Patricia Deloyer

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

BackgroundThe 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.ResultsIn 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.ConclusionThe 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.

Are milk polyamines preventive agents against food allergy

Insufficient polyamine intake could play a role in the induction of sensitization to dietary allergens. This proposal is based essentially on investigations made in sucking rats and in children. In sucking rats it has been established that oral administration of spermine can induce all the modifications occurring in the digestive tract at weaning. In the intestine events occur in two phases. The early event consists of desquamation of the epithelium resulting from an activation of apoptosis. The late event appears to involve an hormonal cascade in which adrenocorticotropic hormone, cytokines, bombesin and corticosterone are included. Observations in human subjects show that: (1) the spermine and spermidine concentrations are generally lower in infant formulas than in human breast milk. Mothers seem consistently to have relatively high or relatively low concentrations of spermine and spermidine in their milk. These individual variations may be due to diet, lifestyle or genetic background; (2) the probability of developing allergy can reach 80 % if the mean spermine concentration in the milk is lower than 2 nmol/ml milk. It is approximately 0 % if the mean spermine concentration is higher than 13 nmol/ml milk; (3) preliminary results show that the intestinal permeability to macromolecules differs in premature babies when they are fed on breast milk compared with infant formulas (J Senterre, J Rigo, G Forget, G Dandrifosse and N Romain, unpublished results). This difference does not seem to be present when powdered milk is supplemented with polyamines at the concentration found in breast milk; (4) spermine increases proliferation and differentiation of lymphocytes isolated from the tonsils of children.

Dietary polyamines and non-neoplastic growth and disease.

This review presents the data that are now available concerning the effects of dietary polyamines at either postnatal or adult stages in non-neoplastic growth and disease. Polyamines provided by food have a potential role in growth and development of the digestive system in neonatal mammals (and fishes). In humans, this property could be of importance in preventing the appearance of food allergies. Dietary polyamines also seem necessary for the maintenance of normal growth and general properties of adult digestive tract. Their possible therapeutic effects have been investigated in gastric, intestinal, and, more recently, whole-body healing.

Intestinal development in suckling rats: direct or indirect spermine action?

The present investigation addresses the question of whether spermine orally given to unweaned rats directly or indirectly exerts its effects on the intestinal brush border disaccharidases and if the adrenal gland secretions play a role in this phenomenon. The results showed that spermine, surgically placed in the lower part of the distal small intestine, induced sucrase, stimulated maltase-specific activity and decreased lactase-specific activity in both proximal and distal segments of the small intestine. Introduction of spermine into the lumen of the large intestine stimulated the specific activities of disaccharidases in the whole small intestine. Intraperitoneal injection had no effect except a slight reduction of lactase-specific activity in the distal intestine. Adrenalectomy prevented the oral effect of spermine on sucrase- and maltase-specific activity but not on lactase-specific activity. Addition of spermine to intestinal explants in organ cultures fails to reproduce any of these effects. It even reduced maltase-specific activity. These findings suggest that dietary polyamines have either direct and indirect effects on properties of rat immature intestine.

Role of interleukin-1β, interleukin-6, and TNF-α in intestinal maturation induced by dietary spermine in rats

In the present investigation, the authors aimed to evaluate the role of cytokines in intestinal postnatal maturation induced by dietary polyamines. Neonatal rats were administered either saline or spermine (8 μmol) orally. Spermine increased interleukin-1β (IL-1β), IL-6, and TNF-α plasma concentration. The maximum concentrations of IL-1β, IL-6, and TNF-α were, respectively, observed at 4, 4, and 8 h posttreatment. Intraperitoneal (ip) injection of IL-1β increased the specific activity of sucrase in whole small intestine, whereas the specific activities of maltase and lactase were significantly enhanced only in the jejunum. IL-6 elicited sucrase and increased maltase specific activity in the whole small intestine, but lactase specific activity was not affected. TNF-α had no effect on sucrase and maltase specific activity, but a slight augmentation of lactase specific activity was detected in the jejunum. Spermine and spermidine content in the intestine was increased by ip injection of IL-1β and IL-6. Corticosterone secretion was elevated by single ip injection of IL-1β, IL-6, or TNF-α. These findings suggest that spermine could induce postnatal intestinal development and corticosterone secretion through a cytokine-dependent mechanism.

Analysis of structural and biochemical events occurring in the small intestine after dietary polyamine ingestion in suckling rats

In the present investigation, we analyzed the mechanism involved in spermine-induced intestinal maturation in suckling rats. Spermine was given orally to suckling pups and biochemical as well as morphological parameters were studied at different times after the beginning of the treatment. Eight hours after administration, spermine produced cell elimination at the villus tops and a decrease in intestinal DNA and protein content. In parallel, protein and DNA concentration and disaccharidase activity were enhanced in the chyme. These transitory alterations were not induced by growth inhibition, as DNA synthesis was not modified, although a brief decrease in protein synthesis was observed. Spermine was not metabolized in cytotoxic products: rat pretreatment with MDL72527 (an inhibitor of polyamine oxidase) did not avoid the decrease in disaccharidase activity and in DNA and protein content. Three days after treatment, sucrase and maltase activity was higher in rats treated with spermine and MDL72527 than that in animals receiving spermine alone. Lactulose or acetylspermine ingestion induced intestinal maturation. Our data suggest that dietary polyamines exert a direct and specific maturational effect on rat small intestine and that an early decrease in lactase activity plays an important role in this phenomenon.

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.

Intestinal maturation induced by spermine in young animals

In suckling rats, it has been established that oral administration of spermine, a dietary polyamine, at appropriate doses, induces all the modifications in the digestive tract that occur at weaning, namely, (a) in the intestine: variations in the specific activities of disaccharidases and peptidases, gene expression, the level of receptors to polymeric immunoglobulins (RPIs), tissue histology, and the intestinal permeability to macromolecules; (b) maturation of the intestinal immunological system, an observation confirmed in mice; (c) an increase in the specific activity of enzymes contained in the pancreas; (d) a change in the growth rate and biochemical properties of the liver, e.g., in the synthesis of RPIs. The mechanism of spermine action is partly understood. In the intestine, two phases of events have been recorded. The first phase consists of the desquamation of the epithelium resulting from an activation of apoptosis, probably induced by the entrance of spermine into the enterocytes. The second phase concerns a hormonal cascade in which adrenocorticotrophic hormone, cytokines, bombesin and corticosterone intervene. A general hypothesis taking into account all the results obtained until now is presented as well as other observations recorded on the effect of exogenous spermine on the intestinal maturation of vertebrates other than the rat.

Polyamine and intestinal properties in adult rats

We questioned whether polyamines coming from the diet or produced by intestinal microflora or by intracellular metabolism influence intestinal functions. Therefore, we compared pathogen-free rats and germ-free rats receiving a diet with low polyamine content and either treated or not treated with difluoromethylornithine (DFMO) and/or methylglyoxal bis (guanylhydrazone) (MGBG). Wet weight, protein content, DNA content, sucrase (EC 3.2.1.48), maltase (EC 3.2.1.20) and lactase (EC 3.2.1.23) specific activities, amounts of putrescine, spermidine and spermine were measured in the mucosa of the proximal and distal intestine. Body weight was also determined. Rats without microflora had a higher specific activity of maltase and higher amounts of spermidine and spermine but lower lactase specific activity than pathogen-free animals; the low-polyamine diet given to germ-free rats had little effect on the functional variables measured (decrease of maltase and lactase specific activities) and did not modify the amounts of polyamines. DFMO and/or MGBG administered to germ-free rats receiving a low-polyamine diet induced modifications of most of the variables studied. Body weight and wet weight of proximal and distal intestine decreased, disaccharidase specific activities decreased, and amounts of polyamines changed according to the inhibitor used. Thus, our results showed that the deprivation of polyamine supply from microflora or from the diet failed, under our experimental conditions, to affect the intestinal properties analysed but exogenous and endogenous polyamine restriction altered general properties of the organism as well as intestinal functions.

Maturation of intestinal digestive and immune systems by food polyamines

Publisher Summary Maturation of the intestine occurs during the first few postnatal weeks in many vertebrates. This maturation allows the pups to adapt to the drastic change from milk to solid food. The enzymes located in the mucosal brush border adapt to solid food: lactase specific activity decreases, in contrast to maltase and sucrase activities that increases. Polyamines are involved in gut maturation in rats. Spermine administration leads to an increase of plasma adrenocorticotrophic hormone (ACTH) and corticosterone, and bilateral adrenalectomy drastically reduces the spermine effect on the small intestine. This chapter describes the phenomena observed in the small intestine after ingestion of spermine and compares them to the natural postnatal maturation occurring at weaning. It proposes a mechanism of spermine action in small intestine maturation, describes the spermine-induced maturation of the spleen, and explores the impact of this substance in human health. Spermine ingested by suckling rats induces the secretion of soluble factors in the circulation. These factors are responsible for hypothalamo-hypophyso-adrenal axis activation. Spermine administration provokes cytokine secretion in plasma, cytokine injection induces a precocious maturation of the small intestine, and spermine administration is responsible for immune system maturation. Polyamines are involved in gut maturation and the polyamine concentration in the intestinal mucosa increases at weaning.