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Am J Physiol Gastrointest Liver Physiol 294: G295-G306, 2008. First published November 21, 2007; doi:10.1152/ajpgi.00173.2007
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INFLAMMATION/IMMUNITY/MEDIATORS

Saccharomyces boulardii ameliorates Citrobacter rodentium-induced colitis through actions on bacterial virulence factors

X. Wu,1,2 B. A. Vallance,1,2 L. Boyer,1,2 K. S. B. Bergstrom,1,2 J. Walker,3 K. Madsen,3 J. R. O'Kusky,4 A. M. Buchan,5 and K. Jacobson1,2,5

1Children and Family Research Institute and 2Division of Gastroenterology, BC Children's Hospital, Vancouver, British Columbia; 3Department of Medicine, University of Alberta, Edmonton, Alberta; and 4Department of Pathology and Laboratory Medicine and 5Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada

Submitted 18 April 2007 ; accepted in final form 10 November 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Saccharomyces boulardii has received increasing attention as a probiotic effective in the prevention and treatment of infectious and inflammatory bowel diseases. The aim of this study was to examine the ameliorating effects of S. boulardii on Citrobacter rodentium colitis in vivo and identify potential mechanisms of action. C57BL/6 mice received 2.5 x 108 C. rodentium by gavage on day 0, followed by S. boulardii (25 mg; 5 x 108 live cells) gavaged twice daily from day 2 to day 9. Animal weights were monitored until death on day 10. Colons were removed and assessed for epithelial barrier function, histology, and myeloperoxidase activity. Bacterial epithelial attachment and type III secreted proteins translocated intimin receptor Tir (the receptor for bacterial intimin) and EspB (a translocation apparatus protein) required for bacterial virulence were assayed. In infected mice, S. boulardii treatment significantly attenuated weight loss, ameliorated crypt hyperplasia (234.7 ± 7.2 vs. 297.8 ± 17.6 µm) and histological damage score (0.67 ± 0.67 vs. 4.75 ± 0.75), reduced myeloperoxidase activity (2.1 ± 0.4 vs. 4.7 ± 0.9 U/mg), and attenuated increased mannitol flux (17.2 ± 5.0 vs. 31.2 ± 8.2 nm·cm–2·h–1). The ameliorating effects of S. boulardii were associated with significantly reduced numbers of mucosal adherent C. rodentium, a marked reduction in Tir protein secretion and translocation into mouse colonocytes, and a striking reduction in EspB expression and secretion. We conclude that S. boulardii maintained colonic epithelial barrier integrity and ameliorated inflammatory sequelae associated with C. rodentium infection by attenuating C. rodentium adherence to host epithelial cells through putative actions on the type III secretion system.

infectious colitis; probiotics; bacterial adherence; Tir; EspB

CITROBACTER RODENTIUM is a gram-negative bacterium that colonizes the colons of mice, causing attaching/effacing (A/E) lesions and colonic hyperplasia (1, 42, 43, 51, 53). The lesions produced at the epithelial surface are indistinguishable from those caused by enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) infections, which are among the leading bacterial causes of human diarrheal diseases worldwide (57). While EPEC leads to the deaths of thousands of children in developing countries (14), EHEC is responsible for sporadic diarrheal outbreaks and deaths in North America and Europe (35). C. rodentium, in addition to serving as a small-animal model for EPEC and EHEC infection, can also serve as a murine model for inflammatory bowel disease (32, 33).

C. rodentium, like EPEC and EHEC, attaches to luminal surfaces of host intestinal epithelial cells, effacing localized regions of microvilli and injecting bacterial effector proteins into the host cell via their locus of enterocyte effacement (LEE)-encoded syringelike type III secretion system (TTSS) (21, 23). Translocator proteins including the E. coli secreted proteins EspA, EspB, and EspD are responsible for the assembly of a translocation conduit in the host cell membrane that assists in the translocation of effector proteins into host cells (21, 41). Effector proteins, including the translocated intimin receptor Tir, which acts as the receptor for the bacterial outer membrane adhesin intimin, anchor the bacteria to host cells, modulate host cell functions, induce cytoskeletal rearrangements, and facilitate bacterial proliferation and disease development (9, 20, 28, 40, 48). In C. rodentium infection, Tir and intimin have been shown to be essential virulence factors (23, 25, 32), while EspB has been shown to be an important colonization factor (46) and component of the translocation apparatus necessary in translocation activity (41, 62). Furthermore, LEE-encoded SepL and SepD have been shown to regulate TTSS hierarchy of translocators and effectors, where SepL and SepD are not only necessary for efficient translocator secretion but also control a switch from translocator to effector secretion in response to environmental signals (21, 22). The ability of A/E pathogens to secrete LEE-encoded proteins into culture medium by using the LEE-encoded TTSS provides an excellent opportunity to examine the impact of potential therapies on these virulence factors (22).

C. rodentium, in addition to providing an excellent in vivo model to investigate pathogen-host interactions under physiological conditions, also provides an opportunity to evaluate therapeutic interventions. Probiotics, which are nonpathogenic live microorganisms that promote favorable effects on health by altering indigenous microflora, have been shown to be efficacious in C. rodentium infection. To date, in vivo probiotic studies in C. rodentium infection have focused on lactobacillus strains and their impact on host epithelial cell and inflammatory responses (13, 34, 60). Pretreatment of mice in early life (13), in young adulthood (34, 60), or concurrently with inoculation of C. rodentium (13) has been shown to effectively alter host regulatory immune responses, leading to amelioration of epithelial damage and attenuation of C. rodentium-induced colitis. While a fine balance exists between the effects of probiotics on attenuation of the host's mucosal proinflammatory responses and the induction of intestinal mucosal hyporesponsiveness to pathogenic determinants, a potential concern remains as to whether such modulation can result in adverse consequences such as delayed pathogen clearance. Thus probiotics that target pathogen virulence should also be considered.

Some studies suggest that probiotics modulate pathogen-host epithelial cell interactions through antibacterial actions (3, 59), through competitive binding to sites on epithelial cells (54), through bacterial adherence to the probiotic (27), or through degradation of bacterial toxins (11, 12). While such studies suggest beneficial effects through a reduction in pathogen-host interaction, it remains unclear whether such effects have potential benefit over the potential risks associated with suppression of the host's mucosal immune response.

Saccharomyces boulardii is a thermophilic nonpathogenic yeast that has been used in vivo for both prevention and treatment of infectious and inflammatory intestinal diseases (5, 29, 30, 37, 45). The benefits of S. boulardii are strengthened by its ability to rapidly achieve high concentrations in the gastrointestinal tract after oral administration and to be maintained at stable levels in a viable form. Moreover, S. boulardii does not permanently colonize the colon and does not usually translocate across the intestinal mucosa (2, 4, 6). Several in vitro studies using S. boulardii have shown protective effects against EHEC and EPEC infection through various mechanisms including bacterial adherence to yeast via adhesions (27), preservation of epithelial barrier function, and modulation of signal transduction pathways (17, 18, 20, 55). Interestingly, while beneficial effects were provided in these models, the studies have predominantly focused on host cellular responses, whereas the potential impact of S. boulardii on virulence of these pathogens has received limited attention. Consequently, the aim of the present study was to examine the efficacy of S. boulardii in ameliorating the intestinal disease associated with C. rodentium infection in vivo, and to determine whether its ameliorating effects are due in part to its actions on pathogenic determinants of the enteric pathogen.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice. Six- to eight-week-old C57BL/6 mice weighing ~18–20 g were purchased from Charles River Laboratories (St. Constant, QC, Canada) and were maintained on standard laboratory chow and tap water ad libitum. All protocols were approved by the Animal Research Ethics Boards at the University of British Columbia.

Bacterial strains and infection of mice. Mice were orally inoculated with wild-type C. rodentium (formerly C. freundii biotype 4280), strain DBS100 (50). For inoculations, bacteria were grown overnight in Luria broth (LB). Mice were infected by oral gavage with 100 µl of LB containing 2.5 x 108 colony-forming units (CFU) of C. rodentium. Control mice received 100 µl of fresh LB by oral gavage. The mice were killed on postinfection (p.i.) day 10. Day 10 p.i. was chosen because at this time the infection is well established and colitis is evident (43). For the in vitro studies, the {Delta}escN mutant strain of C. rodentium (type III apparatus mutant that does not secrete Tir or EspB) was generated as previously described (22) and included as a negative control for assessment of these virulence factors.

Probiotic treatment in vivo. S. boulardii was supplied in lyophilized form by MFI Pharma (Richmond Hill, ON, Canada). Each capsule contained 250 mg of viable S. boulardii cells (~5 x 109). Mice received 100 µl of 250 mg/ml S. boulardii in saline by oral gavage twice daily from day 2 until death on day 10. The dose chosen was in keeping with doses used previously in murine models (8, 10, 24). Control mice received saline by oral gavage.

Preparation of S. boulardii culture supernatant. S. boulardii was first cultured in Sabouraud dextrose (SD) broth (100 mg/ml) overnight at 37°C. The yeast culture was then centrifuged at 5,000 rpm for 30 min at 4°C, and the supernatant was collected. The supernatant was then passed twice through a 0.2-µm-pore size filter to remove the yeast cells. The supernatant was included to ascertain whether the in vitro actions of S. boulardii were mediated through a secreted factor(s).

Assessment of colonic response to infection. Mice were weighed every second day and killed on day 10 p.i. The colons were removed and examined for total and mucosal associated C. rodentium (n = 12/group), histological damage score, crypt heights, goblet cell response, myeloperoxidase (MPO) activity (n = 6/group), and epithelial ion transport and permeability (n = 12/group).

Histological analysis: damage score and crypt height. Colons were excised, and distal colonic segments (0.5 cm) were removed, fixed with 10% neutral buffered formalin for 24 h, and then transferred to 70% ethanol. Fixed tissues were embedded in paraffin. Cross sections of the colon (5 µm) were cut and mounted on slides. Tissue sections were stained with hematoxylin and eosin. Cell morphology was observed under light microscopy. Histological damage scoring was determined with criteria adapted from Galeazzi et al. (26). Briefly, the damage score consisted of a score for the severity of epithelial injury (graded 0–3, from absent to mild including superficial epithelial injury, moderate including focal erosions, and severe including multifocal erosions), the extent of inflammatory cell infiltrate (graded 0–3, from absent to transmural), and goblet cell depletion (0–1). In each case a numerical score was assigned. Three tissue sections from each animal were coded and examined by two blinded observers to prevent observer bias. Tissue sections were assessed (each separated by at least 500 µm) under a Nikon Eclipse 400 light microscope and averaged to obtain a mean histological damage score. Crypt heights were measured by micrometry, with 10 measurements taken in distal colon sections of each mouse. Only well-oriented crypts and tissue sections with intact muscularis propria were measured.

Myeloperoxidase activity. The activity of MPO, an enzyme produced by neutrophils, was measured. Distal colonic segments adjacent to tissues retrieved for histological damage were excised, snap-frozen in liquid nitrogen, and stored at –80°C. MPO activity was measured within 7 days by a previously described method (7). Briefly, tissues were homogenized in hexadecyltrimethylammonium bromide buffer (Sigma) and centrifuged. The supernatant was added to a solution containing o-dianisidine (Sigma) and hydrogen peroxide. The absorbance of the colorimetric reaction was measured by a spectrophotometer. MPO is expressed in units per milligram of wet tissue, with 1 unit being the quantity of enzyme able to convert 1 µmol of hydrogen peroxide to water in 1 min at room temperature.

Epithelial function. Colonic epithelial barrier function was measured in accordance with well-established protocols (15, 44). Mice were anesthetized with halothane and then killed by cervical dislocation. Distal colonic segments were removed and mounted in Lucite chambers exposing mucosal and serosal surfaces to 10 ml of oxygenated Krebs buffer (in mM: 115 NaCl, 8 KCl, 1.25 CaCl2, 1.2 MgCl2, 2 KH2PO4, 225 NaHCO3; pH 7.35). The buffer was maintained at 37°C by a heated water jacket and circulated with CO2-O2. Fructose (10 mM) was added to the serosal and mucosal surfaces. For measurement of basal mannitol fluxes, 1 mM mannitol with 10 µCi of 3H was added to the mucosal side and samples of the buffer from both the serosal and mucosal sides were taken at 5-min intervals. The flux was then calculated with a published protocol (44).

Spontaneous transepithelial potential difference (PD) was determined, and the tissue was clamped at zero voltage by continuously introducing an appropriate short-circuit current (Isc) with an automatic voltage clamp (DVC 1000, World Precision Instruments, New Haven, CT), except for 5–10 s every 5 min when PD was measured by removing the voltage clamp. Stimulated increase in Isc was induced by addition of the adenylate cyclase-activating agent forskolin (10–5 M) to the serosal surface. Epithelial responsiveness was defined as the maximal increase in Isc to occur within 5 min of exposure to secretagogue.

Transmission electron microscopy. Segments of colon, measuring 6–8 mm in length, were fixed by immersion in a fixative solution containing 4% paraformaldehyde (PFA) and 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C for 24–48 h. Tissue blocks were washed in 0.1 M phosphate buffer and postfixed for 12 h in 1% buffered osmium tetroxide. After being washed in acetate buffer for 1 h, the blocks were stained with 2% uranyl acetate for 12 h, followed by dehydration in ascending grades of ethanol, equilibration in propylene oxide, and embedding in Epon (Jembed 812, Canemco, Montreal, QC, Canada). Semithin sections were cut at right angles to the long axis of the intestine at a thickness of 0.7 µm, mounted on glass slides, and stained with 1% toluidine blue in 0.4% sodium borate. After the blocks were trimmed, ultrathin sections were cut at a thickness of 60–65 nm, mounted on Formvar-coated slot grids, and stained with lead citrate. Sections were examined with a Morgagni transmission electron microscope (FEI).

Bacterial counts. Colonic tissues including luminal contents (total bacterial counts) and colonic tissues after colonic contents were vigorously flushed away with sterile phosphate-buffered saline (PBS) (mucosal associated bacterial counts) were homogenized in 1.5 ml of sterile PBS at low speed with a Kinematica tissue homogenizer (Brinkmann). Homogenates were then serially diluted and plated onto MacConkey agar plates selective for gram-negative organisms (PML Microbiologicals). Bacterial colonies were enumerated after overnight incubation at 37°C. C. rodentium colonies were easily distinguished from colonies derived from commensal flora by their size and appearance. The validity of this approach was previously verified by PCR analysis for LEE genes (58). Bacterial counts are reported as colony-forming units per gram.

Western blot analysis of Tir in colonic tissues. To investigate the effect of S. boulardii on bacterial attachment to epithelial cells, Western blot analysis of Tir, an essential virulence factor needed for bacterial attachment, was performed (23). Briefly, colons were opened and stool pellets gently removed, followed by vigorous washing with cold PBS to remove nonadherent bacteria and homogenization with lysis buffer [in mM: 150 NaCl, 20 Tris pH 7.5, 1 EDTA, 2.5 sodium pyrophosphate, 1 β-glycerophosphate, 1 phenylmethylsulfonyl fluoride (PMSF), 1 sodium orthovanadate, and 1 sodium fluoride, with 1% Triton X-100, 1% mammalian protease inhibitor cocktail (Sigma), and 1% bacterial protease inhibitor cocktail (Sigma)]. Thirty micrograms of each protein sample was loaded and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after transfer to nitrocellulose and blocking in a 5% solution of dried skim milk. Membranes were then probed with rat polyclonal antibody against His-tagged Citrobacter Tir. Primary antibodies were probed with goat anti-rat horseradish peroxidase-conjugated IgG (Santa Cruz Biotechnology) for 60 min at room temperature. The presence of antibodies was assessed by an enhanced chemiluminescence detection system (Amersham, Little Chalfont, UK). The intensity of bands was quantified with NIH Scion Image software.

Immunofluorescence staining of LPS and translocated Tir in colonic tissues. To further examine the association between bacterial attachment to epithelial cells and Tir translocation, immunofluorescence staining of colonic tissues was performed with standard techniques as described previously (23). Briefly, colonic tissues were rinsed with ice-cold PBS, embedded in Shandon cryomatrix (Thermo Electron, Pittsburgh, PA), frozen with isopentane (Sigma) and liquid N2, and stored at –80°C. Serial sections were cut at a thickness of 6 µm and fixed in 4% PFA for 15 min at room temperature. Tissue sections were directly blocked with 1% bovine serum albumin for 1 h at room temperature, followed by the addition of the rat polyclonal anti-His-tagged Citrobacter Tir sera (kindly provided by Dr. W. Deng, Biotechnology Laboratory, University of British Columbia, Vancouver, BC, Canada) as well as the rabbit polyclonal anti-LPS (E. coli Poly8, Biotech Laboratories) and incubated at 4°C for 4 h. After extensive washing with PBS, Alexa Fluor 568-conjugated goat anti-rat IgG (Molecular Probes, Eugene, OR) as well as Alexa Fluor 488-conjugated goat-anti-rabbit IgG (Molecular Probes) were added and incubated for 1 h at room temperature. The tissues were again washed and mounted with ProLong Gold Antifade (Invitrogen) mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) for staining host cell DNA. Coverslips were viewed at 350, 488, and 594 nm on a Zeiss AxioImager microscope. Images were obtained with a Zeiss AxioImager microscope equipped with an AxioCam HRm camera operating through AxioVision software (version 4.4).

Western blot analysis of total and secreted proteins from C. rodentium in vitro. To investigate the effect of S. boulardii on secretion of type III proteins by C. rodentium, total and secreted Tir and EspB proteins were assessed as described previously (23) but modified with the addition of S. boulardii. Briefly, C. rodentium strain DBS100 was grown overnight in LB at 37°C. Eighty microliters of C. rodentium culture was cocultured with 80 µl of 100 mg/ml viable S. boulardii or S. boulardii supernatant in 4 ml of plain DMEM (preincubated overnight in a 5% CO2 incubator) to a 1:50 dilution. Cocultures were incubated in upright test tubes in a tissue culture incubator for 6 h to an optical density at 600 nm (OD600) of >0.7. Cocultures were then centrifuged at 13,000 rpm. for 20 min at 4°C to remove bacteria, and the supernatant was precipitated with 10% trichloroacetic acid (TCA) to concentrate proteins secreted into the culture medium. The remaining bacteria pellets were dissolved in 2x SDS-PAGE buffer and designated as total bacterial proteins. The secreted proteins precipitated from the supernatant were also dissolved in 2x SDS-PAGE buffer, and the residual TCA was neutralized with 1 µl of saturated Tris. The volumes of buffer used to resuspend bacterial pellets and secreted proteins were normalized to the OD600 of the subcultures (when cultured with S. boulardii supernatant) or to bacterial numbers counted by the MacConkey agar plate method described above (when cultured with S. boulardii) to ensure equal loading of samples. The proteins were analyzed in 10% SDS-PAGE and processed for Western blotting analysis with rat polyclonal antibodies against the His-tagged Citrobacter Tir and mouse monoclonal antibody against Citrobacter EspB (kindly provided by Dr. W. Deng).

RT-PCR and quantitative real-time PCR analysis of expression of tir and espb genes. The effect of S. boulardii on the expression of tir and espb genes in C. rodentium was assessed by RT-PCR as described previously (21) and by real-time PCR analysis (16, 38). C. rodentium was treated similarly to the growth conditions described above for total and secreted protein assays. After induction in DMEM in the absence or presence of S. boulardii or its supernatant for 6 h in a 5% CO2 tissue culture incubator, bacterial cultures were centrifuged and the bacterial pellet used for isolating total RNA. Bacterial RNA was stabilized with RNAprotect Bacteria Reagent (Qiagen). Bacteria were lysed with lysozyme (Sigma) in Tris-EDTA (TE) buffer to exclude interference with S. boulardii, and total RNA was extracted with the RNeasy mini kit (Qiagen) according to the manufacturer's instructions. The RNA samples were treated with DNase I (1 U of DNase I per 1 µg of RNA) to eliminate any contaminating DNA as described previously (38). Equal amounts of the total RNA (1 µg) for each sample were then used for reverse transcription (RT) with Superscript reverse transcriptase and random primers from Invitrogen according to the supplier's instructions. No contamination by genomic DNA was detectable by RT-PCR and real-time PCR for Tir and EspB after DNase I treatment performed with two negative (no RT and no cDNA) control reactions (data not shown). Real-time PCR was validated by comparing Tir expression by C. rodentium grown in LB and DMEM media as described previously (38), with a 4.5-fold increase in DMEM over that observed in LB medium.

Quantitative real-time PCR was performed with IQ SYBR Green Supermix (Bio-Rad) and a Bio-Rad MJ Mini-Opticon Real-Time PCR System (Bio-Rad). Primers were designed by Primer3 (version 4.0) and tested for degeneracy with the BLAST program. The primers used were EspB: forward 5'-AAACTGATGCGTGAGATGGTC-3' and reverse 5'-CTTCAGAGGCGGTATTGACAG-3'; Tir: forward 5'-GCCGACAGAACAGACAATAGC-3' and reverse 5'-ACATCCAACCTTCAGCATACG-3'; and 16S forward 5'-aggccttcgggttgtaaagt-3' and reverse 5'-gactcaagcctgccagtttc-3'. Cycling conditions were as follows: denaturation for 5 min at 95°C and amplification for 40 cycles at 95°C for 30 s, 60°C for 30 s, and at 72°C for 40 s. After completion of the cycling process, samples were subjected to a temperature ramp (from 53 to 95°C) with continuous fluorescence monitoring for melting curve analysis. For each PCR product, a single narrow peak was obtained by melting curve analysis at the specific melting temperature, indicating specific amplifications. Primer pair efficiency was tested by looking at how {Delta}Ct [the difference between the 2 threshold cycle (Ct) values of 2 PCRs for the same template amount] varied with template dilution, as suggested by the manufacturer's instruction guide. Quantification was carried out with Gene Ex Macro OM 3.0 software (Bio-Rad) where PCR efficiencies for each of the primer sets were incorporated into the final calculation. The {Delta}{Delta}Ct method was used to calculate the relative amount of specific RNA present in a sample, from which the fold induction of transcription of the gene was estimated by comparison to values relative to the wild-type strain grown in DMEM medium. Data are expressed as means ± SD.

Statistical analysis. Results are expressed as means ± SE of separate experiments, except for real-time PCR analysis, where results are expressed as means ± SD. Analyses were conducted with Graph Pad Prism 4 statistical software for Windows (GraphPad Software, San Diego, CA). Differences between means were calculated by one-way analysis of variance or paired t-tests where appropriate. Specific differences were tested with the Student-Neuman-Keuls test. A P value <0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
S. boulardii treatment ameliorates C. rodentium-induced morbidity. C. rodentium-infected mice showed overt clinical signs of infection, including a hunched posture, rapid loss of body weight, and defecation of soft stool. Significant weight loss was evident by day 2 p.i. (P < 0.004, Fig. 1), with weights reaching their lowest levels by day 4 p.i. (P < 0.001, Fig. 1). Thereafter, animal weights increased but still remained significantly below control levels at day 10 p.i. (P = 0.001). Treatment with S. boulardii abrogated most clinical signs of infection and significantly attenuated the weight loss such that on day 4 p.i. animals demonstrated a weight reduction of only 13.2% compared with 31.5% in infected untreated mice (Fig. 1, P < 0.01). Moreover, weight recovery was significantly enhanced in S. boulardii-treated animals, with levels increasing more rapidly, resulting in weights that were not significantly different from those in control animals by day 8 p.i. In contrast, infected untreated animals demonstrated poor weight recovery, with significant weight differences still evident on day 10 p.i. compared with control (P < 0.01) and S. boulardii treated (P < 0.01) animals. Weight gain in mice given S. boulardii alone paralleled that seen in control animals.


Figure 1
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  		Fig. 1. Effect of Citrobacter rodentium infection and Saccharomyces boulardii treatment on body weight. Body weights were monitored every other day until death on day 10 in noninfected control animals (CTL), in animals that received S. boulardii (Sb), in C. rodentium-infected animals (CR), and in C. rodentium-infected animals treated with S. boulardii (CR+Sb). Compared with noninfected control animals, C. rodentium-infected mice demonstrated significant weight loss by day 2 postinfection (p.i.), with weights decreasing further to reach their lowest levels at day 4 p.i. and increasing thereafter but remaining significantly lower at day 10 p.i. Treatment with S. boulardii in C. rodentium-infected animals abrogated weight loss such that weights were significantly higher than those observed in C. rodentium-infected animals by day 4 p.i. By day 8 p.i. body weight had returned to levels that were not significantly different from those in control animals. S. boulardii treatment in control animals had no adverse effects on weight gain. Results are expressed as means ± SE for 12 animals/group. *P < 0.05, **P < 0.004 compared with control; +P < 0.01 compared with S. boulardii-treated C. rodentium-infected animals.

 
S. boulardii treatment ameliorates C. rodentium-induced colitis. In keeping with previous studies (1, 43), C. rodentium infection on day 10 p.i. was associated with superficial epithelial damage, goblet cell depletion, colonic crypt hyperplasia, and the presence of a mild transmural inflammatory infiltrate comprised of neutrophils and mononuclear cells (Fig. 2 and Fig. 3, A and B). These histological changes were more marked in the distal than proximal colon (data not shown), consistent with the predominance of bacterial colonization in the distal colon (58, 61).


Figure 2
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  		Fig. 2. Histological appearance of tissue sections for noninfected control (A), S. boulardii-treated (B), and C. rodentium-infected (C) animals and S. boulardii treatment in C. rodentium-infected animal (D). A: normal histological appearance. B: normal histological appearance. C: superficial epithelial damage, colonic crypt hyperplasia and goblet cell depletion, thickening of the muscularis propria, and mild transmural infiltration of neutrophils and mononuclear cells. D: evidence of a well-organized and intact epithelial cell layer, presence of goblet cells, absence of crypt hyperplasia and thickening of the muscularis propria, and no increase in inflammatory cells.

 

Figure 3
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  		Fig. 3. A: histological damage score measured on day 10 in noninfected control animals (CTL), in animals that received S. boulardii alone (Sb), in C. rodentium-infected animals (CR), and in C. rodentium-infected animals treated with S. boulardii (CR+Sb). C. rodentium infection was associated with a significant increase in histological damage score compared with control. S. boulardii treatment in C. rodentium-infected animals significantly attenuated the increase in damage score such that the damage score was not significantly different from control levels. S. boulardii treatment alone was not associated with a significant change in damage score. Results are expressed as means ± SE for 6 animals/group. **P < 0.001 compared with control; ++P < 0.001 compared with C. rodentium-infected animals. B: myeloperoxidase (MPO) activity measured on day 10. C. rodentium infection was associated with a significant increase in MPO activity compared with control. S. boulardii treatment in C. rodentium-infected animals significantly attenuated the increase in MPO activity compared with C. rodentium-infected animals; however, MPO activity remained significantly elevated above control levels. S. boulardii treatment alone was not associated with a significant change in MPO activity. Results are expressed as means ± SE for 6 animals/group. **P < 0.001 compared with control; ++P < 0.001 compared with C. rodentium-infected and control animals. C: crypt height measured on day 10. C. rodentium infection was associated with a significant increase in crypt height compared with control animals. S. boulardii treatment in C. rodentium-infected animals significantly attenuated the increase in crypt height; however, crypt height remained significantly elevated above control levels. S. boulardii treatment alone was not associated with a significant change in crypt height. Results are expressed as means ± SE for 6 animals/group. *P < 0.05, **P < 0.01 compared with control; +P < 0.05 compared with C. rodentium-infected animals.

 
S. boulardii treatment attenuated the colitic response associated with C. rodentium infection. S. boulardii treatment ameliorated infection-induced epithelial damage and reduced transmural infiltration of neutrophils and lymphocytes (Fig. 2, C and D). Where C. rodentium infection was associated with a marked increase in histological damage score (P < 0.001 compared with control, Fig. 3A), S. boulardii treatment significantly attenuated the damage score to a level that was not significantly different from that in control animals. Moreover, the 47-fold increase in MPO activity observed with C. rodentium infection (P < 0.001 compared with control, Fig. 3B) was significantly abrogated by S. boulardii treatment (P < 0.001); however, MPO levels remained significantly elevated above control values (22-fold increase, P < 0.01). In contrast to the typical goblet cell distribution along the length of colonic crypts, infected tissues displayed only a few mature goblet cells scattered predominantly at the surface of crypts, whereas S. boulardii treatment abrogated goblet cell depletion, resulting in preservation of goblet cells with the majority having a well-differentiated phenotype and containing abundant mucus (data not shown). In addition, S. boulardii treatment significantly attenuated C. rodentium-associated crypt hyperplasia (P < 0.01, Fig. 3C); however, crypt lengths still remained significantly elevated above control levels (P < 0.05). Notably, on its own, S. boulardii had no demonstrable effects on any of the parameters assessed (Figs. 2B and 3). Together, these data demonstrated that oral treatment with S. boulardii significantly attenuated C. rodentium-induced colitis.

S. boulardii treatment attenuates epithelial pathophysiology. Considering that A/E pathogens cause diarrhea, we sought to assess colonocyte function during infection and the impact of S. boulardii on the pathophysiology. Infected mice showed significant increases in unidirectional mannitol fluxes compared with control mice (P < 0.01, Fig. 4A). In contrast, treatment with S. boulardii prevented the infection-induced increase in mannitol fluxes, resulting in levels that were not significantly different from those in control mice (Fig. 4A). Similarly, the increase in mannitol flux observed with infection in the proximal colon was attenuated by treatment with S. boulardii (data not shown). S. boulardii treatment on its own had no effect on mannitol fluxes.


Figure 4
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  		Fig. 4. Colonic barrier function and electrical parameters in noninfected control animals (CTL), S. boulardii-treated animals (Sb), C. rodentium-infected animals (CR), and C. rodentium-infected animals treated with S. boulardii (CR+Sb). C. rodentium infection was associated with a significant increase in unidirectional mannitol flux (A) and a significant reduction in short-circuit current (Isc; B) and {Delta}Isc response to forskolin (C). Treatment with S. boulardii in C. rodentium-infected animals prevented the increase in unidirectional mannitol flux (A) and the reduction in Isc (B) and {Delta}Isc response to forskolin (C) such that these parameters were not significantly different from control. S. boulardii treatment alone resulted in a trend toward increased basal Isc (B) and {Delta}Isc response to forskolin (C). Results are expressed as means ± SE for 12 animals/group. *P < 0.05, **P < 0.01 compared with control animals; +P < 0.05 compared with C. rodentium-infected animals.

 
Additionally, C. rodentium caused a significant reduction in basal Isc (P < 0.05, Fig. 4B), a trend toward a decrease in PD (3.55 ± 0.24 mV infected vs. 4.24 ± 0.61 mV control animals), and a significant 37% reduction in Isc response to forskolin (P < 0.05, Fig. 4C), suggesting impairment in adenosine 3',5'-cyclic monophosphate-dependent active chloride secretion and significant impairment in physiological function. Treatment with S. boulardii prevented infection-induced alteration in colonic ionic transport function such that baseline Isc and {Delta}Isc response to forskolin remained at levels similar to those in control mice (Fig. 4, B and C). Similarly, PD remained at levels similar to baseline (4.28 ± 0.43 infected + S. boulardii treated vs. 4.24 ± 0.61 control animals). Likewise, in the proximal colon, the above parameters were reduced with infection, while this reduction was attenuated by treatment with S. boulardii (data not shown). Interestingly, S. boulardii treatment in control mice resulted in a trend toward increased basal Isc and {Delta}Isc responses to forskolin (Fig. 4, B and C).

On transmission electron microscopy, as demonstrated previously, C. rodentium infection was associated with marked microvillus effacement and intimate attachment of C. rodentium to epithelial cells (A/E lesions, Fig. 5). S. boulardii treatment reduced bacterial attachment accompanied by attenuation in epithelial changes associated with infection (Fig. 5). While the length of microvilli appeared quite variable in each animal in part because of the angle of specimen orientation, S. boulardii treatment led to an obvious attenuation in microvillus effacement (Fig. 5).


Figure 5
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  		Fig. 5. Transmission electron micrographs of representative mouse colonic epithelium from (from top to bottom) a normal, uninfected mouse, a mouse treated with S. boulardii, a mouse infected with C. rodentium, and a C. rodentium-infected S. boulardii-treated mouse. While the normal uninfected and S. boulardii-treated mice showed healthy microvilli, infected mice demonstrated intimately attached bacteria and effacement of microvilli. In contrast, C. rodentium-infected and S. boulardii-treated mice showed an obvious attenuation in microvillus effacement (bottom). Calibration bars, 0.3 µm.

 
S. boulardii treatment reduces mucosal adherence of C. rodentium. We next sought to determine whether S. boulardii had an effect on the numbers of total and/or mucosal adherent C. rodentium in the colon. To address this question total colonic and mucosal associated bacterial CFUs were enumerated after overnight incubation of homogenates on MacConkey agar plates. As shown in Table 1, S. boulardii treatment was associated with a 2.4-fold decrease in total C. rodentium counts compared with untreated infected mice; however, the decrease was not significant. In contrast, S. boulardii treatment significantly reduced the number of mucosal adherent C. rodentium, associated with a 5.9-fold decrease compared with untreated infected animals (P < 0.05, Table 1).


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  		Table 1. Total and mucosal associated bacterial counts in the colons of C57BL/6 mice with or without Saccharomyces boulardii treatment

 
S. boulardii had no bactericidal effect. To examine whether the reduction of mucosal adherent bacteria was due to a bactericidal effect of S. boulardii, we investigated its impact on bacterial growth in vitro. C. rodentium was cocultured with S. boulardii in DMEM for 6 h, after which bacteria was serially diluted and plated onto MacConkey agar plates. The number of bacteria was enumerated after overnight incubation at 37°C. The number of bacteria did not differ significantly between C. rodentium cocultured with or without S. boulardii (7.19 ± 0.87 x 108 vs. 7.04 ± 0.63 x 108 CFU/ml, means ± SE, P = 0.45). Similarly, culture with S. boulardii supernatant prepared with SD broth did not affect bacterial viability (data not shown).

S. boulardii treatment reduces Tir translocation in vivo. It was apparent that while high numbers of C. rodentium remained within the lumen of infected mouse colon after treatment with S. boulardii, proportionally fewer bacteria were found adherent to the mucosa. Moreover, S. boulardii was not found to reduce bacterial numbers through a bactericidal action. Thus we next sought to determine whether the action of S. boulardii was mediated through an effect on C. rodentium attachment, through effects on the expression of the TTSS effector Tir. Western blot analysis of C. rodentium-infected mouse colon lysates (after washing away nonadherent bacteria and homogenizing with lysis buffer containing 1% Triton X-100 to determine total Tir) (50) indicated that protein levels of total Tir were markedly downregulated, by approximately threefold, in the presence of S. boulardii (Fig. 6A), suggesting that Tir expression was reduced in the presence of S. boulardii.


Figure 6
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  		Fig. 6. A: colonic lysates (after washing away nonadherent bacteria) were assessed for total Tir protein by Western blot analysis in control (CTL), S. boulardii-treated (Sb), C. rodentium-infected (CR), and S. boulardii-treated and C. rodentium-infected (CR+Sb) animals. β-Actin antibody was used as the loading control. Treatment with S. boulardii was associated with a marked reduction in protein levels of translocated Tir, with levels decreasing by ~3-fold. B–G: immunostaining of C. rodentium LPS (green), translocated Tir (red), and DAPI-stained epithelial cell nuclei (blue). B and C represent the colon of a CR mouse, with the merged image in D, and E–G provide the corresponding images in a CR+Sb animal. In the C. rodentium-infected tissue, note the bacteria lining the crypt epithelial cells (B) with Tir staining similarly positioned (C) but situated beneath LPS-stained bacteria (D). In contrast, in the CR+Sb tissue, note the bacteria within the lumen (E) and with minimal Tir expression (G, H). Images were taken at x400 magnification, except for insets in D and F, which were taken at x1,000 magnification. Tir was stained for 4 h without any detergent to permeabilize the tissues. As a result, only translocated Tir was labeled.

 
While Tir is expressed within bacteria, it only impacts on bacterial attachment after its translocation into host cells. To determine whether translocated Tir was reduced during S. boulardii treatment, immunofluorescent staining for translocated Tir in the colonic tissues of C. rodentium-infected mice with or without S. boulardii treatment was performed. To selectively stain for translocated Tir, rather than Tir within bacteria, we stained for Tir for 4 h without using detergent to permeabilize bacteria as previously described (23). In the absence of S. boulardii, we found large numbers of adherent C. rodentium coating the luminal surface of epithelial cells (Fig. 6, B–D). Where antisera raised against LPS visualized adherent bacteria (Fig. 6B) attached to epithelial cells, anti-Tir staining demonstrated translocated Tir within colonic epithelial cells (Fig. 6, C and D) beneath the adherent bacteria. In contrast, S. boulardii treatment in most C. rodentium-infected tissues section was associated with a marked reduction in the number of C. rodentium that were attached to the epithelium, as well as the detection of translocated Tir (Fig. 6, E–G). Patchy areas where C. rodentium was attached to epithelial cells in association with translocated Tir were occasionally evident; however, most C. rodentium were found within the lumen or mucous layer (confirmed on gram stain, data not shown) and were not associated with Tir expression (Fig. 6, E–G). The antisera raised against LPS did not demonstrate adherent bacteria in uninfected mice (data not shown). These results confirm and advance earlier results indicating that S. boulardii, while not associated with a significant reduction in total bacteria, significantly reduced adherent bacterial numbers and translocated Tir.

S. boulardii treatment reduces total and secreted EspB protein and secreted Tir protein in vitro. Since S. boulardii treatment reduced the number of attached C. rodentium, we next reasoned that the observed action of S. boulardii was mediated through effects on the TTSS. To address this question, we took advantage of previous methodologies using a defined in vitro culture method for assessment of total and secreted Tir and EspB proteins (23). Western blot analysis confirmed Tir protein expression by C. rodentium (Fig. 7, A and C) and Tir protein secretion into the culture medium (Fig. 7, B and D). In contrast, coculture of S. boulardii with C. rodentium was associated with a significant reduction in secreted Tir protein to almost undetectable levels (Fig. 7, B and D), whereas no effect on bacterial Tir protein expression was observed (Fig. 7, A and C). Consistent with previous studies, the type III secretion system mutant {Delta}escN demonstrated expression of Tir but no evidence of secretion (Fig. 7) (21, 23). Similarly, C. rodentium was shown to express (Fig. 7, A and C) and secrete (Fig. 7, B and D) EspB protein, whereas coculture of S. boulardii with C. rodentium was associated with a significant reduction in the secretion of EspB protein (Fig. 7, B and D) but also accompanied by a significant reduction in bacterial expression of EspB (Fig. 7, A and C). The type III secretion system mutant {Delta}escN demonstrated expression of total EspB protein, but again no evidence of secretion.


Figure 7
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  		Fig. 7. Effect of S. boulardii on total and secreted C. rodentium Tir and EspB in vitro. A and B: Western blot analysis of total (A) and secreted (B) Tir and EspB proteins in C. rodentium in the presence (CR+Sb) or absence (CR) of S. boulardii and {Delta}escN (type III apparatus mutant that does not secrete Tir or EspB). C and D: quantification of total (C) and secreted (D) Tir (filled bars) and EspB (open bars) Western blots (representing a mean of 3 separate runs). Protein levels are expressed as % of untreated C. rodentium control. Coculture of C. rodentium with S. boulardii did not significantly alter total Tir expression in C. rodentium (A, C), however, S. boulardii treatment significantly inhibited Tir secretion in the culture medium even at low concentrations (B, D). In contrast, S. boulardii treatment significantly downregulated total EspB expression (A, C), in addition to significantly inhibiting EspB secretion into the culture medium (B, D). *P < 0.001 compared with corresponding C. rodentium control.

 
To determine whether the action of S. boulardii on the TTSS was mediated by a secreted S. boulardii factor(s), S. boulardii culture supernatant was added to C. rodentium in DMEM. The culture of S. boulardii supernatant with C. rodentium resulted in a reduction in EspB protein expression and secretion (Fig. 8) and in Tir secretion (Fig. 8, B and D) comparable to that observed when C. rodentium was cocultured with S. boulardii (Fig. 7). The effect of the S. boulardii culture supernatant was dose dependent and evident at low concentrations (Fig. 8). Boiling the supernatant abolished the inhibitory action (Fig. 8), suggesting the presence of a heat-labile protein(s). Together, these data suggest that S. boulardii via a secreted protein(s) attenuated attachment of C. rodentium to host epithelial cells through effects on key virulence factors, namely, a reduction in expression and secretion of EspB and a reduction in secretion and translocation of Tir.


Figure 8
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  		Fig. 8. Effect of S. boulardii supernatant (SBS) on total and secreted C. rodentium Tir and EspB in vitro by Western blot analysis. C. rodentium was cocultured with S. boulardii supernatant (SBS) in serial dilutions, from 1:20 to 1:400 with DMEM. Total (A, C) and secreted (B, D) Tir (filled bars) and EspB (open bars) Western blots were quantified (representing a mean of 3 separate runs) and protein levels expressed as % of untreated C. rodentium control. First and second lanes, C. rodentium cultured in DMEM and C. rodentium cultured in DMEM and S. boulardii culture medium [Sabouraud dextrose (SD) broth] at a dilution of 1:50. Total and secreted Tir and EspB proteins were similarly detected in both culture media (A, B). Consistent with the observations in Fig. 7, SBS in culture with C. rodentium significantly reduced Tir secretion in culture medium even at low concentrations (3rd–6th lanes, B; D) but failed to affect total Tir expression (3rd–6th lanes, A; C). In contrast, SBS significantly downregulated total EspB expression (3rd–6th lanes, A; C) and secretion into the culture medium (3rd–6th lanes, B; D). Boiling the supernatant obliterated the inhibitory action of SBS. *P < 0.001 compared with corresponding C. rodentium cultured in DMEM.

 
S. boulardii influences secretion but not transcription of EspB or Tir. To further explore the effects of S. boulardii on total and secreted Tir and EspB proteins, RT-PCR and real-time PCR analysis of Tir and EspB in wild-type C. rodentium in the absence or presence of S. boulardii were performed. No significant difference in transcription of EspB or Tir was evident in C. rodentium in the absence or presence of S. boulardii (Fig. 9A, Table 2). Similarly, S. boulardii supernatant had no effect on the transcription of EspB and Tir in C. rodentium (Fig. 9B, Table 2). These results suggest that S. boulardii or its supernatant exerts a modulatory effect on EspB at a posttranscriptional level and further confirmed that S. boulardii or its supernatant influences secretion but not transcription and translation of Tir.


Figure 9
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  		Fig. 9. Effect of S. boulardii on transcription of EspB and Tir in C. rodentium. No significant difference in transcription of EspB and Tir was found in C. rodentium in the absence and presence of S. boulardii (A). Similarly, S. boulardii supernatant had no effect on transcription of EspB and Tir (B).

 

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  		Table 2. Real-time PCR analysis of EspB and Tir gene expression in wild-type C. rodentium in absence or presence of S. boulardii or S. boulardii supernatant

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
To date, the studies of probiotics in ameliorating C. rodentium infection have focused on their impact on host responses, namely, intestinal epithelial cell, mucosal, and systemic inflammatory responses (13, 17, 18, 24, 34). While the present study assessed host mucosal responses, the study also evaluated the impact of S. boulardii on C. rodentium pathogenic determinants. Specifically, the study demonstrated a potentially novel action of S. boulardii through modulation of the effector protein Tir and the translocator protein EspB with resultant reduction of bacterial attachment to host intestinal epithelial cells. In addition, S. boulardii treatment ameliorated bacteria-induced barrier disruption and attenuated the mucosal inflammatory response.

S. boulardii treatment markedly reduced bacterial adherence to host epithelial cells, enabling us to examine the relationship between epithelial barrier function and bacterial epithelial attachment in vivo. In keeping with recent observations (31), C. rodentium infection was associated with marked disruption in epithelial barrier function that was more pronounced in the distal colon, consistent with the degree of bacterial epithelial adherence and host mucosal response. It was apparent that the effective reduction in bacterial attachment to host epithelia and associated preservation of epithelial barrier function were likely largely responsible for the observed attenuation in mucosal inflammatory response associated with C. rodentium infection. However, the additive effects of S. boulardii-mediated enhancement of epithelial barrier function and a shift from a primarily proinflammatory Th1 cellular response to a more balanced Th1/Thr (regulatory) host immune response cannot be excluded (13, 17–19, 34, 55, 60).

The study demonstrated that S. boulardii treatment significantly reduced Tir translocation into colonocytes of mice infected with C. rodentium in association with a reduction in mucosal associated bacteria, suggesting a potential mechanism of action through reduction in receptor-mediated attachment and anchoring of bacteria to host epithelial cells (23). While a bactericidal effect was ruled out as accounting for these changes, several additional mechanisms of action can be considered, including adherence of C. rodentium to luminal S. boulardii (27), physical interference at the epithelial surface as demonstrated in vitro with lactobacillus strains (54), or potential actions on innate defenses, such as increasing effectiveness of the mucus layer and thus reducing and/or preventing C. rodentium from reaching the epithelium, where it is thought that C. rodentium receives the signals to turn on its TTSS. Consistent with observations in conventional and gnotobiotic mice treated with S. boulardii and exposed to enteropathogens (49), S. boulardii treatment in the C. rodentium model was associated with an insignificant reduction in total colonic C. rodentium counts. The reduction in total colonic C. rodentium might be explained on the basis of reduced bacterial attachment to epithelial cells and increased expulsion of nonadherent bacteria in the stool.

The in vitro culture system used to induce C. rodentium TTSS (23, 39) demonstrated a differential expression and secretion of Tir and EspB in the presence of S. boulardii. The significant reduction in protein levels of secreted Tir together with a significant reduction in expression and secretion of EspB protein suggest that the therapeutic benefits of S. boulardii were mediated through actions on A/E pathogen virulence factors (23, 41, 46, 52). The observation that these actions were inhibited and the secretion profile restored by boiling the supernatant suggests the presence of heat-sensitive protein(s) secreted by S. boulardii.

A key observation of our study was the demonstration that S. boulardii reduced EspB expression, in association with diminished secretion. The lack of effect on EspB transcription suggests posttranscriptional control at a level of translational regulation. Furthermore, S. boulardii may have modified the equilibrium between protein synthesis and degradation, leading to reduced intracellular levels of EspB associated with reduced secretion. Although SepL and SepD have been shown to modulate secretion of translocators including EspB, the action of S. boulardii was likely not mediated through actions on these proteins because in vitro studies with SepL and SepD mutants demonstrate normal expression of EspB but low levels of secreted EspB (21, 22).

Another important finding in our study was that S. boulardii abolished Tir secretion. While S. boulardii may have altered the equilibrium between Tir protein synthesis and degradation with resultant reduced secretion, additional evidence suggests that the reduction in secreted Tir may be secondary to yeast protease digestion (11, 12, 56).

Our observations also suggest that the effect of S. boulardii on EspB contributed to the reduced translocation of Tir into host cells in infected and treated mice. The potential mechanism of action is further supported by the observation that EspB in C. rodentium infection is required for colonization (46) and is an essential component of the translocation apparatus necessary in translocation activity (36, 41, 62). Moreover, in vitro studies indicate that while effector molecules can be expressed and secreted by bacteria in the absence of translocator proteins (23), translocation of effectors into host cells does not occur (41, 46, 47) and in vivo colonization fails (46).

In conclusion, using a murine model of bacteria-induced colitis, we have demonstrated that S. boulardii is effective in attenuating virulence of C. rodentium by reducing bacterial attachment to host epithelial cells potentially through actions on the translocator protein EspB and the effector protein Tir. Further studies are required to determine the specific mechanisms involved.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a grant from the Crohn's Colitis Foundation of Canada (awarded to K. Jacobson). K. Jacobson is a Senior Clinician Scientist supported by the Children with Intestinal and Liver Disorders (CHILD) and British Columbia Children's Hospital Foundations. B. A. Vallance is the CHILD Foundation Research Scholar, a Michael Smith Foundation Health Research Scholar, and the Canada Research Chair in Pediatric Gastroenterology.


   ACKNOWLEDGMENTS
 
The authors thank B. Davis from MFI Pharma for the unrestricted supply of S. boulardii and the technical staff of the Morphometry Laboratories (Nutrition Research Program, Child and Family Research Institute) for assistance with the electron microscopy.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Jacobson, Div. of Gastroenterology, BC Children's Hospital, 4480 Oak St., Rm. K4-181, Vancouver, BC, Canada V6H 3V4 (e-mail: kjacobson{at}cw.bc.ca)

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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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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