Wasting, ischemia, and lymphoid abnormalities in mice expressing T cell-targeted human tumor necrosis factor transgenes.

To evaluate the biologic potential of T cell-specific TNF production in vivo, we have generated transgenic mice constitutively expressing TNF in their T cell compartment. This was achieved by placing a wild-type or a 3'-UTR modified fragment of the human TNF gene under the influence of the T cell-specific, locus control region of the human CD2 gene. Transgenic mice that express human TNF mRNA in T cells develop marked histologic and cellular changes locally in their lymphoid organs and a lethal wasting syndrome associated with widespread vascular thrombosis and tissue necrosis. The extent of pathologic changes and their time of onset appear to reflect levels of transgene expression. Thus, transgenic lines that express the transgene at high levels show both lymphoid organ and systemic abnormalities with wasting. In one transgenic line, mice express lower levels of the transgene and develop normally despite pronounced local lymphoid organ defects, confirming in vivo, the differential potential of localized and systemic TNF action. All pathologic changes could be neutralized by the administration of mAb specific for human TNF. These results demonstrate the important role of T cell-specific TNF production in the development of specific pathology and provide a means by which to evaluate the role of TNF in thymocyte development. Transgenic mice that express TNF constitutively in the T cell compartment offer a unique in vivo system by which to analyze the molecular character of systemic vs contact-dependent and paracrine modes of TNF action. Furthermore, given the species-specific nature of the mouse p75 TNF receptor, it is assumed that the pathology induced by human TNF in these transgenic mice is associated exclusively with p55 TNF receptor signaling. Conceivably, the differential contribution of each of the two TNF receptors in thymus development and TNF-mediated disease can be assessed by comparison of the biologic potential of human vs mouse TNF in the transgenic system developed.

The c-Ha-ras oncogene induces increased expression of beta-galactoside alpha-2, 6-sialyltransferase in rat fibroblast (FR3T3) cells.

Alteration in cell surface carbohydrates, and in particular cell surface sialylation, have been known to occur during oncogenic transformation. To examine the basis for such changes, we have transformed the rat fibroblast cell line FR3T3 with the oncogenes c-Ha-ras EJ, v-mycOK10, v-src, polyoma virus middle T or the transforming bovine papilloma virus 1 (BPV1), and measured the sialytransferase activities of cellular lysates. We found that, in contrast to all other oncogenes examined, c-Ha-ras induced a striking increase in beta-galactoside alpha-2,6-sialytransferase (Gal alpha-2,6-ST) activity in FR3T3 cells. This increase in Gal alpha-2,6-ST activity resulted in the increased expression of cell surface alpha-2,6-linked sialic acid on cell surface glycoconjugates, as determined by cell staining with fluorescein-labelled Sambucus nigra agglutinin. Immunoprecipitation and immunofluorescence experiments revealed that the increase in Gal alpha-2,6-ST activity was due to an elevation of expression of the enzyme. Moreover, Northern analysis suggested that the increased expression of this enzyme was the result of an increase in the steady-state mRNA level of the Gal alpha-2,6-ST gene. These results support the notion that alterations seen in cell surface glycoconjugates during oncogenic transformation can be the result of altered expression of glycosyltransferases.

Comparison of sialyl- and alpha-1,3-galactosyltransferase activity in NIH3T3 cells transformed with ras oncogene: increased beta-galactoside alpha-2,6-sialyltransferase.

Previous studies have indicated that transfection of NIH3T3 cells with the ras oncogene induced modifications of the terminal glycosylation of N-linked glycans which appeared in the early stage after transfection. These changes affected especially the terminal part of N-linked glycans which is substituted with alpha-1,3-Gal residues in NIH3T3 and with Neu5Ac residues in the ras-transformed counterpart. We have transformed NIH3T3 cells with the human c-Ha-ras oncogene, evaluated tumorigenicity and metastatic capacity in vivo and compared alpha-1,3-galactosyltransferase, alpha-2,3- and alpha-2,6-sialyltransferases activities. By using different specific acceptors, we detected the enhancement of sialic acid transfer in transformed cells while the activity of alpha-1,3-galactosyltransferase remained unchanged. We showed that the higher sialyltransferase activity was due to the increase of beta-galactoside alpha-2,6-sialyltransferase in ras-transfectant although alpha-2,3-sialyltransferase was weakly expressed in these cells. On the basis of binding of different lectins, we correlated these observations with changes of protein glycosylation. We concluded that altered glycosylation of ras-transformed NIH3T3 is the result of a competitive effect of the enzymes acting for terminal glycosylation of N-linked glycans and the reflection of the higher expression of alpha-2,6-sialyltransferase.

Transgenic mice expressing human tumour necrosis factor: a predictive genetic model of arthritis.

We have generated transgenic mouse lines carrying and expressing wild-type and 3'-modified human tumour necrosis factor (hTNF-alpha, cachectin) transgenes. We show that correct, endotoxin-responsive and macrophage-specific hTNF gene expression can be established in transgenic mice and we present evidence that the 3'-region of the hTNF gene may be involved in macrophage-specific transcription. Transgenic mice carrying 3'-modified hTNF transgenes shows deregulated patterns of expression and interestingly develop chronic inflammatory polyarthritis. Treatment of these arthritic mice with a monoclonal antibody against human TNF completely prevents development of this disease. Our results indicate a direct involvement of TNF in the pathogenesis of arthritis. Transgenic mice which predictably develop arthritis represent a novel genetic model by which the pathogenesis and treatment of this disease in humans may be further investigated.

[Modifications of sialylation in BHK 21/C13 cells after in vitro transfection by c-Ha-ras human oncogene]. / Modifications de la sialylation des cellules BHK 21/C13 après transfection in vitro par l'oncogène humain c-Ha-ras.

A Hamster kidney fibroblast cell line (BHK 21/C13) has been transfected by c-Ha-ras human oncogene. The expression of the oncogene significantly modified the global sialytransferase activity of the cell extract. This activity is enhanced on an average 2.5 fold whatever the acceptor. In addition, the proportion of cell-surface associated N-glycolylneuraminic acid is enhanced in transfectants, the ratio N-acetylneuraminic acid/N-glycolylneuraminic acid decreases from 7.10 to 2.80. These results suggest that a tight relationship exists, in c-Ha-ras transfected BHK cells, between the expression of the oncogene and the neuraminic acid metabolism as well as a gene regulation of glycoconjugate sialylation.