Human Large Bowel Research Articles

Human intestinal development in a severe-combined immunodeficient xenograft model

The present work describes a severe-combined immunodeficient murine xenograft model used to investigate human gastrointestinal ontogenesis. Specifically, the study has tested whether carefully selected regions of human fetal gut are able to undergo region-specific morphogenesis and epithelial cytodifferentiation when transplanted subcutaneously into immunodeficient mice. In addition, double-label in situ hybridisation techniques, utilising specific human and mouse DNA probes, have been adopted to characterise host and donor cell types and to investigate the potential developmental roles for non-epithelial cells in the regulation of epithelial differentiation pathways in vivo. Human fetal small and large bowel developed to form a characteristic mucosa 10 weeks after transplantation, which displayed clear region-specific structural and functional gradients. The initial phase of xenograft epithelialisation closely resembled the stratified type of epithelium which is present during early fetal gastrointestinal development. Idiosyncratic epithelial differentiation pathways were recorded during xenograft regeneration, with an absence of Paneth cells and an abundance of enteroendocrine cells when compared with developed xenograft and paediatric intestine. Such differences may, therefore, be important in ensuring rapid and region-specific development in the absence of conventional luminal stimuli and hormonal changes that occur normally during pregnancy. In situ hybridisation demonstrated an exclusively human origin for the intestinal xenograft epithelium and muscularis mucosa and externa. Although the submucosa and lamina propria were comprised of a chimeric mixture, murine cells were rarely seen to contact with the epithelium, which interacted primarily with human myofibroblasts and human intraepithelial lymphocytes. It is proposed that a ‘selection’ process operates to maintain species-specific cellular interactions, and this mechanism may subsequently play an important role in regulating epithelial cell differentiation, orchestrated in part by juxtaposed non-epithelial cell types.

Expression of plasminogen activator inhibitor type 1 (PAI-1) by HT-29di human large bowel carcinoma cells is modulated as a function of epithelial differentiation

Highly differentiated epithelial populations (36% mucin-producing cells; sixfold increase in alkaline phosphatase activity; development of flat, substrate-adherent, entero-cytic foci) were induced upon in vitro exposure of HT-29di human colon carcinoma cells to sodium butyrate (NaB). 1,25-dihydroxyvitamin D 3 [1,25-(OH) 2D 3] (10 −7 M) and the ionophore A23187 (0.5 μM) significantly augmented (two to threefold) NaB-induced HT-29di differentiation, whereas 1,25-(OH) 2D 3 or A23187 alone were not effective. Induction reflected specific changes in protein abundance, involving, most notably, a differentiation-associated increase in the expression and substrate-deposition of a 47-kDa protein with pI/mw two-dimensional map coordinates and immunochemical properties identical to that of plasminogen activator inhibitor type 1 (PAI-1), a major regulator of the pericellular proteolytic cascade. Culture of HT-29di cells in medium of either high (2.5 mM) or low (0.25 mM) Ca 2+ concentration did not affect the incidence of ‘spontaneous’ differentiation, although NaB-induced goblet cell and enterocytic maturation was Ca 2+-dependent. The inability of 1,25-(OH) 2D 3, A23187 or modulated Ca 2+ levels alone (i.e., in the absence of NaB) to effect differentiation of HT-29di cells and the Ca 2+-dependence of the NaB response indicate that NaB and Ca 2+ act co-operatively to induce colonic epithelial maturation in vitro.

Sodium butyrate induces apoptosis in human colonic tumour cell lines in a p53-independent pathway: implications for the possible role of dietary fibre in the prevention of large-bowel cancer.

The purpose of this study was to determine whether cultured colonic adenoma and carcinoma cells undergo apoptosis (programmed cell death) in vitro and whether specific growth and dietary factors, thought to be involved in the control of growth and differentiation of human colonic cells, could induce cell death through apoptosis. In cell lines originating from 6 colorectal adenomas and 7 carcinomas, spontaneous apoptosis was observed. Sodium butyrate, a naturally occurring fatty acid, is present in the human large bowel in millimolar amounts as a result of bacterial fermentation of dietary fibre. Sodium butyrate, at physiological concentrations, induced apoptosis in 2 adenoma cell lines, RG/C2 and AA/Cl, and in the carcinoma cell line PC/JW/FI. In contrast, transforming growth factor beta 1, which is thought to have an important role in the control of growth in colonic epithelium, did not induce apoptosis. Neither RG/C2 nor PC/JW/FI contain wild-type p53, therefore this tumour-suppressor gene is not required to mediate signals for the induction of apoptosis in colonic tumour cells. Our studies report the induction of apoptosis in colonic tumour cells by the naturally occurring fatty acid sodium butyrate. Since sodium butyrate is produced by bacterial fermentation of dietary fibre, the observation that this fatty acid can induce apoptosis could, in part, explain why a high-fibre diet appears to be protective against colon cancer. Escape from the induction of programmed cell death may be an important event in colorectal carcinogenesis.

Immunohistochemical study of NCAM expression in the developing human large bowel

Immunohistochemical study of NCAM expression in the developing human large bowel

Proliferation in human gastrointestinal epithelium using bromodeoxyuridine in vivo: data for different sites, proximity to a tumour, and polyposis coli.

The distribution of DNA synthesising cells in the crypts of the epithelium in human small and large bowel after injection of bromodeoxyuridine into patients has been studied in relation to the position of the cells in the crypt using immunohistochemistry. Different sites of normal epithelium have been studied. The ileum has a shorter crypt and a very significantly smaller total cell population size. However, it has similar peak labelling index (LI) values to the colon, while the rectum has a lower peak LI value. The mean position of the label occurs at the 17th cell position in the ileum and at about the 22nd position in both the colon and rectum. The overall mean LI is significantly higher in the ileum at 17.8%, intermediate in the colon at 10.3%, and lowest in the rectum at 8.5%. There is thus an inverse relation between the likelihood of developing a tumour and the rate of cell proliferation as measured by the LI. Assuming a value of 8.6 hours for the duration of S, the data suggest that the cell cycle time in the mid crypt region is about 30 hours for the ileum and colon and about 37 hours for the rectum. Samples taken adjacent (within 1 cm) to a tumour show a general dampening of proliferative activity at all cell positions compared with samples taken more than 5 cm from a tumour. This is illustrated by the average LI, which is about 5.4% in the colon adjacent to a tumour compared with 10% distant; comparable values for the rectum are 4.6% and 8.5%. Samples taken from two patients with polyposis coli show distributions with a significant difference in skewness compared with normal colon and a general shifting of the distribution to the right, that is to higher cell positions. There is a significant increase in the mean cell position and the position of the peak LI in the polyposis coli samples.

Measurement of in vivo proliferation in human colorectal mucosa using bromodeoxyuridine.

In vivo bromodeoxyuridine (BrdUrd) labelling of the human large bowel was performed and a detailed histochemical localisation of label in sections of crypts was undertaken using a monoclonal antibody to BrdUrd containing DNA. Flow cytometric studies on extracted nuclei were also performed (data presented elsewhere). The average crypt in the human large bowel (excluding the rectum) was 82 cells in height and 41 cells in circumference, with a total of about 2000 cells (assuming a topographical correction factor of 0.6). Ten per cent of the cells were replicating their DNA--that is, were in the S phase of the cell cycle--and 0.4% were in mitosis. The median position for the labelling index versus cell position frequency plot is at the 20th cell position--at a quarter of the crypt height. The lower and upper limits of the cell proliferation are given by the 5th and 95th percentiles at cell positions 4 and 43 respectively. The peak labelling index is about 30% and it occurs at cell position 15. The labelling index at the crypt base, the probable stem cell zone, is about 14%, suggesting that these cells have a longer cell cycle. Taking a value of 8.6 hours for the duration of the S phase (deduced from the flow cytometric data) and assuming a growth fraction of 1.0 for the mid-crypt, these data provide an estimate of about 30 hours for the cell cycle time. The rectal crypts are about the same size but contain about 30% fewer S phase cells. The data also yielded a per cent BrdUrd labelled mitosis curve.

IgG subclasscontaining cells in the human large bowel of normal controls, non-IBD colitis, and ulcerative colitis

IgG subclass-containing cells in colonic mucosa were examined in three groups; 1) normal controls 2) cases of ulcerative colitis (UC) 3) cases of colitis excluding UC and Crohn's disease (non-IBD colitis) by indirect immunoperoxidase staining method using mouse anti-IgG subclass monoclonal antibodies. The numbers (and proportions) of IgG1, IgG2, IgG3, and IgG4-containing cells in normal colonic mucosa was 80 +/- 29/mm2 (44.6%), 44 +/- 21 (24.1%), 44 +/- 24 (23.7%), 13 +/- 10 (7.7%), respectively. The proportion of IgG subclass-containing cells in normal colonic mucosa was different from the known proportion of IgG subclass in serum. In UC, the numbers of all IgG subclasses-containing cells were significantly increased compared to controls and non-IBD colitis. However, only IgG1-containing cells were increased in proportion (50.3%) compared to normal controls. In non-IBD colitis, the numbers of IgG1- and IgG2-containing cells were increased compared to the controls, but the increases were less than UC, and there was no difference in the proportion of IgG subclass compared to normal controls. The differences in the numbers and in the proportions of IgG subclass-containing cells between UC and non-IBD colitis may reflect differences in the underlying disease process.

Cimetropium bromide: in vitro and in vivo evaluation of spasmolytic activity on human and dog colon.

The present study investigates the spasmolytic properties of cimetropium bromide, compared to atropine, on human and canine large bowel. The drug behaved as a competitive antagonist of muscarinic-mediated contractions in isolated colonic preparations from both species, with affinity values (pA2) ranging between 7.41 and 7.82. When administered intravenously to conscious dogs provided with a colonic Thiry fistula, cimetropium was a potent inhibitor of large bowel motility evoked by both exogenous and endogenous stimuli. The compound (10-100 micrograms/kg) counteracted colonic motor response to neostigmine administration with an ID50 of 27.9 micrograms/kg; both tonic and phasic components of contractile response were affected. In a comparable range of doses (3-100 micrograms/kg), the drug inhibited motor activity elicited by intraluminal distension.

Quantitative electron microscopic study on grades of atypia in adenomas of the human large bowel.

Clarification of the ultrastructural features of the three different grades of histologic atypia of adenomas (i.e., mild, moderate, and severe atypia) with reference to carcinoma of the human colon and rectum was undertaken using electron microscopic quantitative analysis. Ten normal epithelia, 36 adenomas (14 with mild atypia, 13 with moderate atypia, and 9 with severe atypia) and 14 carcinomas (well-differentiated adenocarcinoma) were examined with light and electron microscopes. Although it has been generally accepted that malignant transformation of an adenoma gradually proceeds with increasing atypia, using light microscopy, ultrastructural quantitative analysis clearly revealed that fine features of individual atypia did not correspond with histologic grades of atypia. Ultrastructural changes occurred abruptly in moderate atypia and showed similar cellular characteristics to carcinoma cells. Mild atypia was almost similar in appearance to normal epithelial cells. Striking changes in the moderate atypic cells were plane apposition of the cell membrane, decrease of the desmosomes, and increase of the lysosomes. These atypical cells were assumed to play a significant role in the adenoma-carcinoma sequence. Therefore, radical surgical therapy would be required for moderate atypic adenoma of the large bowel that is difficult to remove endoscopically.

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.

Ring-like distribution of IgG-containing cells around the lymph follicle in the human intestine.

Immunoglobulin (Ig)-containing cells were studied in the lamina propria of the normal human large bowel and around the lymph follicle, including Peyer's patches, in the normal intestine. Ig-containing cells were identified by the indirect immunoperoxidase staining method, using mouse anti-human Ig monoclonal antibodies. In the lamina propria in the large bowel, the mean percentage of IgA+ (IgA1+ cells and IgA2+ cells), IgM+, IgD+, IgG+ and IgE+ cells was 75.9, 8.5, 7.3, 5.8, 2.5, respectively (total 100), namely there was a marked preponderance of IgA+ cells in comparison to IgG+ cells. However, IgG+ cells were observed not only on the epithelial but also in the serosal side of lymph follicles, showing a ring-like pattern. Ig+ cells of the other four classes did not show such a pattern. The ring-like distribution of IgG+ cells around lymph follicles was observed in both the large and small intestine including Peyer's patches. This tendency was observed in 9 out of 14 follicles (64.3 per cent). A large number of IgG+ cells were observed outside lymph follicles, while a small number of IgG+ cells were observed at the most outer rim of lymph follicles which suggested a local maturation of IgG+ cells. The significance and the role of the newly recognized IgG+ cells in the vicinity of lymph follicles remain to be resolved.

Pharmacokinetics and anti-tumour activity of LM985 in mice bearing transplantable adenocarcinomas of the colon.

LM985 is one of a series of compounds based on the flavone ring structure and selected for clinical trial primarily for its activity in colon 38 as part of the NCI screen. We have investigated the anti-tumour activity against three differing transplantable adenocarcinomas of the mouse colon (MAC). Single i.p. injection at maximum tolerated dose showed no activity against the ascitic tumour MAC 15A, moderate activity against subcutaneous tumours MAC 13 and MAC 15A and produced a significant growth delay against MAC 26. These responses against s.c. tumours were considerably enhanced by repeated injection 7 days later when greater than 90% tumour inhibition was seen in MAC 13 and cures were achieved in MAC 26. Pharmacokinetic studies confirm the rapid degradation of LM985 to LM975, the possible active principle. Analysis of area under the curve for LM975 indicated a good relationship with administered doses of LM985 and tumour responses. The MAC system shows a good correlation with human large bowel cancer and these preliminary observations with LM985 would suggest that it or its metabolite LM975 may have a value in the management of large bowel cancer.

Lectin reactivity in human large bowel

The affinity of six lectins (Arachis hypogaea, Concanavalin A, Bandeiraea simplicifolia I. Dolichus biflorus, Triticum vulgaris and Lotus tetragonolobus) was studied in 10 normal human large intestines. The results showed that each lectin had its own characteristic staining pattern. They differed in their affinity for different cellular components, local distribution within crypts and regional distribution between right (ascending colon) and left (rectum) segments of the large bowel. Arachis hypogaea stained predominantly the supranuclear golgi cisternae and Concanavalin A the cytoplasm. Triticum vulgaris stained preferentially the mucin of the well differentiated cells of the upper crypt while Lotus tetragonolobus characteristically stained the glycocalyx and apical portion of the less differentiated basal cells of the lower crypt. Goblet-cell mucin of the ascending colon had greater affinity for Bandeiraea simplicifolia I and that of the rectum for Triticum vulgaris. Lotus tetragonolobus stained more frequently and extensively the goblet-cell mucin of the upper crypts of the ascending colon than that of the rectum. The goblet cell mucins of the upper crypts were consistently stained in both the right and left segments of the large bowel by Dolichus biflorus while the mucins of the less differentiated cells of the lower crypts were less consistently stained in the ascending colon.

A comparison of ornithine decarboxylase and S-adenosylmethione decarboxylase activity in human large bowel mucosa, polyps, and colorectal adenocarcinoma

A comparison of ornithine decarboxylase and S-adenosylmethione decarboxylase activity in human large bowel mucosa, polyps, and colorectal adenocarcinoma

Definite spontaneous cell-mediated cytotoxicity and HNK-1 cells in the human large intestine.

Spontaneous cell-mediated cytotoxicity (SCMC) and the marker of natural killer (NK) cells mediating SCMC of the human large intestine were studied. Lamina proprial lymphoid cells (LPL) were isolated by sequential dithiothreitol-EDTA-collagenase treatment of the gut specimen. SCMC was measured by the chromium release method. Target cells included P4788 in monolayer, a cell line derived from colon cancer, Chang cells in monolayer, and K562 in suspension. Target cells in monolayer including colon cancer cell line were chosen because they were thought to be more appropriate to assess SCMC for lymphoid cells in the solid organ. While lower compared to cytotoxicities (CT) by peripheral blood lymphoid cells (PBL), define CT were observed in LPL against all three targets. NK cells marker was studied both on LPL by an indirect fluorescent antibody method and on the gut tissue by indirect immunoperoxidase staining using anti HNK-1 monoclonal antibody which defines virtually all NK cells. HNK-1 positive (HNK-1 +) cells were identified in both methods. HNK-1 + cells were observed in the epithelium, lamina propria, and lymph follicle with or without germinal centers. These results clearly demonstrated the presence of SCMC and HNK-1 + cells in the human large bowel.

Nerve mediated responses to drugs and electrical stimulation in aganglionic muscle segments in Hirschsprung's disease

The activity of isolated muscle strips from normal and aganglionic human large bowel was studied in vitro. The intrinsic nerves were stimulated electrically and by nicotinic agonists. The ganglionic preparations displayed a strong inhibitory response due to the release of both norepinephrine and a noncholinergic, nonadrenergic inhibitory neurotransmitter. In the aganglionic strips (obtained from patients with Hirschsprung's disease), nerve activation tended to evoke contraction, apparently due to enhancement in the release of acetylcholine. At the same time, the release of norepinephrine appeared to be less than normal. A particularly interesting finding in the aganglionic muscle strips was the presence of a substantial inhibitory response due to the release of a noncholinergic, nonadrenergic substance. These results provide further evidence for the importance of the innervation of the aganglionic segment in Hirschsprung's disease.

Bacterial fermentation in the human large bowel. Time to change from the roughage model of dietary fibre?

Bacterial fermentation in the human large bowel. Time to change from the roughage model of dietary fibre?

Preservation of cytometric DNA distribution and epithelial marker expression after tumor progression of human large bowel carcinomas.

Tumor specimens were obtained from seven patients with large bowel carcinomas at operation of the primary neoplasm and by resection of local recurrences or metastases 2 1/2 to 36 months later. All specimens were evaluated with regard to nuclear DNA distribution as measured by flow cytometry and expression of carcinoembryonic antigen (CEA), secretory component, epithelial IgA, and HLA-DR antigens, as determined by immunofluorescence staining of tissue sections. Both the DNA distributions and the immunohistochemical staining patterns were similar in the primary and secondary tumors. These findings are in keeping with a monoclonal or oligoclonal tumor progression in advanced large bowel carcinoma.

Isolation and culture of colonic epithelial cells in serum-free medium

Techniques are described for the dissociation, fractionation through Percoll, and in vitro maintenance of mucosal epithelia from the human or guinea pig large bowel. Tissue recovered after surgery is predigested with trypsin-citrate and treated subsequently with a mixture of trypsin, citrate, and collagenase. The resulting suspension of single cells, cell clusters, and partially digested crypts is suspended over a Percoll solution and enriched in multicellular elements by two sequential centrifugations. The recovered multicellular complexes are inoculated to specially treated culture vessels in a serum-free medium supplemented with epidermal growth factor, insulin, transferrin, selenium, and bovine pituitary extract. Epithelia, characterized as such by transmission electron microscopy, adhere to the substrate, form colonies, and can be maintained routinely for study for at least 10 wk.

Chromosomal banding patterns in human large bowel adenomas.

Chromosome studies were performed on direct preparations of 38 benign human large bowel adenomas by fluorescent banding techniques. Five adenomas had a normal diploid karyotype line. Six adenomas had numerical abnormalities, and seven polypoid lesions exhibited structural and numerical changes. In 20 adenomas without analyzable metaphases, it was presumed that the cells were dividing at a lower rate. Mosaics were observed in 25% of cases. The most common finding was trisomy of 7, 8, or 13.