Fetal Colon Research Articles

A shared and unique epitope(s) on human colon, skin, and biliary epithelium detected by a monoclonal antibody

An immune-complex-mediated inflammation has been suggested in the pathogenesis of skin and biliary complications in ulcerative colitis, although specific antigen-antibody complexes have not been found. We recently identified an Mr 40,000 colonic protein that reacts with tissue-bound immunoglobulin G eluted from colon specimens of patients with ulcerative colitis and not with immunoglobulin G eluted from colon specimens of patients with other diseases (J Clin Invest 1985;7:311). Murine monoclonal antibodies to the purified Mr 40,000 colonic protein are developed. The presence of the Mr 40,000 protein was examined with a monoclonal antibody (7E12H12, immunoglobulin M isotype) by an immunoperoxidase assay against various human epithelial tissues (74 specimens) including colon, ileum, jejunum, duodenum, stomach, esophagus, pancreas, liver, respiratory, urinary, and genital tracts. In addition, gall bladder, bile duct, hepatic ducts, skin, synovial membrane, and eye tissue were also examined for any cross-reactivity with 7E12H12 and another anti-Mr 40,000 monoclonal antibody (7E6A5, immunoglobulin G1 isotype). 7E12H12 reacted only with colonicepithelial cells mainly along the basolateral domains of plasma membrane and apical areas. The reactivity was present in both adult and fetal colon including the appendix. Among all the noncolonic tissue specimens, 7E12H12 reacted only with the gall bladder, the bile duct, the hepatic ducts, and the skin. All other epithelial tissues from 15 different organs, including the small intestine, synovium, and eye tissue, did not react. In the skin and biliary tract the immunoreactivity was exclusively present in the epidermal epithelial cells and mucosal epithelium respectively. 7E6A5 stained colonic mucosal cells but did not react with skin, biliary epithelium, synoyium and eye tissue. These results indicate the presence of a unique epitope or epitopes on Mr 40,000 colonic epithelial protein that is shared by the skin and biliary tract epithelial cells. Future studies of this shared epitope or epitopes and its immune recognition may provide further understanding in the pathogenesis of extraintestinal complications involving these organs in patients with ulcerative colitis.

Morphogenesis as a parallel of invasion: the epithelial-mesenchymal boundary and basal lamina in foetal rat colon

In order to determine whether neoplastic invasion, as it occurs in colonic carcinoma, represents a recapitulation of embryonic events, the development of the crypts and basal lamina of the rat colon was examined in foetuses between 16 and 21 days gestation by light and transmission electron microscopy, autoradiography and morphometric techniques. The thickness of the basal lamina increased from days 16 to 18 of gestation. On days 19 and 20 the basal lamina was found to be significantly thicker in the intercryptal areas than at the base of the crypt. This difference disappeared at 21 days gestation. It was also demonstrated that cell migration and renewal occur in the same manner in the foetal colon as in the adult colon. These features do not resemble closely those seen in neoplastic invasion.

Differential Effects of Epidermal Growth Factor and Hydrocortisone in Human Fetal Colon

The influence of epidermal growth factor (EGF) and hydrocortisone on the functional development of human fetal colon was studied in organ cultures. Fetal colon (14 to 17 weeks gestation) was cultured for 5 days at 37°C in serum‐free Leibovitz L‐15 medium alone or supplemented with 1, 10, and 100 ng of EGF/ml or with 50 ng of hydrocortisone/ml of culture medium. The overall morphology of the colonic explants was not altered by the hormonal addition. In the continuous presence of EGF (1, 10, and 100 ng/ml) for 5 days, a significant decrease of [3H]thymidine incorporation into DNA was observed. At the brush border level, the addition of EGF induced a significant drop in sucrase, maltase, and alkaline phos‐phatase activities. These enzymic modifications occurred between the third and fifth day of culture, whereas variation in DNA synthesis was already evident within 24 h. The addition of hydrocortisone at a dose affecting the small intestine (50 ng/ml) did not significantly influence colonic DNA synthesis nor the digestive enzymic activities. These observations show for the first time that EGF, but not hydrocortisone, influences the proliferation and differentiation of human fetal colonic mucosa.

Differential Effects of Epidermal Growth Factor and Hydrocortisone in Human Fetal Colon

The influence of epidermal growth factor (EGF) and hydrocortisone on the functional development of human fetal colon was studied in organ cultures. Fetal colon (14 to 17 weeks gestation) was cultured for 5 days at 37 degrees C in serum-free Leibovitz L-15 medium alone or supplemented with 1, 10, and 100 ng of EGF/ml or with 50 ng of hydrocortisone/ml of culture medium. The overall morphology of the colonic explants was not altered by the hormonal addition. In the continuous presence of EGF (1, 10, and 100 ng/ml) for 5 days, a significant decrease of [3H]thymidine incorporation into DNA was observed. At the brush border level, the addition of EGF induced a significant drop in sucrase, maltase, and alkaline phosphatase activities. These enzymic modifications occurred between the third and fifth day of culture, whereas variation in DNA synthesis was already evident within 24 h. The addition of hydrocortisone at a dose affecting the small intestine (50 ng/ml) did not significantly influence colonic DNA synthesis nor the digestive enzymic activities. These observations show for the first time that EGF, but not hydrocortisone, influences the proliferation and differentiation of human fetal colonic mucosa.

T CELL-MEDIATED DAMAGE IN THE HUMAN COLON IN ORGAN CULTURE A NEW IN-VITRO MODEL OF COLITIS

Immune mechanisms may be important in the pathogenesis of colonic inflammation To test this hypothesis, an organ-culture model was used to study the effects of T cell activation in human colonic mucosa.Lamina propria T cells in expant cultures of human fetal colon (16-20 weeks gestation, n=10) were activated in-situ using poke-weed mitogen or anti-CD atibody, and compared with controls.After 3 days of culture, immunohistochemistry demonstrated a 2-4 fold increase in crypt epithelial cell proliferation in T cell stimulated explants compared with controls; this was associated with a decrease in crypt goblet cell numbers of up to 20 fold. After 5 days, the surface epithelium in stimulated explants appeared flttened with severe loss of goblet cells, and by day 7, severe mucosal damage was observed under both light and electron microscopy. A fall in crypt goblet cells without hyperplasia had occurred in control cultures by day 7; however, the epithelium remained intact, and the degree of mucous depletion, and the rate at which it occurred, were less than in stimulated explants. Goblet cell depletion, crypt hyperplasia, and epithelial damage are common histological findings in colonic inflammation (particularly ulcerative colitis) These experiments suggest that T cells may have an important role in the pathogenesis of colitis. The model provides a new system whereby the mechanisms of T cell-mediated damage in the human colon can be studied.

Structural and enzymatic changes during colonic maturation in the fetal and suckling rat

To assess correlations between cellular differentiation and enzymatic maturation in the developing rat colon, tissue from fetal, suckling, weanling, and adult rats was analyzed by electron microscopy and assayed for lactase, alkaline phosphatase, and sodium-potassium-stimulated adenosine triphosphatase activities. The proximal and distal colon were analyzed independently at all ages. All three enzymes were detected in the fetal colon when the cells were highly undifferentiated. Postnatally, significant regional differences in cellular ultrastructure appeared, only some of which were directly paralleled by enzymatic changes. Each enzyme had a distinct region-specific developmental pattern. Lactase and sodium-potassium-stimulated adenosine triphosphatase were significantly enhanced at birth, decreasing to adult levels by 15 days postnatal. Regional differences were present, but the patterns were similar. These patterns did not parallel the increase in microvillar height and number and basolateral interdigitations of the surface columnar cells, the structural correlates of lactase, and sodium-potassium-stimulated adenosine triphosphatase, respectively. In contrast, developmental changes in alkaline phosphatase activity paralleled structural maturation, at least in part. The activity levels in the distal colon did not change significantly with age and few major structural changes were noted. In the proximal colon, activity increased markedly after birth, and after 10 days decreased rapidly to adult levels, a pattern that coincided with the transient appearance of villi and specialized cells with apical tubules and vesicles known to have alkaline phosphatase activity. The results show age- and region-related changes in cellular ultrastructure and enzymatic activities, only some of which appear to be directly correlated.

Reactivity of a human monoclonal antibody against carcinomas and other lesions of the colon.

To identify tumor-associated antigens that may be immunogenic to man, human monoclonal antibodies (human mAb) were generated by fusing nonsecreting mouse myeloma cells with lymphocytes from regional mesenteric nodes of patients with adenocarcinomas of the colon. One IgG1 human mAb, designated as 14-31-10, was identified by its reactivity against human tumor xenografts. We have studied the reactivity of mAb 14-31-10 with formalin-fixed, paraffin-embedded specimens of human colon. A total of 86 cases were studied, including normal adult and fetal colons, adenocarcinomas of the colon, and a variety of colonic inflammatory diseases and preneoplastic lesions. Intense reactivity was found in 15 of 18 adenocarcinomas of the colon, but not in 10 specimens of normal adult or 4 specimens of fetal colonic mucosa. Interestingly, in four cases of carcinoma, reactivity was also observed in histologically normal mucosa situated 10 cm or more from the primary lesion. On the other hand, no staining was detected in any of the 16 inflammatory lesions. Of the 38 preneoplastic lesions, only 6 showed staining by the mAb: 1 of 5 benign tubular adenomatous polyps, 3 of 9 villous adenomas and tubovillous polyps, 1 of 5 specimens of ulcerative colitis and 1 of 19 specimens of familial polyposis. However, the intensity of staining was only moderate in those cases. Our data, therefore, suggest that the epitope identified by the human mAb 14-31-10 shows preferential expression in preneoplastic and neoplastic lesions of the colon, and in ostensibly normal mucosa at some distance from a primary colonic carcinoma. In all instances, the staining was cytoplasmic, suggesting a cytoplasmic or internal membrane location of the target antigen. This antigen appeared to be distinct from carcinoembryonic antigen, since staining by 14-31-10 was consistently different from that of a mouse monoclonal antibody to carcinoembryonic antigen in serial sections of the same specimens. The restricted reactivity of 14-31-10 suggests its potential application in immunohistochemistry. Moreover, the epitope identified by mAb 14-31-10 may be expressed during the progression of normal mucosa to neoplasia.

Current Technique of Intraperitoneal Transfusion: Do Not Throw Away the Renografin

The original description of intraperitoneal transfusion calls for contrast-dye infusion and X-ray confirmation of placement of the needle free in the peritoneal cavity. Ultrasound methods, using air or saline as contrast media, have been described to replace the X-rays. This report illustrates at least one way in which the ultrasound findings at intraperitoneal transfusion may be illusory, allowing misplaced transfusion into the fetal colon. In specific instances, use of radiopaque dye at intraperitoneal transfusion may still be helpful.

Cell proliferation during morphogenesis of the human colon.

Cell proliferation during morphogenesis of human colon from 8 to 18 weeks of gestation was investigated using radioautography. The sites of [3H]-thymidine uptake were localized in the epithelium, the mesenchyme, and the muscularis externa. As long as the epithelium was stratified, the proliferative pool was very large, and the highest labeling indices (up to 28%) were found during this period. With the formation of villi, the proliferative pool decreased and focused exclusively in the developing crypts by 14-15 weeks. Between 8 and 11 weeks, the labeling index was similar both in the epithelium and the mesenchyme. By 13 weeks of gestation, the small intestinal cell proliferation pattern (epithelium greater than mesenchyme greater than muscular layer) was established in the fetal colon. The biochemical quantitation of the [3H]-thymidine uptake into the total colonic DNA supported the decreasing cell proliferation pattern determined by radioautography. The present investigation establishes basic quantitative data regarding cell proliferation during a particular developmental phase of the human colon.

Expression of O-linked (mucin type) Tn and sialosyl Tn antigens in 22 colorectal carcinomas and fetal colon tissue : N. Harving, T.F. Ørntoft. The Danish Cancer Society, Dep. Exp. Clin. Oncology, Aarhus, DK

Expression of O-linked (mucin type) Tn and sialosyl Tn antigens in 22 colorectal carcinomas and fetal colon tissue : N. Harving, T.F. Ørntoft. The Danish Cancer Society, Dep. Exp. Clin. Oncology, Aarhus, DK

Intestinal mucin antigens in ulcerative colitis and their relationship with malignancy.

The expression of two intestinal mucin-associated antigens large intestine mucin antigen (LIMA) and small intestine mucin antigen (SIMA) were investigated by indirect immunoperoxidase staining of rectal mucosa from patients suffering from ulcerative colitis with (n = 6) and without (n = 31) associated carcinoma and in noncolitic controls (n = 40). The aim was to assess the relationship between antigen patterns and malignant change. SIMA, which is localised predominantly in the small intestine, is virtually undetectable in the normal adult colonic mucosa. However, this antigen is present in the foetal colon and colonic carcinoma. LIMA is expressed in normal colonic mucosa, but absent from the small intestine. LIMA staining patterns were not significantly different among the three groups. In contrast, expression of SIMA was significantly higher in the patients who had developed carcinoma (6/6) than in the noncancer group (7/71) (P less than 0.001). The presence of SIMA was also significantly related to areas of dysplasia compared to normal (P = .03) or inflammation (P less than .05), but it did not differ from mucosa showing "indefinite" atypia. The finding of 31% SIMA-positive biopsies associated with severe inflammation in colitis with active disease, but no evidence of malignancy, is difficult to explain at the present stage. A followup study would be necessary to determine its significance. Perhaps the most important finding is the increased frequency of SIMA-positive foci in histologically normal mucosa in carcinoma patients compared with the noncancer group (P less than .001), suggesting a field change. These observations may be prove useful for the identification of patients who may be at risk of developing carcinoma.

Anatomic evidence for coexistence of cholinergic and adrenergic neurons in the developing human intestine: New aspects in the pathogenesis of developmental neuronal abnormalities

In order to interpret the interrelation of cholinergic and adrenergic myenteric neural elements in the developing human, histochemical methods were used to demonstrate cholinergic and adrenergic activity independently in consecutive cryostat sections of the esophagus, ileocecal region, and colon of fetuses of 9 to 22 weeks ovulation age. At least some of the neural cells remained plastic, with respect to their transmitter choice, and showed both cholinergic and adrenergic function. These cells were shown to be present first in the ileocecal region (11 weeks) then in the esophagus (12 weeks) and lastly in the colon (14 weeks). Our findings support the hypothesis of a dual gradient of maturation of enteric neurons. The choice of transmitters is influenced by a "microenvironmental" factor, which may also arrest the maturation or cause the death of neurons.

Presence and characteristics of epidermal growth factor receptors in human fetal small intestine and colon

In the present study, we demonstrate for the first time the presence of important concentrations of EGF binding sites in isolated epithelial cells of both human fetal small intestine and colon as early as 12 weeks gestation. The pattern of EGF binding in the small intestine between 12 and 17 weeks show that binding was significantly higher (2.5-fold) in younger fetuses than in older fetuses. Moreover, the fetal colon exhibited a much higher binding capacity (1.5–2.5 times) than corresponding intestinal cells for all age groups studied. Analysis of Scatchard representations reveal that the concentration of high- and low-affinity binding sites in colonic epithelial cells are twice the values observed in corresponding intestinal cells. The present data raise interesting possibilities as to the role of this growth factor in human fetal gut development.

The presence and significance of intraepithelial mesenchymal cells in human foetal colon.

During an investigation of the morphogenesis of the human foetal colon, breaks in the basal lamina underlying the surface epithelium were frequently observed at 10 1/2-11 weeks. These occurred at those sites where the mesenchyme was sweeping up into the epithelium prior to the transformation of the epithelium from stratified to a single layer. At the same time numbers of mesenchymal cells appeared among the epithelial cells and some were observed actually in the process of passing through the gaps in the basal lamina. Close contact was apparent between some mesenchymal cells and basal epithelial cells through extended breaks in the basal lamina. Many of the mesenchymal cells within the epithelium contained numbers of apoptotic bodies. This suggests that one of the functions of the intra-epithelial mesenchymal cells is to remove the debris resulting from cell death which occurs in association with the re-arrangement of cells during development of the colon.

Serum- and cell-mediated immune protection of mouse placenta and fetus against a Brucella abortus challenge: Expression of barrier effect of placenta

When mice are intravenously inoculated with a virulent Brucella abortus strain at day 12 to 14 of pregnancy and killed three to five days later, colonization of placentae, fetuses and spleens can be estimated by the frequency and level (bacterial count) of infection and by linkage between individual placental and paired fetal infections. This linkage indicates the placental barrier effect, defined as the number of non-infected fetuses linked to loo colonized placentae. Immune mice serum raised against two Brucella fractions injected one day before challenge (1) restricted the placental colonization (the dose required to infect 50 per cent placentae was increased by so to 70 times compared to controls), (2) decreased the level of splenic and placental infection, and (3) increased the barrier effect so that most fetuses were protected even when linked to a heavily infected placenta. Immune (B + T) spleen cells from mice vaccinated with a Brucella cell-wall fraction transferred to recipients seven to eight days before mating, that is, 22 days before challenge (1) restricted the frequency of placental and fetal colonization, (2) decreased the level of splenic, placental and fetal infections, and (3) increased the barrier effect. However, separated B- and T-cells were less active, in particular on the level of fetal infection. In contrast with serum, the cells did not decrease infection of the fetuses linked to heavily infected placentae.

Human fetal colon in organ culture.

Human fetal colon (14-16 weeks gestation) was cultured as explants for 15 days in serum-free Leibovitz L-15 medium at 37 degrees C. The overall morphology of the colonic explants was well maintained throughout the culture period and all epithelial cell types retained their ultrastructural characteristics. The incorporation of [3H]-leucine continued and even increased, reflecting sustained synthesis of proteins. Even though the incorporation of [3H]-thymidine into the total DNA decreased during culture, the synthesis of DNA continued. The sites of [3H]-thymidine incorporation into the different layers of the colonic wall were studied by radioautography. The incorporation of the radioactive precursor occurs mainly in the epithelium and to lesser degrees in the mesenchyme and the muscular layer. Labeled epithelial nuclei were located in the intervillous areas but not on the villi. The labeling index of the epithelial cells remained constant throughout the culture period indicating the preservation of the proliferative capacity of the epithelium. Brush-border hydrolytic activities, namely those of sucrase, maltase, lactase, trehalase, glucoamylase and alkaline phosphatase, were assayed in the colonic tissue. These enzymic activities generally decreased in the tissue and increased in the medium during the course of culture. These observations clearly demonstrate that fetal colon can be maintained viable for at least 15 days in a serum-free medium. Organ culture now provides the opportunity to study the normal function and metabolism of human colon during its development.

Oncofetal expression of Lex carbohydrate antigens in human colonic adenocarcinomas. Regulation through type 2 core chain synthesis rather than fucosylation.

The mechanism of expression of a series of glycolipid antigens carrying the Lex determinant structure, Gal beta 1----4[Fuc alpha 1----3]GlcNAc beta 1----, and characterized by oncofetal expression in fetal colon and colonic adenocarcinomas has been studied in human fetal and adult proximal colon tissue. Results presented from TLC immunostain analysis of neutral glycolipids isolated from normal adult colonic mucosa have indicated the presence of only barely detectable quantities of both an Lex-active glycolipid that co-migrated with III3V3Fuc2nLc6 and its precursor nLc6. These structures were found in large quantities in glycolipid fractions from human adenocarcinoma tumors and human small cell lung carcinoma NCI-H69 cells. In contrast, type 1 chain-based Lea antigen structures were found in both normal mucosa and adenocarcinomas. Analysis of gangliosides of normal colonic mucosa by TLC immunostain indicated the presence of a series of type 2 chain-based gangliosides; however, sialyl-Lex was not detected. The ability of normal colonic mucosa to synthesize type 2 chain core structures was demonstrated by the presence of a beta 1----4 galactosyltransferase activity with Lc3 as an acceptor in an amount equivalent to 60-65% of the total galactosyltransferase activity. An alpha 1----3 fucosyltransferase was also found to be expressed in significant quantity in adult colonic mucosa. Kinetic studies indicated that this is most probably the alpha 1----3/4 fucosyltransferase suggested to be a product of the Lewis gene (Le). Thus, although normal adult colonic mucosa contained the enzymes to synthesize Lex and sialyl-Lex structures, these antigens were not found. Tissue immunofluorescence studies indicated that type 2 chain precursors and the alpha 1----3/4 fucosyltransferase were found in different cell populations in adult proximal colonic mucosa. However, both type 2 chain core structures and their fucosylated derivatives were found to be associated with epithelial cells of fetal colon. These results indicate that oncofetal expression of Lex antigens in fetal colonic epithelium and in adenocarcinomas but not in normal adult mucosa is due to the retrogenetic expression of type 2 chain precursors which are not found in normal adult colonic epithelial cells.

Differential Distribution of Digestive Enzymes in Isolated Epithelial Cells from Developing Human Fetal Small Intestine and Colon

Summary: The human fetal colon has morphological and biochemical characteristics similar to that of the small intestine during development. A comparative study of these two organs was undertaken by selectively isolating their respective epithelium at different ages of gestation. Histological and biochemical analysis revealed a complete and selective removal of the epithelium from the underlying tissue regardless of their stage of development. Intestinal and colonic epithelial cells showed substantial differences with respect to protein content and distribution. All brush border membrane enzymes studied were present in the colon, although in much lower quantities than in the corresponding intestine. Between 90 and 99% of total disaccharidases. glucoamylase and γ‐glutamyl transpeptidase activities were recovered in the isolated cells. Epithelial content and distribution of alkaline phosphatase (ALP) activity differed markedly, however, between these two tissues. More than 45% of intestinal ALP activity was of nonepithelial origin at 15 weeks, and this percentage increased in a craniocaudal fashion to reach >93% in the 18‐week‐old colon. Inhibition studies using phenylalanine and levamisole showed that these intestinal and colonic isoenzymes are similar in nature although they differ in their respective distribution. The use oi pure fractions of epithelial cells provides an ideal system in which to compare specifically the functional development of intestinal and colonic epithelium.

The production and characterization of monoclonal antibodies to a human colonic antigen associated with ulcerative colitis: cellular localization of the antigen by using the monoclonal antibody.

We detected in human colon extracts a 40 kDa protein(s) that specifically reacts with tissue-bound IgG obtained from the colon of patients with ulcerative colitis or CCA-IgG. Using the hybridoma technology, we developed monoclonal antibodies to this 40 kDa protein. The specific immunoreactivity of one of the monoclonal antibodies (7E12H12, IgM isotype) against the 40 kDa protein was demonstrated both by ELISA and by immunotransblot. Competitive binding experiments showed that CCA-IgG inhibits the binding of 7E12H12 to the 40 kDa protein, suggesting the recognition of common epitope(s) on the 40 kDa protein by the monoclonal antibody and CCA-IgG. 7E12H12 was used to determine cellular localization of the 40 kDa protein. Biopsy tissue specimens from colon, esophagus, stomach, duodenum, jejunum, ileum, liver, pancreas, lungs, kidneys, salivary, and mammary glands were obtained. Tissue specimens were fixed in 4% paraformaldehyde or in 10% formalin. Sections were sequentially incubated with the hybridoma supernatant, biotinylated anti-mouse IgM, avidin-biotin-peroxidase complex, and 3,3'-diaminobenzidine. An unrelated hybridoma supernatant was used as control. The monoclonal antibody exclusively recognized colonic epithelial cells both in the crypt and on the luminal surface. Immunoreactivity was present on the plasma membrane chiefly along the basolateral areas of the cells. Plasma membrane localization of the 40 kDa protein was confirmed by immunoelectron microscopy. All colonic mucosal biopsy specimens from both adult and fetal colon reacted with the monoclonal antibody. None of the biopsy specimens from stomach, duodenum, jejunum, ileum, liver, pancreas, or non-gastrointestinal tissue reacted with the antibody, confirming the organ specificity of the 40 kDa protein. The interaction between this colonic epithelial membrane protein and the CCA-IgG may play an important role in the pathogenesis of ulcerative colitis.

Common characteristics for Na+-dependent sugar transport in Caco-2 cells and human fetal colon.

The recent demonstration that the human colon adenocarcinoma cell line Caco-2 was susceptible to spontaneous enterocytic differentiation led us to consider the question as to whether Caco-2 cells would exhibit sodium-coupled transport of sugars. This problem was investigated using isotopic tracer flux measurements of the nonmetabolizable sugar analog alpha-methylglucoside (AMG). AMG accumulation in confluent monolayers was inhibited to the same extent by sodium replacement, 200 microM phlorizin, 1 mM phloretin, and 25 mM D-glucose, but was not inhibited further in the presence of both phlorizin and phloretin. Kinetic studies were compatible with the presence of both a simple diffusive process and a single, Na+-dependent, phlorizin- and phloretin-sensitive AMG transport system. These results also ruled out any interaction between AMG and a Na+-independent, phloretin-sensitive, facilitated diffusion pathway. The brush-border membrane localization of the Na+-dependent system was inferred from the observations that its functional differentiation was synchronous with the development of brush-border membrane enzyme activities and that phlorizin and phloretin addition 1 hr after initiating sugar transport produced immediate inhibition of AMG uptake as compared to ouabain. Finally, it was shown that brush-border membrane vesicles isolated from the human fetal colonic mucosa do possess a Na+-dependent transport pathway(s) for D-glucose which was inhibited by AMG and both phlorizin and phloretin. Caco-2 cells thus appear as a valuable cell culture model to study the mechanisms involved in the differentiation and regulation of intestinal transport functions.