Preliminary characterization of jejunocyte and colonocyte cell lines isolated by enzymatic digestion from adult and young cattle.

In the present study we developed an enzymatic approach (through the use of collagenase and dispase) to isolate bovine intestinal epithelial cells. Using this method, freshly isolated jejunocytes could be distinguished from simultaneously isolated colonocytes, as the jejunocytes specifically exhibited the small intestinal peptidase gene transcript, as well as an active alkaline phosphatase. The transformation of both types of cell suspension was performed by retroviral infection, using reproduction-defective viruses bearing the gene coding for the large T antigen of the leukaemia simian virus (SV40). The success of the transfection was demonstrated by (1) a significant increase in cell passage numbers (52-53 vs. 7 passages for non-transfected cells), (2) the detection of both the large T transcript and the large T antigen in transformed cells. Possible contamination and progressive substitution of bovine primocultures by non-bovine lineages available in the laboratory was excluded, as the transformed cells presented a bovine typical karyotype. Most transfected cells kept an epithelial morphology after transformation. They also maintained the expression of FABP and enterocyte specific enzymes (brush-border associated maltase and IAP). However, levels of specific activity of these enzymes were low, suggesting that cell differentiation is not completely achieved under the applied culture conditions.

Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21).

BACKGROUND: The absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. The cell lineage Caco-2 is a model commonly used to reproduce the features of the bowel epithelium. However, there is a strong debate regarding the value of Caco-2 cell culture to mimick in vivo situation. Indeed, some authors report in Caco-2 a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. The HT29-5M21 intestinal cell lineage is a mucin-secreting cellular population. A co-culture system carried out in a serum-free medium and comprising both Caco-2 and HT29-5M21 cells was developed. The systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure. RESULTS: In this study, we investigated the activity and localization of the alkaline phosphatase and the expression of IAP and MUC5AC genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of Caco-2 and HT29-5M21 cells. We observed that the culture conditions used (serum-free medium) did not change the phenotype of each cell type, and produced a reproducible model. The alkaline phosphatase expression characterizing Caco-2 cells was influenced by the presence of HT29-5M21 cells. CONCLUSION: The culture formed by 75% Caco-2 and 25% HT29-5M21 produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules.

Immunochemical, biomolecular and biochemical characterization of bovine epithelial intestinal primocultures.

BACKGROUND: Cultures of enterocytes and colonocytes represent valuable tools to study growth and differentiation of epithelial cells. In vitro models may be used to evaluate passage or toxicity of drugs, interactions of enteropathogenes bacteria strains with intestinal epithelium and other physiologic or pathologic phenomenon involving the digestive tract. RESULTS: Cultures of bovine colonocytes and jejunocytes were obtained from organoid-enriched preparations, using a combination of enzymatic and mechanical disruption of the intestine epithelium, followed by an isopicnic centrifugation discarding most single cells. Confluent cell monolayers arising from plated organoids exhibited epithelium typical features, such as the pavement-like structure, the presence of apical microvilli and tight junctions. Accordingly, cells expressed several markers of enterocyte brush border (i.e. maltase, alkaline phosphatase and fatty acid binding protein) as well as an epithelial cytoskeleton component (cytokeratin 18). However, enterocyte primocultures were also positive for the vimentin immunostaining (mesenchyme marker). Vimentin expression studies showed that this gene is constitutively expressed in bovine enterocytes. Comparison of the vimentin expression profile with the pattern of brush border enzymes activities, suggested that the decrease of cell differentiation level observed during the enterocyte isolation procedure and early passages of the primoculture could result from a post-transcriptional de-repression of vimentin synthesis. The low differentiation level of bovine enterocytes in vitro could partly be counteracted adding butyrate (1-2 mM) or using a glucose-deprived culture medium. CONCLUSION: The present study describes several complementary approaches to characterize bovine primary cultures of intestinal cells. Cultured cells kept their morphologic and functional characteristics during several generations.

Intestinal effects of long-lasting spermine ingestion by suckling rats.

Spermine ingestion induces the precocious maturation of the small intestine in suckling rats. Previous observations suggest that spermine-induced intestinal maturation is a two-step phenomenon. The first step is the elimination of immature enterocytes (4-10 h post spermine ingestion) and the second step is the replacement of previous immature cells by adult-type enterocytes (2-3 days post initial spermine administration). The spermine-induced maturation is reversible when spermine administration is stopped. This work was undertaken in order to check whether the extension of polyamine administration (for 3-7 days) after the appearance of spermine-induced maturation can retain the mature state of the small intestine. Our results indicate that extension of spermine administration does not prevent some parameters (sucrase and maltase specific activities) reverting to a typical 'immature' value while others remain at a typical 'mature' level (mucosal weight and lactase specific activity). Our results show that there are at least two different mechanisms in required for the control of spermine-induced maturation of the small intestine.

Modulation of intestinal urea cycle by dietary spermine in suckling rat.

Argininosuccinate synthetase, an ubiquitous enzyme in mammals, catalyses the formation of argininosuccinate, the precursor of arginine. Arginine is recognised as an essential amino acid in foetuses and neonates, but also as a conditionally essential amino acid in adults. Argininosuccinate synthetase is initially expressed in enterocytes during the developmental period, it disappeared from this organ then appeared in the kidneys. Although the importance of both intestinal and renal argininosuccinate synthetases has been recognised for a long time, nutrients have not yet been identified as inducers of the gene expression. In the context of a proteomic screening of intestinal modifications induced by dietary spermine in suckling rats, we showed that argininosuccinate synthetase and carbamoyl phosphate synthase disappeared from enterocytes after this treatment. The disappearance of argininosuccinate synthetase in small intestine was confirmed by immunodetection. Expression of carbamoyl phosphate synthase and argininosuccinate synthetase coding genes decreased also after spermine administration. Expression of other urea cycle enzyme coding genes was modulated by spermine administration: argininosuccinate lyase decreased and arginase increased. Our results fit with the developmental variation of argininosuccinate synthetase and carbamoyl phosphate synthase. Modulation of the gene expression for several urea cycle enzymes suggests a coordination between all the pathway steps and switch toward polyamine (or proline and glutamate) biosynthesis from ornithine.

Enhancement of lysozyme stability and activity by polyamines.

Spermine, a low molecular weight polyamine, administered orally to suckling rats induces the maturation of the small intestine. In this organ, lysozyme is an important component of the innate immunity. In this report, we analysed the binding of spermine to lysozyme and its effect on thermal inactivation of the protein by spectroscopy techniques. The activity of the enzyme was analysed in presence of spermine by lysoplate technique. We studied the effects of spermine ingestion by suckling rats on intestinal lysozyme activity and gene expression. We reported that spermine binds to lysozyme and increases in vitro the thermal stability and the activity of the protein. When administered orally to suckling rats, spermine increases the lysozyme activity in jejunum, but not in ileum. This increase is not due to a modification of the gene expression. The observed effects lead us to postulate that spermine could be used in some mammals as a promoter of the innate immunity.

Short-term effects of spermine ingestion on the small intestine: a comparison of suckling and weaned rats.

We have previously shown that spermine, shortly after its ingestion, can induce the alteration of the morphology of the small intestine of suckling rats. It was proposed that this alteration is due to polyamine accumulation inside the epithelial cells. This could also be related to the fact that the intestine of the suckling rat is in an immature state. To shed light on this issue, disaccharidase and alkaline phosphatase activity assays, protein, DNA and RNA content measurements and polyamine concentration analysis were performed on the small intestine of suckling and weaned Wistar rats treated with spermine. Spermine did not induce the same intestinal alterations in weaned rats compared to suckling animals. Indeed, in sucklings, spermine administration induced a decrease of the protein, DNA, putrescine and spermidine intestinal content, suggesting a cell loss. The cell loss impaired the activity of intestinal enzymes: lactase, maltase and alkaline phosphatase. In weaned rats, the same treatment did not alter these parameters. Exogenous spermine by itself is not sufficient to induce the alterations described here and previously. The maturity degree of the small intestine could be the basis of this process.

Enhancement of transfection efficiency through rapid and noncovalent post-PEGylation of poly(dimethylaminoethyl methacrylate)/DNA complexes.

PURPOSE: The aim of this work was to develop a new strategy to introduce poly(ethylene glycol) (PEG) into methacrylate-based polymer/ DNA complexes in order to produce hemocompatible particles able to transfect cells in the presence of serum. METHODS: Atom transfer radical polymerization was used to synthesize a well-defined poly(2-(dimethylamino)ethyl methacrylate) homopolymer (PDMAEMA) and a poly(2-(dimethylamino)ethyl methacrylate-b-poly(ethylene glycol) alpha-methyl ether, omega-methacrylate) palm-tree-like copolymer (P(DMAEMA-b-MAPEG)). The complexes obtained by self assembly of the pCMVbeta plasmid and the polymers were used to transfect Cos-7 cells. Their physical properties--particle size and zeta potential--were characterized respectively by dynamic light scattering and electrophoretic mobility measurements. Ex vivo hemocompatibility was also determined. RESULTS: The PDMAEMA/pCMVbeta complexes transfected Cos-7 cells exclusively in the absence of serum. Although the P(DMAEMA-bMAPEG) copolymer had no transfection activity per se, the addition of the latter to pre-formed PDMAEMA/DNA complexes significantly enhanced the activity and allowed transfection even in the presence of serum. The presence of palm-tree-like copolymers also improved the hemocompatibility properties of the complexes. No effect on platelet counts was observed for P(DMAEMA-b-MAPEG)/pCMVbeta complexes, whereas a decrease of platelets was clearly observed when blood cells were incubated with PDMAEMA/pCMVbeta complexes. CONCLUSIONS: Such a synergistic effect of noncovalent PEGylation of poly(amino methacrylate)/DNA complexes allows a new and versatile approach to tune up transfection efficiency.

Differential effect of dietary spermine on alkaline phosphatase activity in jejunum and ileum of unweaned rats.

Spermine is a low molecular weight polyamine involved in the postnatal maturation of the gut. When it is administered orally to suckling rats it induces the maturation of their spleen, liver, pancreas, and small intestine. We showed that this polyamine modulates differently the activity of alkaline phosphatase in jejunum and ileum in suckling rat. In 14-day-old rat which had received spermine orally for 3 days, once daily, an increase of alkaline phosphatase activity in the jejunum and a decrease of this activity in the ileum was observed. Alkaline phosphatase was located at the bottom of the villus in the control jejunum and in the whole length of the villus in spermine-treated rats. On the contrary, in ileum of controls, this enzyme was present in the whole length of the villus but disappeared in the spermine-treated animals. An enzyme mass shift was observed in the small intestine after spermine administration. Spermine administration did not change the expression of genes coding for alkaline phosphatase, suggesting a post-transcriptional modification.

Spermine-induced maturation in wistar rat intestine: a cytokine-dependent mechanism.

OBJECTIVES: Polyamines are of great importance in biologic processes such as cell proliferation and differentiation. The ingestion of spermidine or spermine by suckling rats induces the precocious maturation of the small intestine. In a previous article, the authors hypothesized that this phenomenon could be mediated by interleukins. This work was performed to examine the role of IL-1, IL-2, and tumor necrosis factor (TNF)- alpha in the spermine-induced maturation of the small intestine. METHODS: Wistar suckling rats were treated with spermine, FR167653 (inhibitor of IL-1beta/TNF-alpha production), IL-1beta/TNF-alpha neutralizing antibodies, lipopolysaccharide, or IL-2. Intestinal disaccharidase-specific activities, polyamine content, and IL-2 plasma concentration were analyzed. Comparisons were made with untreated control animals. RESULTS: Spermine-induced maturation of the small intestine was decreased by FR167653 but not by the neutralizing antibodies. Lipopolysaccharide injection induced an increase in disaccharidase-specific activity. IL-2 induced a decrease of the intestinal lactase-specific activity. Spermine administration led to a similar decrease of lactase activity and to an increase of IL-2 plasma concentration. CONCLUSIONS: The authors conclude that IL-1beta and TNF-alpha are involved in the spermine effects on maltase- and sucrase-specific activities and suggest that IL-2 is involved in the spermine-induced decrease of lactase activity.