Search

We tested whether rat and human MPO have similar antigenic determinants using 36 human MPO-ANCA positive sera, one mouse anti-rat MPO and four mouse anti-human MPO monoclonal reagents. Purified rat and human MPO were used in ELISA, with or without crossinhibition by preincubation with human MPO or irrelevant antigen in the liquid phase. Only one human MPO ANCA positive serum exhibited significant binding in rat MPO ELISA. This binding was poorly inhibited by preincubation with human MPO in the liquid phase, but was conserved after adsorption of non specific anti-rat activity in a chromatography column. Three mouse anti-human MPO IgG monoclonal antibodies did not recognize rat MPO. Only one mouse anti-human MPO IgA monoclonal antibody bound to rat MPO. This binding was poorly inhibited by preincubation with human MPO (35% at 2 µg/ml). Conversely, the mouse anti-rat MPO monoclonal did not bind human MPO. We have concluded that: (1) Most human MPO-ANCA recognize antigenic determinants on human MPO which are absent on rat MPO. Therefore, human auto-antibodies bind to epitopes which recently appeared after species evolution; (2) Inversely, the mouse anti-rat MPO monoclonal do not bind human MPO. Therefore, rat MPO epitopes have been altered during species evolution; (3) Mice injected with human MPO preferentially develop antibodies against xeno-epitopes which are not present in rodents. Therefore, human MPO may not be the best antigen to raise ANCA in animal models and (4) A comparison of the amino acid sequences of rat and human MPO may help elucidate the major antigenic epitopes.

National audience

International audience

International audience

Two therapeutic assays associating sodium butyrate (NaB) and interleukin-2 (IL-2) were applied on rats after intraperitoneal (i.p.) injection of colon carcinoma cells. The first assay began at day 1 after cell injection, and the second one at day 10 after injection, i.e. when carcinomatosis was established. IL-2 was given i.p. three times per day and NaB one time per day, for five and seven days respectively. The survival median of IL-2 treated rats significantly increased versus those of untreated ones, and was much higher with NaB in supplement. Moreover, using this associative treatment, we observed some cases of complete curacy, even in rats bearing established carcinomatosis.

In an attempt to characterise the mode of dissemination of colorectal carcinoma cells in host tissues, we established in vitro 3 human cancer cell lines isolated from a single patient: ALT-I from a primary colorectal tumour, ALT-F from the corresponding hepatic metastasis, and ALT-G from the lymphatic metastasis. The three cell lines exhibit variations in morphology, karyotype, antigens expression, anchorage-independent cell growth and tumorigenicity in nude mice related to their origin. Studies of the biological properties of these cell lines showed that the ALT tumour cells maintain, in vitro, some biochemical expressions, morphological properties and cytogenetic characteristics largely described for colon carcinoma in vivo. Significant increases of carcinoembryonic and CA19.9 antigens expressions were noted in the primary tumour cells as well as in the comparative metastatic ones. The karyotypes shared structural rearrangements and chromosome losses frequently described in fresh colorectal cancers, and revealed increasing alterations from the primary to the hepatic and lymph node tumours. Although the metastatic potential of the ALT cell lines was not demonstrated in the present paper, significant differences in the tumorigenic properties between the primary and the corresponding metastatic tumour cells were evident using in vitro and in vivo investigations. The present data support the hypothesis that, in our model, the hepatic metastasis might occur before or independently of the proximal lymph node metastasis originating from the colorectal carcinoma.

The presence in tumors of numerous cytokines suggests that they potentially modulate tumor cell activities and host tissue remodelling. To investigate the possible involvement of transforming growth factor type beta (TGF beta) in the metastatic process of cancer development, we have studied the effect of this factor on two rat colon carcinoma cell lines. These cell clones had been previously tested and selected for their ability to develop metastases in syngenic animals or lack of it. The two cell lines were characterized for their production of TGF beta. Production of active and latent forms of TGF beta 1 in the medium conditioned by the rat colon cancer cells were quantified using a bioassay. The presence of active TGF beta 1 was demonstrated in conditioned medium from the progressive tumor (PROb) cells and significant expression of latent forms of TGF beta 1 were found in the conditioned media from both cell clones. TGF beta 1 slightly inhibited proliferation of PROb cells which had been previously described as moderately differentiated, and significantly stimulated proliferation of the regressive (REGb) cells, described as poorly differentiated. On the basis of our observations, we suggest that this endogenous factor could be involved in autocrine regulation of tumor cell activities and in paracrine regulation of stroma cell and immune responses. Active and/or latent expression of TGF beta 1 by the two rat colon carcinoma cell lines, and their variable responses to the growth factor, strongly suggest that this polypeptide is involved in the regulation of tumorigenic expression of adenocarcinoma cells.

International audience

International audience

Sera from BDIX rats bearing the syngeneic colon tumors PROb or REGb were analysed by Western blotting in order to detect a possible humoral response against the grafted tumor. The PROb clone grows progressively in syngeneic hosts and metastasizes, whereas the REGb clone grows slowly and then is rejected. This model was developed by F Martin and his group in Dijon, France. We observed that rats bearing PROb tumors only develop an antibody response against a water-soluble protein of 105 kDa (p105) which is expressed by both tumor clones. This antibody response has never been detected in rats bearing REGb tumors. The antigen p105 was also expressed by normal adult colon as well as some other foetal or adult tissues. It is also present in extracts from several tumor cell lines including human colorectal carcinoma cell lines. Moreover, the titer of detected antibody at day 30 was inversely correlated with the survival of rats after tumor inoculation, suggesting a possible facilitating role of this antibody response.

The presence of some characteristics of normal rat intestinal epithelial cells was studied on two clones originating from a single rat colon carcinoma. These clones differed by their tumorigenic properties in the syngeneic host. However, they grow similarly in vitro and in immuno-deprived animals. The PROb clone which had the ability to form progressive tumors in the syngeneic host appeared to possess more features of differentiated cells than the REGb clone which was immunologically rejected by syngeneic hosts. Indeed, the morphology of the cells was different, the REBb cells having a more fibroblastic appearance while the PROb cells had the capacity to form domes characterizing the functional polarization of the cell layer. The two clones could also be distinguished by their expression of proteins of intermediate filaments. Both expressed cytokeratins showing their epithelial origin, but only REGb cells displayed vimentin which is characteristic of mesenchymal or poorly differentiated epithelial cells. Furthermore, analysis of the expression of a series of glycoconjugate tissue antigens and an unknown protein (p120) showed that the PROb cells resembled more the normal adult digestive epithelium than the REGb cells did. In conclusion, it appears that in this model, the most aggressive cells, those resisting to the constraints imposed by the immune system, are also the more differentiated ones.