Biomedical subjects

M Kedinger

Publications and source records attributed to M Kedinger.

At least 73 records · Page 4Linked to original sources

PubMed1988

Expression of brush border calmodulin-binding proteins during human small and large bowel differentiation.

The expression and immunocytochemical localization of three brush border cytoskeletal calmodulin-binding proteins, caldesmon, fodrin, and the 110 kDa subunit of the 110 kDa calmodulin complex, have been studied in human intestinal epithelial cells as a function of their ontogenic differentiation. At immature stages (fetal week 8), caldesmon and fodrin were present in undifferentiated intestinal epithelial cells. However, no 110 kDa protein was detectable except a 135 kDa immunoreactive species. The 110 kDa form appeared at week 12, when microvilli differentiate, and became prominent at week 14 simultaneously with the disappearance of the 135 kDa species. Finally at week 14, the calmodulin-binding protein pattern was identical to that found in adults. Immunocytochemical experiments revealed that at week 8, antibodies to caldesmon and fodrin gave a fluorescence lining at the periphery of the cells, whereas the 110 kDa immunoreactive species was hardly detectable. Then, as early as week 12 of gestation, with the three antisera, a bright fluorescence lined the apex of the cells, as in adults. In the colon, the events were delayed. This study demonstrates that the developmental pattern of the three calmodulin-binding proteins investigated, caldesmon, fodrin and the 110 kDa subunit, parallels the temporal differentiation of human intestinal brush borders and the proximo-distal morphological intestinal maturation.

Calmodulin-Binding Proteins↗

PubMed1988

A novel marker glycoprotein for the microvillus membrane of surface colonocytes of rat large intestine and its presence in small-intestinal crypt cells.

Murine mAbs were produced against purified microvillus membranes of rat colonocytes in order to establish a marker protein for this membrane. The majority of antibodies binding to the colonic microvillus membrane recognized a single protein with a mean apparent Mr of 120 kD in both proximal and distal colon samples. The antigen is membrane bound as probed by phase-partitioning studies using Triton X-114 and by the sodium carbonate extraction procedure and is extensively glycosylated as assessed by endoglycosidase F digestion. Localization studies in adult rats by light and electron microscopy revealed the microvillus membrane of surface colonocytes as the principal site of the immunoreaction. The antigen was not detectable in kidney or liver by immunoprecipitation but was present in the small intestine, where it was predominantly confined to the apical membrane of crypt cells and much less to the microvillus membrane of differentiated enterocytes. During fetal development, the antigen appears first in the colon at day 15 and 1-2 d later in the small intestine. In both segments, it initially covers the whole luminal surface but an adult-like localization pattern develops soon after birth. The antibodies were also used to develop a radiometric assay for the quantification of the antigen in subcellular fractions of colonocytes in order to assess the validity of a previously developed method for the purification of colonic brush-border membranes (Stieger, B., A. Marxer, and H.P. Hauri. 1986. J. Membr. Biol. 91:19-31.). The results suggest that we have identified a valuable marker glycoprotein for the colonic microvillus membrane, which in adult rats may also serve as a marker for early differentiation of enterocyte progenitor cells in small-intestinal crypt cells.

Age Factors↗

PubMed1988

Calmodulin in normal and cystic fibrosis human intestine at different developmental stages.

Calmodulin concentrations and localisation have been analysed as a function of development in human intestinal epithelial cells from normal and cystic fibrosis individuals. In normal fetuses up to eight weeks of gestation intestinal epithelial cells which were still undifferentiated were not immunoreactive and their calmodulin content was low. From eight weeks onwards there was a significant overall increase in calmodulin content concomitant with its segregation to the apical side of epithelial cells. At 14 weeks of gestation calmodulin concentrations and localisation closely resembled those of adults. The developmental pattern of calmodulin appeared to parallel the morphological and functional maturation of brush borders which occurs during the first trimester of pregnancy. In the intestinal epithelial cells from a 19 weeks cystic fibrosis fetus and a cystic fibrosis newborn infant neither calmodulin concentration, nor its localisation were affected. Similarly, brush border calmodulin binding proteins and enzymatic activities were similar in normal subjects and the cystic fibrosis intestine.

Adult↗

PubMed1988

Epithelial-mesenchymal interactions in intestinal epithelial differentiation.

The complex morphogenetic events and the concomitant structural and functional differentiation of intestinal progenitor cells are dependent on tissue interactions. Several experimental models of hetero-species or -topic recombinants between epithelial and mesenchymal anlagen are described. They enabled us to elucidate the respective roles of these tissue components in morphogenesis, epithelial differentiation, and hormone-elicited responses. Among the mechanisms of tissue interactions, the possible mediation of permissive and instructive information via the extracellular matrix is postulated. Arguments in favor of this are provided by the observation of compositional changes in matrix molecules during intestinal development and differentiation. On the other hand, in vitro experimental data emphasize the role of actual contacts between epithelial and mesenchymal cell populations and the importance of the mesenchyme for basement membrane formation.

Animals↗

PubMed1987

Synthesis of glycosaminoglycans by undifferentiated and differentiated HT29 human colonic cancer cells.

Among the extracellular matrix components which have been suggested to be involved in developmental and neoplastic changes are glycosaminoglycans (GAGs). To try to correlate their amount and nature with the process of enterocytic differentiation, we studied glycosaminoglycan synthesis of human colonic adenocarcinoma cells (HT29 cell line) by [3H]glucosamine and [35S]sulfate incorporation. Enterocytic differentiation of the cells obtained in a sugar-free medium (for review, see A. Zweibaum et al. In: Handbook of Physiology. Intestinal Transport of the Gastrointestinal System, in press, 1987) resulted in a marked increase in total incorporation of labeled precursors (20-fold for [3H]glucosamine, 4.5-fold for [35S]sulfate) as well as in uronic acid content (5-fold); most of the synthesized GAGs were found associated with the cell pellet. Chromatographic and electrophoretic analysis of the labeled GAGs revealed that undifferentiated cells synthesized and secreted hyaluronic acid, heparan sulfate, and one class of chondroitin sulfate. Differentiation of HT29 cells because associated with the synthesis of an additional class of chondroitin sulfate (CS4) concomitant to a decrease in heparan sulfate which is no longer found secreted in the medium. Furthermore, the charge density of this latter GAG component varied as assessed by a shift of its affinity on ion-exchange chromatography.

Adenocarcinoma↗

PubMed1987

Intestinal tissue and cell cultures.

The culture of animal cells and tissues is a widely used technique in the field of cellular and molecular biology; one of the most interesting aspect being linked to the study of the mechanisms of cell differentiation. In the specific case of intestinal epithelial cells, various tissue culture technologies have proved to be important tools for the study of precise facets related to intestinal function, pathology and differentiation. Concerning this latter aspect, organ culture experiments have brought about interesting data on the hormonal or nutritional control of intestinal maturation. Nevertheless, the study of the precise mechanisms underlying epithelial proliferation and/or differentiation at the cellular level needs more adequate cell culture model systems. One of them has been described for two cell lines derived from human colonic adenocarcinomas, in which the cells can be induced to achieve enterocytic-like differentiation. Up to date, none of the continuous cell lines starting from normal undifferentiated cells have allowed generation of morphological or functional enterocytic polarity. In contrast, primary cell cultures which allow maintenance of a more physiological environment for the epithelial cells like contacts with their in vivo counterparts, mesenchymal cells or extracellular matrix molecules, have proved to be promising approaches.

Animals↗

PubMed1987

Growth and differentiation of intestinal endodermal cells in a coculture system.

To investigate the role of epithelial-mesenchymal interactions on intestinal maturation, we cultured embryonic epithelial cells in several experimental conditions. Microexplants of 14-15 days fetal rat intestinal endoderm, separated from the mesenchyme by collagenase, were seeded on dishes coated with different extracellular matrix components (collagens I, III, IV, fibronectin, laminin) or on confluent monolayers of intestinal mesenchymal cells or of fetal skin fibroblasts. Only small variations in the attachment or spreading of the endodermal cells could be observed when they were cultured on the different substrata and their survival never exceeded one week. When cocultured with intestinal or skin fibroblasts, however, endodermal cells grew, formed a monolayer, survival time was prolonged up to two to three weeks, and differentiation occurred. This differentiation was assessed by cell polarisation, morphological maturation of apical brush borders, synthesis of microvillar digestive enzymes and of extracellular matrix molecules seen immunocytochemically. Finally, glucocorticoids which are known to stimulate or induce brush border enzymes, accelerated the morphological and enzymatic maturation only in the cocultures.

Animals↗

PubMed1986

Calmodulin in epithelial intestinal cells during rat development.

Calmodulin was immunocytochemically localized in the brush borders of rat intestinal epithelial cells from the tip to the base of the villi, from day 18 of fetal life up to the adult stage. The early (14th day) fetal cells, like the adult crypt cells, were not immunoreactive, although their calmodulin content was equal to that of the mature cells from the tips of the villi.

Animals↗

PubMed1986

Fetal gut mesenchyme induces differentiation of cultured intestinal endodermal and crypt cells.

An experimental model was designed to analyze the effect of fetal gut mesenchyme on the cytodifferentiation of crypt cells and of embryonic progenitor cells. The cells used were the rat intestinal crypt cell line, IEC-17, and primary cell cultures prepared form isolated 14-day-old fetal intestinal endoderm (EC). Both cultures prepared from isolated 14-day-old fetal rat intestinal endoderm (EC). Both types of cells were associated with 14-day-old fetal rat gut mesenchyme (Rm) and grafted under the kidney capsule of adult rats. Seventy percent of the Rm/EC and ten percent of the Rm/IEC recombinants, recovered after 9 days, exhibited well-vascularized structures in which the mesenchyme had induced morphogenesis of the cells into a villus epithelium. The four main intestinal epithelial cell types, absorptive, goblet, endocrine, and Paneth cells, were identified using electron microscopy. Biochemical determinations of enzyme activities associated with brush border membranes revealed that alkaline phosphatase, lactase, sucrase, and maltase were expressed in both types of associations. These results were confirmed by immunofluorescence staining using monoclonal antibodies to brush border enzymes. Both enzyme assays and immunocytochemistry showed that the amount of enzymes present in the brush border membrane of Rm/IEC grafts was in general lower than that of the Rm/EC recombinants. The results indicate that fetal rat gut mesenchyme enables morphogenesis and cytodifferentiation of both crypt and embryonic progenitor cells.

Aminopeptidases↗

PubMed1986

Maturation of brush border hydrolases in human fetal intestine maintained in organ culture.

This investigation was undertaken to study the effects of hormones, sugars and amniotic fluid on the maturation of brush border enzymes in the human fetal intestine, at early stages of gestation. Intestinal explants from 8-13-weeks fetuses were maintained in organ culture for 3 days in the presence of the agents to be tested. The data show that the explanation of human fetal gut in a serum free culture medium elicits a significant maturation (2-4-fold increase above preculture levels) of lactase and aminopeptidase whatever the gestational stage studied and of sucrase and alkaline phosphatase at specific stages of development. To be expressed, the overall maturation needs the presence of sugar (in particular glucose) in the culture medium. The addition of dexamethasone, insulin or amniotic fluid to the medium did not further enhance brush border enzyme activities except for lactase whose levels were doubled by the dexamethasone. The present data suggest that in addition to the differences which exist among mammalian species in the timing of enzyme development, there may be a species specificity in the factors involved in fetal enzymatic maturation.

Alkaline Phosphatase↗

PubMed1986

Immunocytochemical localization of extracellular-matrix proteins in relation to rat intestinal morphogenesis.

Various extracellular-matrix proteins were detected by indirect immunofluorescence in rat intestine at various stages of development ranging from 14 days of gestation to the adult stage. At the earliest stage studied, laminin, nidogen and type-IV collagen were present at the epithelial/mesenchymal interface, whereas fibronectin and type-III procollagen were found throughout the whole mesenchyme. We were able to relate some changes in the staining patterns of extracellular-matrix proteins to morphogenetic processes. As early as 15 days of gestation, i.e. before villus formation, modifications in the distribution or in the staining intensity of all of the antigens within the mesenchyme paralleled the orientation and segregation of mesenchymal cells in the region surrounding the basal membrane and in the presumptive peripheral muscular layers. During villus outgrowth, the transient disappearance of fibronectin and particularly type-III procollagen from the top of the protruding villus core was evident. During the perinatal period, i.e. when crypts develop, the linear staining for the basal-membrane proteins became restricted to the base of the villi, their labelling along the remaining portion of the villi being more irregular. In mature rat intestine, no major modifications in matrix proteins along the crypt-villus axis in relation to epithelial differentiation were found, except that the labelling for fibronectin and type-III procollagen, which are at this stage more closely related to the basement membrane, was less pronounced in the upper part of villi.

Animals↗

PubMed1986

[Development of the digestive function: regulation of the maturation of intestinal brush border enzymes].

During the suckling period in mammals, the immaturity of the digestive function as well as several functional pecularities lead the gastrointestinal physiology to adapt to milk digestion and absorption. The dietary transition which occurs at weaning is accompanied by wide modifications as neonatal properties are lost and mature digestive functions are acquired. The involvement of factors like tissue interaction, hormones and nutritional substrates in the onset and subsequent maturation of intestinal brush border hydrolases is discussed.

Alkaline Phosphatase↗

PubMed1985

Enzymatic response to glucocorticoids of the chick intestinal endoderm associated with various mesenchymal cell types.

The aim of the present study was to test the morphological and functional maturation of recombinants composed of chick intestinal endoderms associated to different mesenchymal supports and their enzymatic response to glucocorticoids. For this purpose 5.5-day chick embryonic intestinal endoderm has been associated to 14-day fetal rat gut mesenchyme, to rat intestinal fibroblasts (6-day neonatal rat intramucosal fibroblasts) or to rat control fibroblasts, originating from 20-day fetal rat skin and lung and from 6-day neonatal rat intestinal muscle. The recombinants were grown as intracoelomic grafts either for 12 days or for 10 days plus 2 days in organ culture in the presence of dexamethasone. The data show that heterospecific recombinants achieve subnormal morphogenesis and enzymatic maturation. The organ culture experiments further reveal that sucrase activity is insensitive to dexamethasone in all types of recombinants whereas, alkaline phosphatase is highly stimulated over the levels present in the intestine developed in situ whatever the stromal support, except when this support is provided by rat gut mesenchyme. These results support the view that in the intestine the hormonal response is mediated by epithelial-mesenchymal interactions.

Animals↗

PubMed1984

Ability of L-triiodothyronine to modulate glucocorticoid-evoked brush border enzyme activities in cultured fetal rat intestine.

In vitro organ culture of developing fetal rat intestine emphasizes the absence of direct effect of thyroid hormone on several brush border enzymes in contrast with its ability to inhibit or enhance dexamethasone (DX) -evoked enzyme activities. Indeed, the presence of triiodothyronine in the culture medium leads mainly to a significant decrease of DX-stimulated lactase and to a further stimulation of DX-induced sucrase activities.

Animals↗

PubMed1984

Developmental pattern of brush border enzymes in the human fetal colon. Correlation with some morphogenetic events.

The present study is concerned with a multilevel approach to human colon organogenesis, involving scanning and transmission electron microscopy together with brush border enzymology. The results emphasize the particular developmental pattern of sucrase activity which appears towards 11 weeks, increases at 14 weeks, begins to decrease around 28 weeks and disappears totally at term. In contrast, other enzymes like aminopeptidase and alkaline phosphatase persist in the adult colon. The correlation, in the fetal large bowel, of enzyme activities and villus structures similar to those found in the small intestine is discussed.

Alkaline Phosphatase↗

PubMed1984

Effects of human fetal gastroenteric mesenchymal cells on some developmental aspects of animal gut endoderm.

Human intestinal and gastric mesenchymal cells were associated with chick and rat intestinal endoderm in order to test their species-specific capacity on epithelial differentiation. Primary cell cultures were established from human intestinal and gastric mesenchyme. Animal intestinal endoderms were associated with both cell types, grafted in ovo and allowed to develop for 12 days. The morphologic and enzymatic differentiation of the recombinants demonstrated two types of inductive properties exerted by human fetal intestinal and gastric mesenchymal cells, respectively. Firstly, human intestinal mesenchymal cells triggered intrinsic developmental capacities in chick and rat endoderm, i.e. enhanced structural brush-border maturation in both species and precocious sucrase induction in rat endoderm. Secondly, human gastric mesenchymal cells provoked the partial conversion of chick intestinal endoderm into gastric structures. Such properties were not found in homologous animal mesenchymes.

Alkaline Phosphatase↗

PubMed1984

Early organogenesis of human small intestine: scanning electron microscopy and brush border enzymology.

Human small bowel early organogenesis was studied by scanning electron microscopy and found to be correlated to brush border enzymology. The appearance of the brush border enzymes sucrase, lactase, and aminopeptidase (measured in a purified apical membrane fraction) coincides with the first outgrowth of villi (eight weeks). Alkaline phosphatase was detected at seven weeks. The content of these enzymes furthermore increased up to the 14th week when both sucrase and aminopeptidase activities were comparable with adult values.

Alkaline Phosphatase↗

PubMed1983

Sucrase-isomaltase: a marker of foetal and malignant epithelial cells of the human colon.

The presence of sucrase-isomaltase (SI), a glycoprotein hydrolase normally restricted to the brush border membrane of the enterocytes of the small intestine, was investigated in tumours which developed in nude mice inoculated with six human colon carcinoma cell lines (HT-29, Caco-2, HRT-18, HCT-8R, SW-480, and CO-115). Foetal and normal adult human small intestines and colons were used as controls. SI was studied by (1) immunofluorescence with rabbit antibodies raised against purified human small intestine SI; (2) polyacrylamide gel electrophoresis and immunoblotting; and (3) determination of the enzyme activity. SI was antigenically present, and enzymatically active, in all the tumours derived from Caco-2 and HT-29 cells. The presence of the enzyme was associated with that of typical brush borders at transmission electron microscopy examination. SI was absent from the tumours developed with the other four cell lines, as well as from the normal adult colon mucosa. SI was also present and active in the colons of mid-gestation foetuses, ranging in ages between 20 and 28 weeks; it was absent from the colons of late-gestation foetuses. The presence of SI in tumours derived from two cell lines suggests that this enzyme is a marker, so far unsuspected, of certain human colon cancers, and that the differentiation pattern of these particular cancers closely resembles that of the foetal colon.

Animals↗