Intraepithelial gamma delta T cells are closely associated with apoptotic enterocytes in the bovine intestine

The T cell population in the intestinal epithelium, comparable in size to the T cell pool in the spleen, is characterized by the predominant distribution of T cells bearing gamma delta T cell receptors. To determine the functional significance of the intraepithelial lymphocytes, we observed gamma delta T cells present in the jejunal epithelium in cattle, in which there is predominance of gamma delta T cells. Immunohistochemistry of frozen sections demonstrated that gamma delta T lymphocytes were densely distributed in the villous epithelium but there were fewer in the lamina propria and they were not present in the crypt epithelium. Ultrastructurally, intraepithelial gamma delta T cells were characterized by possessing electron-dense granules and interdigitating with enterocyte cytoplasm. Enterocytes, which were inserted by processes of intraepithelial lymphocytes or contacted by their cell bodies, showed morphologic changes seen in apoptotic cell death, such as elevated electron density of the cytoplasm and condensation of the chromatin. Apoptotic cells and cell debris were found in macrophages, which gathered in the subepithelial region of villus tips. These findings suggest that in the small intestine of cattle, gamma delta T cells are involved in the renewal of epithelial cells by inducing apoptosis of epithelial cells.     

Activation and peripheral expansion of murine T-cell receptor gamma delta intraepithelial lymphocytes

BACKGROUND & AIMS: The intestinal epithelial compartment is populated by CD8(+) alpha beta and gamma delta intraepithelial lymphocytes (IELs), which monitor the integrity of the epithelial barrier. alpha beta IELs are activated by peptide antigens presented by class I major histocompatibility complex (MHC) molecules, but it is unclear how gamma delta IELs are activated. METHODS: G8 T-cell receptor (TCR) gamma delta transgenic (Tg) mice (specific for the class I MHC alloantigen, T22/10(b)) were crossed to class I MHC-deficient beta2-microglobulin-knockout (beta2m degrees) mice, and Tg+ IELs were examined for relative yields and surface and functional phenotype. RESULTS: Evidence for class I MHC-induced activation of Tg+ IELs was supported by the detection of 4-fold greater numbers of Tg+ IELs in G8 x beta2m+ mice that proliferated at 15-fold higher levels than IELs from G8 x beta2m degrees mice. However, expression of CD69, production of cytokine (interleukin 2 and interferon gamma), and detection of cytolytic function for IELs in G8 x beta2m degrees mice suggested that class I MHC was not required for gamma delta IEL development or maturation. CONCLUSIONS: These results suggest that CD8(+) TCR gamma delta IELs do not require class I MHC for development but support the notion that antigens presented by class I MHC molecules are involved in the peripheral expansion and differentiation of this subset.     

Apoptotic process in the monkey small intestinal epithelium: I. Association with glutathione level and its efflux

The apoptotic process in the normal gastrointestinal mucosa is of interest due to its possible role in physiological cell renewal. The aim of this study was to identify the apoptotic process in the monkey small intestine and the association of glutathione level and its efflux in this process. Monkey small intestinal epithelial cells were separated in to different fractions consisting of villus, middle and crypt cells. Apoptosis was identified by DNA ladder pattern and Hoechst staining. The level of glutathione, its efflux and the enzymes involved in its metabolism were quantitated in these fractions. Apoptotic cells were identified predominantly in the villus tip cell fractions by both DNA ladder pattern and Hoechst dye staining. Glutathione level was 7 fold higher in the crypt cells as compared to villus tip cells the middle cells showing a gradual decrease. A similar pattern was seen in mitochondrial content of glutathione. As the cells mature from crypt to villus, there is increased efflux of GSH, which may be responsible for the decreased level of GSH in apoptotic villus cells. In the monkey small intestine, apoptotic cells are seen in the villus tip fractions and the glutathione level and its efflux may play a role in this process.     

Apoptotic process in the monkey small intestinal epithelium: 2. Possible role of oxidative stress

Recent findings suggest that intracellular oxidants are involved in the induction of apoptosis and this type of cell death can be inhibited by various antioxidants. In our accompanying paper, we have shown apoptosis in the villus tip cells of the monkey small intestinal epithelium. The aim of the present study was to evaluate the possible relationship between oxidative stress, antioxidant levels and the apoptotic process in the monkey small intestinal epithelium. Monkey small intestinal epithelial cells were isolated into different fractions consisting of villus, middle and crypt cells. Mitochondrial function was assessed by the reduction of the tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), with and without succinate. The extent of lipid peroxidation was assessed by measuring the formation of conjugated diene, depletion of polyunsaturated fatty acids and alpha-tocopherol. Level of antioxidant enzymes like, superoxide dismutase (SOD), catalase, glutathione S-transferase (GST), glutathione peroxidase (GPx) and glutathione reductase were also quantitated in various cell fractions. MTT reduction was significantly decreased in villus cells as compared to the cells from other fractions and this was evident even in presence of the respiratory substrate, succinate. Increased formation of conjugated diene and depletion of polyunsaturated fatty acids were seen in villus and crypt cells as compared to middle fraction cells. The alpha-tocopherol level was decreased in both villus and crypt cells as compared to cells from middle region. Significant decrease of SOD activity was seen in the villus tip cells and a slight decrease was seen in the crypt fractions. Glutathione dependent enzymes like GST, GPx and GSH reductase showed higher activity in the villus fractions. A similar observation was also seen in the catalase activity. This study has shown that although oxidative stress is seen in both villus and crypt cells, decreased mitochondrial function was seen in villus tip cells which may be responsible for apoptotic process in the intestinal epithelium.     

Immunohistochemical characterization of intraepithelial and subepithelial mononuclear cells of the upper airways

The upper airway is the first site of exposure to inhaled antigens and the site of initiation of mucosal immunity to certain antigens; however, the intraepithelial lymphoid populations of this region have not been well characterized. We studied 6-mu frozen tissue sections from tonsils, adenoids, and nasal mucosae using immunohistochemistry and a panel of antibodies to mononuclear antigens to determine whether nasal mucosa contained distinctive populations of mononuclear cells. Intraepithelial lymphocytes (IELs) of nasal mucosa were CD3+, CD8+, and mainly CD5+. Tonsil and adenoid both showed diffuse CD8+ IELs; clusters of CD4+ IELs were associated with B cells within the crypt epithelium. All nasal IELs were uniformly negative for Leu8 (homing receptor analog of Mel14). Scattered Leu8-positive cells were present within tonsil and adenoid crypt epithelium only. Nasal IELs rarely expressed HML1 and were often CD7-, whereas the majority of tonsillar and adenoidal IELs were HML1+ and variably CD7+. In nasal mucosa and in deep submucosa of tonsil and adenoid, 80 to 90% of T cell receptor expression was of alpha/beta type. There was a concentration of gamma/delta T cell receptor-positive cells in intraepithelial and subepithelial zones of tonsil and adenoid, with areas of up to 30% gamma/delta T cell receptor positivity. A population of intraepithelial dendritic cells was identified in all three tissues expressing mononuclear phagocyte system antigens CD14 and KiM1P, but lacking CD1a. Virtually no B cells and no organized subepithelial lymphoid tissue were identified in nasal mucosa. Nasal mucosal lymphoid tissue seems to differ from that of endodermally derived mucosae, tonsil, and adenoids to share similarities with both mucosa-associated lymphoid tissue and peripheral lymph nodes.     

Rat intestinal glycolipids. II. Distribution and biosynthesis of glycolipids and ceramide in villus and crypt cells

Intestinal epithelial cells were isolated from rat intestine and grouped into villus and crypt cell fractions. Glycolipids were purified from each cell fraction and quantitated by fluorimetric determination of glycolipid sphingosine. Significant quantities of ceramide were found in all cell fractions and accounted for approximately 15% of total glycolipid sphingosine. While villus and crypt cell fractions quantitatively contained differing amounts of sphingosine, all cell fractions contained proportionally similar quantities of sphingosine when compared to cellular cholesterol or phospholipid. Individual glycolipids, however, showed significant differences in distribution between villus and crypt cells. Hematoside and glucosylceramide were proportionally increased in villus cells, while crypt cells showed an increase in trihexosylceramide and ceramide content. The rate of UDPglucose : hydroxy fatty acid ceramide glucosyltransferase was higher in villus cells while the rate of UDPgalactose : lactosylceramide galactosyltransferase was 3--4 times increased in crypt cells. These studies demonstrate that significant differences in both the distribution and biosynthesis of individual glycolipids occur in crypt and villus cells of rat intestine and are of possible importance in the process of intestinal cell differentiation.     

The role of IL-7 in thymic and extrathymic development of TCR gamma delta cells

IL-7-deficient (IL-7(-/-)) mice have reduced numbers of B and TCR alpha beta cells, but lack mature TCR gamma delta cells. Although most T cell development occurs in the thymus, some intestinal intraepithelial lymphocytes (IEL), including TCR gamma delta cells, can develop extrathymically. Epithelial cells in both thymus and intestine synthesize IL-7, suggesting that TCR gamma delta cell development could occur in either site. To evaluate the role of thymic IL-7 in development of TCR gamma delta cells, newborn TCR beta-deficient (TCR beta(-/-)) thymi were grafted to IL-7(-/-) mice. Donor- and host-derived TCR gamma delta cells were recovered from thymus grafts, spleen, and IEL. However, when IL-7(-/-) thymi were grafted to TCR beta(-/-) mice, no development of graft-derived TCR gamma delta cells occurred, indicating that extrathymic IL-7 did not support TCR gamma delta IEL generation from newborn thymic precursors. In contrast, TCR gamma delta IEL development occurred efficiently in adult, thymectomized, irradiated C57BL/6J mice reconstituted with IL-7(-/-) bone marrow. This demonstrated that extrathymic development of TCR gamma delta IEL required extrathymic IL-7 production. Thus, intrathymic IL-7 was required for development of thymic TCR gamma delta cells, while peripheral IL-7 was sufficient for development of extrathymic TCR gamma delta IEL.     

The effect of transposition to jejunum on epithelial cell kinetics in an ileal segment

Epithelial cell kinetics were studied in an ileal segment after transposition to proximal jejunum. The number of cells per villus column in the transposed ileum increased after 4--7 days to reach values normal for jejunum after 14--30 days. This increase was accompanied by a simultaneous increase in the number of cells per crypt column up to 130% of values in jejunum and ileum in situ. The percentage of labelled crypt cells, after labelling with 3H-thymidine, and the relative size of the proliferative cell compartment in the crypt in the transposed ileum did not differ from values in the ileum in situ at any time interval after surgery. The total proliferative activity per crypt, which was determined by scintillation counting of isolated crypts after 3H-thymidine labelling, increased two-fold from 7 days after surgery. Cell migration studies showed that the increase in the number of villus cells was probably not caused by a change in the life span of the epithelial cells. It seems that the increase in the number of villus cells in ileal epithelium after transposition to proximal jejunum is brought about by an enlargement of the crypt, while the relative size of the proliferative cell compartment in the crypt remains unchanged.     

Differential effects of IL-2 and IL-15 on the death and survival of activated TCR gamma delta+ intestinal intraepithelial lymphocytes

TCR gamma delta+ cells are enriched in the intestine mucosa and constitute approximately half of the intestinal intraepithelial lymphocytes (iIEL) in mice. They are likely activated by self and foreign Ags in situ, but little is known about how the activated gamma delta iIEL are regulated. In the iIEL compartment, IL-2 is produced by activated TCR alpha beta+ iIEL, and IL-15 message is detected in iIEL and in the epithelial cells. We found surface expression of IL-2 as well as IL-15Rs on activated gamma delta iIEL, and examined the effects of IL-2 and IL-15 on the survival and death of gamma delta iIEL during secondary stimulation through TCR. We found that both cytokines supported growth of the restimulated gamma delta iIEL, but exerted different effects on their survival. A significant higher number of live cells were recovered from the gamma delta iIEL cultures restimulated in IL-15 than in IL-2. Quantitation of apoptotic cells showed more cell death in the IL-2 group than in the IL-15 group. The cell death was associated with restimulation through TCR and was not caused by insufficient growth factor, thus representing activation-induced cell death. Western blot analyses found no difference in the levels of Bcl-2 and Bax proteins between the two groups. However, the level of Bcl-xL protein diminished with time in the IL-2 group whereas the level was sustained in the IL-15 group, which may contribute to the pro-survival effect of IL-15. These results demonstrated that the survival of activated gamma delta iIEL is differentially regulated by IL-2 and IL-15.     

Genetic regulation of enterocyte function: a quantitative in situ hybridisation study of lactase-phlorizin hydrolase and Na(+)-glucose cotransporter mRNAs in rabbit small intestine

The enterocyte undergoes sequential changes in its structure and function as it migrates rapidly from the small intestinal crypts to the villus tip. The mechanisms by which these changes are regulated "in tune" with ontogenic and dietary changes in the luminal environment are currently under investigation. This study has employed oligonucleotide probes to follow the expression of the lactase-phlorizin hydrolase (LPH) and Na(+)-glucose cotransporter (SGLT1) genes in rabbit small intestine using quantitative in situ hybridisation histochemistry. The profiles of LPH mRNA and SGLT1 mRNA accumulation along the crypt-villus axis were found to be very similar. Although mRNA was undetectable in the crypt. LPH and SGLT1 mRNA levels rose rapidly at the crypt-villus junction, reaching a maximum between 210 microns and 330 microns above this point. Further up the villus the level of mRNAs declined. SGLT1 mRNA was present in all small intestinal segments (duodenum, jejunum and ileum), whereas LPH mRNA was absent from the ileum. LPH activity rose and fell in conjunction with mRNA, but SGLT1 activity was greatest at the villus tip where mRNA levels were considerably reduced. These data have been used to discuss the genetic regulation of enterocyte differentiation and function.     

The impact of primary and modular nursing delivery systems on perceptions of caring behavior

BACKGROUND: The somatostatin analogue octreotide impairs intestinal regeneration and the adaptive response to intestinal resection by inhibition of enterocyte migration and proliferation and increased apoptosis. Epidermal growth factor (EGF) stimulates regeneration and adaptation by increasing proliferation and reducing apoptosis. The aim of this study was to determine the effect of EGF on octreotide-induced enterocyte apoptosis. METHODS: Twenty-four rabbits underwent patch enteroplasty in the distal ileum to stimulate the mucosa. There were four study groups: octreotide 250 microgram/kg/day, EGF 40 microgram/kg/day, EGF plus octreotide, and control. Normal ileal mucosa adjacent to the patch was evaluated at 7 days for villus height, crypt depth, crypt cell production rate (CCPR), and in situ end labeling of DNA fragmentation. RESULTS: Octreotide alone increased apoptosis compared with controls at the villus tip (40 +/- 7% vs 18 +/- 7%, P < 0.05), lateral villus (9 +/- 2% vs 3 +/- 2%, P < 0.05), and crypt (15 +/- 3% vs 10 +/- 3%, P < 0. 05). EGF decreased apoptosis in the crypt (2 +/- 1%) and villus (6 +/- 1% villus tip and 1 +/- 1% lateral villus, P < 0.05) compartments. EGF inhibited octreotide-induced apoptosis in the crypt (5 +/- 2%) but not the villus (31 +/- 5% villus tip and 6 +/- 2% lateral villus, P < 0.05). Mean DNA fragmentation was significantly greater in octreotide-treated animals (P < 0.05). The octreotide-treated animals had reduced crypt depth and villus height but normal CCPR compared with controls. EGF increased CCPR and crypt depth compared with controls. Combining EGF and octreotide resulted in crypt depth and CCPR similar to those of controls but reduced villus height. CONCLUSIONS: EGF inhibits octreotide-induced apoptosis. This effect is greater in crypt than in villus enterocytes. Octreotide appears to have both direct and indirect effects on enterocyte apoptosis.     

In vitro polarographic studies of rat brain oxygen uptake in simulated uraemia and hepatic coma

The villi and crypts of the small intestine of the albino rat (Rattus norvigicus) have been enumerated and measured at different ages from 2 weeks to 8 months. The number of villi increased up to the third month of age and the number of crypts up to the eighth month. In the proximal intestine, the mean length of the villus base increased up to the fifth month as ridge-shaped villi were formed. Villus height was greater proximally than distally and this, and the crypt depth, remained constant from the end of the first month of age. The total villus circumference per unit area of intestine, and the villus circumference per crypt was the same proximally and distally, and was relatively constant after the first month of age. The total circumference of the crypt mouths per square millimeter of intestine was the same proximally and distally and, at all ages, was greater than the total villus circumference. The villus surface area per square millimeter of intestine, or per crypt, remained relatively constant after the first month and was greater proximally than distally, due only to the taller villi proximally. The change from finger-shaped to ridge-shaped villi did not affect the villus mucosal surface area. The changes in villus shape are probably not determined by differences in the rate of crypt cell production.     

SNS Na+ channel expression increases in dorsal root ganglion neurons in the carrageenan inflammatory pain model

In contrast to conventional T cells, natural killer (NK) 1.1+ T cell receptor (TCR)-alpha/beta+ (NK1+T) cells, NK cells, and intestinal intraepithelial lymphocytes (IELs) bearing CD8-alpha/alpha chains constitutively express the interleukin (IL)-2 receptor (R)beta/15Rbeta chain. Recent studies have indicated that IL-2Rbeta/15Rbeta chain is required for the development of these lymphocyte subsets, outlining the importance of IL-15. In this study, we investigated the development of these lymphocyte subsets in interferon regulatory factor 1-deficient (IRF-1-/-) mice. Surprisingly, all of these lymphocyte subsets were severely reduced in IRF-1-/- mice. Within CD8-alpha/alpha+ intestinal IEL subset, TCR-gamma/delta+ cells and TCR-alpha/beta+ cells were equally affected by IRF gene disruption. In contrast to intestinal TCR-gamma/delta+ cells, thymic TCR-gamma/delta+ cells developed normally in IRF-1-/- mice. Northern blot analysis further revealed that the induction of IL-15 messenger RNA was impaired in IRF-1-/- bone marrow cells, and the recovery of these lymphocyte subsets was observed when IRF-1-/- cells were cultured with IL-15 in vitro. These data indicate that IRF-1 regulates IL-15 gene expression, which may control the development of NK1+T cells, NK cells, and CD8-alpha/alpha+ IELs.     

Scanning electron microscopy of mouse intestinal mucosa after cobalt 60 and D-T neutron irradiation

The stem-cell population of the intestinal crypt is an important model system in experimental radiobiology. Standardized techniques have been developed to allow quantitation of the response of crypt cells to radiation injury following doses of 0-2 krad of D-T neutrons or 60Co gamma rays. These techniques rely on the identification of regenerating crypt cells three-and-a-half days after irradiation. The results are expressed as the number of regenerating crypts per circumference of small intestine, as determined by conventional histological examination; the more profound the injury, the smaller the crypt count. The practical relevance of crypt-counting techniques to clinical radiotherapy is limited by their relative insensitivity; the dose levels commonly used in fractionated radiotherapy produce no detectable response. Scanning electron microscopy of the mucosal surface provides a more sensitive measure of radiation injury. The earliest detectable changes occur at the level of 300 rad of gamma radiation, well below the threshold of the crypt-counting technique. At around 1,000 rad, where the first drop in crypt counts occurs, there are well-marked morphological changes which become more severe with increasing dose levels. Some differences have been observed between the morphological effects of gamma and neutron irradiation at points of radiobiological equivalence in terms of crypt counts (using an RBE value of about 2). The changes observed may reflect more than the disruption of epithelial cell kinetics. Mucosal morphology is the total expression of many different biological parameters of which the regenerative ability of the crypt cells is only one. The surface microanatomy of the gut may be the most sensitive indicator of radiation injury which is conveniently available for study.     

Morphological and molecular processes of polyp formation in Apc(delta716) knockout mice

Mutations in the human adenomatous polyposis coli (APC) gene are responsible for not only familial adenomatous polyposis but also many sporadic cancers of the digestive tract. Using homologous recombination in embryonic stem cells, we recently constructed Apc gene knockout mice that contained a truncation mutation at codon 716 (Apc(delta716)). The heterozygous mice developed numerous intestinal polyps. All microadenomas dissected from nascent polyps had already lost the wild-type allele, indicating the loss of heterozygosity (M. Oshima et al., Proc. Natl. Acad. Sci. USA, 92: 4482-4486, 1995). We also demonstrated that cyclooxygenase 2 is induced in the polyps at an early stage and plays a key role in polyp development (M. Oshima et al., Cell 87: 803-809, 1996). We have analyzed the process of polyp development in these mice both at morphological and molecular levels. A small intestinal microadenoma is initiated as an outpocketing pouch in a single crypt and develops into the inner (lacteal) side of a neighboring villus forming a double-layer nascent polyp. The microadenoma then enlarges and gets folded inside the villus. When it fills the intravillous space, it expands downward and extends into adjoining villi, rather than rupturing into the intestinal lumen. During this course of development, the basement membrane remains intact, and the labeling index of the microadenoma cells is similar to that of the normal crypt epithelium. As in the crypt cells, neither transforming growth factor beta1 nor its receptor type II is expressed in the microadenoma cells. No hot spot mutations in the K-ras gene are found in the microadenoma tissue during these early stages of polyp development. Essentially, the same results have been obtained for the colonic polyps as well. These results suggest that early adenomas in the Apc(delta716) polyps are very similar to the normal proliferating cells of the crypt except for the lack of directed migration along the crypt-villus axis.     

Cytokine gene expression in intestine of rat during the postnatal developmental period: increased IL-1 expression at weaning

In the present study we have investigate whether cytokines are constitutively and differently expressed in intestine during the differentiative processes that take place at weaning. We have analyzed the expression of IL-1 beta, IL-2, IL-4 and IFN gamma by polymerase chain reaction in Peyer's patches (PP) and in intestine deprived of PP (I-PP) of rats from 16 to 30 days of age. The results showed a constitutive and marked expression of the cytokines already before weaning, with the exception of IL-2 in PP and IFN gamma in I-PP. IL-beta was the only cytokine to show a different expression at various ages with an initial increase at 19 days and a further elevation at 21 days when intestinal epithelium passes through major differentiative stages, suggesting an involvement of this cytokine in intestinal development. We have also tested whether treatment of rats with the immunosuppressor cyclosporin A (CsA) could affect intestinal differentiation. The results showed that only some markers of differentiation were affected (proliferation of staminal crypt cells and length of crypts). This was probably due to a direct effect rather than an immunomediated effect of CsA, since treatment of three intestinal cell lines (Caco-2, HT-29, FRIC) with CsA indicated that this drug can exert a cytostatic activity on intestinal cells.     

The regulation and functional consequence of proinflammatory cytokine binding on human intestinal epithelial cells

Products of an activated immune system may affect cells within the immune system as well as nonlymphoid cells in the local environment. Given the immunologically activated state of the intestinal tract, it is conceivable that locally produced cytokines could regulate epithelial cell function. To assess whether epithelial cells are targets for particular cytokines, we initiated studies on the binding of a panel of proinflammatory cytokines in freshly isolated epithelial cells from normal and inflammatory bowel disease (IBD) patients as well as in cell lines. Isolated intestinal epithelial cells (IEC) were stained with phycoerythrin-conjugated or biotinylated cytokines to determine the expression and density of receptors for IL-1beta, IL-6, granulocyte-macrophage CSF (GM-CSF), and TNF-alpha. Receptors for IL-1beta, IL-6, and GM-CSF were readily detectable in all epithelial cell preparations at levels equal to (GM-CSFR) or lower than those seen on monocytes. However TNFalpha-R were not detectable on freshly isolated IECs. Receptor density was greater in surface vs crypt epithelial cells, but no significant differences were seen between normal and IBD epithelial cells. Expression of IL-1R and IL-6R was enhanced by LPS and IFN-gamma. Functionally, IL-1beta enhanced proliferation of the IEC cell line, DLD1, whereas GM-CSF treatment of de-differentiated crypt-like DLD1 and HT29 cells resulted in enhanced expression of ICAM-1. Furthermore, TNF-alpha treatment enhanced the secretion of IL-8 and GRO-alpha in HT29 cells, but not in freshly isolated IEC cultures. The differential binding and function of proinflammatory cytokines on IEC support the hypothesis that these cytokines may be involved in normal physiological processes as well as in regulating mucosal immune responses.     

Paneth cell differentiation in the developing intestine of normal and transgenic mice

Paneth cells represent one of the four major epithelial lineages in the mouse small intestine. It is the only lineage that migrates downward from the stem-cell zone located in the lower portion of the crypt of Lieberkühn to the crypt base. Mature Paneth cells release growth factors, digestive enzymes, and antimicrobial peptides from their apical secretory granules. Some of these factors may affect the crypt stem cell, its transit-cell descendants, differentiating villus-associated epithelial lineages, and/or the gut microflora. We used single and multilabel immunocytochemical methods to study Paneth cell differentiation during and after completion of gut morphogenesis in normal, gnotobiotic, and transgenic mice as well as in intestinal isografts. This lineage emerges coincident with cytodifferentiation of the fetal small intestinal endoderm, formation of crypts from an intervillus epithelium, and establishment of a stem-cell hierarchy. The initial differentiation program involves sequential expression of cryptdins, a phospholipase A2 (enhancing factor), and lysozyme. A dramatic increase in Paneth cell number per crypt occurs during postnatal days 14-28, when crypts proliferate by fission. Accumulation of fucosylated and sialylated glycoconjugates during this period represents the final evolution of the lineage's differentiation program. Establishment of this lineage is not dependent upon instructive interactions from the microflora. Transgenic mice containing nucleotides -6500 to +34 of the Paneth cell-specific mouse cryptdin 2 gene linked to the human growth hormone gene beginning at its nucleotide +3 inappropriately express human growth hormone in a large population of proliferating and nonproliferating cells in the intervillus epithelium up to postnatal day 5. Transgene expression subsequently becomes restricted to the Paneth cell lineage in the developing crypt. Cryptdin 2 nucleotides -6500 to +34 should be a useful marker of crypt morphogenesis and a valuable tool for conducting gain-of-function or loss-of-function experiments in Paneth cells.     

Suppression of preneoplastic changes in the intestine of rats fed low levels of polyamines

Administration for 7 days of an enteral diet that is naturally deficient in polyamines strikingly reduces the preneoplastic changes observed in the intestines of adult Wistar rats previously treated with the carcinogen 1,2-dimethylhydrazine. On the contrary, supplementing the enteral diet with spermidine favors preneoplastic development. The effects of the low-polyamine diet included a 40% decline in the putrescine content of the intestinal mucosa, a significant decrease in the turnover rate of the epithelial cells from the crypts to villus tip in the ileum, and a 2-fold reduction in the number of abnormal colonic crypts. The experimental data support the view that it might be of interest to control the dietary intake of polyamines in the clinical management of cancer patients.