Effects of laxative and N-acetylcysteine on mucus accumulation, bacterial load, transit, and inflammation in the cystic fibrosis mouse small intestine

The accumulation of mucus in affected organs is characteristic of cystic fibrosis (CF). The CF mouse small intestine has dramatic mucus accumulation and exhibits slower interdigestive intestinal transit. These factors are proposed to play cooperative roles that foster small intestinal bacterial overgrowth (SIBO) and contribute to the innate immune response of the CF intestine. It was hypothesized that decreasing the mucus accumulation would reduce SIBO and might improve other aspects of the CF intestinal phenotype. To test this, solid chow-fed CF mice were treated with an osmotic laxative to improve gut hydration or liquid-fed mice were treated orally with N-acetylcysteine (NAC) to break mucin disulfide bonds. Treatment with laxative or NAC reduced mucus accumulation by 43% and 50%, respectively, as measured histologically as dilation of the intestinal crypts. Laxative and NAC also reduced bacterial overgrowth in the CF intestine by 92% and 63%, respectively. Treatment with laxative normalized small intestinal transit in CF mice, whereas NAC did not. The expression of innate immune response-related genes was significantly reduced in laxative-treated CF mice, whereas there was no significant effect in NAC-treated CF mice. In summary, laxative and NAC treatments of CF mice reduced mucus accumulation to a similar extent, but laxative was more effective than NAC at reducing bacterial load. Eradication of bacterial overgrowth by laxative treatment was associated with normalized intestinal transit and a reduction in the innate immune response. These results suggest that both mucus accumulation and slowed interdigestive small intestinal transit contribute to SIBO in the CF intestine.     

Bacteria-lectin interactions in phytohemagglutinin-induced bacterial overgrowth of the small intestine

The mechanism of phytohemagglutinin-induced bacterial overgrowth of the small bowel in the rat was studied. Interaction of the lectin with bacterial isolates selected at random from those that comprised the major population of the overgrowth was determined. In both bacterial agglutination assays and glycocalyx stabilization, no specific association between lectin and bacteria was seen. In three independent binding assays phytohemagglutinin was not found to increase bacterial adherence to washed intestinal mucosa. Phytohemagglutinin would not appear to act, therefore, as a direct ligand to mediate bacterial adherence or to modify the mucosal surface to increase bacterial adherence.     

Disrupted tight junctions in the small intestine of cystic fibrosis mice

The tight junction (TJ) is the major determinant of paracellular permeability, which in the gut protects the body from entry of harmful substances such as microbial components. In cystic fibrosis (CF), there is increased permeability of the small intestine both in humans and in CF mice. To gain insight into the mechanisms of increased intestinal permeability in CF, I analyze the composition of the TJ in a cystic fibrosis transmembrane conductance regulator (Cftr) knockout mouse model. Significant changes in TJ gene expression in the CF intestine were found for Cldn1, Cldn7, Cldn8 and Pmp22, which were expressed at lower levels and Cldn2 that was expressed at a higher level. Protein levels of claudin-2 were increased in the CF intestine as compared to wild-type, while other TJ proteins were not significantly different. In the villus epithelium of the CF intestine, all TJ components analyzed were mislocalized to the basal cytoplasm and showed varying degrees of loss from the TJ and apico-lateral surfaces. The pore-forming claudin-2 in the CF intestine showed more intense staining but was correctly localized to the TJ, principally in the crypts that are enlarged in CF. The cytokine TNFα, known to affect TJ, was elevated to 160 % of wild-type in the CF intestine. In summary, there is a dramatic redistribution of claudin proteins from the TJ/lateral membrane to the basal cytoplasm of the villus epithelium in the CF intestine. These changes in TJ protein localization in CF are likely to be involved in the increased permeability of the CF small intestine to macromolecules and TNFα may be a causative factor.     

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.     

Cellular localization of cystic fibrosis transmembrane regulator protein in piglet and mouse intestine

Antibodies raised against the cystic fibrosis transmembrane regulator protein (CFTR) were used to localize CFTR in intestinal tissues of piglets and mice. Positive staining for CFTR was detected in goblet cells of both species. A second population of epithelial cells of unknown phenotype was also labeled by anti-CFTR antibodies. The labeling pattern was abolished by preincubation of anti-CFTR antibodies with the immunogen or when non-immune IgG was used in place of anti-CFTR antibodies. These results support other studies that suggest that alterations in goblet cell function may be involved in the intestinal abnormalities associated with cystic fibrosis. Received: 4 May 1995 / Accepted: 6 September 1995     

Altered intestinal chloride transport in cystic fibrosis

Sodium ion and chloride transport was studied in vitro in small intestinal and colonic tissue from patients with cystic fibrosis (CF) and from non-CF control subjects matched as to age and sex. Normal histological appearance and substantial response to mucosal glucose (5 mM, ileum) or mucosal amiloride (10(-5) M, colon) indicated normal tissue viability in both control and CF tissues. Electroneutral NaCl absorption was demonstrated in the small intestine of control subjects and CF patients. Small intestinal and colonic tissues of control subjects responded to four secretagogues (theophylline, 5 mM; prostaglandin E2, 10(-6) M; calcium ionophore (A23187), 10(-5) M; bethanechol, 5 x 10(-5) M), with electrogenic chloride secretion. The tissues of CF patients, however, did not respond to any of the test secretagogues. These studies demonstrate that an abnormality in chloride transport is present in the small intestinal and colonic epithelia of CF patients. Unlike airway epithelia, which secrete chloride in response to Ca ionophore, the intestinal epithelia of CF patients do not respond to either cAMP- or Ca-mediated secretagogues. This abnormality in intestinal electrolyte transport may play a role in the pathogenesis of meconium impactions in CF patients.     

Prolonged small-intestinal transit time in cystic fibrosis.

A lactulose hydrogen breath test was performed on 10 patients with cystic fibrosis and 15 control subjects matched for age and sex. All normal subjects had a fasting breath hydrogen concentration of less than 20 ppm. In contrast, seven of the patients with cystic fibrosis had high concentrations (25-170 ppm), which fell to 20 ppm or below on prolonged fasting (14-23 hours). Two patients showed no rise in breath hydrogen concentrations after lactulose, and in one patient the breath hydrogen concentration rose at 15 minutes, suggesting bacterial colonisation of the small bowel. Seven of the patients had prolonged small-bowel transit times (160-390 minutes) compared with those in the control group (50-150 minutes).     

Ceramide in bacterial infections and cystic fibrosis

Ceramide is formed by the activity of sphingomyelinases, by degradation of complex sphingolipids, reverse ceramidase activity or de novo synthesized. The formation of ceramide within biological membranes results in the formation of large ceramide-enriched membrane domains. These domains serve the spatial and temporal organization of receptors and signaling molecules. The acid sphingomyelinase-ceramide system plays an important role in the infection of mammalian host cells with bacterial pathogens such as Neisseria gonorrhoeae, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium and Pseudomonas aeruginosa. Ceramide and ceramide-enriched membrane platforms are also involved in the induction of apoptosis in infected cells, such as in epithelial and endothelial cells after infection with Pseudomonas aeruginosa and Staphylococcus aureus, respectively. Finally, ceramide-enriched membrane platforms are critical regulators of the release of pro-inflammatory cytokines upon infection. The diverse functions of ceramide in bacterial infections suggest that ceramide and ceramide-enriched membrane domains are key players in host responses to many pathogens and thus are potential novel targets to treat infections.     

Microecological aspects of small intestinal bacterial overgrowth syndrome

Small intestinal bacterial overgrowth syndrome (SIBOS) means chronic recurrent diarrhea with malabsorption, intoxication and increased risk of endogenous infection. This syndrome are accompanied by increase of overall bacterial burden in biotope >10(5) CFU/ml in adults and >10(4) CFU/ml in children, emergence of different species of enterobacteria, bacteroides, clostridia and fusobacteria et al. in small intestine. Such characteristics of the syndrome allow to consider it as syndrome of disturbances of intestinal microflora (dysbacteriosis). Microecological changes are accompanied by B12 vitamin deficiency anemia, hypovitaminosis, protein deficiency, translocation of bacteria and their toxins from intestine in blood, emergence of endotoxinemia and possible generalization of infection. SIBOS is diagnosed by concentration of hydrogen in expiratory flow (lactulosa load test) or by bacteriological study of aspirate from proximal part of small intestine. Complex treatment includes containing lacto- and bifidobacteria probiotics and, in more severe cases, antimicrobial agents (vancomycine, metronidazole, aminoglycosides, amoxicillin clavulanate, tetracycline, and cephalosporines of 2nd generation) with following correction of disturbed microbiocenosis by different probiotics.     

Stem-cell organization in mouse small intestine

We have investigated stem-cell organization in mouse small intestine (SI) by using a cellular marker induced by somatic mutation. In small intestinal whole mounts from heterozygous Dlb-1b/Dlb-1a mice stained with a peroxidase conjugate of Dolichos biflorus agglutinin (DBA-Px), mutations of Dlb-1b in stem cells result in loss of DBA-Px binding and so are recognizable as wholly or partly unstained crypts. The frequency of these clonal patterns can be measured during the accumulation of spontaneous mutations in untreated mice, or after treatment with ethylnitrosourea (ENU). The results show that there is a single infrequently dividing stem cell that maintains the epithelium of each crypt through a population of transit stem cells. The entire crypt epithelium is renewed approximately every 12 weeks.     

幽门螺杆菌与小肠细菌过生长的关系分析

目的探讨幽门螺杆菌(Helicobacter pylori,H.pylori)感染与小肠细菌过生长(small intesitinal bacterial overgrowth,SIBO)之间的关系。方法将52例因腹痛伴腹胀不适住院的患者,分为H.pylori阳性组和H.pylori阴性组,采用葡萄糖氢呼气试验检(Glucose hydrogen breath test,GHBT)测SIBO的情况。结果 H.pylori阳性组有22人为SIBO阳性(75.86%),H.pylori阴性组有6人为SIBO阳性(21.74%),两组之间差异有统计学意义(r=0.538,P0.05),表明二者存在关联性。结论 H.pylori的感染与SIBO密切相关。     

Altered cholesterol homeostasis in cultured and in vivo models of cystic fibrosis

Determining how the regulation of cellular processes is impacted in cystic fibrosis (CF) is fundamental to understanding disease pathology and to identifying new therapeutic targets. In this study, unesterified cholesterol accumulation is observed in lung and trachea sections obtained from CF patients compared with non-CF tissues, suggesting an inherent flaw in cholesterol processing. An alternate staining method utilizing a fluorescent cholesterol probe also indicates improper lysosomal storage of cholesterol in CF cells. Excess cholesterol is also manifested by a significant increase in plasma membrane cholesterol content in both cultured CF cells and in nasal tissue excised from cftr(-/-) mice. Impaired intracellular cholesterol movement is predicted to stimulate cholesterol synthesis, a hypothesis supported by the observation of increased de novo cholesterol synthesis in lung and liver of cftr(-/-) mice compared with controls. Furthermore, pharmacological inhibition of cholesterol transport is sufficient to cause CF-like elevation in cytokine production in wild-type cells in response to bacterial challenge but has no effect in CF cells. These data demonstrate via multiple methods in both cultured and in vivo models that cellular cholesterol homeostasis is inherently altered in CF. This perturbation of cholesterol homeostasis represents a potentially important process in CF pathogenesis.     

应用质子泵抑制剂导致小肠细菌过度生长发生的可能性

目的观察应用质子泵抑制剂(PPI)患者,在强效抑酸状态下小肠细菌过生长(SIBO)的发生情况。方法用葡萄糖呼气H2试验,检测57例服用PPI制剂患者,30名健康志愿者作为对照。结果 PPI制剂组中呼气H2检测SIBO阳性28例,阴性29例;健康对照组中SIBO阳性3例,阴性27例。PPI组与健康对照组比较差异有统计学意义(P0.01),PPI制剂使用时间与SIBO阳性率存在正相关性。结论长期服用PPI制剂的患者可并发SIBO,且SIBO的发病率与PPI制剂使用时间呈正相关。     

Primary inflammation in human cystic fibrosis small airways

Most cystic fibrosis (CF) patients die of lung failure, due to the combined effects of bacterial infection, neutrophil-mediated inflammation, and airway obstruction by hyperviscous mucus. To this day, it remains unclear where and how this pathological vicious circle is initiated in vivo. In particular, it has proven difficult to investigate whether inflammatory pathways are dysregulated in CF airways independently of infection. Also, the relative involvement of large (tracheobronchial) vs. small (bronchiolar) airways in CF pathophysiology is still unclear. To help address these issues, we used an in vivo model based on the maturation of human fetal CF and non-CF small airways in severe combined immunodeficiency mice. We show that uninfected mature CF small airway grafts, but not matched non-CF controls, undergo time-dependent neutrophil-mediated inflammation, leading to progressive lung tissue destruction. This model of mature human small airways provides the first clear-cut evidence that, in CF, inflammation may arise at least partly from a primary defect in the regulation of neutrophil recruitment, independently of infection.     

Altered intracellular pH regulation in neutrophils from patients with cystic fibrosis

Cystic fibrosis (CF) is a condition characterized by neutrophil-mediated lung damage and bacterial colonization. The physiological basis for reported functional alterations in CF neutrophils, including increased release of neutrophil elastase, myeloperoxidase, and oxidants, is unknown. These processes are, however, regulated by intracellular pH (pH(i)). We demonstrate here that pH(i) regulation is altered in neutrophils from CF patients. Although resting pH(i) is similar, pH(i) after acid loading and activation (N-formyl-methionyl-leucyl-phenylalanine and phorbol 12-myristate 13-acetate) is more acidic in CF cells than in normal cells. Furthermore, patients with non-CF-related bronchiectasis handle acid loading and activation in a fashion similar to subjects with normal neutrophils, suggesting that chronic pulmonary inflammation alone does not explain the difference in pH(i). This is further supported by data showing that normal neutrophils exposed to the CF pulmonary milieu respond by increasing pH(i) as opposed to decreasing pH(i) as seen in activated CF neutrophils. These pH(i) differences in activated or acid-loaded CF neutrophils are abrogated by ZnCl(2) but not by amiloride and bafilomycin A(1), suggesting that passive proton conductance is abnormal in CF. In addition, DIDS, which inhibits HCO(3)(-)/Cl(-) exchange, causes alkalinization of control but not of CF neutrophils, suggesting that anion transport is also abnormal in CF neutrophils. In summary, we have shown that pH(i) regulation in CF neutrophils is intrinsically abnormal, potentially contributing to the pulmonary manifestations of the condition.     

Altered O-glycosylation and sulfation of airway mucins associated with cystic fibrosis

Cystic fibrosis (CF) is the most lethal genetic disorder in Caucasians and is characterized by the production of excessive amounts of viscous mucus secretions in the airways of patients, leading to airway obstruction, chronic bacterial infections, and respiratory failure. Previous studies indicate that CF-derived airway mucins are glycosylated and sulfated differently compared with mucins from nondiseased (ND) individuals. To address unresolved questions about mucin glycosylation and sulfation, we examined O-glycan structures in mucins purified from mucus secretions of two CF donors versus two ND donors. All mucins contained galactose (Gal), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), fucose (Fuc), and sialic acid (Neu5Ac). However, CF mucins had higher sugar content and more O-glycans compared with ND mucins. Both ND and CF mucins contained GlcNAc-6-sulfate (GlcNAc-6-Sul), Gal-6-Sul, and Gal-3-Sul, but CF mucins had higher amounts of the 6-sulfated species. O-glycans were released from CF and ND mucins and derivatized with 2-aminobenzamide (2-AB), separated by ion exchange chromatography, and quantified by fluorescence. There was nearly a two-fold increase in sulfation and sialylation in CF compared with ND mucin. High performance liquid chromatography (HPLC) profiles of glycans showed differences between the two CF samples compared with the two ND samples. Glycan compositions were defined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Unexpectedly, 260 compositional types of O-glycans were identified, and CF mucins contained a higher proportion of sialylated and sulfated O-glycans compared with ND mucins. These profound structural differences in mucin glycosylation in CF patients may contribute to inflammatory responses and increased pathogenesis by Pseudomonas aeruginosa.