Randomized Clinical Trial Open Access
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. May 21, 2015; 21(19): 5985-5994
Published online May 21, 2015. doi: 10.3748/wjg.v21.i19.5985
Effectiveness of probiotic therapy for the prevention of relapse in patients with inactive ulcerative colitis
Yasushi Yoshimatsu, Akihiro Yamada, Ryuichi Furukawa, Koji Sono, Aisaku Osamura, Kentaro Nakamura, Hiroshi Aoki, Yukiko Tsuda, Nobuo Hosoe, Nobuo Takada, Yasuo Suzuki
Yasushi Yoshimatsu, Akihiro Yamada, Ryuichi Furukawa, Koji Sono, Aisaku Osamura, Kentaro Nakamura, Hiroshi Aoki, Yukiko Tsuda, Nobuo Hosoe, Nobuo Takada, Yasuo Suzuki, Department of Internal Medicine, Faculty of Medicine, Toho University, Toho University Sakura Medical Center, Chiba 285-8741, Japan
Author contributions: Yoshimatsu Y and Suzuki Y contributed equally to this work; Yoshimatsu Y and Suzuki Y designed research; Yoshimatsu Y, Yamada A, Furukawa R, Sono K, Osamura A, Nakamura K, Aoki H, Tsuda Y, Hosoe N and Takada N performed research; Yoshimatsu Y and Suzuki Y contributed new reagents/analytic tools; Yoshimatsu Y and Suzuki Y analyzed data; and Yoshimatsu Y and Suzuki Y wrote the paper.
Ethics approval: The study was reviewed and approved by the ethical committee of Toho University Sakura Medical Center.
Informed consent: All study participants, or their legal guardian, provided informed written consent prior to study enrollment.
Conflict-of-interest: Yasushi Yoshimatsu has no conflict of interest.
Data sharing: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Yasushi Yoshimatsu, Assistant Professor, Department of Internal Medicine, Faculty of Medicine, Toho University, Toho University Sakura Medical Center, 564-1, Shimoshizu, Sakura, Chiba 285-8741, Japan. 600184yy@sakura.med.toho-u.ac.jp
Telephone: +81-43-4628811 Fax: +81-43-4874246
Received: October 27, 2014
Peer-review started: October 28, 2014
First decision: November 14, 2014
Revised: December 15, 2014
Accepted: February 11, 2015
Article in press: February 11, 2015
Published online: May 21, 2015

Abstract

AIM: To evaluate the effectiveness of probiotic therapy for suppressing relapse in patients with inactive ulcerative colitis (UC).

METHODS: Bio-Three tablets, each containing 2 mg of lactomin (Streptococcus faecalis T-110), 10 mg of Clostridium butyricum TO-A, and 10 mg of Bacillus mesentericus TO-A, were used as probiotic therapy. Sixty outpatients with UC in remission were randomly assigned to receive 9 Bio-Three tablets/day (Bio-Three group) or 9 placebo tablets/day (placebo group) for 12 mo in addition to their ongoing medications. Clinical symptoms were evaluated monthly or on the exacerbation of symptoms or need for additional medication. Fecal samples were collected to analyze bacterial DNA at baseline and 3-mo intervals. Terminal restriction fragment length polymorphism and cluster analyses were done to examine bacterial components of the fecal microflora.

RESULTS: Forty-six patients, 23 in each group, completed the study, and 14 were excluded. The relapse rates in the Bio-Three and placebo groups were respectively 0.0% vs 17.4% at 3 mo (P = 0.036), 8.7% vs 26.1% at 6 mo (P = 0.119), and 21.7% vs 34.8% (P = 0.326) at 9 mo. At 12 mo, the remission rate was 69.5% in the Bio-Three group and 56.6% in the placebo group (P = 0.248). On cluster analysis of fecal flora, 7 patients belonged to cluster I, 32 to cluster II, and 7 to cluster III.

CONCLUSION: Probiotics may be effective for maintaining clinical remission in patients with quiescent UC, especially those who belong to cluster I on fecal bacterial analysis.

Key Words: Ulcerative colitis, Probiotics, Inflammatory bowel disease, Cluster analysis

Core tip: We conducted a single-center, randomized, double-blind, placebo-controlled study to examine whether 12 mo of probiotic therapy was useful for preventing relapse of ulcerative colitis (UC) in patients who were already in remission. The relapse rates in the probiotic therapy group and placebo group were respectively 0.0% vs 17.4% at 3 mo (P = 0.036), 8.7% vs 26.1% at 6 mo (P = 0.119), and 21.7% vs 34.8% (P = 0.326) at 9 mo. At 12 mo, the remission rate was 69.5% in the probiotic therapy group and 56.6% in the placebo group (P = 0.248). Therefor probiotics may be effective for maintaining clinical remission in patients with quiescent UC.



INTRODUCTION

Ulcerative colitis (UC) is a chronic, idiopathic, refractory, inflammatory bowel disease (IBD) characterized by inflammatory mucosal injury of the colon, with repeated periods of remission and relapse. The cause and etiology of UC remain unclear. The mainstay of treatment for UC is sulfasalazine- or mesalazine-based therapy. In patients with moderate to severe UC, steroids are used concurrently to attempt to induce remission. However, a considerable number of cases are resistant to steroids. Patients with steroid-resistant disease are given immunosuppressants and newly developed biological preparations to promote remission induction. Although these new treatments have enhanced the remission induction rate as compared with conventional therapy, achievement of a high long-term rate of remission maintenance remains a largely unattained goal. Steroids are very effective for the induction of remission, but do not contribute to remission maintenance. In addition, long-term treatment with high doses of steroids is associated with high rates of various adverse effects, seriously impairing the quality of life of patients. Sulfasalazine, mesalazine, and immunomodulators promote remission maintenance, but are not adequately effective. Moreover, an appreciable number of patients cannot tolerate these drugs, and immunomodulators can cause serious adverse events, necessitating close follow-up. Therefore, the development of new remission maintenance treatments that are very effective and safe with good compliance when used on a long-term basis has been eagerly awaited.

Recently, probiotic therapy has been acknowledged to be potentially effective and safe in patients with UC. Probiotics are defined as a live microbial feed nutritional supplement that beneficially affects the host by improving the balance of the intestinal flora. Studies of animal models of colitis have suggested that the intestinal flora has an important role in the pathogenesis of colitis. In IBD-sensitive knockout or transgenic mice, colitis develops in the presence of a normal intestinal flora, but not in mice raised in a germ-free environment, strongly suggesting that the intestinal flora participates in the development of colitis[1,2]. Therefore, probiotic therapy designed to correct the intestinal flora is expected to be useful for preventing colitis.

Many studies have examined the effects of specific bacterial strains or species in active UC. However, very few studies have reported on the relation between the intestinal flora as a whole (including microorganisms that are difficult or impossible to culture) and the pathological characteristics of UC[3-7]. In a previous study, we therefore gave probiotic or synbiotic therapy for 4 wk to 20 patients with mild to moderate UC who did not respond to, or could not tolerate, standard therapy [oral mesalamine preparations, sulfasalazine, azathiopurine (AZA)/6-mercaptopurine (6-MP), and mesalamine enemas]. Our results confirmed that such therapy can improve clinical symptoms and endoscopic findings and provided evidence that remission induction is promoted by a certain improvement in the intestinal flora. We also reported that probiotic therapy might be effective for maintenance of remission[8]. On the basis of the results of our previous study, we conducted a single-center, randomized, double-blind, placebo-controlled study to examine whether 12 mo of probiotic therapy is useful for preventing relapse of UC in patients who were already in remission.

MATERIALS AND METHODS
Patients

The study group comprised patients with UC in remission who were receiving treatment on an outpatient basis at Sakura Medical Center, Toho University. UC was diagnosed in accordance with the diagnostic criteria proposed by the Survey Research Group of Intractable Inflammatory Intestinal Disorders/Specified Diseases, Japanese Ministry of Health, Labour and Welfare. Patients 13 years or older in whom the CAI was maintained at 5 or less while receiving drugs such as mesalazine, salazosulfapyridine, or steroids, with no change in treatment regimens within 4 wk before study entry, were enrolled in this randomized, double-blind, placebo-controlled study.

Patients were excluded if they had serious cardiac disease, serious renal disease, hypotension (systolic blood pressure, ≤ 80 mmHg), a history of shock during extracorporeal circulation, serious infections such as sepsis or pneumonia, or a serum hemoglobin concentration of less than 10 g/dL. We also excluded patients who newly began treatments such as leukocytapheresis, granulocyte adsorptive apheresis, or immunosuppressant therapy with drugs such as 6-mercaptopurine, azathioprine, and cyclosporine to improve symptoms, as well as patients who had milk allergy or a CAI of 6 or higher. Pregnant women were also excluded. All procedures were in accordance with the Helsinki Declaration of 1975, as revised in 1983.

Study probiotic

Bio-Three tablets (Toa Pharmaceutical Co., Ltd., Toyama, Japan), a live microbial preparation, and matching placebo tablets (Toa Pharmaceutical Co., Ltd.) were used as the study preparations. Bio-Three tablets were granted manufacturing approval in 1963. Each tablet contains 2 mg of lactomin (Streptococcus faecalis T-110), 10 mg of Clostridium (Clostridium butyricum TO-A), and 10 mg of Bacillus (Bacillus mesentericus TO-A), combined with potato starch and lactose. This preparation is effective for resolving various symptoms caused by abnormal intestinal flora and mainly improves bowel movement disorders. Placebo tablets were prepared by substituting equivalent amounts of starch for the probiotic powder. Placebo tablets were identical to Bio-Three tablets and could not be distinguished from the active preparation on the basis of appearance.

Study design and treatment

At the start of the study, 30 outpatients were randomly assigned to the Bio-Three group and 30 to the placebo group by means of a computer-generated scheme. The study protocol was reviewed and approved by the Ethics Committee of Sakura Medical Center, Toho University.

In both the Bio-Three group and the placebo group, patients orally received 3 tablets 3 times daily. In principle, the duration of treatment was 12 mo. Fecal samples were collected immediately before and 3, 6, 9, and 12 mo after the start of treatment. Fecal samples for measurement of organic acids were preserved by freezing, without modification. Fecal samples used for DNA extraction were suspended in GTC solution (100 mmol/L Tris-HCl, pH 9.0; 40 mmol/L Tris-EDTA, pH 8.0; and 4 mol/L guanidine thiocyanate) and were preserved at 4 °C. As for concomitant medication (therapy), the use of mesalazine and salazosulfapyridine was unrestricted, but steroids could not be used as remission maintenance therapy. The use of drugs with similar effects as the study drug, potentially affecting the evaluation of effectiveness (i.e., other active live microbial preparations, laxatives, etc.) was prohibited from 1 wk before study entry to the completion of the study. In principle, the use of oral antibiotics was also prohibited, but the use of topical antibiotics other than oral preparations was not particularly restricted. If a patient received a new treatment in addition to their basic therapy with drugs such as mesalazine or salazosulfapyridine, relapse was diagnosed, and the study treatment and fecal sample collection were discontinued.

Analysis of intestinal microflora

DNA extraction: About 800 μL of the fecal sample suspension preserved at 4 °C was transferred to a tube containing zirconia beads (Nippon Gene Co., Ltd., Tokyo, Japan), and the cells were processed with the use of FastPrep FP120A cell disruptor (MP Biomedicals, Irvine, CA). After cooling on ice, the specimen was centrifuged at 5000 rpm for 1 min. DNA was automatically extracted from the processed supernatant with the use of a 12GC and GC series Magtration-MagaZorb DNA Common Kit 200N (Precision System Science, Chiba, Japan). The final concentration of the extracted DNA was adjusted to 10 ng/μL.

Terminal restriction fragment length polymorphism analysis: Terminal restriction fragment length polymorphism (T-RFLP) analysis was performed as described by Nagashima et al[9]. The 16S rRNA gene was amplified with the use of primer sets516F (5′-TGCCAGCAGCCGCGGTA-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The 5′-end of the forward primer 516F was labeled with 6′-carboxyfluorescein. Amplified polymerase chain reaction (PCR) products were refined with the use of MultiScreen® PCRμ96 filter plates (Millipore, Tokyo, Japan).

The refined PCR products (about 3 μL) were digested for 3 h at 55 °C with 10 U of Bsl I restriction enzyme (New England Biolabs, Inc., Ipswich, MA, United States). The length of the separated fluorescent PCR fragment was determined with an ABI PRISM 3130xl genetic analyzer (Applied Biosystems, Tokyo, Japan), and the data were analyzed with GeneMapper® software. MapMarker® X-Rhodamine Labeled 50-1000bp (BioVentures, Inc., Murfreesboro, TN, United States) was used as a size standard marker.

Cluster analysis: To objectively interpret differences in T-RFLP patterns, NTSYSpc software (Exeter Software, Setauket, NY, United States) was used to perform cluster analysis. Each terminal restriction fragment (T-RF) was expressed as a percentage of the peak area of all T-RFs. Disparity in similarity among fecal samples in individual patients was calculated using a correlation matrix and was presented graphically on tree diagrams with the use of a weighted pair-group method with arithmetic mean (WPGMA) clustering[10].

High-performance liquid chromatography analysis of fecal organic acids: Organic acid concentrations in fecal samples were measured as follows. About 0.1 g of fecal sample was measured and combined with trans-crotonic acid as an internal standard substance, and extraction was performed twice with 0.6 mL of 0.25% ammonia solution. After adding a 0.3-fold dilution of 10% (w/v) perchloric acid, deproteinization was performed by centrifugation. The solution was filtered and analyzed with a post-column high-performance liquid chromatography (HPLC) system (Waters, Milford MA, United States). Organic acids in the sample were separated with an ion exchange column (organic acid column, 7. 8 mm i.d. × 300 mm long; Waters). The reaction temperature in the column and post-column was 60 °C. The mobile phase was 0.08% perchloric acid delivered at a flow rate of 0.8 mL/min. The solution eluted from the column was allowed to react with BTB solution (0.2 mmol/L bromothymol blue, 5.2 mmol/L sodium hydroxide, and 15 mmol/L disodium hydrogen phosphate), delivered at a flow rate of 0.8 mL/min. The absorbance was quantitatively measured at 445 nm using an ultraviolet visible spectrophotometer (2487 Dual λ UV/Vis Detector; Waters).

Statistical analysis

Clinical data were statistically analyzed with SAS software (version 8.2, SAS Institute Inc., Cary, NC, United States). For patients with UC in remission, the Kaplan-Meier method was used to compare the cumulative non-relapse rate over the course of 12 mo between the Bio-Three group and placebo group. The statistical significance of differences between groups was evaluated with the log-rank test and the generalized Wilcoxon test, and 95% confidence intervals were calculated.

RESULTS
Patient characteristics

At the start of the study, 30 patients each were randomly assigned to the Bio-Three group and the placebo group. After randomization, the baseline characteristics of sex, age, age at disease onset, disease duration, disease extent, and concomitant treatment did not differ between the groups (Table 1). Among the enrolled subjects, 7 patients in each group were excluded because they met the exclusion criteria specified in the protocol, such as the use of prohibited drugs or refusal to participate in the study. Treatment was actually begun in 23 patients in the Bio-Three group and 23 in the placebo group (Figure 1).

Table 1 Baseline characteristics of the study group.
Bio-three (n = 23)Placebo (n = 23)
Male/female16/712/11
Age (yr, mean ± SD)44.8 ± 13.842.9 ± 15.9
Age of onset (yr)37.1 ± 14.436.0 ± 14.2
Disease duration (yr, mean ± SD)8.0 ± 6.36.7 ± 5.9
Left colon69
Proctosigmoiditis65
Total/subtotal119
Concomitant drug
Pentasa1113
Salazopyrin109
Pentasa + salazopyrin10
Nothing11
Figure 1
Figure 1 Clinical outcomes of patients according to treatment received.
Clinical results

After the study began, the number of patients who had relapse was 2 at 6 mo, 5 at 9 mo, and 7 at 12 mo among the 23 patients in the Bio-Three group and 4 at 3 mo, 6 at 6 mo, 8 at 9 mo, and 10 at 12 mo among the 23 patients in the placebo group. Kaplan-Meier curves were plotted, and relapse rates at each time point were compared between the groups with the use of the χ2 test (two-tailed, α = 0.05). The P value was 0.0363 at 3 mo, 0.1197 at 6 mo, and 0.3259 at 9 mo. The cumulative remission maintenance rate at 12 mo was 56.6% (n = 12) in the placebo group and 69.5% (n = 16) in the Bio-Three group (Figure 2). When remission maintenance rates were compared between the treatment groups with the use of the log-rank test, the P value was 0.302, with a hazard ratio of 0.607 (95%CI: 0.23-1.59). On the generalized Wilcoxon test, the P value was 0.248. During our study, no adverse changes were observed in the Bio-Three group, as compared with the placebo group, indicating that there is no problem with the safety of Bio-Three.

Figure 2
Figure 2 Cumulative remission maintenance rate in the Bio-Three group and placebo group.
Bacteriological analysis

In our previous study evaluating the effectiveness of Bio-Three for inducing remission in patients with active UC[8], a cluster analysis was performed to evaluate fluctuations in fecal flora, using the T-RF data derived by digestion of the PCR products with Bsl I restriction enzyme, and several findings were obtained. In the present study of the effectiveness of Bio-Three for remission maintenance, the PCR products were similarly digested with Bsl I restriction enzyme, and cluster analysis of the T-RF data showed that the fecal flora can be divided into 3 clusters, consistent with the results of our previous study. To confirm whether the 3 clusters in the present study correspond to the 3 clusters in our previous study, the fecal sample data used in our previous study of remission induction were linked to the fecal sample data in the present study, and another cluster analysis was performed. The results showed that each of the fecal samples from our previous study belonged to the same clusters as those in our previous analysis. Therefore, for convenience the same names of the clusters in our previous study were used in the present study, i.e., the cluster in the upper part of the figure was named cluster II, the cluster in the middle part was named cluster III, and the other cluster was named cluster I (Figure 3, Table 2).

Table 2 Comparison of characteristics of fecal samples (according to cluster).
Group(sample number)OTU124/all T-RFs (%)Butyrate/acetate ratio (mol)
Cluster I2.8 ± 3.60.254 ± 0.172
Cluster II (n = 138)27.6 ± 12.00.184 ± 0.147
Cluster III (n = 28)18.0 ± 10.80.075 ± 0.126
Figure 3
Figure 3 Alteration of intestinal microflora after treatment in the Bio-Three group and placebo group. The dendrogram indicates the similarity of individual intestinal microflora T-RFLP patterns of fecal samples obtained after 0, 3, 6, 9, and 12 mo of treatment. Solid line and circles, Bio-Three group and remission; double line and open circles, Bio-Three group and relapse; solid dotted line and arrows, placebo group and remission; double dotted line and arrows, placebo group and relapse. The first and last samples from the same individuals are connected with lines. The tables indicate clinical effectiveness according to the cluster that the subjects belonged to at the start of this study.

A total of 138 fecal samples belonged to cluster II. The clinical outcomes of the 32 subjects who belonged to cluster II at the start of treatment were remission maintained in 11 of the 16 patients in the Bio-Three group and 10 of the 16 patients in the placebo group. This difference was not significant. As a characteristic of T-RF in cluster II, OTU124 accounted for a significantly higher proportion of total T-RF peak area in cluster II than in the other clusters.

A total of 28 fecal samples belonged to cluster III. The clinical outcomes of the 7 subjects who belonged to cluster III at the start of treatment were remission maintained in 2 of the 4 patients in the Bio-Three group and 2 of the 3 patients in the placebo group. The difference between the groups was not significant.

A total of 39 fecal samples belonged to cluster I. The clinical outcomes in the 7 subjects who belonged to cluster I at the start of treatment were remission maintained in all 3 patients in the Bio-Three group, as compared with only 1 of the 4 patients in the placebo group. Among the 39 fecal samples that belonged to cluster I, 19 fecal samples were derived from patients in the Bio-Three group, and 17 of these samples were from patients who had a final evaluation of remission maintained. The other 20 fecal samples were from patients in the placebo group, and 8 of these samples were from patients who had a final evaluation of remission maintained. When these data were compared with the use of Pearson’s χ2 test, the P value was 0.0013, indicating that the rate of remission maintenance was significantly higher in the Bio-Three group than in the placebo group, and the relapse rate was significantly lower in the Bio-Three group than in the placebo group.

HPLC analysis of fecal organic acids

The fecal concentrations of short-chain fatty acids did not differ significantly between the Bio-Three group and the placebo group at any time during treatment. On comparison of the clusters derived by T-RFLP analysis, the ratio of the concentration (mmol/L) of butyrate to that of acetate (Bu/Ac ratio) was significantly higher in cluster I than in clusters II and III. When fecal organic acids were compared according to clinical outcomes (i.e., between fecal samples obtained at each of the specified times from patients in whom remission was maintained for 1 year and fecal samples from patients who had relapse within 6 mo after fecal collection), the concentrations of butyric acid and other short-chain fatty acids did not differ significantly between the groups. However, the Bu/Ac ratio at each of the sampling times was significantly higher in fecal samples obtained from patients who had relapse within 6 mo after fecal collection than in those obtained from patients who remained in remission (Table 3).

Table 3 Comparison of characteristics of fecal samples (according to clinical outcomes).
Group (sample number)OTU124/all T-RFs (%)Butyrate/acetate ratio (mol)
Remission (before treatment) (n = 29)22.7 ± 13.80.165 ± 0.143
Remission (months 3 and 6) (n = 58)22.0 ± 14.60.145 ± 0.136
Remission (months 9 and 12) (n = 56)21.7 ± 14.10.160 ± 0.146
Relapse (within 6 mo) (n = 28)20.7 ± 14.70.260 ± 0.185
DISCUSSION

T-RFLP is a molecular technique that allows the diversity and colony structure of microbial complexes to be promptly compared and the diversity of ecosystems to be evaluated[11,12]. Recently, several studies have performed T-RFLP in patients with UC. However, few studies have examined time-series samples obtained from the same subjects. Most previous investigations have evaluated fecal samples obtained at a specific time[13-17].

We previously studied the effectiveness of probiotic therapy for inducing remission in patients with flare-ups of UC. On T-RFLP cluster analysis, fecal flora could be divided into 3 clusters, designated as clusters I, II, and III. The flora of patients whose UC disease activity index (UCDAI) was improved by probiotic therapy was centered around cluster II. Cluster II flora was characterized by a high ratio of OTU124[8]. Nagashima et al[9], who proposed the T-RFLP technique used in our study, reported that OTU124 is derived from Bifidobacteria. Many studies have examined the relation between Bifidobacteria and UC, and most have reported that Bifidobacteria mitigates inflammation associated with UC[18-21]. On the basis of these findings, we previously concluded that cluster II flora is “healthy intestinal flora (appropriate intestinal flora)” in patients with UC. Probiotic therapy may therefore be less effective for improving flora in patients who initially have cluster II flora, leading only to marginal improvement clinically[8].

The results of the present study similarly suggest that cluster II represents “healthy intestinal flora”. Consequently, probiotic therapy is expected to be of the greatest potential benefit for intestinal flora that is most dissimilar to cluster II. This speculation is supported by the following two points. First, in our previous study of patients with “flare-ups” of UC, only 6 (30%) of the 20 subjects had intestinal flora belonging to cluster II before treatment[8]. In the present study of patients with UC “after induction of remission”, 32 (70%) of the 46 subjects had intestinal flora belonging to cluster II. Given that the proportion of patients with “appropriate intestinal flora” is expected to be higher among patients with remission induction and remission maintenance than among those with relapse, the fact that the majority of patients in the present study, who were already in remission, belonged to cluster II supports our speculation that probiotic therapy is potentially most beneficial for patients with intestinal flora most dissimilar to cluster II. Second, among the 32 subjects who belonged to cluster II at the start of treatment in the present study, remission was maintained in 11 of the 16 patients in the Bio-Three group and 10 of the 16 patients in the placebo group. There was no difference in the remission maintenance rate between these groups. In contrast, among the 7 subjects who belonged to cluster I (i.e., the cluster farthest from cluster II) before treatment, remission was maintained in all 3 patients in the Bio-Three group, whereas relapse occurred in 3 of the 4 patients in the placebo group. These findings indicate that probiotic therapy was less effective in patients who initially belonged to cluster II (“appropriate intestinal flora”), with no difference from placebo. In contrast, probiotic therapy was potentially most beneficial for patients with intestinal flora belonging to cluster I flora before treatment, the cluster that is farthest from cluster II. Consequently, probiotic therapy was more effective than placebo for maintaining remission in this subgroup of patients. These results were very interesting and are consistent with the findings of our previous study[8].

The fecal Bu/Ac ratio differed between patients with relapse and those in whom remission was maintained for 12 mo and was significantly higher within 6 mo before relapse than at other times (Table 3). Interestingly, the Bu/Ac ratio tended to be higher in feces belonging to cluster I than in the other clusters (Table 2). These findings may be attributed to the following mechanism. Butyrate serves as an energy source for intestinal epithelial cells and is known to induce apoptosis of colorectal cancer cells and the differentiation of intestinal epithelial cells. In addition, butyrate has been shown to inhibit the activation of nuclear factor kappa B (NF-κB) and to have anti-inflammatory properties[11,22,23]. On the other hand, the utilization efficiency of butyrate has been reported to be low in the colonic mucosa of patients with refractory UC. The anti-inflammatory activity of butyrate has prompted several studies of its effectiveness and mechanism of action in patients with UC[24-28].

In our previous study evaluating the effectiveness of Bio-Three for inducing remission in patients with UC, the decrease in the UCDAI after treatment (i.e., the improvement in symptoms of UC) correlated with the decrease in the fecal butyrate concentration[8]. On the basis of this finding, we performed breath tests after administration of [1-13C]-butyrate enemas in 10 patients with active UC and 12 with quiescent UC and confirmed that the utilization efficiency of butyrate was decreased in patients with high inflammatory activity[29-33]. These findings suggest that an increased Bu/Ac ratio resulting from decreased absorption of butyrate, an indicator of anti-inflammatory activity, and an increase in fecal butyrate concentrations is associated with a higher risk of relapse in patients with UC. The mean rate of remission maintenance at 12 mo in patients who receive mesalazine preparations alone is estimated to be about 61% (range: 45%-71%) on the basis of the results of previous randomized controlled trials[34-41]. This rate is similar to the relapse rate among patients who received mesalazine preparations alone (56.6%) in the placebo group of our study.

We used the Kaplan-Meier method to compare relapse rates between the treatment groups. The relapse rate after 12 mo did not differ significantly between the Bio-Three group and the placebo group on either the log-rank test or generalized Wilcoxon test. However, detailed analysis showed that clinical effectiveness differed between the Bio-Three group and placebo group among patients who belonged to cluster I. As mentioned above, probiotic therapy is most likely to be effective in patients with intestinal flora belonging to cluster I, which is characterized by both a low ratio of OTU124 (which tended to be high in patients belonging to cluster II, classified as “healthy intestinal flora”) and a high fecal Bu/AC ratio (which was significantly higher in patients within 6 mo before relapse than in patients without relapse).

Combining probiotics or synbiotics with conventional drugs has been recommended as a safe and effective treatment for patients with active UC. For more than 10 years considerable attention has focused on the effectiveness of probiotic therapy for UC[42]. Nearly all studies have reported that probiotics such as VSL#3[43-45], BIFICO[46], and E. coli Nissle 1917[47] and prebiotics such as GBF[48-50] and BGS[51] are useful for maintaining remission maintenance and preventing relapse in patients with UC. In comprehensive Cochrane reviews of clinical studies evaluating the effectiveness of probiotics for UC, Mallon et al[52] and Naidoo et al[53] concluded that although probiotics are ineffective for the induction of remission, probiotics combined with conventional therapy are expected to be effective for maintenance of remission. A meta-analysis conducted by Sang et al[54]. reported that probiotics are slightly but not significantly effective for remission induction, but significantly contribute to remission maintenance.

The results of our study suggest that cluster analysis of patients’ intestinal flora before treatment can contribute to the effective use of probiotic therapy. To our knowledge, our study represents an unprecedented attempt to define factors related to the effectiveness of Bio-Three for the prevention of relapse in patients with inactive UC. Not only the fecal flora, but also the fecal concentration of short-chain fatty acids differed between patients who had relapse within 1 year and those in whom remission was maintained for 1 year. This finding suggests that patient profiling on the basis of factors such as the results of cluster analysis of fecal flora and the fecal short-chain fatty acid concentration might facilitate prediction of the response to treatment and future clinical status in patients with UC.

ACKNOWLEDGMENTS

The T-RFLP analysis and cluster analysis were performed by TOA Pharmaceutical Co., Ltd. and TechnoSuruga Laboratory Co., Ltd. This study was supported by in part by a grant from the Japan Ministry of Health and Welfare.

COMMENTS
Background

Ulcerative colitis (UC) is a chronic, idiopathic, refractory, inflammatory bowel disease (IBD) characterized by inflammatory mucosal injury of the colon, with repeated periods of remission and relapse. Recently, probiotic therapy has been acknowledged to be potentially effective and safe in patients with UC. Probiotics are defined as a live microbial feed nutritional supplement that beneficially affects the host by improving the balance of the intestinal flora.

Research frontiers

Studies of animal models of colitis have suggested that the intestinal flora has an important role in the pathogenesis of colitis. In IBD-sensitive knockout or transgenic mice, colitis develops in the presence of a normal intestinal flora, but not in mice raised in a germ-free environment, strongly suggesting that the intestinal flora participates in the development of colitis. Therefore, probiotic therapy designed to correct the intestinal flora is expected to be useful for preventing colitis.

Innovations and breakthroughs

The results confirmed that probiotic therapy can improve clinical symptoms and endoscopic findings and provided evidence that remission induction is promoted by a certain improvement in the intestinal flora. The authors also reported that probiotic therapy might be effective for maintenance of remission.

Applications

The authors conducted a single-center, randomized, double-blind, placebo-controlled study to examine whether 12 mo of probiotic therapy is useful for preventing relapse of UC in patients who were already in remission.

Peer-review

In this study, the authors designed the randomized double-blind controlled study to evaluate the effectiveness of probiotic therapy for suppressing relapse in patients with UC. They concluded that probiotics may be effective for maintaining clinical remission in patients with quiescent UC, especially those who belong to cluster I on fecal bacterial analysis. This paper is well written and the results of the study are clinically interesting.

Footnotes

P- Reviewer: Iizuka M, Perakath B, Thompson JR S- Editor: Ma YJ L- Editor: A E- Editor: Zhang DN

References
1.  Furrie E, Macfarlane S, Kennedy A, Cummings JH, Walsh SV, O’neil DA, Macfarlane GT. Synbiotic therapy (Bifidobacterium longum/Synergy 1) initiates resolution of inflammation in patients with active ulcerative colitis: a randomised controlled pilot trial. Gut. 2005;54:242-249.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 450]  [Cited by in F6Publishing: 295]  [Article Influence: 28.1]  [Reference Citation Analysis (0)]
2.  Campieri M, Gionchetti P. Bacteria as the cause of ulcerative colitis. Gut. 2001;48:132-135.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 122]  [Cited by in F6Publishing: 99]  [Article Influence: 6.1]  [Reference Citation Analysis (0)]
3.  Danese S, Sans M, Fiocchi C. Inflammatory bowel disease: the role of environmental factors. Autoimmun Rev. 2004;3:394-400.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 244]  [Cited by in F6Publishing: 183]  [Article Influence: 14.4]  [Reference Citation Analysis (0)]
4.  Macfarlane GT, Furrie E, Macfarlane S. Bacterial milieu and mucosal bacteria in ulcerative colitis. Novartis Found Symp. 2004;263:57-64; discussion 64-70, 211-218.  [PubMed]  [DOI]  [Cited in This Article: ]
5.  Cummings JH, Kong SC. Probiotics, prebiotics and antibiotics in inflammatory bowel disease. Novartis Found Symp. 2004;263:99-111; discussion 111-114, 211-218.  [PubMed]  [DOI]  [Cited in This Article: ]
6.  Ohkusa T, Okayasu I, Ogihara T, Morita K, Ogawa M, Sato N. Induction of experimental ulcerative colitis by Fusobacterium varium isolated from colonic mucosa of patients with ulcerative colitis. Gut. 2003;52:79-83.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 161]  [Cited by in F6Publishing: 129]  [Article Influence: 8.9]  [Reference Citation Analysis (0)]
7.  Kanauchi O, Matsumoto Y, Matsumura M, Fukuoka M, Bamba T. The beneficial effects of microflora, especially obligate anaerobes, and their products on the colonic environment in inflammatory bowel disease. Curr Pharm Des. 2005;11:1047-1053.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 48]  [Cited by in F6Publishing: 37]  [Article Influence: 3.0]  [Reference Citation Analysis (0)]
8.  Tsuda Y, Yoshimatsu Y, Aoki H, Nakamura K, Irie M, Fukuda K, Hosoe N, Takada N, Shirai K, Suzuki Y. Clinical effectiveness of probiotics therapy (BIO-THREE) in patients with ulcerative colitis refractory to conventional therapy. Scand J Gastroenterol. 2007;42:1306-1311.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 38]  [Cited by in F6Publishing: 28]  [Article Influence: 3.2]  [Reference Citation Analysis (0)]
9.  Nagashima K, Hisada T, Sato M, Mochizuki J. Application of new primer-enzyme combinations to terminal restriction fragment length polymorphism profiling of bacterial populations in human feces. Appl Environ Microbiol. 2003;69:1251-1262.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 173]  [Cited by in F6Publishing: 58]  [Article Influence: 9.6]  [Reference Citation Analysis (0)]
10.  Kibe R, Sakamoto M, Yokota H, Ishikawa H, Aiba Y, Koga Y, Benno Y. Movement and fixation of intestinal microbiota after administration of human feces to germfree mice. Appl Environ Microbiol. 2005;71:3171-3178.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 38]  [Cited by in F6Publishing: 19]  [Article Influence: 2.4]  [Reference Citation Analysis (0)]
11.  Liu WT, Marsh TL, Cheng H, Forney LJ. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl Environ Microbiol. 1997;63:4516-4522.  [PubMed]  [DOI]  [Cited in This Article: ]
12.  Sakamoto M, Hayashi H, Benno Y. Terminal restriction fragment length polymorphism analysis for human fecal microbiota and its application for analysis of complex bifidobacterial communities. Microbiol Immunol. 2003;47:133-142.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 83]  [Cited by in F6Publishing: 58]  [Article Influence: 4.6]  [Reference Citation Analysis (0)]
13.  Sepehri S, Kotlowski R, Bernstein CN, Krause DO. Microbial diversity of inflamed and noninflamed gut biopsy tissues in inflammatory bowel disease. Inflamm Bowel Dis. 2007;13:675-683.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 162]  [Cited by in F6Publishing: 121]  [Article Influence: 11.6]  [Reference Citation Analysis (0)]
14.  Andoh A, Sakata S, Koizumi Y, Mitsuyama K, Fujiyama Y, Benno Y. Terminal restriction fragment length polymorphism analysis of the diversity of fecal microbiota in patients with ulcerative colitis. Inflamm Bowel Dis. 2007;13:955-962.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 81]  [Cited by in F6Publishing: 63]  [Article Influence: 5.8]  [Reference Citation Analysis (0)]
15.  Johnson MW, Rogers GB, Bruce KD, Lilley AK, von Herbay A, Forbes A, Ciclitira PJ, Nicholls RJ. Bacterial community diversity in cultures derived from healthy and inflamed ileal pouches after restorative proctocolectomy. Inflamm Bowel Dis. 2009;15:1803-1811.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 12]  [Cited by in F6Publishing: 11]  [Article Influence: 1.0]  [Reference Citation Analysis (0)]
16.  Kohyama A, Ogawa H, Funayama Y, Takahashi K, Benno Y, Nagasawa K, Tomita S, Sasaki I, Fukushima K. Bacterial population moves toward a colon-like community in the pouch after total proctocolectomy. Surgery. 2009;145:435-447.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 27]  [Cited by in F6Publishing: 22]  [Article Influence: 2.3]  [Reference Citation Analysis (0)]
17.  Nishikawa J, Kudo T, Sakata S, Benno Y, Sugiyama T. Diversity of mucosa-associated microbiota in active and inactive ulcerative colitis. Scand J Gastroenterol. 2009;44:180-186.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 83]  [Cited by in F6Publishing: 68]  [Article Influence: 6.9]  [Reference Citation Analysis (0)]
18.  Ishikawa H, Matsumoto S, Ohashi Y, Imaoka A, Setoyama H, Umesaki Y, Tanaka R, Otani T. Beneficial effects of probiotic bifidobacterium and galacto-oligosaccharide in patients with ulcerative colitis: a randomized controlled study. Digestion. 2011;84:128-133.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 115]  [Cited by in F6Publishing: 67]  [Article Influence: 11.5]  [Reference Citation Analysis (0)]
19.  Wildt S, Nordgaard I, Hansen U, Brockmann E, Rumessen JJ. A randomised double-blind placebo-controlled trial with Lactobacillus acidophilus La-5 and Bifidobacterium animalis subsp. lactis BB-12 for maintenance of remission in ulcerative colitis. J Crohns Colitis. 2011;5:115-121.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 83]  [Cited by in F6Publishing: 57]  [Article Influence: 8.3]  [Reference Citation Analysis (0)]
20.  Fujimori S, Gudis K, Mitsui K, Seo T, Yonezawa M, Tanaka S, Tatsuguchi A, Sakamoto C. A randomized controlled trial on the efficacy of synbiotic versus probiotic or prebiotic treatment to improve the quality of life in patients with ulcerative colitis. Nutrition. 2009;25:520-525.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 130]  [Cited by in F6Publishing: 80]  [Article Influence: 10.8]  [Reference Citation Analysis (0)]
21.  Groeger D, O’Mahony L, Murphy EF, Bourke JF, Dinan TG, Kiely B, Shanahan F, Quigley EM. Bifidobacterium infantis 35624 modulates host inflammatory processes beyond the gut. Gut Microbes. 2013;4:325-339.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 183]  [Cited by in F6Publishing: 116]  [Article Influence: 22.9]  [Reference Citation Analysis (0)]
22.  Andoh A, Shimada M, Araki Y, Fujiyama Y, Bamba T. Sodium butyrate enhances complement-mediated cell injury via down-regulation of decay-accelerating factor expression in colonic cancer cells. Cancer Immunol Immunother. 2002;50:663-672.  [PubMed]  [DOI]  [Cited in This Article: ]
23.  Andoh A, Fujiyama Y, Hata K, Araki Y, Takaya H, Shimada M, Bamba T. Counter-regulatory effect of sodium butyrate on tumour necrosis factor-alpha (TNF-alpha)-induced complement C3 and factor B biosynthesis in human intestinal epithelial cells. Clin Exp Immunol. 1999;118:23-29.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 54]  [Cited by in F6Publishing: 46]  [Article Influence: 2.5]  [Reference Citation Analysis (0)]
24.  Den Hond E, Hiele M, Evenepoel P, Peeters M, Ghoos Y, Rutgeerts P. In vivo butyrate metabolism and colonic permeability in extensive ulcerative colitis. Gastroenterology. 1998;115:584-590.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 72]  [Cited by in F6Publishing: 27]  [Article Influence: 3.1]  [Reference Citation Analysis (0)]
25.  Steinhart AH, Hiruki T, Brzezinski A, Baker JP. Treatment of left-sided ulcerative colitis with butyrate enemas: a controlled trial. Aliment Pharmacol Ther. 1996;10:729-736.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 129]  [Cited by in F6Publishing: 106]  [Article Influence: 5.4]  [Reference Citation Analysis (0)]
26.  Roda A, Simoni P, Magliulo M, Nanni P, Baraldini M, Roda G, Roda E. A new oral formulation for the release of sodium butyrate in the ileo-cecal region and colon. World J Gastroenterol. 2007;13:1079-1084.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in CrossRef: 41]  [Cited by in F6Publishing: 22]  [Article Influence: 2.9]  [Reference Citation Analysis (0)]
27.  Hamer HM, Jonkers DM, Renes IB, Vanhoutvin SA, Kodde A, Troost FJ, Venema K, Brummer RJ. Butyrate enemas do not affect human colonic MUC2 and TFF3 expression. Eur J Gastroenterol Hepatol. 2010;22:1134-1140.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 17]  [Cited by in F6Publishing: 7]  [Article Influence: 1.5]  [Reference Citation Analysis (0)]
28.  Hamer HM, Jonkers DM, Vanhoutvin SA, Troost FJ, Rijkers G, de Bruïne A, Bast A, Venema K, Brummer RJ. Effect of butyrate enemas on inflammation and antioxidant status in the colonic mucosa of patients with ulcerative colitis in remission. Clin Nutr. 2010;29:738-744.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 91]  [Cited by in F6Publishing: 69]  [Article Influence: 8.3]  [Reference Citation Analysis (0)]
29.  Hosoe N, Suzuki Y, Shirai K. [1-13C] sodium butyrate breath test in patients with active and quiescent ulcerative colitis by colonoscopic examination. Dig Absorpt. 2008;31:43-47.  [PubMed]  [DOI]  [Cited in This Article: ]
30.  Mitsui R, Ono S, Karaki S, Kuwahara A. Neural and non-neural mediation of propionate-induced contractile responses in the rat distal colon. Neurogastroenterol Motil. 2005;17:585-594.  [PubMed]  [DOI]  [Cited in This Article: ]
31.  Mitsui R, Ono S, Karaki S, Kuwahara A. Propionate modulates spontaneous contractions via enteric nerves and prostaglandin release in the rat distal colon. Jpn J Physiol. 2005;55:331-338.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 18]  [Cited by in F6Publishing: 7]  [Article Influence: 1.1]  [Reference Citation Analysis (0)]
32.  Ono S, Karaki S, Kuwahara A. Short-chain fatty acids decrease the frequency of spontaneous contractions of longitudinal muscle via enteric nerves in rat distal colon. Jpn J Physiol. 2004;54:483-493.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 30]  [Cited by in F6Publishing: 15]  [Article Influence: 1.9]  [Reference Citation Analysis (0)]
33.  Maslowski KM, Vieira AT, Ng A, Kranich J, Sierro F, Yu D, Schilter HC, Rolph MS, Mackay F, Artis D. Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43. Nature. 2009;461:1282-1286.  [PubMed]  [DOI]  [Cited in This Article: ]
34.  Prantera C, Kohn A, Campieri M, Caprilli R, Cottone M, Pallone F, Savarino V, Sturniolo GC, Vecchi M, Ardia A. Clinical trial: ulcerative colitis maintenance treatment with 5-ASA: a 1-year, randomized multicentre study comparing MMX with Asacol. Aliment Pharmacol Ther. 2009;30:908-918.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 53]  [Cited by in F6Publishing: 39]  [Article Influence: 4.4]  [Reference Citation Analysis (0)]
35.  Green JR, Gibson JA, Kerr GD, Swarbrick ET, Lobo AJ, Holdsworth CD, Crowe JP, Schofield KJ, Taylor MD. Maintenance of remission of ulcerative colitis: a comparison between balsalazide 3 g daily and mesalazine 1.2 g daily over 12 months. ABACUS Investigator group. Aliment Pharmacol Ther. 1998;12:1207-1216.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 62]  [Cited by in F6Publishing: 48]  [Article Influence: 2.8]  [Reference Citation Analysis (0)]
36.  Miner P, Hanauer S, Robinson M, Schwartz J, Arora S. Safety and efficacy of controlled-release mesalamine for maintenance of remission in ulcerative colitis. Pentasa UC Maintenance Study Group. Dig Dis Sci. 1995;40:296-304.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 99]  [Cited by in F6Publishing: 24]  [Article Influence: 3.8]  [Reference Citation Analysis (0)]
37.  Courtney MG, Nunes DP, Bergin CF, O’Driscoll M, Trimble V, Keeling PW, Weir DG. Randomised comparison of olsalazine and mesalazine in prevention of relapses in ulcerative colitis. Lancet. 1992;339:1279-1281.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 68]  [Cited by in F6Publishing: 51]  [Article Influence: 2.3]  [Reference Citation Analysis (0)]
38.  Giaffer MH, Holdsworth CD, Lennard-Jones JE, Rodrigues CA, McIntyre PB, Manjunatha S, Baron JH, Barrison IG, Polson RJ, Hoare AM. Improved maintenance of remission in ulcerative colitis by balsalazide 4 g/day compared with 2 g/day. Aliment Pharmacol Ther. 1992;6:479-485.  [PubMed]  [DOI]  [Cited in This Article: ]
39.  Mulder CJ, Tytgat GN, Weterman IT, Dekker W, Blok P, Schrijver M, van der Heide H. Double-blind comparison of slow-release 5-aminosalicylate and sulfasalazine in remission maintenance in ulcerative colitis. Gastroenterology. 1988;95:1449-1453.  [PubMed]  [DOI]  [Cited in This Article: ]
40.  Paoluzi OA, Iacopini F, Pica R, Crispino P, Marcheggiano A, Consolazio A, Rivera M, Paoluzi P. Comparison of two different daily dosages (2.4 vs. 1.2 g) of oral mesalazine in maintenance of remission in ulcerative colitis patients: 1-year follow-up study. Aliment Pharmacol Ther. 2005;21:1111-1119.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 60]  [Cited by in F6Publishing: 42]  [Article Influence: 3.8]  [Reference Citation Analysis (0)]
41.  Järnerot G, Ström M, Danielsson A, Kilander A, Lööf L, Hultcrantz R, Löfberg R, Florén C, Nilsson A, Broström O. Allopurinol in addition to 5-aminosalicylic acid based drugs for the maintenance treatment of ulcerative colitis. Aliment Pharmacol Ther. 2000;14:1159-1162.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 15]  [Cited by in F6Publishing: 12]  [Article Influence: 0.7]  [Reference Citation Analysis (0)]
42.  Kanauchi O, Mitsuyama K, Araki Y, Andoh A. Modification of intestinal flora in the treatment of inflammatory bowel disease. Curr Pharm Des. 2003;9:333-346.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 58]  [Cited by in F6Publishing: 27]  [Article Influence: 3.2]  [Reference Citation Analysis (0)]
43.  Gionchetti P, Lammers KM, Rizzello F, Campieri M. VSL#3: an analysis of basic and clinical contributions in probiotic therapeutics. Gastroenterol Clin North Am. 2005;34:499-513, ix-x.  [PubMed]  [DOI]  [Cited in This Article: ]
44.  Bibiloni R, Fedorak RN, Tannock GW, Madsen KL, Gionchetti P, Campieri M, De Simone C, Sartor RB. VSL#3 probiotic-mixture induces remission in patients with active ulcerative colitis. Am J Gastroenterol. 2005;100:1539-1546.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 473]  [Cited by in F6Publishing: 356]  [Article Influence: 29.6]  [Reference Citation Analysis (0)]
45.  Mimura T, Rizzello F, Helwig U, Poggioli G, Schreiber S, Talbot IC, Nicholls RJ, Gionchetti P, Campieri M, Kamm MA. Once daily high dose probiotic therapy (VSL#3) for maintaining remission in recurrent or refractory pouchitis. Gut. 2004;53:108-114.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 623]  [Cited by in F6Publishing: 442]  [Article Influence: 36.6]  [Reference Citation Analysis (0)]
46.  Cui HH, Chen CL, Wang JD, Yang YJ, Cun Y, Wu JB, Liu YH, Dan HL, Jian YT, Chen XQ. Effects of probiotic on intestinal mucosa of patients with ulcerative colitis. World J Gastroenterol. 2004;10:1521-1525.  [PubMed]  [DOI]  [Cited in This Article: ]
47.  Kruis W, Fric P, Pokrotnieks J, Lukás M, Fixa B, Kascák M, Kamm MA, Weismueller J, Beglinger C, Stolte M. Maintaining remission of ulcerative colitis with the probiotic Escherichia coli Nissle 1917 is as effective as with standard mesalazine. Gut. 2004;53:1617-1623.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 767]  [Cited by in F6Publishing: 514]  [Article Influence: 45.1]  [Reference Citation Analysis (0)]
48.  Kanauchi O, Suga T, Tochihara M, Hibi T, Naganuma M, Homma T, Asakura H, Nakano H, Takahama K, Fujiyama Y. Treatment of ulcerative colitis by feeding with germinated barley foodstuff: first report of a multicenter open control trial. J Gastroenterol. 2002;37 Suppl 14:67-72.  [PubMed]  [DOI]  [Cited in This Article: ]
49.  Bamba T, Kanauchi O, Andoh A, Fujiyama Y. A new prebiotic from germinated barley for nutraceutical treatment of ulcerative colitis. J Gastroenterol Hepatol. 2002;17:818-824.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 84]  [Cited by in F6Publishing: 37]  [Article Influence: 4.4]  [Reference Citation Analysis (0)]
50.  Mitsuyama K, Saiki T, Kanauchi O, Iwanaga T, Tomiyasu N, Nishiyama T, Tateishi H, Shirachi A, Ide M, Suzuki A. Treatment of ulcerative colitis with germinated barley foodstuff feeding: a pilot study. Aliment Pharmacol Ther. 1998;12:1225-1230.  [PubMed]  [DOI]  [Cited in This Article: ]
51.  Suzuki A, Mitsuyama K, Koga H, Tomiyasu N, Masuda J, Takaki K, Tsuruta O, Toyonaga A, Sata M. Bifidogenic growth stimulator for the treatment of active ulcerative colitis: a pilot study. Nutrition. 2006;22:76-81.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 40]  [Cited by in F6Publishing: 25]  [Article Influence: 2.5]  [Reference Citation Analysis (0)]
52.  Mallon P, McKay D, Kirk S, Gardiner K. Probiotics for induction of remission in ulcerative colitis. Cochrane Database Syst Rev. 2007;CD005573.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 67]  [Cited by in F6Publishing: 53]  [Article Influence: 4.8]  [Reference Citation Analysis (0)]
53.  Naidoo K, Gordon M, Fagbemi AO, Thomas AG, Akobeng AK. Probiotics for maintenance of remission in ulcerative colitis. Cochrane Database Syst Rev. 2011;CD007443.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 54]  [Cited by in F6Publishing: 38]  [Article Influence: 5.4]  [Reference Citation Analysis (0)]
54.  Sang LX, Chang B, Zhang WL, Wu XM, Li XH, Jiang M. Remission induction and maintenance effect of probiotics on ulcerative colitis: a meta-analysis. World J Gastroenterol. 2010;16:1908-1915.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in CrossRef: 74]  [Cited by in F6Publishing: 50]  [Article Influence: 6.7]  [Reference Citation Analysis (0)]