© Inštitut za sanitarno inženirstvo, 2009. ■ Professional paper
UDC: 616.3
Inflammatory bowel disease, intestinal microflora, prebiotics and probiotics
Rok OREL*
abstract:
Inflammatory bowel disease (IBD) is a group of intestinal conditions characterized by chronic relapsing course of uncontrolled inflammation within the gastrointestinal tract. The two main types of IBD are ulcerative colitis (UC) and Crohn's disease (CD).
Both genetic and environmental factors are involved in etiopathogenesis. According to most recent theories, IBD is probably a consequence of abnormal mucosal immune response to antigens of gut bacterial microflora in geneticaly susceptable individuals. If tolerance to commensal bacteria is lost, an immune response may be elucidated against non-pathogenic bacteria, leading to increased production of inflammatory cytokines and chemokines. Consequently different subsets of inflammatory cells are activated.
Growing knowledge about implication of gut microflora into the pathogenesis of IBD encouraged scientific word to search for new therapeutic strategies concentrated on changing the microenvironmental factors. Nutritional therapy has been advocated in CD patients, especially for children and adolescents. The rationale behind prebiotic use is to elevate the populations of certain beneficial bacteria and thereby quantitatively changing the composition of microflora. Although several prebiotic compounds possess promising properties to have beneficial effect on IBD, only few of them (Plantago ovata, germinated barley foodstuff) have been clinically tested.
Multiple mechanisms of action have been suggested to explain the effect of probiotics in IBD. A great number of basic, animal model and human studies have revealed the great potential of probiotic use in treatment of IBD patients. However, clinical use of probiotics has been proved effective only in a therapy of pouchitis and maintenance of remission in ulcerative colitis, while their effectiveness in a therapy of Crohn's disease is not firmly proved.
key words:
Inflammatory bowel disease, Crohn's disease, Ulcerative colitis, Pouchitis, Intestinal microflora, Prebiotics, Probiotics
Received: 25. 1. 2009. Accepted: 7. 3. 2009.
University Children's Hospital, University Medical Center Ljubljana Medical Faculty, University of Ljubljana Vrazov trg 1, 1000 Ljubljana, Slovenia Phone: 00386 1 5229 276 E-mail: orel.orel@siol.net
* corresponding author
INTRODUCTION
Inflammatory bowel disease (IBD) is a group of idiopathic intestinal conditions characterized by chronic relapsing course of uncontrolled inflammation within the gastrointestinal tract. The two main types of IBD are ulcerative colitis (UC) and Crohn's disease (CD). UC and CD share many characteristics, but they also differ in certain aspects. UC is characterised by mucosal inflammation of large bowel while small bowel mucosa is spared. Rectal mucosa is regularly inflamed and inflammation spreads continuously to the proximal parts of the colon. On the contrary in Crohn's disease not only colon, but any part of gastrointestinal tract from mouth to anus can be involved. Inflammation spreads through all layers of intestinal wall and inflamed and healthy parts of intestine can follow each other (so called skip lesions). Moreover, CD can present not only with intestinal inflammation but also with penetrating lesions such as intestinal fistulas to other parts of intestine, adjacent hollow organs such as urinary bladder or vagina and skin, and with fibrozating course resulting in intestinal stenoses.
Abdominal pain and tenderness, diarrhoea often containing blood and mucus in the stools, as well as fatigue, low grade fever, weight loss and growth retardation in the case of early onset disease in the childhood are most frequent symptoms and signs of IBD. Although IBD are primary diseases of the intestinal tract, involvement of many extraintestinal organs such as joints, skin, eyes, liver, biliary tract or urinary organs is not unusual.
The prevalence of IBD varies in different populations across the world. The prevalence of CD and UC are high in the most developed countries of Northern and Western Europe and North America, reaching 214 and 243, and 198 and 229 per 105, respectively, but they are low in the developing countries of Asia, Africa and South America [1]. Moreover, the incidences of UC and CD are still growing. IBD manifests during childhood and adolescence in approximately 25% to 40% of all patients [2,3].
Etiopathogenesis of IBD is not completely understood. Both genetic and environmental factors are involved. Increased sanitation and the lifestyles within developed countries appear to increase the risk of IBD. It has been proposed that the exposure to unhygienic conditions during childhood can prime the intestinal environment which will lead to optimal mucosal immune development and regulation, preventing a future immune response [4]. According to most recent theories, IBD is probably a consequence of abnormal mucosal immune response to antigens of gut bacterial microflora in genetically susceptible individuals.
Previous studies focused on identifying specific pathogenic infections responsible for IBD. Mycobacterium paratuberculosis, Mycobacterium kansasii, paramixovirus, Listeria monocytogenes, Chlamydia trachomatis, RNA reovirus and Pseudomonas multophilia infection were regarded associated with Crohn's disease, and Escherichia coli, diplostreptococcus, Fusobacterium necrophorum, Shigella, Helicobacter hepaticus and RNA viruses were linked to ulcerative colitis [5]. However, further studies have not confirmed the role of specific infections in the pathogenesis of IBD. Moreover, there is a growing evidence that normal bacterial microflora
Moreover, CD can present not only with intestinal inflammation but also with penetrating lesions such as intestinal fistulas to other parts of intestine, adjacent hollow organs such as urinary bladder or vagina and skin, and with fibrozating course resulting in intestinal stenoses.
According to most recent theories, IBD is probably a consequence of abnormal mucosal immune response to antigens of gut bacterial microflora in genetically susceptible individuals.
Several observations in humans implicate microbial factors in the pathogenesis of IBD. Bowel lesions in IBD occur predominantly in areas with highest bacterial counts like terminal ileum and colon.
In the case of IBD however, genetically predisposed individuals appear to lose the normal tolerance to commensal bacteria, leading to elevated inflammatory response.
can trigger harmful immune reactions in susceptible hosts. The most convincing evidence supporting the role of enteric microflora in the pathogenesis of IBD comes from animal models. Animals with genetically engineered dysregulation of the immune response develop spontaneous colitis when grown in normal conditions resembling IBD in humans. However, they do not develop intestinal inflammation when grown in germ free environment, indicating that bacterial exposure and colonisation are essential for the development of colitis [6-8]. Interleukin-10 deficient mice displayed a significant higher number of mucosal adherent bacteria and lower levels of protective bacteria like Lactobacillus compared with healthy mice. Both the proportion of mucosal adherent bacteria and the development of colitis were significantly decreased by nutritional supplementation with lactose or enema delivery of Lactobacillus reuteri [9]. Similarly, Lactobacillus plantarum when given in feedings to the IL-10 knock-out mice attenuated established colitis, corresponding to reduction in intestinal permeability and anti-endotoxin core antibody levels [10,11].
Several observations in humans implicate microbial factors in the pathogenesis of IBD [12]. Bowel lesions in IBD occur predominantly in areas with highest bacterial counts like terminal ileum and colon [13]. Diversion of faecal stream is associated with distal improvement in patients with CD and relapse occurs after restoration of faecal stream [14]. UC patients who undergo ileal pouch-anal anastomosis surgery develop mucosal inflammation after bacterial colonisation of the pouch [15]. Early IBD lesions can be induced in susceptible individuals by the direct installation of faecal material into non-inflamed excluded loops of intestine [16]. Antibiotic treatment appears to provide clinical benefit in patients with CD and inflammation of ileal pouch [17].
Pathogenic events in IBD may be associated with different alterations in the intestinal flora in the ileum and colon. More bacteria were detected on the mucosal surface of IBD patients than on those of healthy controls and bacterial invasion of mucosa was evident in up to 83% of biopsies from IBD patients but no bacteria were detected in tissue of controls [18]. Moreover, IBD patients have altered composition of commensal enteric bacteria with increased Bacteroides, adherent/invasive Escherichia coli, Enterococci, and decreased Bifidobacterium and Lactobacillus species [19].
INTESTINAL MICROFLORA, IMMUNE SYSTEM, GENES AND INFLAMMATION
Optimal development of intestinal immune system is determined in part by environmental contact with the commensal gut microflora [20]. Changes in the microbial flora may alter mucosal immune development. In the healthy gut, there is a symbiotic relationship between the host and the commensal bacteria in which exposure leads to down-regulation of inflammatory genes, inhibiting the immune response of the gut (4). In the case of IBD however, genetically predisposed individuals appear to lose the normal tolerance to commensal bacteria, leading to elevated inflammatory response. The microbiota then provides a constant stimulus for the host immune system [21].
Majority of genes found to be associated with the increased risk for development of IBD are encoding proteins functioning in preservation of mucosal barrier function or in regulation of mucosal immune system. The major breakthrough of understanding a linkage between genetic predisposition and IBD development was made in 2001 by 3 independent groups which reported the identification of the first Crohn's disease susceptibility gene, NOD2, subsequently renamed CARD15 by the International Nomenclature Committee, on chromosome 16q12 [22-24]. The CARD15 gene encodes a protein that contains 2 caspase rich domains (CARDs), a central nucle-otide-binding domain (NBD), and a leucine-rich repeat region [25]. There are 3 common genetic variants of CARD15 associated with CD, Arg702Trp, Gly908Arg, and Leu1007fsinsC, and many other less common putative variants [22,26]. 10% to 30% of CD patients are heterozygotes for one of these common mutations, whereas 3% to 15% of patients are either ho-mozygotes or compound heterozygotes [27]. The relative risk of development CD associated with carriage of one CARD15 variants is between 1.5 and 3, increasing to 20 to 40 in people carrying two mutations [27]. CARD15 belongs to the family of pattern-recognition receptors which are responsible for microbial recognition, induction of anti-microbial genes and control of adaptive immune response [28]. While CARD15 is an intracellular pattern-recognition protein capable of recognizing peptidoglycans from gram-negative and gram-positive bacteria through the detection of mu-ramyl dipeptide, the minimal motif in all peptidoglycans [29,30], other pattern-recognition receptors, named toll-like receptors (TLRs), recognize other microbial components like lipoproteins (TLR1, 2, 6), double-stranded RNA (TLR3), lipopolysaccharide (TLR4), flagellin (TLR5), single-stranded RNA (TLR7, 8), and CpG DNA (TLR9) [28,31]. Mutations in genes for tolllike receptors as well as for CARD4/NOD1 receptor may be also associated with increased susceptibility for IBD [32-35].
Binding of specific microbial components to the pattern-recognition receptors in most cases, including CARD15, leads to intracellular signalling pathways which result in most occasions to nuclear factor-kB (NF-kB) activation [30,36,37]. NF-kB is a key intracellular signalling molecule in a variety of inflammatory pathways and its elevated in IBD tissues [38]. However, common mutations of CARD15 gene lead to a decrease in NF-kB activation and not to its overactivation [30]. It has been proposed that failure to trigger protective pathways by bacterial components mediated by CARD15 results in defective bacterial eradication [39] resulting in a NF-kB activation by CARD15 independent mechanisms. CARD15 receptors originally thought to be confined to monocytic and dentritic cells [40] have been recently found also in intestinal epithelial cells [41] and Paneth cells [42]. Paneth cells play an important antibacterial role in the gut, secreting potent antimicrobial substances such as lyzozyme, phospholipase A2 and a and ß defensins [27,43].
Pattern-recognition receptors are required to discriminate between pathogenic and commensal microorganisms. If tolerance to commensal bacteria is lost, an immune response may be elucidated against non-pathogenic bacteria, leading to increased production of inflammatory cytokines and chemokines (4). Consequently different subsets of inflammatory cells are activated. Mucosa of patients with CD is dominated by CD4+ lymphocytes with a type 1 helper-T-cell (Th 1) phenotype, characterized by
Mutations in genes for tolllike receptors as well as for CARD4/NOD1 receptor may be also associated with increased susceptibility for IBD.
Paneth cells play an important antibacterial role in the gut, secreting potent antimicrobial substances such as lyzozyme, phospholipase A2 and a and ß defensins.
However, growing knowledge about implication of gut microbial environment into the pathogenesis of IBD encouraged scientific word to search for new therapeutic strategies concentrated on changing the microenvironmental factors that play an important role in the pathogenesis.
the production of interferon-g and interleukin-2, while the mucosa of patients with UC is dominated by CD4+ lymphocytes with an atypical type 2 helper-T-cell (Th 2) phenotype, characterized by production of transforming growth factor ß (TGF-ß) and interleukin-5 [44]. Th1 cytokines activate macrophages to produce a potent mix of broadly active cytokines as interleukin-12, interleukin-18, macrophage migration inhibitor factor, tumour necrosis factor (TNF), interleukin-1, and interleukin-6 [45].
In conclusion, intolerance to intestinal microorganisms because of geneti-cal susceptibility in addition to possible dysbiosis of gut microflora may together lead to broad spectrum inflammation of the gut.
INFLUENCING GUT MICROFLORA - THERAPEUTIC OPTION
IN IBD
Traditionally, therapy of IBD has been directed against inflammatory response of gut immune system. Corticosteroids, 5-aminosalicylates, immunosuppressive and immunoregulatory agents have been used for over a half of a century to treat active disease and to maintain remission. In the last decade new biologic agents, such as anti-TNF antibodies, emerged as therapeutical options targeting immune system components most important for intestinal inflammation. However, growing knowledge about implication of gut microbial environment into the pathogenesis of IBD encouraged scientific word to search for new therapeutic strategies concentrated on changing the microenvironmental factors that play an important role in the pathogenesis.
Not surprisingly, nutritional therapy has been advocated in IBD patients, especially for children and adolescents, for many years, as the disease often results in weight lost, poor growth and development and numerous nutrient deficiencies [46-49]. However, early studies using enteral nutrition with so called elemental formulas, in which proteins were degraded to amino acids, showed that they were not efficacious only in restoring patient's nutritional status but also in reducing activity of intestinal inflammation in CD patients [50-52]. Because of their bitter taste and high cost, elemental formulas have been gradually replaced by semielemental formulas, using short peptides instead of amino acids, and by polymeric formulas containing whole protein molecules. Interestingly, several meta-analy-ses revealed that therapy with enteral nutrition has efficiency comparable of that of corticosteroid treatment in active CD, especially in childhood population and that polymeric formulas are as efficient as the elemental ones [53-57]. Although several mechanisms of an action could be important in therapeutical use of enteral nutrition, its influence on the intestinal microflora may play a crucial role [58].
Experimental evidence of the central role of the luminal flora as an essential factor for the development of IBD provided an impetus to the development of alternative strategies to manipulate the intestinal flora by prebiot-ics and probiotics.
PREBIOTICS AND IBD
Prebiotics are non-digestible food constituents that beneficially affect the host by selectively stimulating the growth or activity of one, or limited
number of bacterial species in the gut, thus improving host health [59]. The rationale behind prebiotic use is to elevate the populations of certain endogene beneficial bacteria such as Lactobacillus and Bifidobacterium and thereby quantitatively changing the composition of microflora. This change may act beneficially by causing luminal production of short chain fatty acids (SCFA), which are important nutrients for the intestinal cells and induce acidic environment, by preventing of pathogenic bacteria adherence and by production of anti-bacterial substances [60]. Some examples of prebiotics are dietary fiber and some types of oligosaccharides. Although several prebiotic compounds possess promising properties to have beneficial effect on IBD, only few of them have been clinically tested.
Inulin and oligofructose are composed of multiple saccharide units, which are indigestible by the human enzymes. They stimulate the growth of lactic acid bacteria and the generation of SCFA [61]. In dextran sodium sulphate-induced colitis animal model, inulin attenuated gut inflammation [61].
Similarly, fructooligosaccharide was shown to decrease the severity of damage in experimental model of rat trinitrobenzene sulfonic acid induced colitis [62].
Psyllium, also called Ispaghula husk or Plantago ovata, is a water soluble dietary fiber [63]. Hallert and co-workers reported that Ispaghula husk significantly attenuates symptoms in patients with UC [64] and Spanish Group for the Study of Crohn's Disease and Ulcerative Colitis (GETECCU) found it as efficient as sulphasalazine in maintaining remission [65].
Germinated barley foodstuff (GBF) is derived from aleurone layer and scutellum fractions of germinated barley and consists mainly of dietary fiber and glutamine-rich protein [66]. It induces intestinal microflora to produce SCFA [67]. Treatment of rat experimental colitis with GBF led to improvement of the clinical and pathological signs of colitis and decrease serum IL-8 and alpha 1-acid-glycoprotein. GBF was comparable effective against mucosal inflammation and more effective against diarrhoea when compared with sulphasalazine [68]. The same Japanese group proved the effectiveness of GBF in several studies on patients with active UC and UC in remission [69,70]. Patients revealed both clinical and endoscopic improvement of colitis. Therefore, GBF is registered as a special foodstuff for UC by Japan's Ministry of Health, Labour and Welfare.
PROBIOTICS AND IBD
Probiotics are defined as live microorganisms which, when consumed in adequate quantities, confer a health benefit on the host [21]. Multiple mechanisms of action have been suggested to explain the effect of probiotics in IBD. Potential mechanisms include suppression of growth or epithelial binding and invasion by pathogenic bacteria, production of antimicrobial substances, improved epithelial barrier function, and immunoregulation [60,71]. A great number of basic, animal model and human studies have revealed the great potential of probiotic use in treatment of IBD patients.
The effects of probiotic are probably both strain-dependent and dose dependent. For example, probiotic Lactobacillus rhamnosus GG (LGG) at-
The rationale behind prebiotic use is to elevate the populations of certain endogene beneficial bacteria such as Lactobacillus and Bifidobacterium and thereby quantitatively changing the composition of microflora.
Treatment of rat experimental colitis with GBF led to improvement of the clinical and pathological signs of colitis and decrease serum IL-8 and alpha 1-acid-glycoprotein.
The effects of probiotic are probably both strain-dependent and dose dependent.
The effect of probiotics on barrier function was studied in T84 cell monolayers. It was demonstrated that VSL#3 prevented the decrease in trans-epithelial resistance following incubation with pathogenic bacteria.
The reviewers concluded that probiotics in combination with conventional therapy do not improve overall remission rates in patients with mild to moderate ulcerative colitis however; they may provide some benefits in terms of reduction of disease activity.
tenuated the TNF-a induced IL-8 production at doses 106"8 by the Caco-2 intestinal cell line, but on the contrary, at higher doses LGG actually increased IL-8 levels [72]. This finding indicated that determining the correct dose of probiotic for treatment is vital. The same study demonstrated that heat-killed LGG was also able to decrease IL-8 production, conflicting the paradigm that viability of probiotics is essential for their efficacy. Similarly, bacterial DNA from VSL#3, a high dose mixture of three strains of Bifidobacteria, four strains of Lactobacilli, and one strain of Streptococcus salivarius ssp. thermophilus, was able to decrease IL-8 secretion, delay NF-kB activation and stabilise IkB levels [73]. However, in another study using Lactobacillus reuteri on HT-29 and T84 cells only live but not deactivated bacteria reduced TNF-a induced IL-8 production and induced production of anti-inflammatory factors [74]. The effect of probiotics on barrier function was studied in T84 cell monolayers. It was demonstrated that VSL#3 prevented the decrease in trans-epithelial resistance following incubation with pathogenic bacteria [75].
The most convincing evidence of probiotic efficacy and mechanisms came from animal studies. More than 20 different animal models of IBD are available [76]. In IL-10 knockout mice, L. plantarum 299V [77], L. salivarius subspecies salivarius 433118 and UCC118 [78,79] , B. infantis 35624 [78], and VSL#3 (80) have been shown to attenuate intestinal inflammation [76]. In HLA-B27 transgenic rats LGG prevented recurrent colitis after antibiotic treatment, whereas L. plantarum had no effect (81). Amelioration of inflammation was observed after administration of L. salivarius subspecies salivarius CECT5713 [82] and L. plantarum NCIMB8826 [83] in trinitrobenzene sulfonic acid-induced colitis, and after L .reuteri and oat fiber application in acetic acid and methotrexate induced colitis in rats [84,85]. Similarly, VSL#3 and LGG significantly improved sulphhydril-blocker iodoacetamide-induced colitis in rats, but had no effect on dinitrobenzene sulfonic acid-induced colitis [86]. These experiments clearly demonstrated that the effect of probiotic treatment depends both on probiotic strain and on type of inflammation.
Not only live bacteria, but also soluble bacterial antigens extracted from E. coli in dextran sulphate sodium-induced colitis (DSS) model [87] and bacterial DNA from VSL#3 preparation in DSS and IL-10 knockout mouse models [73,88] showed the ability to reduce inflammation. Therefore, viability of probiotic bacteria was not proven to be a prerequisite for their effect.
Many clinical trials have been performed to investigate the efficacy of pro-biotics in achieving and maintaining remission of different forms of IBD. However, only four which investigated the efficacy of probiotics for induction of remission in ulcerative colitis met the criteria of randomised controlled trials and were reviewed in recent Cochrane Collaboration review [89]. The reviewers concluded that probiotics in combination with conventional therapy do not improve overall remission rates in patients with mild to moderate ulcerative colitis however; they may provide some benefits in terms of reduction of disease activity.
Rembacken et al. [90] reported that probiotics (E. coli Nissle 1917) with steroids had similar effectiveness to mesalazine with steroids in achieving
remission, however relapse rate was slightly higher in the mesalazine group compared to probiotic group (73% vs. 67%, P < 0.05).
Kato et al. [91] reported that probiotics (Bifidobacterium breve, B. bifi-dum, Lactobacillus acidophilus) with 5-aminosalicilates (5-ASA) were as effective as placebo with 5-ASA when they compared the proportion of patients achieving the remission. However, when they took into account not only remission data but also clinical improvement data, clinical activity index (CAI) was found to be significantly lover in the probiotic group compared to placebo group after 12 weeks of treatment (3.7+/-0.4 vs. 5.8+/-0.8, P < 0.05). They also found that mean endoscopic index score and mean histological score was significantly reduced in the probiotic group (P < 0.01) but not in the placebo group after therapy.
Although Tursi et al. [92] did not found statistical significant difference in proportion of patients who achieved remission in probiotic (VSL#3)+5-ASA group in compare with placebo+5-ASA group, the mean time to remission was significantly shorter for probiotic group (4 vs. 7 days, P < 0.01).
In the trial of Furrie and colleagues [93] no significant differences were found between synbiotic (probiotic Bifidobacterium longum and fructooli-gosaccharide) group and placebo group in numbers of patients improved, CAI, endoscopy and histology scores. However, synbiotic treatment in conjunction with standard therapy caused significant reduction in TNF-a and IL-1a compared to standard therapy and placebo.
Several controlled studies showed that some probiotics can be used in the maintenance therapy of UC. Rembacker et al. [94] randomized UC patients who entered remission with conventional therapy to receive mesala-zine or probiotic (E. coli Nissle 1917) for maintenance treatment for one year. At the end of the trial 73% of 5-ASA-treated patients relapsed as compared with 67% of those assigned to the probiotic. Authors concluded that the two strategies were of equivalent efficacy.
Same can be concluded from the studies of Kruis et al. They found that 11.3% of patients treated with 5-ASA relapsed in 12-week follow-up period as compared with 16% treated with E. coli Nisssle 1917 [95]. In their later study they found that during one year follow-up relapse occurred in 36.4% of the E. coli group and 33.9% of the mesalazine group [96].
Ishikawa et al. found out that bifidobacteria-fermented milk supplemented as a dietary adjunct was effective in maintaining remission of UC. After 1 year treatment, exacerbation of symptoms was observed in 27% of probi-otic group and in 90% of the control group [97].
Zocco et al. [98] compared three groups of therapeutic regimen for maintenance of remission in UC patients. They found no significant difference in relapse rate at 6 and 12 month between the groups that received Lactobacillus rhamnosus GG or 5-ASA or both of them.
In an open uncontrolled study Venturi et al. [99] with VSL#3, 75% of patients with UC remained in remission during one-year follow-up.
Pouchitis, chronic inflammation of ileal pouch created after proctocolectomy, is usually treated by antibiotics. However, several controlled trials have revealed that probiotic use can be highly effective. Gionchetty et al.
Several controlled studies showed that some probiotics can be used in the maintenance therapy of UC.
In contrast to these results, probiotic Lactobacillus rhamnosus GG was ineffective in preventing relapses of chronic pouchitis.
In conclusion, clinical use of probiotics has been proved effective in a therapy of pouchitis and maintenance of remission in ulcerative colitis, while their effectiveness in a therapy of Crohn's disease is not firmly proved.
[100] compared the efficacy of VSL#3 with placebo in maintenance of remission of pouchitis. The patients in the probiotic group relapsed in 15% as compared with 100% in the placebo group.
These results were practically replicated by Mimura et al. [101], who found that the relapse rate in one year after beginning of the therapy was 15% for VSL#3 group versus 94% for the placebo group.
Gionchetti et al. [102] studied prophylactic role of probiotic treatment in patients undergoing colectomy and pouch surgery. During the first year after operation only 10% of the patients in VSL#3 group but 40% of the patients in placebo group developed pouchitis.
In contrast to these results, probiotic Lactobacillus rhamnosus GG was ineffective in preventing relapses of chronic pouchitis [103].
Efficacy of probiotics for preventing recurrence of active disease has been studied in patients who rich the remission after medical or surgical therapy of CD. Malchow [104] who treated small number of patients with colonic CD in remission with either E. coli Nissle 1917 or placebo for 3 months, observed relapse rate 33% in the probiotic group and 63% in the placebo group.
Campieri et al. [105] reported that VSL#3 in combination with 5-ASA were more efficient than 5-ASA alone in prevention of post-operative recurrence of CD. They observed endoscopic recurrence in 40% of patients treated with 5-ASA alone but only in 10% of patients on combined therapy with 5-ASA and probiotics.
However, in two clinical studies using probiotic Lactobacillus rhamnosus GG, no differences were found in compare to placebo in CD relapse prevention [106,107]. French GETAID group [108] reported slightly lower en-doscopic recurrence in a group of patients that was treated post-opera-tively with probiotic Lactobacillus johnsonii strain LA1 than in the placebo group (49% vs. 64%), but the difference was not statistically significant.
Guslandi et al. [109] reported that maintenance therapy with Saccharo-myces boulardii and 5-ASA was significantly more effective in preventing relapse of CD than 5-ASA alone.
In the meta-analysis by Rolfe et al. for Cochrane Collaboration [110] seven studies were identified to reach the inclusion criteria for review of maintenance therapy in CD. The authors concluded that there was no statistical significant benefits of E. coli for reducing the risk of relapse compared to placebo (RR 0.43, 95% CI 0.15 to 1.20), or Lactobacillus GG after surgically-induced remission (RR 1.58, 95% CI 0.30 to 8.40) or medically-induced remission (RR 0.83, 95% CI 0.25 to 2.80). There were no statistically significant benefits for reducing the risk of relapse compared to maintenance therapy employing aminosalicylates or azathioprine (RR 0.67, 95% CI 0.13 to 3.30). In children, there was no statistically significant difference between Lactobacillus GG and placebo for reducing the risk of relapse (RR 1.85, 95% CI 0.77 to 4.40).
In conclusion, clinical use of probiotics has been proved effective in a therapy of pouchitis and maintenance of remission in ulcerative colitis, while their effectiveness in a therapy of Crohn's disease is not firmly proved.
REFERENCES
[1]	Loftus EV, Sandborn WJ. Epidemiology of inflammatory bowel disease. Gastroenterol Clin N Am 2002; 31: 1-20.
[2]	Griffiths AM, Hugot J-P. Crohn Disease. In: Walker WA, Goulet O, Kleinman RE, Sherman PM, Schneider BL, Sanderson (eds.). Pediatric Gastrointestinal Disease. Fourth edition. Hamilton, Ontario, Decker Inc, 2004: 789-824.
[3]	Leichtner AM, Higuchi L. Ulcerative colitis. In: Walker WA, Goulet O, Kleinman RE, Sherman PM, Schneider BL, Sanderson (eds.). Pediatric Gastrointestinal Disease. Fourth edition. Hamilton, Ontario, Decker Inc, 2004: 825-49.
[4]	Geier MS, Butler RN, Howarth GS. Inflammatory bowel disease: current insights into pathogenesis and new therapeutic options: probiotics, prebiotics and synbiotics. International J Food Microbiol 2007; 115: 1-11.
[5]	Sartor RB. Microbial factors in the pathogenesis of Crohn's disease, ulcerative colitis, and experimental intestinal inflammation. In: Kirschner JB, ed. Inflammatory bowel disease. Philadelphia, WB Saunders, 2000: 153-78.
[6]	Contractor NV, Bassiri H, Reya T, et al. Lymphoid hyperplasia, autoimmunity, and compromised intestinal intraepithelial lymphocyte development in colitis-free gnotobiotic IL-2-deficient mice. J Immunol 1998 1; 160: 385-94.
[7]	Dianda L, Hanby AM, Wright NA, Sebesteny A, Hay-day AC, Owen MJ. T cell receptor-alpha beta-deficient mice fail to develop colitis in the absence of a microbial environment. Am J Pathol 1997; 150: 91-7.
[8]	Sellon RK, Tonkonogy S, Shultz M, et al. Resident enteric bacteria are necessary for development of spontaneous colitis and immune system activation in in-terleukin-10-deficient mice. Infect Immunol 1998; 66: 5224-31.
[9]	Madsen KL, Doyle IS, Jewell D, et al. Lactobacillus species prevent colitis in interleukin 10 gene-deffi-cient mice. Gastroenterology 1999; 116: 1107-14.
[10]	Schultz M, Veltcamp C, Dieleman LA, et al. Lactobacillus plantarum 299V in the treatment and prevention of spontaneous colitis in interleukin-10-deficient mice. Inflamm Bowel Dis 2002; 8: 71-80.
[11]	Kennedy RJ, Hoper M, Deodhar K, Erwin PJ, Kirk SJ, Gardiner KR. Interleukin 10-deficient colitis: new similarities to human inflammatory bowel disease. Br J Surg 2000; 87: 1346-51.
[12]	Kwon JH, Farrell RJ. Probiotics and inflammatory bowel disease. Biodrugs 2003; 17: 179-86.
[13]	Sartor RB. The influence of normal microbial flora on the development of chronic mucosal inflammation. Res Immunol 1997; 148: 567-76.
[14]	Rootgeerts P, Geboes K, Peeters M, et al. Effect of faecal stream diversion on reccurence of Crohn's disease in a neoterminal ileum. Lancet 1991; 338: 7714.
[15]	Sandborn WJ, Landers CJ, Tremaine WJ, Targan SR. Antineutrophil cytoplasmic antibody correlates with chronic pouchitis after ileal pouch-anal anastomosis. Am J Gastroenterol 1995; 90: 740-7.9.
[16]	D'Haens GR, Geboes K, Peeters M, Baert F, Ectors N, Rutgeerts P. Early lesions of recurrent Crohn's disease caused by infusion of intestinal contents in excluded ileum. Gastroenterology 1998; 114: 262-7.
[17]	Guslandi M. Antibiotics for inflammatory bowel disease: do they work? J Gastroenterol Hepatol 2005; 17: 145-7.
[18]	Kleesen B, Kroesen AJ, Buhr HJ, Blaut M. Mucosal and invading bacteria in patients with inflammatory bowel disease compared with controls. Scand J Gas-troenterol 2002; 37: 1034-41.
[19]	Neut C, Bulois P, Desreumaux P, et al. Changes in the bacterial flora of the neoterminal ileum after ileoco-lonic resection for Crohn's disease. Am J Gastroen-terol 2002; 97: 939-46.
[20]	Rock GAW, Stanford JL. Give us this day our daily germs. Immunol Today 1998; 19: 112-6.
[21]	Shanahan F. Probiotics in inflammatory bowel disease-therapeutic rationale and role. Advanced Drug Delivery Rew 2004; 56: 809-18.
[22]	Hugot JP, Chamaillard M, Zouali H, et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease. Nature 2001; 411: 599-603.
[23]	Ogura Y, Bonen DK, Inohara N, et al. A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease. Nature 2001; 411: 603-6.
[24]	Hampe J, Cuthbert A, Croucher PJ, et al. Association between insertion mutation in NOD2 gene and Crohn's disease in German and British populations. Lancet 2001; 357: 1925-8.
[25]	Ogura Y, Inohara N, Benito A, Chen FF, Yamaoka S, Nunez G. Nod2, a Nod1/Apaf-1 family member that is restricted to monocytes and activates NF-kappaB. J Biol Chem 2001; 276: 4812-8.
[26]	Lesage S, Zouali H, Cezard JP, et al. CARD15/NOD2 mutational analysis and genotype-phenotype correlation in 612 patients with inflammatory bowel disease. Am J Hum Genet 2002; 70: 845-57.
[27]	Ahmad T, Tamboli CP, Jewell D, Colombel J-F. Clinical relevance of advances in genetics and pharmacogenetics of IBD. Gastroenterology 2004; 126: 153349.
[28]	Cario E. Bacterial interactions with cells of the intestinal mucosa: toll-like receptors and NOD2. Gut 2005; 54: 1182-93.
[29]	Girardin SE, Boneca IG, Viala J, et al. Nod2 is a general sensor of peptidoglycan through muramyl dipep-tide (MDP) detection. J Biol Chem 2003; 278: 836972.
[30]	Inohara N, Ogura Y, Fontalba A, et al. Host recognition of bacterial muramyl dipeptide mediated through NOD2: implication for Crohn's disease. J Biol Chem 2003; 278: 5509-12.
[31]	O'Neill LA. How toll-like receptors signal: what we know and what we not know. Cur Opin Immunol 2006; 18: 3-9.
[32]	Girardin SE, Boneca IG, Carneiro LA, et al. Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science 2003; 300:1584-7.
[33]	Torok HP, Glas J, Tonenchi L, et al. Polymorphism of the lipopolysaccharide-signaling complex in inflammatory bowel disease: association of a mutation in the toll-like receptor 4 gene with ulcerative colitis. Clin Immunol 2004; 112: 85-91.
[34]	McGovern DP, Hysi P, Ahmad T, et al. Association between a complex insertion/deletion polymorphism in NOD1 (CARD4) and suspectibility to inflammatory bowel disease. Hum Mol Genet 2005; 14: 1245-50.
[35]	Rosenstiel P, Sina C, End C, et al. Regulation of DMBT1 via NOD2 and TLR4 in intestinal epithelial cells modulates bacterial recognition and invasion. J Immunol. 2007; 178: 8203-11.
[36]	Chamaillard M, Girardin SE,Viala J, Philpott DJ. Nods, Nalps and Naip: intracellular regulators of bacterial-induced inflammation. Cell Microbiol 2003;5:581-92.
[37]	Girardin SE, Boneca IG, Viala J, et al. Nod2 is a general sensor of peptidoglycan through muramyl dipep-tide (MDP) detection. J Biol Chem 2003; 278: 886972.
[38]	Neurath MF, Fuss I, Schürmann G, et al. Cytokine gene transcription by NF-kappa B family members in patients with inflammatory bowel disease. Ann N Y Acad Sci 1998; 859: 149-59.
[39]	Hisamatsu T, Suzuki M, Reinecker HC, Nadeau WJ, McCormick BA, Podolski DK. CARD15/NOD2 functions as an antibacterial factor in human intestinal epithelial cells. Gastroenterology 2003; 124: 9931000.
[40]	Gutierrez O, Pipaon C, Inohara N, et al. Induction of Nod2 in myelomonocytic and intestinal epithelial cells via nuclear factor-kappa B activation. J Biol Chem. 2002; 277: 41701-5.
[41]	Rosenstiel P, Fantini M, Bräutigam K, et al. TNF-al-pha and IFN-gamma regulate the expression of the NOD2 (CARD15) gene in human intestinal epithelial cells. Gastroenterology 2003; 124: 1001-9.
[42]	Lala S, Ogura Y, Osborne C, et al. Crohn's disease and the NOD2 gene: a role for Paneth cells. Gastroenterology 2003; 125: 47-57.
[43]	Ayabe T, Satcheli DP, Wilson CL, Parks WC, Selsted ME, Quellette AJ. Secretion of microbicidal alpha-de-fensins by intestinal Paneth cells in response to bacteria. Nat Immunol 2000; 1: 113-8.
[44]	Fuss IJ, Neurath M, Boirivant M, et al. Disparate CD4+ lamina propria (LP) lymphokine secretion profiles in inflammatory bowel disease: Crohn's disease LP cells manifest increased secretion of INF-gamma, whereas ulcerative colitis LP cells manifest increased secretion of IL-5. J Immunol 1996; 157: 1261-70.
[45]	Podolsky DK. Inflammatory bowel disease. N Engl J Med 2002; 347: 417-29.
[46]	Hill RJ, Cleghorn GJ, Withers GD, Lewindon PJ, Ee LC, Connor F, Davies PSW. Resting energy expediture in children with inflammatory bowel disease. J Pedi-atr Gastroenterol Nutr 2007; 45: 342-6.
[47]	Kleinman RE, Baldassano RN, Caplan A, Griffiths AM, Heyman MB, Issenman RM, Lake AM. Nutrition support for pediatric patients with inflammatory bowel disease: a clinical report of the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition. J Pediatr Gastroenterol Nutr 2004; 39: 1527.
[48]	Kirschner BS. Growth and development in chronic inflammatory bowel disease. Acta Pediatr Scand 1990; 366: 98-104.
[49]	Kugathasan S, Nebel J, Skelton JA, Markowitz J, Kel-jo D, et al. Body mass index in children with newly diagnosed inflammatory bowel disease: observations from two multicenter North American inception cohorts. J Pediatr 2007; 151: 523-7.
[50]	Vointk AJ, Echave V, Feller JH, Brown RA, Gurd FN. Experience with elemental diet in the treatment of inflammatory bowel disease. Is this primary therapy? Arch Surg 1973; 107: 329-33.
[51]	Navarro J, Vargas J, Cezard JP, Charritat JL, Polonovski C. Prolonged constant rate elemental en-teral nutrition in Crohn's disease. J Pediatr Gastroenterol Nutr 1982; 1: 541-6.
[52]	Morin CL, Roulet M, Roy CC, Weber A. Continous elemental enteral alimentation in children with Crohn's disease and growth failure. Gastroenterology 1980: 79; 1205-10.
[53]	Griffiths AM, Ohlsson A, Sherman PM, Sutherland LR. Meta-analysis of enteral nutrition as a primary treatment of active Crohn's disease. Gastroenterology 1995; 108: 1056-67.
[54]	Fernandez-Banares F, Cabre E, Esteve-Comas M, Gassull MA. How efective is enteral nutrition in inducing clinical remission in active Crohn's disease? A meta-analysis of the randomized clinical trials. JPEN 1995; 19: 356-64.
[55]	Messori A, Trallori G, D'Albasio G, Milla M, Vanozzi G, Pacini F. Defined-formula diets versus steroids in the treatment of active Crohn's disease: A meta-anal-ysis. Scand J Gastroenterol 1996; 31: 267-72.
[56]	Zachos M, Tondeur M, Griffiths AM. Enteral nutritional therapy for induction of remission in Crohn's disease. Cochrane Database of Systematic Reviews 2007; Issue 1, Art. No. CD000542. DO1: 10.1002/14651858.
[57]	Heuschkel RB, Menache CC, Megerian JT, Baird AE. Enteral nutrition and corticosteroids in the treatment of acute Crohn's disease in children. J Pediatr Gastroenterol Nutr 2000; 31: 8-15.
[58]	Lionetti P, Callegari ML, Ferrari S, Cavicchi MC, Pozzi E, de Martino M, Morelli L. Enteral nutrition and microflora in pediatric Crohn's disease. JPEN 2005; 29: S173-5.
[59]	Bengmark S,Martindale R. Prebiotics and synbiotics in clinical medicine. Nutr Clin Pract 2005; 20: 24461.
[60]	Sartor RB. Therapeutic manipulation of the enteric microflora in inflammatory bowel disease: antibiotics, probiotics and prebiotics. Gastroenterology 2004; 126: 1620-33.
[61]	Videla S, Vilaseca J, Antolin M, et al. Dietary inulin improves distal colitis induced by dextran sodium sulfate in the rat. Am J Gastroenterol. 2001; 96: 148693.
[62]	Cherbut C, Michel C, Lecannu G. The prebiotic characteristics of fructooligosaccharides are necessary for reduction of TNBS-induced colitis in rats. J Nutr 2003;133: 21-7.
[63]	Kanauchi O, Mitsuyama K, Araki Y, Andoh A. Modification of intestinal flora in treatment of inflammatory bowel disease. Curr Pharm Des 2003; 9: 333-46.
[64]	Hallert C, Kadma M, Petersson BG. Ispaghula husk may relieve gastrointestinal symptoms in ulcerative colitis in remission. Scand J Gastroenterol 1991; 26: 747-50.
[65]	Fernandez-Banares F, Hinojosa J, Sanchez-Lombrana JL, Navarro E, Martinez-Salmeron JF, Garcia-Puges A, et al. Randomized clinical trial of Plantago ovata seeds (dietary fiber) as compared with mesalamine in maintaining remission in ulcerative colitis. Spanish Group for the Study of Crohn's Disease and Ulcerative Colitis (GETECCU). Am J Gastroenterol 1999; 94: 427-33.
[66]	Kanauchi O, Agata K. Protein, and dietary fiber-rich new foodstuff from brewer's spent grain increased excretion of feces and jejunum mucosal protein content in rats. Biosci Biotechnol Biochem 1997; 61: 29-33.
[67]	Kanauchi O, Fujiyama Y, Mitsuyama K, et al. Increased growth of Bifidobacterium and Eubacterium by germinated barley foodstuff, accompanied by enhanced butyrate production in healthy volunteers. Int J Mol Med 1999; 3: 175-9.
[68]	Kanauchi O, Nakamura T, Agata K, Mitsuyama K, Iwanaga T. Effects of germinated barley foodstuff on dextran sulfate sodium-induced colitis in rats. J Gastroenterol 1998; 33: 179-88.
[69]	Kanauchi O, Mitsuyama K, Homma T, et al. Treatment of ulcerative colitis patients by long-term administration of germinated barley foodstuff: multicenter open trial. Int J Mol Med 2003;12: 701-4.
[70]	Hanai H, Nakamuchi O, Mitsuyama K, et al. Germinated barley foodstuff prolongs remission in patients with ulcerative colitis. Int J Mol Med 2004; 13: 6437.
[71]	Mitsuyama K, Tonoyaga A, Sata M. Intestinal microflora as a therapeutic target in inflammatory bowel disease. Gastroenterology 2002; 37 (Suppl XIV): 737.
[72]	Zhang L, Li N, Calcedo R, Neu J. Alive and dead Lactobacillus rhamnosus GG decrease tumor necrosis factor-alpha-induced interleukin-8 production in Caco-2 cells. J Nutr 2005; 135: 1752-6.
[73]	Jijon H, Becker J, Diaz H, et al. DNA from probiotic bacteria modulates murine and human epithelial and immune function. Gastroenterology 2004; 126: 1358-73.
[74]	Ma D, Forsythe P, Bienenstock J. Live Lactobacillus reuteri is essential for the inhibitory effect on tumor necrosis factor alpha-induced interleukin-8 expression. Infect Immun 2004; 72: 5308-14.
[75]	Otte JM, Podolsky DK. Functional modulation of en-terocytes by gram-positive and gram-negative microorganisms. Am J Physiol 2004; 286: G613-26.
[76]	Evaschuk JB, Dieleman LA. Probiotics and prebiotics in chronic inflammatory bowel diseases. World J Gas-troenterl 2006; 12: 5941-50.
[77]	Schultz M, Veltkamp C, Dieleman LA, et al. L plantarum 299V in the treatment and prevention of spontaneous colitis in interleukin-10-deficient mice. Inflamm Bowel Dis 2002; 8: 71-80.
[78]	McCarthy J, O'Mahony L, O'Callaghan L, et al. Double blind, placebo controlled trial of two probiotic strains in interleukin 10 knockout mice and mechanistic link with cytokine balance. Gut 2003; 52: 975-80.
[79]	O'Mahony L, Feeney M, O'Halloran S, et al. Probiotic impact on microbial flora inflammation and tumor development in IL-10 knockout mice. Aliment Pharmacol Ther 2001; 15: 1219-25.
[80]	Madsen K, Cornish A, Soper P, et al. Probiotic bacteria enhance murine and human intestinal epithelial barrier function. Gastroenterology 2001; 121: 580-91.
[81]	Dielman LA, Goerres MS, Arends A, et al. Lactobacillus GG prevents reccurence of colitis in HLA-B27 transgenic rats after antibiotic treatment. Gut 2003; 52: 370-6.
[82]	Peran L, Camuesco D, Comalda M, et al. Preventive effect of a probiotic, Lactobacillus salivarius ssp. sali-varius, in the TNBS model of rat colitis. World J Gas-troenterol 2005; 11: 5185-92.
[83]	Pavan S, Desreumaux P, Mercenier A.Use of mouse models to evaluate the persistence, safety, and immune modulation capacities of lactic acid bacteria. Clin Diagn Lab Immunol 2003; 10: 696-701.
[84]	Fabia R, Ar'Rajab A, Johansson ML, et al. The effect of exogenous administration of Lactobacillus reuteri R2LC and oat fiber on acetic acid-induced colitis in the rat. Scand J Gastroenterol 1993; 28: 155-62.
[85]	Mao Y, Nobaek S, Kasravi B, et al. The effect of lactobacillus strains and oat fiber on methotrexate-in-duced enterocolitis in rats. Gastroenterology 1996; 111: 334-44.
[86]	Shibolet O, Karmeli F, Eliakim R, Swennen E, et al. Variable response to probiotics in two models of experimental colitis in rats. Inflamm Bowel Dis 2002; 8: 399-406.
[87]	Konrad A, Mahler M, Flogerzi B, et al. Amelioration of murine colitis by feeding a solution of lysed Escherichia coli. Scand J Gastroenterol 2003; 38: 172-9.
[88]	Rachmilewitz D, Katakura K, Karmeli F, et al. Toll-like rceptor 9 signaling mediates the antiinflammatory effects of probiotics in murine experimental colitis. Gastroenterology 2004; 126: 520-8.
[89]	Mallon P, McKay D, Kirk S, Gardiner K. Probiotics for induction of remission in ulcerative colitis. Cochrane Database of Systematic Reviews 2007; Issue 4, Art. No.: CD005573. DOI: 10.1002/14651858. CD005573.pub2.
[90]	Rembacken BJ, Snelling AM, Hawkey PM, Chalmers DN, Axon AT. Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomized trial. Lancet 1999; 354: 635-9.
[91]	Kato K, Mizuno S, Umesaki Y, et al. Randomized placebo-controlled trial assessing the effect of bifidobac-teria-fermented milk on active ulcerative colitis. Aliment Pharmacol Ther 2004; 20: 1133-41.
[92]	Tursi A, Brandimarte G, Giorgetti GM, Forti G, Modeo ME, Gigliobianco A. Low dose balsalazide plus a high potency probiotic preparation is more effective than balsalazide alone or mesalazine in the treatment of acute mild-to-moderate ulcerative colitis. Med Sci Monit 2004; 10: 126-31.
[93]	Furrie E, MacFerlane S, Kennedy A, et al. Synbiotic therapy (Bifidobacterium longum/Synergy 1) initiates resolution of inflammation in patients with active ulcerative colitis: a randomised controlled trial. Gut 2005; 54: 242-9.
[94]	Rembacker BJ, Snelling AM, Hawkey PM, Dixon ATR. A double blind trial on non pathogenic E. coli vs mesalazine for the treatment of ulcerative colitis. Gut 1997; 41: 3911.
[95]	Kruis W, Schutz E, Fric P, Fixa B, Judmaier G, Stolte M. Double-blind comarison of oral Escherichia coli preparation and mesalazine in maintaining remission of ulcerative colitis. Alliment Pharmacol Ther 1997; 11: 853-8.
[96]	Kruis W, Fric P, Pokrotnieks J, et al. Maintaining remission of ulcerative colitis with the probiotic Escherichia coli Nissle 1917 is as effective as with standard mesalazine. Gut 2004; 53: 1617-23.
[97]	Ishikawa H, Akedo I, Umesaki Y, Tanaka R, Imaoka A, Otani T. Randomized controlled trial on the effect of bifidobacteria-fermented milk on ulcerative colitis. J Am Coll Nutr 2003; 22: 56-63.
[98]	Zocco MA, dal Verme LZ, Cremonini F, et al. Efficacy of Lactobacillus GG in maintaining remission of ulcer-ative colitis. Aliment Pharmacol Ther 2006; 23: 1567-4.
[99]	Venturi A, Gioncherti P, Rizzelo P, et al. Impact on the composition of the faecal flora by a new probiotic prparation: preliminary data on maintenance treatment of patients with ulcerative colitis. Alliment Pharmacol Ther 1999; 13: 1103-8.
[100]	Gionchetti P, Rizzelo F, Venturi A, et al. Oral bacterio-therapy as maintenance treatment in patients with chronic pouchitis: a double-blind, placebo-controlled trial. Gastroenterology 2000; 119: 305-9.
[101]	Mimura T, Rizzelo F, Helwig U, et al. Once daily high dose probiotic therapy (VSL#3) for maintaining remission in recurrent or refractory pouchitis. Gut 2004; 53: 108-14.
[102]	Gionchetti P, Rizzelo F,Helwig U, et al. Prophylaxis of pouchitis onset with probiotic therapy: a double-blind placebo-controlled trial. Gastroenterology 2003; 124:1202-9.
[103]	Kuisma J, Mentula S, Jarvinen H, Kahri A, Saxelin M, Farkkila M. Effect of Lactobacillus rhamnosus GG on ileal pouch inflammation and microbial flora. Aliment Pharmacol Ther 2003; 17: 509-15.
[104]	Malchow HA. Crohn's disease and Escherichia coli. A new aproach in therapy to mantain remission of colonic Crohn's disease? J Clin Gastroenterol 1997; 25: 653-8.
[105]	Campieri M, Rizzelo F, Venturi A, et al. Combination of antibiotic and probiotic treatment is efficious in prophylaxis of post-operative recurrence of Crohn's disease: a randomized controlled study vs mesalasi-ne. Gastroenterology 2000; 118: A781.
[106]	Prantera C, Scribano ML, Falasco G, Andreoli A, Luzi C. Ineffectiveness of probiotics in preventing recurrence after curative resection for Crohn's disease: a randomised controlled trial with Lactobacillus GG. Gut 2002; 51: 405-9.
[107]	Schultz M, Timmer A, Herfath H, et al. Lactobacillus GG in inducing and maintaining remission of Crohn's disease. BMC Gastroenterol 2004; 4: 5.
[108]	Marteau P, Lemann M, Seksik P, et al. Ineffectiveness of Lactobacillus johnsonii LA1 for prophylaxis of postoperative recurrence in Crohn's disease: a randomised, double-blind, placebo-controlled GETAID trial. Gut 2006; 55: 842-7.
[109]	Guslandi M, Mezzi G, Sorghi M, Testoni PA. Saccha-romyces boulardii in maintenance treatment of Crohn's disease. Dig Dis Sci 2000; 45: 1462-4.
[110]	Rolfe VE, Fortun PJ, Hawkey CJ, Bath-Hextall F. Probiotics for maintenance of remission in Crohn's disease. Cochrane Database Systematic Reviews 2006, Issue 4, Art. No.: CD004826. DOI: 10.1002/ 14651858. CD004826.pub2.