatoryactivity on thymocytes (see our other Abstract in this Conference). We demonstrated that HTE cell culture supernatant contained interleukin-6 (IL-6) biologic activity, as determined by the a b i l i t y to support proliferation of Bg and by inhibition of its activity by anti human IL-6 antibodies. HTE-CS contained also two CSF activities, G-CSF and M-CSF, as determined by proliferation of the murine cell lines 32D and 14MI.4 which are dependent on the respective factors and by specific inhibition of their activity with anti G-CSF and anti M-CSF antibodies. We did not find activities on cell lines dependent on IL-I or l l - 2 (DIO and CTLL, respectively). No antiviral, interferon activity was found either. Thus, using biological assays, we demonstrated that cultured human epithelial cells produce both immunoregulatoryand hematopoietic cytokines. Our findings suggest that thymic stromal cells play an important role in proliferation and differentiation of hematopoietic precursor cell which should conceivably be linked to T cell maturation in the thymus.
176.
Imunomodulatory Activities in Culture Supernatant (CS) of Human Thymic Epithelial Cells Grown Under Defined Conditions Licita Schreiber, llana Eshel, Avlram Meilin, Yedida Sharabi and Jacob Shoham. Department Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel
of
We previously reported on the use of extracellular matrix and defined medium for the selective cultivation of murine epithelial (MTE) and mesenchymal thymic stromal cells - (J. Immunol. 144, 1554, 1990) and on the analysis of cytokines secreted by these cells (J. Immunol. 144, 1563, 1990). We applied the same approach for the cultivation of human thymic epithelial (HTE) cells and tested its CS. HTE-CS strongly enhanced the responsiveness of both human and murine thymocytes to mitogens (PHA, Con A), as measured by 3H-thymidine incorporation and by lymphokine (IL-2 and IL-3) production, with a higher effect in the homologouscombination. The responsive thymocyte population was the more mature, PNA-negative, hydrocortisone resistant population. Comparison of HTE-CS activity with that of 3 known thymic factors (thymulin, thymopoietin and thymostimulin) indicated that all the thymic factors had an enhancing effect in the assays, but without the species preference, clearly observed with HTE-CS. The results indicate a strong immunomodulatoryactivity of the CS tested, and calls for further analysis of the factors or cytokines involved.