Jean-Louis Nano

Effects of fatty acids on the growth of Caco-2 cells.

Epidemiological studies suggest that polyunsaturated fatty acids may protect against colorectal neoplasia. In order to explore this observation, cell proliferation and viability, lipid composition, membrane fluidity, and lipid peroxidation were measured in Caco-2 cells after 48h incubation with various fatty acids. Saturated and monounsaturated fatty acids incorporated less well in the membranes than polyunsaturated fatty acids (PUFAs). All of the PUFAs tested had an inhibitory effect on cell proliferation/viability whereas the saturated and monounsaturated fatty acids did not. Addition of palmitic acid had no significant effect on membrane fluidity whereas unsaturated fatty acids increased membrane fluidity in a dose-dependent manner. PUFAs strongly increased tumor cell lipid peroxidation in a dose-dependent manner. Saturated and monounsaturated fatty acids increased lipid peroxidation in this cell line only at high concentration. Preincubation of Caco-2 cells with vitamin E prevented the inhibition of proliferation/viability, the elevation of the MDA concentration and the increased membrane fluidity induced by PUFAs. Our data indicate that PUFAs are potent inhibitors of the growth of colon cancer cells in vitro.

Establishment and characterization of an epithelial intestinal cell line from rat fetus

An epithelial intestinal cell line has been established from explants of fetal rat small intestine. After the 9th passage (approx. 25 population doublings) epithelial-like cells acquired the properties of a permanent cell line. The epithelial nature of this cell line, and of clone IRD 98 subsequently isolated, is supported by morphological and ultrastructural criteria, and also by the presence of enzymes characteristic of enterocytes, such as aminopeptidase, alkaline phosphatase, gamma-glutamyl transferase, lactase and maltase. The occurrence of the triglyceride pathway enzyme monoacylglycerol acyltransferase and of apoproteins (Apo A1 and Apo E) can also be demonstrated. Taken together, the results presented here provide evidence that clone IRD 98 is an epithelial cell line, most likely originating from the relatively differentiated cell layer of fetal rat small intestine.

Regulation of cholesterol synthesis and binding of lipoproteins in cultured rat intestinal epithelial cells

The regulation of cholesterol synthesis has been studied using a rat epithelial intestinal cell line (IRD 98) as a cellular model. As observed in other cell types, mevinolin increases the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) and concomitantly reduces the incorporation of [14C]acetate into cholesterol. Free cholesterol is able to suppress reductase activity. In contrast, when cells are shifted from standard culture medium to lipoprotein-deficient medium, an increase in hydroxymethylglutaryl-CoA reductase specific activity (2-5-fold) is observed. The possible regulatory roles of the different classes of human lipoproteins were thus compared. The effects of a long-term exposure to LDL and HDL vary according to cell density. In actively growing cells, VLDL and LDL cause a decrease in the level of hydroxymethylglutaryl-CoA reductase, whereas HDL do not have a significant effect. In contrast, in subconfluent preresting cells, HDL provoke large decreases in hydroxymethylglutaryl-CoA reductase activity as compared to VLDL and LDL. While LDL binding is constant, the maximal binding capacity of HDL in subconfluent cells is seven times that of actively growing cells. Altogether, these results suggest an important role for HDL in the regulation of intestinal cholesterol synthesis.

Cholesterol uptake in the human intestine. Hypo- and hyperresponsiveness

Cholesterol uptake was studied at the small intestine biopsies taken from patients without intestinal malfunction. Three distinct groups of patients were described: those with low (146 +/- 19) nmol/mm2 per 2 h), medium (455 +/- 18 nmol/mm2 per 2 h) and high (833 +/- 24 nmol/mm2 per 2 h) rates of cholesterol uptake. Positive correlation between cholesterol uptake and intestinal cholesterol synthesis was observed in the last two groups.

Regulation of cholesterol uptake in the rat intestinal cell line

A new model to study cholesterol absorption in the rat intestinal cells is described. Rat intestine epithelial cells IRD98 were incubated with mixed micelles containing bile acid, phospholipid, cholesterol or its nonabsorbable analogue, sitosterol, and trace amounts of [3H]cholesterol or [14C]sitosterol. Cholesterol and sitosterol uptake was then determined following lipid extraction; specific cholesterol uptake was determined as the difference between cholesterol and sitosterol uptake. Cholesterol, but not sitosterol, uptake was time- and dose-dependent and saturable. Loading of cells with non-lipoprotein cholesterol reduced cholesterol, but not sitosterol, uptake in a dose-dependent manner. In contrast, treatment of cells with an inhibitor of cholesterol synthesis, lovastatin, stimulated cholesterol, but not sitosterol, uptake in a dose-dependent manner. Treatment of cells with palmitic, caproic and oleic acids up-regulated specific cholesterol uptake, while linoleic and stearic acids had an opposite effect. None of the fatty acids affected sitosterol uptake.

Comparative study of Nd-YAG laser versus electrocautery for liver metastatic resection in the rat

The thermal, hemostatic and lymphostatic effects of the Nd-YAG laser suggest a benefit in the treatment of multiple liver metastases. The aim of this work was to evaluate experimentally this hypothesis in a comparative study with conventional electrocautery resection of liver metastases. The original animal model was represented by syngeneic BDIX rats inoculated under the liver capsule with 1.5×106 DHD/K12 tumour cells originating from a clone of a 1,2 dimethyl hydrazine induced rat colon cancer. One hundred and ten rats bearing three liver metastases were randomly treated by laser volatilization or electrocautery enucleation. The first group of 60 rats was used as a survival group: the laser treated rats survived significantly longer than rats treated by cautery (49.9 days vs 28.9 days) (mean values;p<0.015). In this group, the temperature elevation during operation at the edge of the treated lesion was found higher in the laser group than in the cautery group (56±4.6°C vs 8±3°C) (mean values±s.d.;p<0.001). Nd-YAG laser was also a faster procedure than cautery resection (21±4.2 s vs 57±6.1 s) (mean values±s.d.;p<0.001). At the time of autopsy, the infection rate with laser was found lower than in the cautery group (p<0.025) while no bile leakage was evident. A peritoneal tumour dissemination with ascites was noted in the majority of dead rats. In the second group of 50 rats, the metastatic recurrence was assessed. At day 7, no metastases were found in the laser treated rats while a mean number of 6.5 was found in the cautery group (p<0.001). At the 15th day, more metastases were present in the cautery group. There was a significant correlation between the total number of metastases and the time of death. Those findings suggest that the Nd-YAG laser destruction of experimental liver metastases by its specific effects on tumour cells delayed the recurrence of metastases when compared to the electrocautery resection, contributing to a longer survival of the laser treated rats.

Purification and biological properties of an epithelial intestinal cell growth inhibitor from a human small intestine

Endogenous mitotic inhibitors act as control-mechanisms in intestinal epithelium proliferation. The presence of an inhibitor of cultured intestinal epithelial cell from a villous extract of rat jejunum has been reported in one of our papers. The object of the study now reported was to find the presence of a growth inhibitor in the villous extract from mans small intestine and to purify and characterize this factor when found. Our results reveal that: (1) Such an inhibitor was found in a supernatant preparation obtained from human intestinal epithelial cells. The inhibition of the proliferation of epithelial cells (IRD-98) it induced was seem to be dose-dependent and non-cytotoxic. (2) After chromatography on hydroxylapatite, on DEAE and then on ACA 54 (gel permeation), a low-molecular-weight protein (15 kDa) called purified intestinal inhibitor (PII) was isolated (purification factor of approx. 50,000 with respect to the supernatant fraction). This fraction proved to inhibit the IRD-98 cells in a reversible manner. When cells are incubated with this protein, cells prove to be arrested in phase G1 of the cell cycle as is revealed by the flow cytometry studies. The results obtained support the hypothesis that regulation of cell proliferation is mediated by endogenous inhibitors at the epithelial level.

W1695 Influence of Gastrointestinal Food Allergy On Colonic Immune Activation and Barrier Integrity in the Irritable Bowel Syndrome

Nematode infection results in a polarized Th2 cytokine response and development of alternatively activated macrophages (AAMΦ).IL-13 upregulation of 5-HT2A and PAR-2 is essential for infection-induced changes in smooth muscle hypercontractile response to 5-HT and the PAR-2 agonist, SLIGRL, respectively. Little is known, however, about the interaction between macrophages and these mediators.Aim:To determine the interaction between IL-13 and 5HT or SLIGRL on macrophage development.Methods: Macrophages were collected from uninfected C57BL/6 mice (naive) by peritoneal lavage 4 days after intraperitoneal injections of thioglycate. Macrophages were plated and exposed to vehicle, PAR-2 agonist, SLIGRL(200μM), or 5-HT(50μM)in the presence or absence of IL-13(10ng/ml)for 24 hours. Macrophages were harvested and real-time PCR was used to measure mRNA expression of specific markers. To determine the persistence of these effects we studied macrophages harvested from mice 20 weeks post H. polygyrus(Hp)infection (primed).Results:In naive macrophages, IL-13 upregulated expression of the AAMΦ marker, arginase-1 (1.0±0.1 vs 4.6±1.3 fold). Exposure of naive macrophages to SLIGRL (1.4±0.9 fold) or 5-HT (0.5±0.01) alone did not affect expression of arginase-1; however, SLIGRL markedly potentiated the effects of IL-13 on arginase-1 (4.6±1.3 vs 23.8±0.9). In macrophages harvested from previously Hp infected mice (primed), expression of AAMΦmakers was not different from control. Exposure to SLIGRL or 5-HT alone significantly upregulated by arginase1 and CD206 (table). The effect of SLIGRL was associated with increased expression of PAR-2 (1±0.9 vs 4.9±1.7 fold) as well as IL-4Rα (1±0.1 vs 2.3±0.5 fold) and IL-13Rα1 (1±0.2 vs 3.1±0.3), the receptor components for the type-2 IL-4 receptor that binds IL-13.Conclusions:During infection, exposure to PAR-2 agonists promotes the development of AAMΦ critical for the infection induced changes in smooth muscle contractility. Long lasting effects of infection include the ability of PAR-2 agonists or 5-HT to induce the AAMΦ phenotype by increasing the sensitivity of macrophages to IL-13. This may be important in the development of a Th2 memory response and the persistent effects of infection on gut function.