Probiotics in Inflammatory Bowel Diseases

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Introduction

The rationale for using probiotics in inflammatory bowel disease (IBD) is based on convincing evidence implicating intestinal bacteria in their pathogenesis.

The distal ileum and the colon are the areas with the highest bacterial concentrations and represent the sites of inflammation in IBD; similarly pouchitis, the non-specific inflammation of the ileal reservoir after ileo-anal anastomosis, appears to be associated with bacterial overgrowth and dysbiosis. Enteric bacteria or their products have been found within the inflamed mucosa of patients with Crohn's disease (CD) [1].

There is evidence of a loss of immunologic toler- ance to commensal bacteria in patients with IBD [2, 3]. Patients with CD respond consistently to diver- sion of the faecal stream, with immediate recurrence of inflammation after restoration of intestinal conti- nuity [4] or infusion of luminal content into the bypassed ileum [5], and pouchitis does not occur before the ileostomy closure.

Patients with IBD have altered composition of enteric flora with increase of aggressive bacteria such as Bacterioides, adherent/invasive Escherichia coli, Enterococci, and decrease of protective Lactobacillus and Bifidobacterium species [6]. However, the most compelling evidence is derived from animal models;

despite great diversity in genetic defects and immunopathology, a consistent feature of many transgenic and knockout mutant murine models of colitis is dependency on the presence of normal enteric flora for full expression of inflammation [7].

All these observations have suggested the possibility of preventing or treating IBD by manipulating intestinal microflora and increasing evidence supports the potential therapeutic role of probiotics in IBD [8].

Probiotics

The first to propose the use of probiotics for the pur- pose of health maintenance and disease prevention

was Elie Metchnikoff, the Russian Nobel Prize win- ner, who at the turn of the last century suggested that high concentration of Lactobacilli in the intestinal flora was important for health and longevity in humans [9]. Probiotics are defined as "living organisms, which upon ingestion in certain numbers, exert health benefits beyond inherent basic nutrition" [10].

Bacteria associated with probiotic activity are most commonly Lactobacilli, Bifidobacteria and Streptococcibut other non-pathogenic bacteria such as some strains of Escherichia coli and non-bacterial organisms such as the yeast Saccharomyces boulardii have been used (Table 1). For clinical application probiotic strains must possess certain characteristics.

They need to be resistant to acid and bile, and must have the ability to be metabolically active within the luminal flora, where, ideally, they should survive, but not persist in the long term. They should be antagonistic against pathogenic bacteria and, obviously, they must be safe for human use and should maintain their viability and beneficial properties during manufacturing processes [11].

Mechanisms of Action

Several mechanisms have been proposed to account for probiotic action including the antagonistic activity against pathogenic bacteria either by inhibition of adherence and translocation or by production of anti-bacterial substances such as anti-microbial pep-

Probiotics in Inflammatory Bowel Diseases

Paolo Gionchetti, Fernando Rizzello, Claudia Morselli, Rosy Tambasco, Massimo Campieri

Table 1.Organisms associated with probiotic activity Lactobacilli

Bifidobacteria Streptococci Others bacterial strains

Enterococci, non-pathogenic E. coli Non bacterial organisms

Yeasts (Saccharomyces boulardii)

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tides (bacteriocins) and hydrogen peroxide. Probi- otics also stimulate mucosal defence both at the level of immune and epithelial function with an increase in sIgA production, blockade of pro-inflammatory cytokines, enhancement of anti-inflammatory cytokines levels, stimulation of intestinal mucin expression and improvement of gut permeability.

Finally they are able to produce nutrients of special importance to the intestine such as short-chain fatty acids and vitamins (Table 2).

Animal Models Studies

Encouraging results have been obtained with probiotic therapy in experimental colitis. Administration of Lactobacillus reuteri was shown to significantly reduce inflammation in acetic acid- and methotrex- ate-induced colitis in rats [12, 13]. More recently Lac- tobacillus sp. was shown to be able to prevent the development of spontaneous colitis in interleukin-10 (IL-10) deficient mice [14], and continuous feeding with Lactobacillus plantarum could attenuate an established colitis in the same knockout model [15].

A strain of Lactobacillus salivarius (subsp. Saliva rius) reduced the rate of progression from inflamma- tion through dysplasia and colonic cancer in IL-10 deficient mice [16], and a strain of Bifidobacterium, infantis and of Lactobacillus salivarius were able to attenuate inflammation with a reduced ability to produce Th1-type cytokines in the IL-10 knockout model [17]. Using a cocktail of probiotic bacteria (VSL#3), Shibolet and colleagues have shown a significant attenuation of inflammation with a decrease of MPO (myeloperoxidase) and NOS (nitric oxide synthase) activity in iodoacetamide-induced colitis in rats [18], while Madsen and colleagues have reported a significant improvement of inflammation together with a reduction in mucosal levels of pro- inflammatory cytokines and a normalization of colonic barrier integrity in IL-10 KO mice [19].

Ulcerative Colitis

The efficacy of a non-pathogenic strain of Escherichia coliNissle 1917 in the treatment of IBD has been tested in three recent trials. In the other three controlled studies, E. coli Nissle 1917 has been found to exhibit efficacy similar to that of mesalazine in maintenance treatment of ulcerative colitis (UC) [20–22].

We have explored another strategy, using a probiotic preparation (VSL#3) characterised by very high bacterial concentration (each packet containing 450 billion viable bacteria) and the presence of a cocktail of eight different bacterial species. This product con- tains viable lyophilised bacteria of four strains of Lactobacilli(L. casei, L. plantarum, L. acidophilus, L.

delbrueckiisubsp. Bulgaricus), three strains of Bifi- dobacteria(B. longum, B. breve, B. infantis) and one strain of Streptococcus salivarius subsp. Ther- mophilus.

We carried-out a pilot study using VSL#3 as maintenance treatment in patients with UC in remission, who are allergic or intolerant to sulphasalazine and mesalazine, to verify the impact on the faecal flora of this preparation. Twenty patients received 6 g per day of VSL#3 (1 800 billion bacteria) for 12 months and were assessed clinically and endoscopically at baseline and at 6 and 12 months or in case of relapse.

Stool culture and determination of faecal pH were also performed at different intervals.

Microbiological determination showed a signifi- cant increase in concentration of Lactobacilli, Bifi- dobacteria and Streptococcus thermophilus, which was already evident after 20 days and persisted through the treatment period with return to basal levels within 15 days after stopping the treatment without modification of the faecal concentration of Bacteroides, Enterococci, Coliforms, Clostridia and the total anaerobes and aerobes. Faecal pH was significantly reduced by the treatment and 15 of the 20 patients (75%) remained in remission [23].

In an uncontrolled pilot study VSL#3, at a very high dose (3 600 billion), was able to induce remission in 63%, with a positive response in another 23%

of patients with active mild-to-moderate disease [24].

Similarly, in an open, uncontrolled 4-week study, the yeast Saccharomyces boulardii induced remission in 71% of patients with mild to moderate ulcerative colitis [25].

Pouchitis

In a double-blind study, we have compared the efficacy of VSL#3 with placebo in the maintenance treat- Table 2.Mechanisms of action of probiotics

Inhibits pathogenic enteric bacteria Decreases luminal pH

Secretes bacteriocidal proteins Colonisation resistance Blocks epithelial binding

Improves epithelial and mucosal barrier function Produces SCFA

Enhances mucus production Increases barrier integrity Alters immunoregulation

Increases IL-10 and TGFβ and decreases TNF Increases IgA production

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ment of chronic pouchitis. Forty patients who obtained clinical and endoscopic remission after 1 month of combined antibiotic treatment (rifaximin 2 g/day plus ciprofloxacin 1 g/day) were randomised to receive VSL#3 6 g daily (1 800 billion bacteria/day) or an identical appearing placebo for 9 months. Clinical assessment was done every month; endoscopic and histological assessment were performed at entry and every 2 months thereafter. Stool culture was done before and after antibiotic treatment and subse- quently every month during maintenance treatment.

Relapse was defined as an increase of at least 2 points in the clinical portion of pouchitis activity index that should be confirmed endoscopically and histologically. All the 20 patients treated with placebo had a relapse in the follow-up period; in contrast 17 of the 20 (85%) patients treated with VSL#3 were still in remission after 9 months. Interestingly, all these 17 patients had a relapse within 4 months after suspen- sion of the active treatment. Faecal concentration of Lactobacilli, Bifidobacteria and Streptococcus sali- varius subsp. Thermophilus were significantly increased within 1 month after starting VSL#3 treatment and remained stable throughout the study.

However, this increase did not affect concentration of the other bacterial groups, suggesting that the effect was not mediated by suppression of endoge- nous luminal bacteria [26]. These results have been recently replicated by a study evaluating the efficacy of VSL#3 in maintaining antibiotic-induced remission (obtained after a 1-month treatment with metronidazole 800 mg/day plus ciprofloxacin 1 g/day) for 1 year in patients with refractory or recurrent pouchitis: 20 patients received VSL#3 1 800 billion bacteria once a day for one year and 16 patients received a placebo during the same period. Clinical, endoscopic and histological evaluations were made before, 2 and 12 months after the randomisation. A parallel assessment of the quality of life (QoL) was obtained with IBDQ. This study has substantially confirmed the observations made previously, with maintenance remission rate at 1 year for 85% in the VSL#3 group and 6% in the placebo group. A high QoL score was obtained by the group treated with VSL#3 [27].

As regards the mechanisms of action, in these patients we found that continuous administration of VSL#3 determined a significant increase of IL-10 tis- sue levels together with a significant decrease of tis- sue levels of the pro-inflammatory cytokines TNF alfa, IL-1 and IFN gamma, and a decrease of matrix metalloproteinase activity [28]. On the other hand, Lactobacillus GG was ineffective in preventing relapses in patients with chronic pouchitis in a placebo-controlled trial [29].

We have also carried-out a double-blind placebo

controlled trial to evaluate the efficacy of VSL#3 in the prevention of pouchitis onset in patients operat- ed for ileal pouch-anal anastomosis (IPAA) for UC.

Within 1 week after ileostomy closure, 40 patients, were randomised to receive VSL#3 3 g per day (900 billion bacteria/day) or an identical placebo for 12 months; patients were assessed clinically endoscopically and histologically at 1, 3, 6, 9, 12 months accord- ing to PDAI.

Patients treated with VSL#3 had a significantly lower incidence of acute pouchitis compared to those treated with placebo during the first year after ileostomy closure (10 vs. 40%; p<0.05). Moreover, IBDQ was significantly improved only in the group treated with VSL#3, and median stool frequency in patients who did not develop pouchitis, was significantly less in the VSL#3 group compared to the placebo group [30].

Crohn’s Disease

Results with probiotics in Crohn’s disease are conflicting. In a small pilot study, treatment with cap- sules containing E. coli Nissle 1917 was compared to the administration of placebo in maintenance of steroid-induced remission of colonic CD. Twelve patients were treated with E. coli Nissle 1917 and 11 with placebo; at the end of the treatment period (12 weeks) relapse rates were 33% in the E. coli group and 63% in the placebo group, but unfortunately, because of the very small number of patients treated this difference did not reach statistical significance [31].

In a small comparative open study, the association of Saccharomyces boulardii 1 g/day plus mesalamine 2 g/day was significantly superior to mesalamine 3 g/day in maintenance of remission in a 6-month trial [32]. Prantera et al. have reported no benefit for Lac- tobacillus GG in preventing post-operative recurrence [33] in 1-year double-blind placebo-controlled trial. Additionally, more recently the same strain was shown not to be superior to placebo as an adjunct to standard treatment in the maintenance of medically induced remission in children [34].

We carried-out a single-blind study to compare a sequential antibiotic-probiotic treatment with mesalazine in the prevention of the post-operative recurrence of CD; 40 patients within 1 week after curative surgery were randomised to receive a high dose of rifaximin, a non-absorbable wide spectrum antibiotic, for 3 months followed by VSL#3 6 g per day for 9 months or mesalazine 4 g per day for 12 months.

Patients were assessed clinically and endoscopically at 3 and 12 months; the combined antibiotic-probiotic treatment determined a significant lower incidence

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of severe endoscopic recurrence both at 3 and 12 months (10% and 20% vs. 40% and 40%, respectively;

p<0.01) [35].

Conclusions

Many clinical and experimental observations do sug- gest an involvement of the intestinal microflora in the pathogenesis and in the perpetuation of IBD. Pro- biotics may provide a simple and attractive way to prevent or treat IBD, and patients find the probiotic concept appealing because it is safe, non-toxic and natural. A highly concentrated cocktail of probiotics (VSL#3) is effective in the prevention of pouchitis onset and relapses. Results in ulcerative colitis are promising both in prevention of relapses and treatment of mild to moderate attacks. Results in Crohn’s disease are not yet clear because of conflicting data and the limited number of well-performed studies.

It is important to select a well-characterised probiotic preparation; in fact viability and survival of bacteria in many available preparations are unproven. It should be remembered that the beneficial effect of one probiotic preparation does not imply the efficacy of other preparations containing different bacterial strains, because each individual probiotic strain has unique biological properties. There is the need to improve our knowledge on the composition of enteric flora or “the neglected organ” and of the intestinal physiology and its relationship with the luminal ecosystem.

References

1. Guarner F, Casellas F, Borruel N et al (2002) Role of microecology in chronic inflammatory bowel diseases.

Eur J Clin Nutr 56(Suppl 4):S34–S38

2. Duchmann R, Kaiser I, Hermann E et al (1995) Toler- ance exists towards resident intestinal flora but is bro- ken in active inflammatory bowel disease (IBD). Clin Exp Immunol 102:104–108

3. MacPherson A, Khoo UY, Forgacs I et al (1996) Mucosal antibodies in inflammatory bowel disease are directed against intestinal bacteria. Gut 38:365–375 4. Janowitz HD, Croen EC, Sachar DB (1998) The role of

the faecal stream in Crohn's disease: an historical and analytic perspective. Inflamm Bowel Dis 4:29–39 5. D'Haens GR, Geboes K, Peeters M et al (1998) Early

lesions of recurrent Crohn's disease caused by infusion of intestinal contents in excluded ileum. Gas- troenterology 114:771–774

6. Neut C, Bulois P, Desreumaux P et al (2002) Changes in the bacterial flora of the neoterminal ileum after ileocolonic resection for Crohn’s disease. Am J Gas- troenterol 97:939–946

7. Sartor RB (1995) Insights into the pathogenesis of inflammatory bowel disease provided by new rodent

models of spontaneous colitis. Inflam Bowel Dis 1:64–75

8. Campieri M, Gionchetti P( 1999) Probiotics in inflammatory bowel disease: new insight to pathogenesis or a possible therapeutic alternative? Gastroenterology 116:1246–1249

9. Metchnikoff E (1907) The prolongation of life: opti- mistic studies. Heinemann, London, pp 161-183 10. Schaafsma G (1996) State of the art concerning probi-

otic strains in milk products. IDF Nutr News l5:23–42 11. Lee YK, Salminen S (1995) The coming age of probi-

otics. Trends Food Sci Technol 6:241–245

12. Fabia R, Ar'rajab A, Johansson M-L et al (1993) The effect of exogenous administration of Lactobacillus reuterii R2LC and oat fiber on acetic acid-induced colitis in the rat. Scand J Gastroenterol 28:155–162 13. Mao Y, Nobaek S, Kasravi B et al (1996) The effects of

Lactobacillus strains and oat fibre on methotrexate- induced enterocolitis in rats. Gastroenterology 111:334–344

14. Madsen KL, Tavernini MM, Doyle JSG, Fedorak RN (1999) Lactobacillus sp prevents development of ente- rocolitis in interleukin-10 gene-deficient mice. Gas- troenterology 116:1107–1114

15. Schultz M, Veltkamp C, Dieleman LA et al (2002) Lac- tobacillus plantarum 299V in the treatment and pre- vention of spontaneous colitis in interleukin-10 deficient mice. Inflamm Bowel Dis 8:71–80

16. O’Mahony L, Feenex M, O’Halloran S et al (2001) Pro- biotic impact on microbial flora, inflammation and tumour development in IL-10 knockout mice. Aliment Pharmacol Ther 15:1219–1225

17. McCarthy J, O’Mahony L, O’Callaghan L et al (2003) Double-blind, placebo-controlled trial of two probiotic strains in interleukin 10 knockout mice and mecha- nistic link with cytokine balance. Gut 52:975–980 18. Shibolet O, Karmeli F, Eliakim R et al (2002) Variable

response to probiotics in two models of experimental colitis in rats. Inflamm Bowel Dis 8:399–408

19. Madsen K, Cornish A, Soper P et al (2001) Probiotic bacteria enhance murine and human intestinal epithelial barrier function. Gastroenterology 121:580–591 20. Kruis W, Schuts E, Fric P (1997) Double-blind com-

parison of an oral Escherichia coli preparation and mesalazine in maintaining remission of ulcerative colitis. Aliment Pharmacol Ther 11:853–858

21. Rembacken BJ, Snelling AM, Hawkey P (1999) Non pathogenic Escherichia coli vs mesalazine for the treatment of ulcerative colitis: a randomised trial.

Lancet 354:635-639

22. Kruis W, Fric P, Potrotnieks J et al (2004)Maintaining remission of ulcerative colitis with Escherichia Coli Nissle 1917 is as effective as with standard mesalazine.

Gut 53:1673–1623

23. Venturi A, Gionchetti P, Rizzello F et al (1999) Impact on the faecal flora composition of a new probiotic preparation. Preliminary data on maintenance treatment of patients with ulcerative colitis (UC) intolerant or allergic to 5-aminosalicylic acid (5 ASA). Aliment Pharmacol Ther 13:1103–1108

24. Bibiloni R, Federak RN, Tannock GW et al (2005) VSL#3 probiotic-mixture induces remission in patients with active ulcerative colitis. AM J Gastroen- terol 100(7):1539–1546

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25. Guslandi M, Giollo P, Testoni PA (2003) A pilot trial of Saccharomyces boulardii in ulcerative colitis. Eur J Gastroenterol Hepatol 15:697–698

26. Gionchetti P, Rizzello F, Venturi A et al (2000) Oral bacteriotherapy as maintenance treatment in patients with chronic pouchitis: a double-blind, placebo-controlled trial. Gastroenterology 119:305–309

27. Mimura t, Rizzello F, Helwig U et al (2004) Once daily high dose probiotic therapy for maintaining remission in recurrent or refractory pouchitis. Gut 53:108–114 28. Ulisse S, Gionchetti P, D’Alò S et al (2001) Increased

expression of cytokines, inducible nitric oxide synthase and matrix metalloproteinases in pouchitis:

effects of probiotic treatment (VSL#3). Gastroenterol- ogy 120(Suppl):A281

29. Kuisma J, Mentula S, Kahri A et al (2003) Effect of Lac- tobacillus rhamnosus GGon ileal pouch inflammation and microbial flora. Aliment Pharmacol Ther 17:509–515 30. Gionchetti P, Rizzello F, Venturi A et al (2000) Pro- phylaxis of pouchitis onset with probiotic therapy: a double blind, placebo controlled trial. Gastroenterolo- gy 118:A190

31. Malchow HA (1997) Crohn's disease and Escherichia coli.a new approach in therapy to maintain remission of colonic Crohn's disease? J Clin Gastroenterol 25:653–658

32. Guslandi M, Mezzi G, Sorghi M et al (2000) Saccha- romyces boulardii in maintenance treatment of Crohn’s disease. Dig Dis Sci 45:1462–1464

33. Prantera C, Scribano ML, Falasco G et al (2002) Inef- fectiveness of probiotics in preventing recurrence after curative resection for Crohn’s disease: a ran- domised controlled trial with Lactobacillus GG. Gut 51:405–409

34. Bousvaros A, Guandalini S, Baldassano RN et al (2005)A randomised, double-blind trial of Lactobacil- lus GGversus placebo in addition to standard maintenance therapy for children with Crohn’s disease.

Inflamm Bowel Dis 11:833–839

35. Campieri M, Rizzello F, Venturi A et al (2000) Combi- nation of antibiotic and probiotic treatment is effica- cious in prophylaxis of post-operative recurrence of Crohn's disease: a randomised controlled study vs.

mesalazine. Gastroenterology 118:A781