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NEUROREGULATION AND MOTILITY

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

Anatomy and Cell Biology, University of Kansas School of Medicine, Kansas City, Kansas

Submitted 2 May 2007 ; accepted in final form 29 June 2007

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
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.

small intestinal bacterial overgrowth

Secreted gel-forming mucins, mainly Muc2 in the intestine, bind bacteria and help carry them aborally for efficient clearance from the small intestine. When mucus clearance is impaired, bacteria can proliferate in the mucus and may use it as an energy source (36). Mucus accumulation in the CF intestine is in part due to the dehydrated, acidic environment as well as the altered glycosylation of mucins (38, 40), which may increase the viscosity of mucus. Our laboratory (32) has demonstrated that bacteria that overgrow the CF mouse small intestine colonize the mucus that accumulates in the intestinal lumen. Even though the major intestinal mucin genes (Muc2 and Muc3) do not have increased levels of expression (15, 21), mucin glycoprotein levels are dramatically increased in the CF mouse small intestine (29). Thus, mucus clearance is reduced rather than there being an increase in synthesis in the CF intestine. Interestingly, reduction of bacterial load with antibiotics decreases the accumulation of mucus in the CF intestine without a major effect on mucin gene expression (15). This suggests that secretion from goblet cells is stimulated by bacterial overgrowth. These results indicate that bacteria not only take advantage of mucus to colonize the small intestine but actively participate in stimulating mucus secretion by the intestinal epithelium.

Another aspect of the CF intestinal phenotype related to bacterial overgrowth is that intestinal transit is impaired (1, 13, 19, 27). The interdigestive intestinal motility (migrating motor complex) has a housekeeping function that "sweeps" the lumen clean and thereby removes mucus and embedded bacteria. Dysmotility is a major cause of SIBO (22). We (14) recently showed that interdigestive transit through the CF mouse small intestine is dramatically slower than in wild-type (WT) mice. Thus, a combination of viscous, dehydrated mucus and slower interdigestive motility likely combine to allow SIBO in the CF intestine.

We have proposed that abnormal mucus accumulation in the CF intestine provides a niche for bacterial overgrowth, and we hypothesized that interventions to improve mucus clearance would be beneficial. We used two different oral treatments of CF mice to improve mucus clearance. The first treatment was to maintain mice on solid mouse chow and treat them by replacing their drinking water with an osmotic laxative [polyethylene glycol (PEG)] in a balanced salt solution. Laxative treatment prevents the lethal intestinal obstruction that otherwise occurs in the CF mouse on solid chow and is believed to do so by improving hydration of the gut lumen (10). Laxative-treated CF mice apparently have less mucus accumulation and less dilation of the intestinal crypt lumen compared with liquid-fed mice (9), although this previously has not been quantitatively compared. PEG-based laxative is also used clinically to treat intestinal obstruction in CF patients (11), and its use is associated with a significant decrease in the occurrence of SIBO (18).

The second treatment used in this study was to orally administer N-acetylcysteine (NAC) by addition to the liquid diet. NAC is a mucolytic that acts as a reducing agent to break disulfide bonds in mucin molecules and thereby decrease the viscosity of mucus (17, 37). The results of this study show that both laxative and NAC can reduce mucus accumulation and bacterial load but that laxative is much more effective at reducing SIBO and the innate immune response and also is associated with normal interdigestive motility.

	MATERIALS AND METHODS

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Animals and treatments. Cftr^+/– mice (cftr^tm1UNC) were originally obtained from the Jackson Laboratory (Bar Harbor, ME). Mice were backcrossed on the C57Bl/6 background until congenic (33), and heterozygous mice were backcrossed regularly with the parental strain to prevent genetic drift (as suggested by the Jackson Laboratory, www.jax.org). Cftr^+/– mice were bred to obtain WT (Cftr^+/+) and CF (Cftr^–/–) mice. Occasional Cftr heterozygous mice were used as WT controls, as they do not have any CF phenotype. Mice of both sexes were used between 6 and 10 wk of age for experiments. To compare body weight gains, mice were weighed at 6 wk of age (given treatment for 3 wk postweaning). Weights of CF mice were calculated as percent body weights of WT mice of the same sex and treatment group. This allowed use of both female and male mouse data to increase the power of statistical comparisons (20).

Groups of WT and CF littermates were fed standard solid chow and treated with an oral laxative solution instead of drinking water (10). The laxative solution was prepared to match the composition of Colyte (in g/l: 60 PEG 3350, 1.46 NaCl, 0.745 KCl, 1.68 NaHCO₃, and 5.698 Na₂SO₄). Unless noted otherwise, all reagents were obtained from Sigma (St. Louis, MO). Additional groups of WT and CF littermates were maintained on a complete elemental liquid diet (Peptamen, Nestle, Deerfield, IL) (16) either alone or treated by supplementation with NAC (5 mg/ml Peptamen). To enhance the survival of CF pups, laxative treatment and liquid feeding were begun at 2 wk of age by placing the mother and pups on treatments. When NAC was used, it was added to the liquid diet beginning at weaning (3 wk of age) and maintained until death. All animal procedures were approved by the University of Kansas Medical Center Institutional Animal Care and Use Committee.

Morphology and morphometric analysis. Mice were fasted overnight with free access to water or laxative solution as appropriate. The entire intestinal tract was resected and placed in ice-cold saline. The mesentery was cut enough to allow extension of the small intestine to its full length. The middle 6 cm of the small intestine was cut into four pieces, and the tissue was fixed overnight in the cold in Carnoy's solution (ethanol-acetic acid-chloroform at a ratio of 6:3:1 by volume). Tissues were processed in paraffin, cross sections (5 µm thick) were prepared, and sections were stained for mucins using periodic acid-Schiff (PAS)-Alcian blue (AB; pH 2.5) by a commercial service (Mass Histology, Worcester, MA). Microscopic images were recorded on a Nikon Diaphot microscope equipped with a SPOT RT-KE digital camera (Diagnostic Instruments, Sterling Heights, MI). Images were analyzed by an individual unaware of sample identities using Photoshop (Adobe, San Jose, CA) to mark areas of interest and National Institutes of Health Image J (http://rsb.info.nih.gov/ij/) to quantify the marked areas. The crypt width was used as an estimate of mucus accumulation as previously described (15). Heights of the villi, depths of crypts, and thicknesses of the inner circular and outer longitudinal muscle layers of the muscularis externa were measured only from sections that had a well-oriented crypt-villus axis.

Measurement of bacterial load by quantitative PCR of the bacterial 16S rRNA gene. After the death of the mice, PBS containing 10 mM DTT was flushed through the lumen of the small intestine and flushed material was processed as previously described (32) to extract microbial genomic DNA. The microbial load was measured by quantitative PCR using universal bacterial 16S rRNA-specific primers as previously described (31). The 16S rRNA PCR product from a laboratory strain of Escherichia coli was cloned and used to generate a standard curve for copy number determinations in the real-time quantitative PCR assays as previously described (32).

Measurements of gastric emptying and small intestinal transit. Gastric emptying and small intestinal transit were measured as previously described (14). Briefly, mice were fasted overnight with free access to water or laxative solution as appropriate. In the morning between 8 and 9 AM, mice were gavaged with a 100-µl bolus of 25 mg/ml rhodamine-dextran prepared in 1.5% methylcellulose in saline. At 20 min postgavage, the entire gastrointestinal tract was removed, placed into ice-cold saline, and divided into the stomach and 10 equal segments of the small intestine. In none of the animals did the tracer reach the cecum/large intestine within 20 min postgavage. The luminal contents of the stomach and intestinal segments were rinsed out into 1.75 ml saline. The released material was centrifuged, and the supernatants were used to determine rhodamine fluorescence. Gastric emptying was calculated as the percentage of tracer remaining in the stomach at 20 min relative to the total amount recovered from the stomach and small intestine. For measurements of small intestinal transit, raw fluorescence data were transformed by calculating the geometric center of the fluorescence (GCF) present in the small intestine only as follows: GCF = [(fraction of fluorescence per segment) x (segment number)].

Measurement of gene expression by quantitative RT-PCR. Total RNA was prepared from the entire small intestine by the TRIzol method as previously described (32). Real-time quantitative RT-PCR was performed with an iCycler instrument (Bio-Rad, Hercules, CA) using a one-step RT-PCR kit (Qiagen, Valencia, CA). Information about the genes and specific primers are shown in Table 1. mRNA for ribosomal protein L26 (Rpl26) was used as a housekeeping gene for normalization. Expression levels were calculated using the C_t method (where C_t is threshold cycle) after correcting for differences in PCR efficiencies (28) and were expressed relative to WT levels on the liquid diet.

	RESULTS

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Two approaches were used to investigate the consequences of mucus accumulation in the CF small intestine. CF mice were treated with an oral laxative, which improves the hydration status of the intestinal lumen and protects mice from lethal intestinal obstruction (10). Second, the chemical NAC, which has reducing activity and can decrease the viscosity of mucus by breaking disulfide bonds between mucin molecules (17, 37), was used. The dose of NAC we used was similar to that employed to reduce Helicobacter pylori infection in mice (24).

Mucus accumulation was estimated morphometrically from the width of intestinal crypts as previously described (15) after tissue had been stained with PAS-AB. Luminal mucus is not always retained in fixed tissues, even when using Carnoy's solution (unpublished observations). Therefore, we used crypt measurements because mucus in this cul-de-sac is better preserved and more accurately measured than mucus in the intestinal tract lumen proper. Because of this, we may have underestimated the effects of the interventions on the reduction of mucus accumulation.

In untreated CF mice raised on the liquid diet, intestinal crypts were greatly dilated and filled with PAS-AB-reactive mucus that extended up along the villus surface into the intestinal lumen (Fig. 1A). In contrast, WT mice on the liquid diet had narrow crypt lumens with minimal PAS-AB staining in either the crypt lumen or intestinal lumen proper, but there was staining of the luminal surfaces of the epithelium (Fig. 1A). In CF mice treated with laxative, the width and PAS-AB reactivity of the crypts were noticeably less than in liquid-fed CF mice, whereas tissue from WT mice treated with laxative was not noticeably different than that from untreated liquid-fed WT mice (Fig. 1A). The decrease in crypt lumen width in CF mice treated with laxative was confirmed by morphometry. Untreated liquid-fed CF mice had an average crypt width more than sevenfold greater than WT mice (Fig. 1B). With laxative, the CF crypt width was significantly less, such that it was now only about threefold wider on average compared with WT mice (Fig. 1B). Comparable with the effect of laxative, treatment of mice with NAC also reduced crypt lumen width of liquid-fed CF mice (Fig. 1) to about threefold that of WT mice (Fig. 1B).

View larger version (111K): [in this window] [in a new window]   Fig. 1. Effects of laxative and N-acetylcysteine (NAC) on mucus accumulation and tissue morphometry in cystic fibrosis (CF) mice. A: representative images of the middle portion of the small intestine from wild-type (WT) and CF mice. After fixation in Carnoy's solution, tissues were processed for periodic acid-Schiff (PAS)-Alcian blue (AB) staining. The following samples are shown: control (untreated with the liquid diet), laxative (laxative treatment with solid chow), and NAC (NAC added to the liquid diet) from WT and CF mice. B: quantitation of the effects of laxative and NAC on mucus accumulation in CF mice. Mucus accumulation was estimated from the widths of intestinal crypts. Random images of PAS-AB-stained crypts were collected using a x20 objective. Crypt lumen widths were measured as described in MATERIALS AND METHODS. Data are means ± SE from 8 liquid-fed WT mice (1,794 crypts), 9 laxative-treated WT mice (3,965 crypts), 6 NAC-treated WT mice (804 crypts), 6 liquid-fed CF mice (1,983 crypts), 3 laxative-treated CF mice (1,612 crypts), and 6 NAC-treated CF mice (933 crypts). Lax, chow-fed mice treated with laxative in drinking water; Liq, liquid-fed untreated mice; NAC, liquid-fed mice treated with NAC. C and D: morphometric analysis of the effects of laxative and NAC on intestinal tissues in CF mice. Sections from each group of mice were analyzed as described in MATERIALS AND METHODS. Only sections showing the entire crypt-villus axis were measured. C: villus height; D: crypt depth. Data are means ± SE from n = 3 WT and 3 CF mice for each treatment group (crypt-villus axes from 93 laxative-treated WT mice, 116 liquid-fed WT mice, 105 NAC-treated WT mice, 54 laxative-treated CF mice, 70 liquid-fed CF mice, and 103 NAC-treated CF mice). NS, not statistically significant.

To investigate whether laxative or NAC treatments would reduce tissue hypertrophy, a morphometric analysis was performed. Heights of villi (Fig. 1C) and depths of crypts (Fig. 1D) were significantly greater in liquid-fed CF mice compared with liquid-fed WT mice, as recently reported (3). There was a tendency for the muscle layers to be thicker in liquid-fed CF mice compared with WT mice, but the differences were not statistically significant (data not shown). The only significant effect observed in laxative- or NAC-treated CF mice was that the crypt depth was less in laxative-treated CF mice compared with untreated liquid-fed CF animals (Fig. 1D). NAC tended to decrease the crypt depth as well, but this effect was not statistically significant (Fig. 1D).

We (32) have shown that the accumulated mucus in the CF mouse small intestine is heavily colonized by bacteria. To test whether a reduction in intestinal mucus would also reduce the bacterial load, we used quantitative PCR for the bacterial 16S rRNA gene as previously described (32). As previously shown, untreated liquid-fed CF mice have SIBO compared with WT mice on the liquid diet (Fig. 2). Treatment of CF mice with laxative reduced bacterial overgrowth by 92%, and the 16S levels in laxative-treated CF mice were not statistically significant compared with WT mice on laxative (Fig. 2). In contrast, treatment with NAC was less effective than laxative, and bacterial load in NAC-treated CF mice was reduced by 63% (Fig. 2).

View larger version (17K): [in this window] [in a new window]   Fig. 2. Effects of laxative and NAC on bacterial load in small intestines of WT and CF mice. Intestines were removed and flushed with DTT in saline, and the luminal material was extracted to isolate genomic bacterial DNA. DNA was used for quantitative PCR with universal 16S rRNA gene primers. A standard curve was prepared using serial dilutions of a plasmid containing the cloned 16S rRNA gene insert. Bacterial load data are expressed as means ± SE of the copy number of 16S per small intestine. The following numbers of mice were used: Liq, n = 8 WT and 6 CF mice; Lax, n = 9 WT and 6 CF mice; and NAC, n = 6 WT and 5 CF mice.

There was a small reduction in gastric emptying in untreated CF mice raised on the liquid diet compared with WT mice on the same diet, but due to high variability the difference was not statistically significant (Fig. 3). There was a trend to greater gastric emptying in laxative-treated WT mice compared with WT mice on chow with plain drinking water, but the difference was not significant (Fig. 3). When CF mice were treated with laxative, gastric emptying was significantly greater than in liquid-diet raised CF mice, and the degree of gastric emptying was not different compared with laxative-treated WT mice (Fig. 3). Treatment of liquid-fed CF mice with NAC also tended to increase gastric emptying, but the difference was not statistically significant compared with untreated liquid-fed CF mice (Fig. 3).

View larger version (17K): [in this window] [in a new window]   Fig. 3. Effects of laxative and NAC on gastric emptying in WT and CF mice. Mice were fasted overnight, and, between 8 and 9 AM, they were gavaged with a bolus of rhodamine-dextran (see MATERIALS AND METHODS). Twenty minutes later, mice were killed, and fluorescence in the stomach and intestines was measured. Gastric emptying was calculated from the fluorescence remaining in the stomach and total fluorescence recovered. CF mice treated with laxative had significantly greater gastric emptying compared with CF mice on the liquid diet; none of the other comparisons were significantly different. Data are means ± SE. The following numbers of mice were used: chow-fed mice with no treatment (Chow), n = 5 WT mice; Lax, n = 15 WT and 10 CF mice; Liq, n = 13 WT and 10 CF; and NAC, n = 9 WT and 7 CF mice. Values for untreated liquid-fed WT and CF mice include data from a recently published study (14).

View larger version (27K): [in this window] [in a new window]   Fig. 4. Effects of laxative and NAC on small intestinal transit in WT and CF mice. These data are from the same mice used for Fig. 3. See Fig. 5 for geometric center of fluorescence (GCF) calculations and statistical analysis. Data are means ± SE of the fluorescence in each segment relative to the total fluorescence in the small intestine. Values for untreated liquid-fed WT and CF mice include data from a recently published study (14).

View larger version (20K): [in this window] [in a new window]   Fig. 5. Effects of laxative and NAC on small intestinal transit in WT and CF mice. Distributions of rhodamine-dextran fluorescence shown in Fig. 4 were used to calculate the GCF in the small intestine using the following formula: GCF = [(fraction of fluorescence per segment) x (segment number)]. Data are means ± SE. Values for untreated liquid-fed WT and CF mice include data from a recently published study (14).

View larger version (23K): [in this window] [in a new window]   Fig. 6. Effects of laxative and NAC on gene expression in small intestines of WT and CF mice. Total RNA was isolated from the entire small intestine and used as a template for quantitative RT-PCR with the indicated gene-specific primers (Table 1). Data are were normalized to the housekeeping gene ribosomal protein L26 (Rpl26) and are expressed relative to liquid-fed WT values. The following numbers of mice were used: Liq, n = 10 WT and 10 CF mice; Lax, n = 11 WT and 6 CF mice; NAC, n = 6 WT and 5 CF mice. Mcpt2, mast cell protease 2; Lrg1, leucine-rich 2-glycoprotein; Hemt1, hematopoietic cell transcript 1; Retnlb, resistin-like molecule-; Socs3, suppressor of cytokine signaling 3.

	DISCUSSION

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The mechanisms by which laxative and NAC can reduce mucus accumulation are distinct. Laxative delivers a nonabsorbable osmolyte into the gut lumen that causes the retention of ingested fluid as well as an influx of water from the body into the lumen. This increases the hydration state of the luminal contents. Better hydration decreases mucus viscosity and allows easier clearance of it from the intestinal lumen. On the other hand, NAC reduces the viscosity of mucus by breaking disulfide bonds between and within mucin molecules.

Besides reducing the viscosity of mucus, NAC has other actions that potentially might contribute to improving the luminal environment in CF. NAC can serve as a precursor for the cellular antioxidant glutathione, and there is strong evidence that oxidative stress is important in tissue damage in CF (8). A benefit of orally administered NAC in CF was demonstrated in a recent phase 1 clinical trail that showed a decrease in airway neutrophil levels (39). This was believed to be due to the antioxidant activities of NAC. Our mouse study did not find a significant effect on expression of the inflammatory marker genes we measured, so there does not seem to be a benefit from the antioxidant properties of NAC on the CF intestinal phenotype. NAC has also been reported to inhibit sodium absorption by nasal epithelial cells (35), and, if this occurs in the intestine, it would tend to improve the hydration state of the intestinal lumen and may have contributed to the reduction in mucus accumulation in NAC-treated CF mice. The efficacy of NAC on hydration would not appear to be very great as the overall result of NAC administration was limited compared with laxative treatment.

In addition to reducing mucus accumulation and SIBO, a major effect of laxative treatment was a normalization of interdigestive small intestinal transit in CF mice. Improved transit is expected to contribute to the eradication of SIBO. The mechanism by which laxative normalized transit in the CF intestine is not known at present. One possibility could be due to the fact that osmotic laxatives cause distension of the intestinal wall and thereby stimulate motility (12, 25). Laxative-treated mice were given access to the laxative solution during the overnight fast prior to transit measurements, so it is possible that there was enough laxative in the gut to have an effect on transit of the tracer. However, the fluorescent tracer used for all these experiments was prepared in a similar osmotically active compound (methylcellulose), which makes an acute effect of PEG seem unlikely. It is also possible that the gut had become adapted to the laxative treatment, which lasted for at least 5 wk before experiments. Chronic exposure of the gut to the osmotic effects of the laxative could cause adaptation and modification of the interdigestive motility behavior.

A third possibility is that laxative treatment, in addition to a numerical reduction in bacteria, also resulted in normalization of the composition of microflora in the intestine. A more normal composition of the microflora may have a positive effect on interdigestive transit. This is a complex issue because there is a two-way interaction between intestinal flora and interdigestive transit (2). Normal interdigestive transit is known to be a major factor in controlling bacterial load in the small intestine (22). At the same time, the composition of bacteria in the intestine affects intestinal motility by mechanisms not yet well defined. Work in germ-free rats versus those colonized with conventional flora or specific bacterial strains has shown that the microbial ecology of the gut has significant effects on gut behavior (23). Animals that are germ free or colonized only with E. coli have slower small intestinal transit compared with rats colonized a conventional species mixture.

It is important to note that the predominant species that overgrows the CF mouse small intestine on the liquid diet is E. coli, which is expected to contribute to the decreased interdigestive transit (14). Although laxative treatment reduced bacterial load in the CF intestine to levels near the WT intestine, it is not known what bacteria remain in the laxative-treated CF intestine and how the bacterial composition compares with that of WT mice. A culture-based analysis of aerobic bacteria in laxative-treated CF mice did not show marked differences in the composition of bacterial species in their small intestine compared with WT mice (9), so it may be that laxative-treated CF mice have a normal composition of bacterial species. However, in that study, CF mice treated with laxative did have about fivefold greater survival in the terminal ileum of orally administered Salmonella typhimurium, demonstrating that laxative treatment did not fully correct the CF intestine's ability to deal with bacteria. We are in the process of determining the composition of bacterial species in the CF mouse treated with laxative using the inclusive and powerful molecular analysis of 16S rRNA genes. This is expected to be informative as to whether the bacterial species composition is more normal and might explain the positive effect of laxative on interdigestive transit in the CF small intestine.

Modification of the bacterial ecology of the gut may be beneficial in CF. It has been reported that there are increased levels of fecal calprotectin and nitric oxide in rectal dialysis fluid from CF patients, indicating inflammation (7). That study also showed that after administration of the probiotic Lactobacillus GG, these inflammatory parameters were significantly decreased. It will be of interest to see if probiotics can improve motility and the other aspects of the CF intestinal phenotype.

Because laxative treatment had such wide-ranging positive effects on the CF intestinal phenotype, it is difficult to know which aspect of its actions is most important. It is interesting to note that the prokinetic drug cisapride (a 5-HT₄ agonist) has shown improvements in gastrointestinal symptoms in CF patients with intestinal obstruction (26). In a more recent study (4), cisapride was also shown to improve gastrointestinal motility in CF patients. It is possible that correcting interdigestive transit by itself may prove to be sufficient to reduce mucus accumulation and eradicate SIBO in the CF intestine, and this deserves further investigation.

In summary, both laxative and NAC significantly reduced mucus accumulation in the CF intestine. However, only laxative was very effective at reducing SIBO and decreasing the expression of innate immune response-related genes. In addition, laxative treatment also normalized small intestinal transit in the CF mouse. These data suggest that the amount of mucus as well as interdigestive small intestinal transit are involved in controlling bacterial load but that normal transit may be the more important factor.

	GRANTS

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This work was supported by Cystic Fibrosis Foundation Grants DELISL05G0 and DELISL06G0.

	ACKNOWLEDGMENTS

 
We thank Lauren Meldi and Racquel Sewell for managing the mouse colony and Maureen Flynn for help with the morphometric analysis.

	FOOTNOTES

 

Address for reprint requests and other correspondence: R. C. De Lisle, Anatomy and Cell Biology, Univ. of Kansas School of Medicine, Kansas City, KS 66160 (e-mail: rdelisle{at}kumc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

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1. Bali A, Stableforth DE, Asquith P. Prolonged small-intestinal transit time in cystic fibrosis. Br Med J 287: 1011–1013, 1983.[Abstract/Free Full Text]
2. Barbara G, Stanghellini V, Brandi G, Cremon C, Di Nardo G, De Giorgio R, Corinaldesi R. Interactions between commensal bacteria and gut sensorimotor function in health and disease. Am J Gastroenterol 100: 2560–2568, 2005.[CrossRef][Web of Science][Medline]
3. Beharry SA, Ackerely C, Corey M, Kent G, Heng YM, Christensen H, Luk C, Yantiss RK, Nasser IA, Zaman M, Freedman SD, Durie PR. Long-term docosahexaenoic acid therapy in a congenic murine model of cystic fibrosis. Am J Physiol Gastrointest Liver Physiol 292: G839–G848, 2006.[CrossRef][Web of Science][Medline]
4. Bentur L, Hino B, Shamir R, Elias N, Hartman C, Eshach-Adiv O, Berkowitz D. Impaired gastric myolectrical activity in patients with cystic fibrosis. J Cyst Fibros 5: 187–191, 2006.[CrossRef][Web of Science][Medline]
5. Borowitz D, Durie PR, Clarke LL, Werlin SL, Taylor CJ, Semler J, De Lisle RC, Lewindon PJ, Lichtman SM, Sinaasappel M, Baker RD, Baker SS, Verkade HJ, Lowe ME, Stallings VA, Janghorbani M, Heubi J. Gastrointestinal outcomes and confounders in cystic fibrosis. J Pediatr Gastroenterol Nutr 41: 273–285, 2005.[CrossRef][Web of Science][Medline]
6. Bouquet J, Sinaasappel M, Neijens HJ. Malabsorption in cystic fibrosis: mechanisms and treatment. J Pediatr Gastroenterol Nutr 7, Suppl 1: S30–S35, 1988.[Web of Science][Medline]
7. Bruzzese E, Raia V, Gaudiello G, Polito G, Buccigrossi V, Formicola V, Guarino A. Intestinal inflammation is a frequent feature of cystic fibrosis and is reduced by probiotic administration. Aliment Pharmacol Ther 20: 813–819, 2004.[CrossRef][Web of Science][Medline]
8. Cantin AM, White TB, Cross CE, Forman HJ, Sokol RJ, Borowitz D. Antioxidants in cystic fibrosis. Conclusions from the CF Antioxidant Workshop, Bethesda, Maryland, November 11–12, 2003. Free Radic Biol Med 42: 15–31, 2007.[CrossRef][Web of Science][Medline]
9. Clarke LL, Gawenis LR, Bradford EM, Judd LM, Boyle KT, Simpson JE, Shull GE, Tanabe H, Ouellette AJ, Franklin CL, Walker NM. Abnormal Paneth cell granule dissolution and compromised resistance to bacterial colonization in the intestine of CF mice. Am J Physiol Gastrointest Liver Physiol 286: G1050–G1058, 2004.[Abstract/Free Full Text]
10. Clarke LL, Gawenis LR, Franklin CL, Harline MC. Increased survival of CFTR knockout mice with an oral osmotic laxative. Lab Anim Sci 46: 612–618, 1996.[Web of Science][Medline]
11. Cleghorn GJ, Stringer DA, Forstner GG, Durie PR. Treatment of distal intestinal obstruction syndrome in cystic fibrosis with a balanced intestinal lavage solution. Lancet 1: 8–11, 1986.[Web of Science][Medline]
12. Da Cunha MJ, Summers RW, Thompson HH, Wingate DL, Yanda R. Effects of intestinal secretagogues and distension on small bowel myoelectric activity in fasted and fed conscious dogs. J Physiol 321: 483–494, 1981.[Web of Science]
13. Dalzell AM, Freestone NS, Billington D, Heaf DP. Small intestinal permeability and orocaecal transit time in cystic fibrosis. Arch Dis Child 65: 585–588, 1990.[Abstract/Free Full Text]
14. De Lisle RC. Altered transit and bacterial overgrowth in the cystic fibrosis mouse small intestine. Am J Physiol Gastrointest Liver Physiol 293: G104–G111, 2007.[Abstract/Free Full Text]
15. De Lisle RC, Roach EA, Norkina O. Eradication of small intestinal bacterial overgrowth in the cystic fibrosis mouse reduces mucus accumulation. J Pediatr Gastroenterol Nutr 42: 46–52, 2006.[CrossRef][Web of Science][Medline]
16. Eckman EA, Cotton CU, Kube DM, Davis PB. Dietary changes improve survival of CFTR S489X homozygous mutant mouse. Am J Physiol Lung Cell Mol Physiol 269: L625–L630, 1995.[Abstract/Free Full Text]
17. Emil S, Nguyen T, Sills J, Padilla G. Meconium obstruction in extremely low-birth-weight neonates: guidelines for diagnosis and management. J Pediatr Surg 39: 731–737, 2004.[CrossRef][Web of Science][Medline]
18. Fridge JL, Conrad C, Gerson L, Castillo RO, Cox K. Risk factors for small bowel bacterial overgrowth in cystic fibrosis. J Pediatr Gastroenterol Nutr 44: 212–218, 2007.[Web of Science][Medline]
19. Gregory PC. Gastrointestinal pH, motility/transit and permeability in cystic fibrosis. J Pediatr Gastroenterol Nutr 23: 513–523, 1996.[CrossRef][Web of Science][Medline]
20. Haston CK, Corey M, Tsui LC. Mapping of genetic factors influencing the weight of cystic fibrosis knockout mice. Mamm Genome 13: 614–618, 2002.[CrossRef][Web of Science][Medline]
21. Hinojosa-Kurtzberg AM, Johansson MEV, Madsen CS, Hansson GC, Gendler SJ. Novel MUC1 splice variants contribute to mucin overexpression in CFTR-deficient mice. Am J Physiol Gastrointest Liver Physiol 284: G853–G862, 2003.[Abstract/Free Full Text]
22. Husebye E. The pathogenesis of gastrointestinal bacterial overgrowth. Chemotherapy 51, Suppl 1: 1–22, 2005.[Web of Science][Medline]
23. Husebye E, Hellstrom PM, Sundler F, Chen J, Midtvedt T. Influence of microbial species on small intestinal myoelectric activity and transit in germ-free rats. Am J Physiol Gastrointest Liver Physiol 280: G368–G380, 2001.[Abstract/Free Full Text]
24. Huynh HQ, Couper RTL, Tran CD, Moore L, Kelso R, Butler RN. N-acetylcysteine, a novel treatment for Helicobacter pylori infection. Dig Dis Sci 49: 1853–1861, 2004.[CrossRef][Web of Science][Medline]
25. Knowles CH, Martin JE. Slow transit constipation: a model of human gut dysmotility. Review of possible aetiologies. Neurogastroenterol Motil 12: 181–196, 2000.[CrossRef][Web of Science][Medline]
26. Koletzko S, Corey M, Ellis L, Spino M, Stringer DA, Durie PR. Effects of cisapride in patients with cystic fibrosis and distal intestinal obstruction syndrome. J Pediatr 117: 815–822, 1990.[Web of Science][Medline]
27. Lewindon PJ, Robb TA, Moore DJ, Davidson GP, Martin AJ. Bowel dysfunction in cystic fibrosis: importance of breath testing. J Paediatr Child Health 34: 79–82, 1998.[CrossRef][Web of Science][Medline]
28. Liu W, Saint DA. A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics. Anal Biochem 302: 52–59, 2002.[CrossRef][Web of Science][Medline]
29. Malmberg EK, Noaksson KA, Phillipson M, Johansson ME, Hinojosa-Kurtzberg M, Holm L, Gendler SJ, Hansson GC. Increased levels of mucins in the cystic fibrosis mouse small intestine and modulator effects of the Muc1 mucin expression. Am J Physiol Gastrointest Liver Physiol 291: G203–G210, 2006.[Abstract/Free Full Text]
30. Mascarenhas MR, Mondick JT, Barrett JS, Zhuang H, Edwards K, Schall JI. Comparison of gastric emptying and small bowel transit in subjects with CF and healthy controls (Abstract). Pediatr Pulmonol Suppl 29: 377, 2006.
31. Nadkarni MA, Martin FE, Jacques NA, Hunter N. Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology 148: 257–266, 2002.[Abstract/Free Full Text]
32. Norkina O, Burnett TG, De Lisle RC. Bacterial overgrowth in the cystic fibrosis transmembrane conductance regulator null mouse small intestine. Infect Immun 72: 6040–6049, 2004.[Abstract/Free Full Text]
33. Norkina O, Kaur S, Ziemer D, De Lisle RC. Inflammation of the cystic fibrosis mouse small intestine. Am J Physiol Gastrointest Liver Physiol 286: G1032–G1041, 2004.[Abstract/Free Full Text]
34. Quigley EM, Quera R. Small intestinal bacterial overgrowth: roles of antibiotics, prebiotics, and probiotics. Gastroenterology 130: S78–S90, 2006.[CrossRef][Web of Science][Medline]
35. Rochat T, Lacroix JS, Jornot L. N-acetylcysteine inhibits Na⁺ absorption across human nasal epithelial cells. J Cell Physiol 201: 106–116, 2004.[CrossRef][Web of Science][Medline]
36. Sonnenburg JL, Angenent LT, Gordon JI. Getting a grip on things: how do communities of bacterial symbionts become established in our intestine? Nat Immun 5: 569–573, 2004.[CrossRef][Web of Science]
37. Sun F, Tai S, Lim T, Baumann U, King M. Additive effect of dornase alfa and Nacystelyn on transportability and viscoelasticity of cystic fibrosis sputum. Can Respir J 9: 401–406, 2002.[Medline]
38. Thomsson KA, Hinojosa-Kurtzberg M, Axelsson KA, Domino SE, Lowe JB, Gendler SJ, Hansson GC. Intestinal mucins from cystic fibrosis mice show increased fucosylation due to an induced fucalpha1-2 glycosyltransferase. Biochem J 367: 609–616, 2002.[CrossRef][Web of Science][Medline]
39. Tirouvanziam R, Conrad CK, Bottiglieri T, Herzenberg LA, Moss RB, Herzenberg LA. High-dose oral N-acetylcysteine, a glutathione prodrug, modulates inflammation in cystic fibrosis. Proc Natl Acad Sci USA 103: 4628–4633, 2006.[Abstract/Free Full Text]
40. Wesley A, Forstner J, Qureshi R, Mantle M, Forstner G. Human intestinal mucin in cystic fibrosis. Pediatr Res 17: 65–69, 1983.[Web of Science][Medline]

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