Showing papers on "Crypt published in 1980"

Response of the rat small-intestine epithelium to methotrexate.

Abstract: We studied jejunal epithelial structure and function in rats 24, 48, 96, and 192 hours after a single intravenous injection of methotrexate (MTX) 30 mg/kg. The acute effect of the drug on the gut at 24 and 48 hours was characterised, as expected, by reduced mitoses in crypts, shortened villi, and depressed activity of thymidine kinase (an enzyme normally confined to intestinal crypt cells). At 96 hours, when MTX was no longer detectable in serum, the intestine had entered a proliferative phase characterised by increased crypt mitoses, accelerated migration of enterocytes along villi, and the presence on villi of epithelial cells with the enzyme profile of crypt cells, decreased disaccharidase, alkaline phosphatase, and Na+-K+ATPase activities and increased thymidine kinase activity. Although the enzyme data suggested that enterocyte maturation was defective during this proliferative phase, glucose-stimulated Na+ transport, normally a function of fully differentiated villus cells, was normal at 96 hours. Measured both in Ussing chambers and in suspensions of enterocytes isolated from villi, Na+ transport responded normally to glucose at 96 hours, although the response had been significantly depressed at 24 hours. These findings cannot be attributed to MTX-induced malnutrition, as all comparisons included pair-fed controls. We conclude that, in the MTX-induced malnutrition, as all comparisons included pair-fed controls. We conclude that, in the small intestine under conditions of altered epithelial renewal, some components of enterocyte function may be affected more than others. Comparing the present experimental model with another intestinal disorder, acute viral enteritis, in which proliferative activity is excessive, it is clear that the nature of the original intestinal injury is a significant determinant of the pattern of enterocyte response.

Cell proliferation at different sites along the length of the rat colon.

Abstract: This study has been undertaken in order to compare in detail cell proliferation in the mucosal crypts at several sites along the length of the large bowel of the rat. The techniques which have been used include simple morphometry, calculation of mitotic and tritiated thymidine labelling indices, metaphase arrest with vincristine, and the fraction of labelled mitoses method. Major differences exist in the size and shape of the crypts at different sites. In particular the distribution of the proliferating cells within the crypt varies. Mean cell cycle time ranges from 58 h in the descending colon to 25 h in the caecum; this variation appears to be brought about largely by changes in the duration of G1, the other phase durations remaining relatively constant. There is also variation in cell cycle time and growth fraction at different levels within the crypt; throughout the bowel cells appear to cycle more slowly at the bottom of the crypt, but changes in growth fraction do not display a similarly consistent pattern. Clearly the organisation of cell proliferation in the normal rat colon is very complex, and strict definition of anatomical location is required in any study of cell proliferation whether in normal or in diseased animals.

Intestinal calcium-binding protein. A protein indicator of enterocyte maturation associated with the terminal web.

Abstract: The vitamin D-dependent calcium-binding protein (CaBP) was studied in relation to the age of the cell, in isolated epithelial cell populations removed from rat duodenum. Alkaline phosphatase and thymidine kinase activities were used as markers to characterize differentiated villus cells and undifferentiated (mitotically active) crypt cells, respectively. CaBP distribution along the length of the villus, as established by radioimmunoassay, appears as a gradient increasing from the crypt to the tip of the villus. CaBP concentration in cells is shown to be (i) negatively correlated with the thymidine kinase activity of cells, and (ii) positively correlated with the alkaline phosphatase activity of cells. This indicates that CaBP is absent in crypt cells and appears in differentiated cells with the development of the brush border. Thus CaBP, like alkaline phosphatase, can be considered as an indicator of enterocyte maturation. These data were also confirmed by studying the cellular localization of the protein. In addition both indirect immunofluorescence and immunoperoxidase staining methods reveal that antibody against CaBP decorates the terminal web, but not the microvilli of the brush border of mature absorptive cells. The results suggest that CaBP may act as a modulator of some Ca2+-mediated biochemical processes at the level of the enterocyte brush border.

Surface-membrane biogenesis in rat intestinal epithelial cells at different stages of maturation.

Abstract: The biosynthesis of membrane proteins and glycoproteins has been studied in rat intestinal crypt and villus cells by measuring the incorporation of L-[5,6-3H] fucose, D-[2-3H] mannose and L-[3,4,5-3H] leucine, given intraperitoneally, into Golgi, lateral-basal and luminal membranes. Incorporation of leucine and mannose was approximately equal in crypt and villus cells, whereas fucose incorporation was markedly higher (3-4 times) in the differentiated villus cells. As previously reported [Quaroni, Kirsch & Weiser (1979) Biochem J. 182. 203-212] most of the fucosylated glyco-proteins synthesized in the villus cells and initially present in the Golgi and lateral-basal membranes were found re-distributed, within 3-4h of label administration, in the luminal membrane. A similar process appeared to occur in the crypt cells, where, however, only few fucose-labelled glycoproteins were identified. In contrast, most of the leucine-labelled and many mannose-labelled membrane components found in the lateral-basal membrane of both crypt and villus cells did not seen to undergo a similar re-distribution process. The fucosylated glycoproteins of the intestinal epithelial cells represent, therefore, a special class of membrane components, most of which appear with differentiation, that are selectively localized in the luminal portion of the plasmalemma. In contrast with the marked differences in protein and glycoprotein patterns between the luminal membrane of villus and crypt cells, only minor differences were found between their lateral-basal membrane components: their protein patterns on sodium dodecyl sulphate/polyacrylamide slab gels, and the patterns of fucose-, mannose- and leucine-labelled components (analysed 3-4h after label administration) were very similar. Although the minor differences detected may be of importance, it appears that most of the surface-membrane changes accompanying cell differentiation in the intestinal epithelial cells are localized in the luminal portion of their surface membrane.

The effect of a single injection of cytosine arabinoside on cell population kinetics in the mouse jejunal crypt

Abstract: The early effects of a single injection of cytosine arabinoside (ara-C) on cell population kinetics in the jejunal crypt of the mouse were studied using autoradiography with tritiated thymidine, and metaphase arrest with vincristine. Ara-C had three main effects on crypt cells: a block of cells near the transition from G1 toS, death of nearly all cells inS, and a temporary block of the survivors, which remained viable and were able to proceed through the cell cycle. Throughout the crypt there was a decrease in cell cycle time and an increase in growth fraction. Although changes in proliferative rate were highest in the lowest part of the crypt it was not possible to show that crypt repopulation originated only from basal crypt cells, and the data are consistent with repopulation from the faster cycling cells in the proliferative compartment.

Cytodifferentiation of the rat Paneth cell: an immunocytochemical investigation in suckling and weanling animals.

Abstract: The cytodifferentiation of the intestinal Paneth cell was investigated in 2- to 50-day-old rats by evaluating the phenotypic expression of bacteriolytic lysozyme. Samples of duodenum, jejunum, and ileum were exposed to anti-lysozyme antiserum and treated by the periodic-acid Schiff (PAS) technique for the demonstration of carbohydrate moieties. The distribution of the Paneth cell population was then assessed. At days 3-6, Paneth cells were present in ≤ 1% of intestinal crypts and exhibited (a) location in the bases of crypts, (b) apical granules positive for lysozyme, and (c) a slender truncated shape extending to the crypt lumen. By days 6-8, Paneth cells had acquired their typical broad, truncated shape and prominent secretory apparatus. Between 1 and 7 weeks postpartum, the frequency of Paneth cellcontaining crypts in duodenum rose from 9% to 79%; in jejunum and ileum the frequency increased through the third week of life and stabilized at 77%–93% thereafter. At all ages, a rostro-caudal gradient of crypts containing Paneth cells was observed, and greater numbers of paneth cells per crypt were seen caudally. In both suckling and weanling animals, two intermediate cell types were encountered. The first predominated at crypt bases through the fourth week of life and contained lysozyme-positive supranuclear granules, concomitant with an apical aggregation of PAS-positive material. The second type, identical to goblet cells in size and shape, predominated along the walls of intestinal crypts and at the bases of villi, and manifested both lysozyme-positive granules and PAS-positive mucigen droplets in the apical cytoplasm. This cell type appeared by day 12 and persisted through day 50. The results of this study provide further evidence that Paneth cells differentiate from a subpopulation of multipotent stem cells residing at crypt bases. The observation of Paneth cell-goblet cell transitional forms supports the view that these two cell types share a common lineage.

Characterization of isolated villus and crypt cells from the small intestine of the adult mouse

Abstract: Suspensions of sequentially isolated villus and crypt cells were obtained in order to study certain biochemical changes associated with differentiation of epithelial cells in the small intestine of the mouse. Microscopic observation of the various cell fractions reveals that the epithelial cells detach as individual cells or small sheets of epithelium from the tip to the base of the villus, whereas cells in the crypt regions are separated as entire crypt units. The isolated cells retain their ultrastructural integrity as judged by electron microscopy. Chemical characterization of the various fractions shows that the total cellular protein content, expressed in activity per cell, remains relatively constant throughout the villus region followed by a noticeable drop in the crypt zone. On the other hand, sharp variations in values of cell DNA content are observed in the crypt zone depending on the reference of activity being used. Activity profiles of several brush border enzymes confirm the biochemical changes that occur during the migration of cells from the crypt to the villus tip, as observed in other species, with maximum activity of sucrase in the mid-villus region, of glucoamylase, trehalase, lactase and maltase in the upper third region, and of alkaline phosphatase at the villus tip. Forty-eight-hour suspension cultures of cell fractions corresponding to cells at the base of the villus and crypt zones show a moderate decrease in protein and enzyme activities to approximately 70% of their original value, with DNA content remaining stable throughout the incubation period. The use of biochemical activities as indicators of cellular integrity during cell culture is discussed.

Increased mucosal damage during parasite infection in mice fed an elemental diet.

Abstract: We have examined the effects of parasite infection on the mucosal architecture of mice maintained on an elemental diet (Vivonex). Techniques used were conventional histology, micro-dissection and measurement of individual villi and crypts, and measurement of crypt cell proliferation rate by a metaphase accumulation technique. In normal, non-parasitised mice the elemental diet caused no change in villus height, crypt depth, or crypt cell proliferation. Likewise, the only effects of chronic protozoal infection or Nippostrongylus brasiliensis infection on the intestine of mice fed a normal diet have been a slight crypt hypertrophy and an increase in crypt cell proliferation rate without villous atrophy. However, the combination of elemental diet and parasite infection resulted in increased mucosal damage when compared with infected mice on a normal diet. Elemental diet mice infected with the nematode Nippostrongylus brasiliensis had significantly reduced villus height and correspondingly raised crypt length and metaphase accumulation rate. Elemental diet mice infected with the protozoan Giardia muris did not have villous atrophy but there was a significant increase in crypt length and metaphase accumulation when compared with infected normal diet mice. These experiments show that in two animal models of enteric infection, elemental diet has altered the host parasite relationship to the detriment of the host.

Amino acid and hexose transport by cultured crypt cells from rat small intestine.

Abstract: The characteristics of amino acid and sugar transport in intestinal crypt epithelial cells have been examined by measuring substrate uptake in an established epithelial cell line. These cells (IEC-6 cells) have been characterized as derived from rat small intestinal crypt cells on the basis of morphological criteria (J. Cell. Biol. 80: 248-265, 1979). Amino acid transport appeared to be mediated by both Na+-dependent and Na+-independent systems. Hexose uptake was stereospecific and Na+ independent, and was markedly inhibited by phloretin and cytochalasin B. Since glucocorticoids are known to have profound effects on maturation of the intestinal epithelium in vivo, their effects on transport properties of the cultured crypt cells were studied. Hydrocortisone, while completely inhibiting cell growth, increased the initial uptake rates of various hexoses, while having little or nor effect on the initial rate of amino acid uptake. The increased hexose uptake appeared to be due to a change in Vmax rather than Km. Appearance of the Na+-dependent hexose transport system, which is present in differentiated enterocytes, was not elicited by in vitro treatment with glucocortcoids.

Influence of restricted diet on the cell cycle in the crypt of mouse small intestine.

Abstract: Previously we reported that the small intestine of mice adapted to dietary restriction had low mitotic acitivty in the crypts. In that case there was no marked difference in the villus and crypt cell numbers, but the migrating speed from crypt to villus decreased, consequently the transit time was prolonged. The present study was undertaken in order to clarify whether or not the cell cycle of the proliferative cells in the crypts alters when the mitotic activity decreases. The results obtained in this study indicate that: 1) There are differences in cell cycle and labeling index between the unrestricted animals and restricted animals. 2) The cycle time of the duodenal and jejunal cells, particularly of G1 phase was prolonged under dietary restriction. 3) The prolongation of cycle time was not found in the ileum. It is thought that the proliferative cell number principally controls mitotic acitivity in the crypts. Still, reduction of the proliferative cell number will be accounted for by the increase of resting cells in G1 phase.

Pattern of crypt cell proliferation in the pre- and post-closure ileum of the neonatal rat: effects of sympathectomy.

Abstract: The present experiments were designed to investigate ileal crypt cell division before and after neonatal closure to macromolecular absorption by measurements of mitotic index, labelling index, colchicine-induced estimation of mitotic rate (mitoses/cell/hour) and crypt depth and villus height on postnatal days 15–23. In addition, the effects of the sympathetic nervous system on crypt cell proliferation were analyzed by guanethidine-induced sympathectomy. Guanethidine treatment resulted in at least a 70 % reduction in superior cervical and coeliac perikarya at 15 days after birth. All cell proliferative indices demonstrated a rather constant rate of cell division in the ileal epithelium between 15 and 17 days after birth with a sudden burst of mitotic activity on day 18. The mitotic rate of control rats increased from day 18 to 23 with 3-week-old rat demonstrating a faster rate of ileal crypt cell proliferation than adult rats. The ileal crypt depth more than doubled between 15 and 23 days while the height of the villus column increased slowly but steadily (36 %) during the period of this study. Cell division is inhibited by guanethidine-induced sympathectomy although there is still an acceleration of mitosis in the post-closure period. The relationship of sympathectomy and ileal crypt cell proliferation is discussed and compared to hormonal effects on closure and related developmental events.

Changes in the synthesis of ribosomal ribonucleic acid and of poly(A)-containing ribonucleic acid during the differentiation of intestinal epithelial cells in the rat and in the chick.

Abstract: Epithelial cells were isolated from rat and chick small intestine by techniques which separated subpopulations of differentiating villus and upper crypt cells from each other and from populations of mitotically dividing lower crypt cells. Incorporation of precursors into epithelial-cell DNA, cytoplasmic rRNA and cytoplasmic poly(A)-containing RNA occurred in the lower crypt cells in vivo when precursor was supplied from the vascular system of the intestine. Incorporation of precursor into 28S and 18S rRNA continued in the upper crypt cells, but occurred to only a very slight extent (if at all) in villus cells, whereas incorporation into poly(A)-containing RNA continued (at a diminishing rate) as the differentiating cells migrated along the villi. When precursor was supplied from the intestinal lumen, its incorporation into DNA and into rRNA of crypt cells was not very different from that observed with the other mode of precursor administration, but incorporation into villus-cell poly(A)-containing RNA then occurred at essentially the same rate in all intestinal epithelial cells in vivo. Cytoplasmic poly(A)-containing RNA appeared to turn over in rat crypt cells with a half-life not exceeding 24 h; crypt-cell rRNA showed no turnover and no evidence could be found for the presence of 'metabolic DNA'.

Study of the kinetics of globule leucocytes in the intestinal epithelium of rats after single or double infection with Trichinella spiralis.

Abstract: In a previous study we suggested that intestinal globule leucocytes (GL) in the mouse represent a cell population independent of the intestinal mast cells (IMC). In this paper we examined the interdependence of IMC and GL in the rat and also the kinetics of GL in the intestinal epithelium. For this purpose rats received a single or double infection with the nematode Trichinella spiralis. After the double infection worms were rapidly expelled. The numbers of IMC and GL were related to the degree of infection. IMC were always observed evenly distributed through the stroma of the villus. During infection, however, GL were shown to shift from a predominantly infra-nuclear position in the crypt of Lieberkuhn and the base of the villus, to a supra-nuclear position in the base and mid-villus region. The data were interpreted as evidence for active migration of GL through the epithelial lining. The fact the GL were first observed in the crypt and basal villus region and the consecutive kinetics could be interpreted as evidence for the independence of GL from IMC. On the other hand a preferential migration of degranulated IMC to the crypt and basal villus region cannot be excluded.

The combined effects of hydroxyurea and X irradiation on murine duodenal mucosa and host survival.

Abstract: The duodenal response to x irradiation, in terms of both crypt survival (D/sub 0/ and 10-clone dose) and host toxicity (LD/sub 50/6/), has been evaluated as a function of time of x irradiation after either a single injection of hydroxyurea (3 mg/g body wt) or five injections (0.5 mg/g followed at 1-h invervals by four injections of 0.25 mg/g each). The data indicate that the five-injection protocol is more effective than a single injection at accumulating proliferating crypt cells at the G/sub 1//S interface and that the initial cohort of S-phase cells after the hydroxyurea release is more radioresistant than the exponentially distributed population of proliferating crypt cells (D/sub 0/ approx. = 220 rad vs control D/sub 0/ approx. = 150 rad). The duodenal crypt cells x-irradiated 1 h after the last hydroxyurea injection (both protocols), when they are still blocked at the G/sub 1//S interface, are also more radiosensitive (D/sub 0/ approx. = 100 rad) than control crypt cells. This is apparently due to a chemical radiosensitization by the hydroxyurea still present in the serum of the mice. A comparison of the 10-clone dose to the LD/sub 50/6/ dose indicates that there is an uncoupling of crypt survival frommore » the so-called clinical endpoint of host toxicity in such a combined-modality protocol. This suggests that, when a systemic cell-cycle phase-specific agent is used in combination with whole-abdomen irradiation, crypt survival per se is only one important variable contributing to the classical gastrointestinal death syndrome.« less

Changes of immunoreactive sucrase-isomaltase complex in rat small intestine during postnatal development and maturation along the villus-crypt axis.

Abstract: Appearance of immunoreactive sucrase-isomaltase complex was observed in rat small intestine during postnatal development and maturation along the villus-crypt axis by single radial immunodiffusion. The immunoreactive sucrase-isomaltase complex in brush-border mem brane increased in parallel with enzyme activities until weaning. After weaning, higher amounts of the immunoreactive enzyme proteins were found as compared with their activities. On the other hand, during cell maturation in adult rat jejunum, the immunoreactive enzyme proteins increased with the activities of sucrase-isomaltase complex. However, a significant amount of the immunoreactive enzyme proteins was observed in the crypt cells with low enzyme activities. Chromatographic profiles on Sephadex G-200 column of the sucrase-isomaltase complex in the upper villus and crypt cells did not change. From these results, it is suggested that the appearance of sucrase and isomaltase activities until weaning is ascribed to the synthesis of an active sucrase-isomaltase complex or the synthesis of an inactive proenzyme followed by rapid conversion to active enzyme, and during cell maturation, it is caused by the synthesis of the inactive proenzyme in the crypt cells followed by its activation in the villus cells.

Surface characteristics of cultured adult rat colon using scanning electron microscopy.

Abstract: The surface characteristics of cultured distal colonic mudosa from adult male Fischer 344 rats have been studied under the scanning electron microscope (SEM). Colonic mucosa, physically separated from the submucosa and muscle, was cultured on a matrix of human fibrin foam. The luminal surface of uncultured colonic mucosa formed repeating circular units of cells around individual crypt openings. After 2 days in organ culture, this normal arrangement of surface epithelium was lost. Circular arrangement of cells surrounding the crypts reappeared after 7 days in culture but did not show the well-demarcated crypt units. By 14 days in vitro fewer crypt units were seen; many of these showed a slightly convex contour. Organ cultures retained a variable number of glandular crypts after 21 to 28 days. Occasional epithelial cells were found attached to the interstitial surfaces of the fibrin foam. The organ culture system described appears to be applicable to experimental investigations into the direct effects of various substances on adult colonic epithelium.