Cystic fibrosis growth retardation is not correlated with loss of Cftr in the intestinal epithelium

Maldigestion due to exocrine pancreatic insufficiency leads to intestinal malabsorption and consequent malnutrition, a mechanism proposed to cause growth retardation associated with cystic fibrosis (CF). However, although enzyme replacement therapy combined with increased caloric intake improves weight gain, the effect on stature is not significant, suggesting that growth retardation has a more complex etiology. Mouse models of CF support this, since these animals do not experience exocrine pancreatic insufficiency yet are growth impaired. Cftr absence from the intestinal epithelium has been suggested as a primary source of growth retardation in CF mice, a concept we directly tested by generating mouse models with Cftr selectively inactivated or restored in intestinal epithelium. The relationship between growth and functional characteristics of the intestines, including transepithelial electrophysiology, incidence of intestinal obstruction, and histopathology, were assessed. Absence of Cftr exclusively from intestinal epithelium resulted in loss of cAMP-stimulated short-circuit current, goblet cell hyperplasia, and occurrence of intestinal obstructions but only slight and transient impaired growth. In contrast, specifically restoring Cftr to the intestinal epithelium resulted in restoration of ion transport and completely protected against obstruction and histopathological anomalies, but growth was indistinguishable from CF mice. These results indicate that absence of Cftr in the intestinal epithelium is an important contributor to the intestinal obstruction phenotype in CF but does not correlate with the observed growth reduction in CF.     

Functional Cftr in crypt epithelium of organotypic enteroid cultures from murine small intestine

Physiological studies of intact crypt epithelium have been limited by problems of accessibility in vivo and dedifferentiation in standard primary culture. Investigations of murine intestinal stem cells have recently yielded a primary intestinal culture in three-dimensional gel suspension that recapitulates crypt structure and epithelial differentiation (Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, Van Es JH, Abo A, Kujala P, Peters PJ, Clevers H. Nature 459: 262-265, 2009). We investigated the utility of murine intestinal crypt cultures (termed "enteroids") for physiological studies of crypt epithelium by focusing on the transport activity of the cystic fibrosis transmembrane conductance regulator Cftr. Enteroids had multiple crypts with well-differentiated goblet and Paneth cells that degranulated on exposure to the muscarinic agonist carbachol. Modified growth medium provided a crypt proliferation rate, as measured by 5-ethynyl-2'-deoxyuridine labeling, which was similar to proliferation in vivo. Immunoblots demonstrated equivalent Cftr expression in comparisons of freshly isolated crypts with primary and passage 1 enteroids. Apparent enteroid differences in mRNA expression of other transporters were primarily associated with villous epithelial contamination of freshly isolated crypts. Microelectrode analysis revealed cAMP-stimulated membrane depolarization in enteroid epithelium from wild-type (WT) but not Cftr knockout (KO) mice. Morphological and microfluorimetric studies, respectively, demonstrated Cftr-dependent cell shrinkage and lower intracellular pH in WT enteroid epithelium in contrast to Cftr KO epithelium or WT epithelium treated with Cftr inhibitor 172. We conclude that crypt epithelium of murine enteroids exhibit Cftr expression and activity that recapitulates crypt epithelium in vivo. Enteroids provide a primary culture model that is suitable for physiological studies of regenerating crypt epithelium.     

Mapping of genetic factors influencing the weight of cystic fibrosis knockout mice

One of the poorly understood clinical manifestations of cystic fibrosis (CF) is low body weight. Mice in which the CF causative gene, cystic fibrosis transmembrane conductance regulator (Cftr), has been knocked out reflect this as they are smaller than age-matched littermates. The variable weight of F2 Cftr -/- (CF) mice derived from a cross between congenic C57BL/6J and BALB/cJ Cftr heterozygotic mice permits the mapping of modifiers of this cystic fibrosis phenotype. In this report, quantitative trait loci (QTL) mapping was used to identify the chromosomal locations of genes that contribute to the body weight of 12-week-old F2 CF mice. Five loci of CF body weight were detected with four of the five acting in a sex-specific manner. Significant linkage of the phenotype to a region of Chromosome (Chr) 13 from D13Mit179 to D13Mit254 (LOD = 4.2) was established in female mice; and suggestive loci on Chrs 7 and 10 were identified. The weights of F2 male CF mice were suggestively linked to regions of Chrs 1 and 6, and to the same locus on Chr 7 as in female mice. The suggestive loci did not influence the weight of the limited set of control mice and thus are presumed to be CF specific in their effects. Further study of these putative CF body weight modifiers may provide insight on the pathogenesis of cystic fibrosis.     

Generation of a conditional null allele for Cftr in mice

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes a cAMP-regulated chloride channel that is important in controlling the exchange of fluid and electrolytes across epithelial cells. Mutation of CFTR can lead to cystic fibrosis (CF), the most common lethal genetic disease in Caucasians. CF is a systemic illness with multiple organ systems affected including pulmonary, gastrointestinal, pancreatic, immune, endocrine, and reproductive systems. To understand the role of CFTR in the various tissues in which it is expressed, we generated a murine conditional null allele of Cftr (Cftr(fl10)) in which loxP sites were inserted around exon 10 of the Cftr gene. The Cftr(fl10) allele was validated by generating constitutive Cftr null (Cftr(Delta10)) mice using the protamine-cre system. The Cftr(Delta10/Delta10) mice displayed almost identical phenotypes to previously published CF mouse models, including poor growth, decreased survival, intestinal obstruction, and loss of Cftr function as assessed by electrophysiology measurements on gut and nasal epithelium. Mice containing the conditional null Cftr allele will be useful in future studies to understand the role of Cftr in specific tissues and developmental time points and lead to a better understanding of CF disease.     

Increased apoptosis and accelerated epithelial migration following inhibition of hedgehog signaling in adaptive small bowel postresection

The intestinal epithelium undergoes a marked adaptive response following loss of functional small bowel surface area characterized by increased crypt cell proliferation and increased enterocyte migration from crypt to villus tip, resulting in villus hyperplasia and enhanced nutrient absorption. Hedgehog (Hh) signaling plays a critical role in regulating epithelial-mesenchymal interactions during morphogenesis of the embryonic intestine. Our previous studies showed that blocking Hh signaling in neonatal mice results in increased small intestinal epithelial crypt cell proliferation and altered enterocyte fat absorption and morphology. Hh family members are also expressed in the adult intestine, but their role in the mature small bowel is unclear. With the use of a model of intestinal adaptation following partial small bowel resection, the role of Hh signaling in the adult gut was examined by determining the effects of blocking Hh signaling on the regenerative response following loss of functional surface area. Hh-inactivating monoclonal antibodies or control antibodies were administered to mice that sustained a 50% intestinal resection. mRNA analyses of the preoperative ileum by quantitative real-time PCR revealed that Indian hedgehog was the most abundant Hh family member. The Hh receptor Patched was more abundant than Patched 2. Analyses of downstream targets of Hh signaling demonstrated that Gli3 was twofold more abundant than Gli1 and Gli2 and that bone morphogenetic protein (BMP)2 was most highly expressed compared with BMP1, -4, and -7. Following intestinal resection, the expression of Hh, Patched, Gli, and most BMP genes was markedly downregulated in the remnant ileum, and, in anti-Hh antibody-treated mice, expression of Patched 2 and Gli 1 was further suppressed. In Hh antibody-treated mice following resection, the enterocyte migration rate from crypt to villus tip was increased, and by 2 wk postoperation, apoptosis was increased in the adaptive gut. However, crypt cell proliferation, villus height, and crypt depth were not augmented. These data indicate that Hh signaling plays a role in adult gut epithelial homeostasis by regulating epithelial cell migration from crypt to villus tip and by enhancing apoptosis.     

Targeted intestinal overexpression of the immediate early gene tis7 in transgenic mice increases triglyceride absorption and adiposity

Following loss of functional small bowel surface area due to surgical resection, the remnant gut undergoes an adaptive response characterized by increased crypt cell proliferation and enhanced villus height and crypt depth, resulting in augmented intestinal nutrient absorptive capacity. Previous studies showed that expression of the immediate early gene tis7 is markedly up-regulated in intestinal enterocytes during the adaptive response. To study its role in the enterocyte, transgenic mice were generated that specifically overexpress TIS7 in the gut. Nucleotides -596 to +21 of the rat liver fatty acid-binding protein promoter were used to direct abundant overexpression of TIS7 into small intestinal upper crypt and villus enterocytes. TIS7 transgenic mice had increased total body adiposity and decreased lean muscle mass compared with normal littermates. Oxygen consumption levels, body weight, surface area, and small bowel weight were decreased. On a high fat diet, transgenic mice exhibited a more rapid and proportionately greater gain in body weight with persistently elevated total body adiposity and increased hepatic fat accumulation. Bolus fat feeding resulted in a greater increase in serum triglyceride levels and an accelerated appearance of enterocytic, lamina propria, and hepatic fat. Changes in fat homeostasis were linked to increased expression of genes involved in enterocytic triglyceride metabolism and changes in growth with decreased insulin-like growth factor-1 expression. Thus, TIS7 overexpression in the intestine altered growth, metabolic rate, adiposity, and intestinal triglyceride absorption. These results suggest that TIS7 is a unique mediator of nutrient absorptive and metabolic adaptation following gut resection.     

Alanyl-glutamine promotes intestinal epithelial cell homeostasis in vitro and in a murine model of weanling undernutrition

Alanyl-glutamine (Ala-Gln) has recently been shown to enhance catch-up growth and gut integrity in undernourished children from Northeast Brazil. We hypothesized that the intestinal epithelial effects of Ala-Gln in malnourished weanling mice and mouse small intestinal epithelial (MSIE) cells would include modulation of barrier function, proliferation, and apoptosis. Dams of 10-day-old suckling C57BL/6 pups were randomized to a standard diet or an isocaloric Northeast Brazil "regional basic diet," moderately deficient in protein, fat, and minerals. Upon weaning to their dam's diet on day of life 21, pups were randomized to Ala-Gln solution or water. At 6 wk of age, mice were killed, and jejunal tissue was collected for morphology, immunohistochemistry, and Ussing chamber analysis of transmucosal resistance and permeability. Proliferation of MSIE cells in the presence or absence of Ala-Gln was measured by MTS and bromodeoxyuridine assays. MSIE apoptosis was assessed by annexin and 7-amino-actinomycin D staining. Pups of regional basic diet-fed dams exhibited failure to thrive. Jejunal specimens from undernourished weanlings showed decreased villous height and crypt depth, decreased transmucosal resistance, increased permeability to FITC-dextran, increased claudin-3 expression, and decreased epithelial proliferation and increased epithelial apoptosis (as measured by bromodeoxyuridine and cleaved caspase-3 staining, respectively). Undernourished weanlings supplemented with Ala-Gln showed improvements in weight velocity, villous height, crypt depth, transmucosal resistance, and epithelial proliferation/apoptosis compared with unsupplemented controls. Similarly, Ala-Gln increased proliferation and reduced apoptosis in MSIE cells. In summary, Ala-Gln promotes intestinal epithelial homeostasis in a mouse model of malnutrition-associated enteropathy, mimicking key features of the human disease.     

Retinoblastoma protein (pRb), but not p107 or p130, is required for maintenance of enterocyte quiescence and differentiation in small intestine

The function of retinoblastoma protein (pRb) in the regulation of small intestine epithelial cell homeostasis has been challenged by several groups using various promoter-based Cre transgenic mouse lines. Interestingly, different pRb deletion systems yield dramatically disparate small intestinal phenotypes. These findings confound the function of pRb in this dynamic tissue. In this study, Villin-Cre transgenic mice were crossed with Rb (flox/flox) mice to conditionally delete pRb protein in small intestine enterocytes. We discovered a novel hyperplasia phenotype as well as ectopic cell cycle reentry within villus enterocytes in the small intestine. This phenotype was not seen in other pRb family member (p107 or p130) null mice. Using a newly developed crypt/villus isolation method, we uncovered that expression of pRb was undetectable, whereas proliferating cell nuclear antigen, p107, cyclin E, cyclin D3, Cdk2, and Cdc2 were dramatically increased in pRb-deficient villus cells. Cyclin A, cyclin D1, cyclin D2, and Cdk4/6 expression was not affected by absent pRb expression. pRb-deficient villus cells appeared capable of progressing to mitosis but with higher rates of apoptosis. However, the cycling villus enterocytes were not completely differentiated as gauged by significant reduction of intestinal fatty acid-binding protein expression. In summary, pRb, but not p107 or p130, is required for maintaining the postmitotic villus cell in quiescence, governing the expression of cell cycle regulatory proteins, and completing of absorptive enterocyte differentiation in the small intestine.     

Temporal and spatial dynamics underlying capacitative calcium entry in human colonic smooth muscle

Angiotensin converting enzyme (ACE) has been shown to be involved in regulation of apoptosis in nonintestinal tissues. This study examined the role of ACE in the modulation of intestinal adaptation utilizing ACE knockout mice (ACE-/-). A 60% small bowel resection (SBR) was used, since this model results in a significant increase in intestinal epithelial cell (EC) apoptosis as well as proliferation. Baseline villus height, crypt depth, and intestinal EC proliferation were higher, and EC apoptosis rates were lower in ACE-/- compared with ACE+/+ mice. After SBR, EC apoptosis rates remained significantly lower in ACE-/- compared with ACE+/+ mice. Furthermore, villus height and crypt depth after SBR continued to be higher in ACE-/- mice. The finding of a lower bax-to-bcl-2 protein ratio in ACE-/- mice may account for reduced EC apoptotic rates after SBR in ACE-/- compared with ACE+/+ mice. The baseline higher rate of EC proliferation in ACE-/- compared with ACE+/+ mice may be due to an increase in the expression of several EC growth factor receptors. In conclusion, ACE appears to have an important role in the modulation of intestinal EC apoptosis and proliferation and suggests that the presence of ACE in the intestinal epithelium has a critical role in guiding epithelial cell adaptive response.     

Increased levels of mucins in the cystic fibrosis mouse small intestine, and modulator effects of the Muc1 mucin expression

The mouse model (Cftr(tm1UNC)/Cftr(tm1UNC)) for cystic fibrosis (CF) shows mucus accumulation and increased Muc1 mucin mRNA levels due to altered splicing (Hinojosa-Kurtzberg AM, Johansson MEV, Madsen CS, Hansson GC, and Gendler SJ. Am J Physiol Gastrointest Liver Physiol 284: G853-G862, 2003). However, it is not known whether Muc1 is a major mucin contributing to the increased mucus and why CF/Muc1-/- mice show lower mucus accumulation. To address this, we have purified mucins from the small intestine of CF mice using guanidinium chloride extraction, ultracentrifugation, and gel filtration and analyzed them by slot blot, gel electrophoresis, proteomics, and immunoblotting. Normal and CF mice with wild-type (WT) Muc1 or Muc1-/- or that are transgenic for human MUC1 (MUC1.Tg, on a Muc1-/- background) were analyzed. The total amount of mucins, both soluble and insoluble in guanidinium chloride, increased up to 10-fold in the CF mice compared with non-CF animals, whereas the CF mice lacking Muc1 showed intermediate levels between the CF and non-CF mice. However, the levels of Muc3 (orthologue of human MUC17) were increased in the CF/Muc1-/- mice compared with the CF/MUC1.Tg animals. The amount of MUC1 mucin was increased several magnitudes in the CF mice, but MUC1 did still not appear to be a major mucin. The amount of insoluble mucus of the large intestine was also increased in the CF mice, an effect that was partially restored in the CF/Muc1-/- mice. The thickness of the firmly adherent mucus layer of colon in the Muc1-/- mice was significantly lower than that of WT mice. The results suggest that MUC1 is not a major component in the accumulated mucus of CF mice and that MUC1 can influence the amount of other mucins in a still unknown way.     

Abnormal Paneth cell granule dissolution and compromised resistance to bacterial colonization in the intestine of CF mice

Paneth cells of intestinal crypts contribute to host defense by producing antimicrobial peptides that are packaged as granules for secretion into the crypt lumen. Here, we provide evidence using light and electron microscopy that postsecretory Paneth cell granules undergo limited dissolution and accumulate within the intestinal crypts of cystic fibrosis (CF) mice. On the basis of this finding, we evaluated bacterial colonization and expression of two major constituents of Paneth cells, i.e., alpha-defensins (cryptdins) and lysozyme, in CF murine intestine. Paneth cell granules accumulated in intestinal crypt lumens in both untreated CF mice with impending intestinal obstruction and in CF mice treated with an osmotic laxative that prevented overt clinical symptoms and mucus accretion. Ultrastructure studies indicated little change in granule morphology within mucus casts, whereas granules in laxative-treated mice appear to undergo limited dissolution. Protein extracts from CF intestine had increased levels of processed cryptdins compared with those from wild-type (WT) littermates. Nonetheless, colonization with aerobic bacteria species was not diminished in the CF intestine and oral challenge with a cryptdin-sensitive enteric pathogen, Salmonella typhimurium, resulted in greater colonization of CF compared with WT intestine. Modest downregulation of cryptdin and lysozyme mRNA in CF intestine was shown by microarray analysis, real-time quantitative PCR, and Northern blot analysis. Based on these findings, we conclude that antimicrobial peptide activity in CF mouse intestine is compromised by inadequate dissolution of Paneth cell granules within the crypt lumens.     

Hyaluronic acid is radioprotective in the intestine through a TLR4 and COX-2-mediated mechanism

The intestinal epithelium is sensitive to radiation injury. Damage to the intestinal epithelium is dose limiting in radiation therapy of abdominal cancers. There is a need for agents that can be given before radiation therapy to protect the intestinal epithelium. C57BL6 mice were subjected to 12 Gy of total body radiation. Some mice received intraperitoneal hyaluronic acid (HA) before radiation. Mice were killed 6 h after radiation to assess radiation-induced apoptosis in the intestine; other mice were killed at 84 h to assess crypt survival. Total body radiation (12 Gy) resulted in increased expression of HA synthases and HA in the intestine and increased plasma HA (5-fold). Intraperitoneal injection of HA (30 mg/kg) before radiation resulted in a 1.8-fold increase in intestinal crypt survival and a decrease in radiation-induced apoptosis. The radioprotective effects of HA were not seen in Toll-like receptor 4 (TLR4)- or cyclooxygenase-2 (COX-2)-deficient mice. Intraperitoneal injection of HA induced a 1.5-fold increase in intestinal COX-2 expression, a 1.5-fold increase in intestinal PGE?, and the migration of COX-2-expressing mesenchymal stem cells from the lamina propria in the villi to the lamina propria near the crypt. We conclude that 1) radiation induces increased HA expression through inducing HA synthases, 2) intraperitoneal HA given before radiation reduces radiation-induced apoptosis and increases crypt survival, and 3) these radioprotective effects are mediated through TLR4, COX-2, and the migration of COX-2-expressing mesenchymal stem cells.     

Proliferation, not apoptosis, alters epithelial cell migration in small intestine of CFTR null mice

Expression of a mutated cystic fibrosis transmembrane conductance regulator (CFTR) has been shown to enhance proliferation within CF airways, and cells expressing a mutated CFTR have been shown to be less susceptible to apoptosis. Because the CFTR is expressed in the epithelial cells lining the gastrointestinal tract and all CF mouse models are characterized by gastrointestinal obstruction, we hypothesized that CFTR null mice would have increased epithelial cell proliferation and reduced apoptosis within the small intestine. The rate of intestinal epithelial cell migration from crypt to villus was increased in CFTR null mice relative to mice expressing the wild-type CFTR. This difference in migration could be explained by an increase in epithelial cell proliferation but not by a difference in apoptosis within the crypts of Lieberkühn. In addition, using two independent sets of CF cell lines, we found that epithelial cell susceptibility to apoptosis was unrelated to the presence of a functional CFTR. Thus increased proliferation but not alterations in apoptosis within epithelial cells might contribute to the pathophysiology of CF.     

Mast Cells and Gastrointestinal Dysmotility in the Cystic Fibrosis Mouse

Background

Cystic fibrosis (CF) has many effects on the gastrointestinal tract and a common problem in this disease is poor nutrition. In the CF mouse there is an innate immune response with a large influx of mast cells into the muscularis externa of the small intestine and gastrointestinal dysmotility. The aim of this study was to evaluate the potential role of mast cells in gastrointestinal dysmotility using the CF mouse (Cftr^tm1UNC, Cftr knockout).

Methodology

Wild type (WT) and CF mice were treated for 3 weeks with mast cell stabilizing drugs (ketotifen, cromolyn, doxantrazole) or were treated acutely with a mast cell activator (compound 48/80). Gastrointestinal transit was measured using gavage of a fluorescent tracer.

Results

In CF mice gastric emptying at 20 min post-gavage did not differ from WT, but was significantly less than in WT at 90 min post-gavage. Gastric emptying was significantly increased in WT mice by doxantrazole, but none of the mast cell stabilizers had any significant effect on gastric emptying in CF mice. Mast cell activation significantly enhanced gastric emptying in WT mice but not in CF mice. Small intestinal transit was significantly less in CF mice as compared to WT. Of the mast cell stabilizers, only doxantrazole significantly affected small intestinal transit in WT mice and none had any effect in CF mice. Mast cell activation resulted in a small but significant increase in small intestinal transit in CF mice but not WT mice.

Conclusions

The results indicate that mast cells are not involved in gastrointestinal dysmotility but their activation can stimulate small intestinal transit in cystic fibrosis.     

The intestinal epithelium compensates for p53-mediated cell death and guarantees organismal survival

Mdm2 is the major inhibitor of the p53 tumor suppressor. Loss of Mdm2 in mice or in specific tissues of the mouse always yields p53-dependent lethal phenotypes. However, the role of Mdm2 in tissues with high turnover capacity is unknown. We have engineered mice lacking Mdm2 in the intestinal epithelium using the Cre/LoxP system. Loss of Mdm2 (Mdm2(intDelta)) results in viable animals, but neonates display multiple intestinal abnormalities such as hyperplasia, enterocyte vacuolization, and inflammation. These defects correlate with a drastic increase in p53-dependent apoptosis in highly proliferative and differentiated cells. Unexpectedly, the observed phenotypes disappear with age. The tissue selects against Mdm2-null cells and increases its proliferative capacity. Additionally, the intestinal stem and progenitor cell populations are enriched leading to an increase in crypt fission events. Enhanced proliferation is achieved by activation of the canonical Wnt and EGFR-mediated Ras/MAPK pathways. While Mdm2 is a critical inhibitor of p53 in the intestinal epithelium, the tissue employs a series of processes that compensate for cell death.     

CpG-Oligodeoxynucleotide Treatment Protects against Ionizing Radiation-Induced Intestine Injury

Background

the bone marrow and the intestine are the major sites of ionizing radiation (IR)-induced injury. Our previous study demonstrated that CpG-oligodeoxynucleotide (ODN) treatment mitigated IR-induced bone marrow injury, but its effect on the intestine is not known. In this study, we sought to determine if CpG-ODN have protective effect on IR-induced intestine injury, and if so, to determine the mechanism of its effect.

Methods and Findings

Mice were treated with CpG-ODN after IR. The body weight and survival were daily monitored for 30 days consecutively after exposure. The number of surviving intestinal crypt was assessed by the microcolony survival assay. The number and the distribution of proliferating cell in crypt were evaluated by TUNEL assay and BrdU assay. The expression of Bcl-2, Bax and caspase-3 in crypt were analyzed by Immunohistochemistry assay. The findings showed that the treatment for irradiated mice with CpG-ODN diminished body weight loss, improved 30 days survival, enhanced intestinal crypts survival and maintained proliferating cell population and regeneration in crypt. The reason might involve that CpG-ODN up-regulated the expression of Bcl-2 protein and down-regulated the expression of Bax protein and caspase-3 protein.

Conclusion

CpG-ODN was effective in protection of IR-induced intestine injury by enhancing intestinal crypts survival and maintaining proliferating cell population and regeneration in crypt. The mechanism might be that CpG-ODN inhibits proliferating cell apoptosis through regulating the expression of apoptosis-related protein, such as Bax, Bcl-2 and caspase-3.     

Mice overexpressing CD97 in intestinal epithelial cells provide a unique model for mammalian postnatal intestinal cylindrical growth

Postnatal enlargement of the mammalian intestine comprises cylindrical and luminal growth, associated with crypt fission and crypt/villus hyperplasia, respectively, which subsequently predominate before and after weaning. The bipartite adhesion G protein–coupled receptor CD97 shows an expression gradient along the crypt–villus axis in the normal human intestine. We here report that transgenic mice overexpressing CD97 in intestinal epithelial cells develop an upper megaintestine. Intestinal enlargement involves an increase in length and diameter but does not affect microscopic morphology, as typical for cylindrical growth. The megaintestine is acquired after birth and before weaning, independent of the genotype of the mother, excluding altered availability of milk constituents as driving factor. CD97 overexpression does not regulate intestinal growth factors, stem cell markers, and Wnt signaling, which contribute to epithelial differentiation and renewal, nor does it affect suckling-to-weaning transition. Consistent with augmented cylindrical growth, suckling but not adult transgenic mice show enlarged crypts and thus more crypt fissions caused by a transient increase of the crypt transit-amplifying zone. Intestinal enlargement by CD97 requires its seven-span transmembrane/cytoplasmic C-terminal fragment but not the N-terminal fragment binding partner CD55. In summary, ectopic expression of CD97 in intestinal epithelial cells provides a unique model for intestinal cylindrical growth occurring in breast-fed infants.     

Anoctamin 1 dysregulation alters bronchial epithelial repair in cystic fibrosis

Cystic fibrosis (CF) airway epithelium is constantly subjected to injury events due to chronic infection and inflammation. Moreover, abnormalities in CF airway epithelium repair have been described and contribute to the lung function decline seen in CF patients. In the last past years, it has been proposed that anoctamin 1 (ANO1), a Ca^2 +-activated Cl^? channel, might offset the CFTR deficiency but this protein has not been characterized in CF airways. Interestingly, recent evidence indicates a role for ANO1 in cell proliferation and tumor growth. Our aims were to study non-CF and CF bronchial epithelial repair and to determine whether ANO1 is involved in airway epithelial repair. Here, we showed, with human bronchial epithelial cell lines and primary cells, that both cell proliferation and migration during epithelial repair are delayed in CF compared to non-CF cells. We then demonstrated that ANO1 Cl^? channel activity was significantly decreased in CF versus non-CF cells. To explain this decreased Cl^? channel activity in CF context, we compared ANO1 expression in non-CF vs. CF bronchial epithelial cell lines and primary cells, in lung explants from wild-type vs. F508del mice and non-CF vs. CF patients. In all these models, ANO1 expression was markedly lower in CF compared to non-CF. Finally, we established that ANO1 inhibition or overexpression was associated respectively with decreases and increases in cell proliferation and migration. In summary, our study demonstrates involvement of ANO1 decreased activity and expression in abnormal CF airway epithelial repair and suggests that ANO1 correction may improve this process.     

热应激对小鼠器官指数、小肠损伤及胃HSP70 mRNA表达的影响

目的探讨热应激对小鼠器官指数、小肠形态、胃黏膜HSP70 mRNA表达量及糖代谢相关激素的影响。方法采用单因子实验设计,将年龄和体重相近的18只KM小鼠随机分为对照组和热应激组,分别测定心、肝、脾、肺、肾重量,小鼠胃黏膜HSP70 mRNA表达量、血浆中胰岛素和胰高血糖素浓度以及十二指肠和空肠的绒毛高度、隐窝深度,并对肝脏、十二指肠和空肠进行病理组织学检查。结果与结论热应激对小鼠器官指数无影响,可显著提高小鼠胃黏膜HSP70 mRNA表达量,降低血浆中胰岛素的含量,并造成小鼠肝脏、十二指肠和空肠严重损伤。