Conclusions In children with active distal ulcerative colitis, rectal infusion of L. reuteri is effective in improving mucosal inflammation and changing mucosal expression levels of some cytokines involved in the mechanisms of inflammatory bowel disease.

Results Thirty‐one patients accomplished the trial (17 males, median age 13 year, range 7–18). Mayo score (including clinical and endoscopic features) decreased significantly in the L. reuteri group (3.2 ± 1.3 vs. 8.6 ± 0.8, P < 0.01) compared with placebo (7.1 ± 1.1 vs. 8.7 ± 0.7, NS); furthermore, histological score significantly decrease only in the L. reuteri group (0.6 ± 0.5 vs. 4.5 ± 0.6, P < 0.01) (placebo: 2.9 ± 0.8 vs. 4.6 ± 0.6, NS). At the post‐trial evaluation of cytokine mucosal expression levels, IL‐10 significantly increased ( P < 0.01) whereas IL‐1β, TNFα and IL‐8 significantly decreased ( P < 0.01) only in the L. reuteri group.

Methods A total of 40 patients (median age: 7.2 years range 6–18) with mild to moderate UC were enrolled in a prospective, randomised, placebo‐controlled study. They received an enema solution containing 10 10 CFU of L. reuteri ATCC 55730 or placebo for 8 weeks, in addition to oral mesalazine. Clinical endoscopic and histological scores as well as rectal mucosal expression levels of IL‐10, IL‐1β, TNFα and IL‐8 were evaluated at the beginning and at the end of the trial.

Aim To assess in children with active distal UC the effectiveness of Lactobacillus (L) reuteri ATCC 55730 enema on inflammation and cytokine expression of rectal mucosa.

Background Intestinal microbiota manipulation, one of the pathogenetic components of inflammatory bowel disease (IBD), has become an attractive therapy for ulcerative colitis (UC).

Background Ulcerative colitis (UC) is a chronic relapsing inflammatory bowel disease (IBD) characterised by periods of acute illness often alternating with periods of remission.1, 2 As in Crohn’s disease (CD), another IBD form, the pathogenesis remains elusive, but an abnormal interaction between intestinal immunity and gut microbiota in individuals genetically predisposed seems to play a critical role.3 The treatment of UC is mostly based on anti‐inflammatory drugs, corticosteroids and immunomodulators, whereas biologics are given in severe cases, refractory to traditional therapy.4 These drugs are useful to reduce the inflammation, yet their systemic activity and side effects can be troublesome and affect quality of life.5 Manipulation of intestinal microbiota, one of the pathogenetic components of the disease, has become an attractive therapy.6 At least three different theories have been suggested to explain the implication of luminal bacteria in IBD pathogenesis: presence of a persistent pathogen; abnormal permeability of the epithelial barrier leading to an excessive bacterial translocation; imbalance in the diversity and magnitude of microbiota, referred as dysbiosis.7, 8 In general, an increased prevalence of mucosa‐associated microbes has been reported in intestinal tissues of adult and paediatric IBD patients.9, 10 Moreover, studies in IBD patients, mainly in CD, have disclosed an increased presence of peculiar Escherichia coli strains capable to adhere to and invade epithelial cells, and to replicate in the latter and in the macrophages.11, 12 Probiotics are live microorganisms that in adequate amount confer a healthy benefit to the host.13 They change gut microbial species composition and diversity, increase mucosal barrier function and elicit a variety of responses from intestinal immune cells due to interactions between surface‐bound or secreted ligands and membrane receptors.14 In the last years, several studies have shown a beneficial effect of different probiotic preparations in inducing and maintaining remission in adults and children with IBD.15, 16 However, many of them have methodological limitations concerning sample size, double‐blinding procedures, inadequate information about random allocation. Two randomised controlled trials have documented safety and efficacy of probiotics in inducing and maintaining remission in children with moderate UC.15, 17, 18 Use of probiotics in CD has produced less convincing results than in UC.19 This study was performed in children with distal active UC to assess the effectiveness of rectal administration of Lactobacillus (L) reuteri ATCC 55730 (Reuflor® Italchimici SpA, Italy), on rectal mucosa inflammation as well as on mucosal expression levels of some cytokines, known to be involved in the inflammatory mechanisms of UC.

Patients and Methods This was a prospective randomised, placebo‐controlled study. Eligible patients were recruited at the Pediatric Gastroenterology and Liver Unit of the Sapienza University of Rome between November 2008 and February 2011. This Unit is a tertiary referral centre for paediatric IBD patients. Forty patients (median age: 7.2 years range 6–18; male:female) were enrolled into the study: all patients had a confirmed endoscopic and histological diagnosis of ulcerative proctitis/proctosigmoiditis with mild to moderate disease activity. Disease activity was assessed by the Mayo Disease Activity Index (DAI).20 The latter is calculated by summing the scores of four variables each of which is graded on a scale from 0 to 3: stool frequency, bleeding, physician’s assessment of disease activity and mucosal appearance. The maximum potential score is 12, with mild to moderate disease activity defined as a score <10. Only patients with mild to moderate active distal UC based on DAI score were selected. All patients underwent ileo‐colonoscopy (Paediatric Videocolonoscope Olympus, PCF 180 AI) under general anaesthesia or conscious sedation using intravenous pethidine and midazolam. Diagnosis and classification of IBD were based on widely agreed criteria21; infectious and immunological diseases as well as malabsorption syndromes and food allergy were excluded in all. Endoscopy was performed prior to the trial and the day after the last enema infusion. Biopsy samples were obtained during colonoscopy from inflamed colonic mucosa. Tissues were fixed in 4% paraformaldehyde, dehydrated, paraffin embedded and stained with hematoxylin and eosin. Endoscopic appearance of rectal mucosa was scored as follows: 0, normal; 1, granularity, lack of vascular pattern, friability (contact bleeding); 2, spontaneous bleeding and exudation; 3, frank ulcers. The histological grading was based on the following features, as previously reported22: grade 1, essentially normal mucosa; grade 2, isolated inflammatory cells or cell aggregates of either lymphoplasmacytic cells or eosinophilic granulocytes; grade 3, marked increase of the inflammatory cells, slight atrophy; grade 4, definite marked atrophy, crypt abscesses, follicle formation in deeper cell layers, severe increase in acute inflammatory cells; grade 5, ulcers with atrophy, crypt abscesses and deep follicles. A higher score indicate more severe inflammation. All patients included into the study received a chronic administration of oral mesalazine at a dose ranging from 50 to 75 mg/kg/day during the last 12 weeks. The median duration of the disease flare‐up before entering into the trial was 6.0 weeks (range: 4–16). Exclusion criteria were: other causes of active proctitis or proctosigmoiditis such as infections, medical drugs and CD; furthermore, patients who had received either oral or topical corticosteroids, topical aminosalicylates, antibiotics during the previous 12 weeks and immunomodulators during the previous 20 weeks were excluded from the trial. At the beginning of the study, patients were randomised to receive before bedtime an enema solution containing 1010 CFU of L. reuteri ATCC 55730 or placebo for 8 weeks. Patients received the enema in the evening, before going to sleep and retaining it in the rectum for as long as possible (at least 10 min in left supine position after administration). The control visit was performed at week 2, 4 and 8. Patients whose clinical condition did not improve after 2 weeks were classified as nonresponders and discontinued. Randomization was performed according to a computer‐generated list by means of sequentially numbered sealed envelopes indicating their medication. Each patient was randomised when all patient recruitment criteria were met. During the trial, all patients maintained oral mesalazine. The Figure 1 shows the study algorithm. Figure 1 Open in figure viewer PowerPoint Algorithm of the study. Specimens obtained at rectal level were also stored in RNA later (Ambion, Austin, TX, USA) before RNA extraction. RT‐PCR Amplification Assay. Total RNA was isolated from biopsies using the RNeasy kit (Qiagen, Hilden, Germany), reverse‐transcribed by SuperScript First‐strand Synthesis System (Invitrogen, Carlsbad, CA, USA) and cDNA was amplified by Platinum Taq Polymerase (Invitrogen). The following primers were used: IL‐1β‐fw: 5′‐AAACAGATGAAGTGCTCCTTCCAGG‐3′ and IL‐1β‐rv: 5′‐TGGAGAACACCACTTGTTGCTCCA‐3′; IL‐8‐fw: 5′‐TCTGCAGCTCTGTGTGAAGG‐3′ and IL‐8‐rv: 5′‐AATTTCTGTGTTGGCGCAG‐3′; IL10‐fw: 5′‐ATGTAGCCGCCCCACACAGA‐3′ and IL‐10‐rv: 5′‐CATCCATCTTTTTCAGCCA‐3′; TNFα‐fw: 5′‐TCTGGCCCAGGCAGTCAGATC‐3′, and TNFα‐rv: 5′‐TCAGCTTGAGGGTTTGCTACAA‐3′. To confirm the use of equal amounts of RNA in each experiment, all samples were checked in parallel for β‐actin mRNA expression (primers: fwd 5′‐TCATCACCATTGGCAATGAG‐3′ and rvs 5′‐ACTGTGTTGGCGTACAGGT‐3′). The annealing temperature was 54 °C for IL‐1β, and 50 °C for β‐actin. The primary outcome was the variation in the disease activity as defined by Mayo DAI. Remission was defined as a final DAI score of <2.0 points; clinical response was defined as a reduction in the DAI of ≥2 points. Clinical relapse was defined as the occurrence or worsening of symptoms, accompanied by an increase in the DAI score to 4 and necessitating a change in therapy. The secondary outcomes were changes in the rectal histology and in the inflammatory cytokine mucosal expression. The study protocol was defined in accordance with the Declaration of Helsinki and approved by the ethical committee of the University Hospital Umberto I in Rome. Written informed consent was obtained from parents of all children; children older than 12 years of age signed a statement of assent. Statistical analysis Results are presented as means (s.d.). We analysed data by Mann–Whitney and Wilcoxon test where appropriate (GraphPad InStat 3.1 for Mac OSX). Values of P < 0.05 (two‐tailed) were considered significant. The primary endpoint used for the sample size definition was variation in the disease activity as defined by Mayo DAI in each group. A sample size of 40 patients would provide 80% power to detect a difference in 2 points in the DAI with a two tailed, alpha = 0.05. Data were also correlated by Pearson’s correlation coefficient, and 95% confidence intervals were calculated. The Chi‐squared test or Fisher’s exact test was used when appropriate.

Results Of the 40 patients enrolled into the study, a premature discontinuation occurred in nine (in four within the first 2 weeks, in five between the weeks 2 and 4). The main reasons were: low compliance with irregular enema administration (seven subjects) and an acute infectious episode (two subjects). Thirty‐one patients accomplished the trial (17 males, median age 13 year, range 7–18); the baseline characteristics of them are shown in the Table 1. During the course of the study the enema infusions were well tolerated and no cases of worsened disease activity or serious adverse events were reported. At baseline, all patients had a moderate active UC and the two groups were comparable for the Mayo DAI. As summarised in Figure 2, the Mayo score decreased significantly in the L. reuteri group (3.2 ± 1.3 vs. baseline 8.6 ± 0.8, P < 0.01) compared with placebo (7.1 ± 1.1 vs. baseline 8.7 ± 0.7, NS). All patients on L. reuteri and 53% on placebo had a clinical response (P < 0.01), whereas clinical remission was reached in 31% of L. reuteri group and in no patient on placebo (P < 0.05); in both groups no flare‐ups occurred. Table 1. Baseline characteristics of the patients Placebo group (n = 15) Reuteri group (n = 16) Age, years, median (and ranges) 13 (7–18) 12.5 (7–18) Male:Female 8:6 9:7 Disease duration, years, median (and ranges) 2 (0.5–4.4) 2 (0.6–4.5) Age at diagnosis, years, median (and ranges) 7.0 (5–16) 7.7 (5–16) Previous medications 1. Anti‐inflammatory drugs, N (%) 8 (53) 9 (56) 2. Immunomodulators, N (%) 3 (20) 3 (19) 3. Biologics, N (%) 0 (0) 1 (6) 4. No therapy, N (%) 4 (27) 3 (19) Figure 2 Open in figure viewer PowerPoint Individual and mean values of Mayo score at baseline and at the end of the trial in the two study groups (31 patients). Figure 3 shows the behaviour of the histological score: the latter significantly decreased only in the L. reuteri group (0.6 ± 0.5 vs. baseline 4.5 ± 0.6, P < 0.01) compared with placebo (2.9 ± 0.8 vs. baseline 4.6 ± 0.6, NS). Figure 3 Open in figure viewer PowerPoint Individual and mean values of the histological score in the two study groups (31 patients), at baseline and after 8 weeks of treatment (L. reuteri: P < 0.01 vs. baseline; placebo: NS vs. baseline). Vertical bars as s.d. The differences in mucosal cytokine levels in the two groups were not significant before treatment. At the post‐trial evaluation there was a significant increase in the mucosal expression levels of IL‐10 and a significant decrease in those of IL‐1β, TNFα and IL‐8 mucosal expression levels only in the L. reuteri group (P < 0.01) (Figure 4). Figure 4 Open in figure viewer PowerPoint Differences in expression levels of rectal mucosa cytokines between study groups (31 patients), at baseline and after 8 weeks of treatment. *P < 0.01, U (arbitrary units). Vertical bars as s.d.

Discussion This randomised placebo‐controlled study provides evidence that inflammation of the rectal mucosa in paediatric UC can be reduced by local administration of L. reuteri ATCC55730 in addition to standard oral mesalazine. It is noticeable that in our trial clinical efficacy of the probiotic administration was paralleled by a marked decrease of mucosal damage as documented by endoscopy and histology and that there were striking changes of the mucosal expression levels of cytokines known to be involved in the mechanisms leading to IBD. We selected a patient population mainly with distal colonic inflammation, as the aim of the study was the evaluation of rectal delivered therapy. However, it is widely known that paediatric UC has a predominant phenotype of extensive colonic inflammation.2 It should also be observed that the trial did not include intermediate evaluation of DAI, whose changes were the main outcome of the study. Thus, it cannot be considered as an ITT basis. The patients that did not complete the trial were lost to follow‐up because of low compliance with the treatment or acute extra‐GI infection episodes, while non flare ups of the disease occurred in them; moreover, they were comparable to the others for baseline demographic variables. The rationale of therapeutic strategies in IBD aimed at manipulating composition of gut microbiota derives from recent views on mechanisms underlying IBD.23 Experimental observations have shown that in a variety of animal models the occurrence of spontaneous colitis seems to entirely depend on the presence of the luminal flora.24 It is also of interest that several human genes thought to be associated with susceptibility to IBD modulate innate immune responses and encode proteins underlying host‐microbial interactions.25 Moreover, both alleviation of mucosal inflammation following diversion of faecal stream and its relapse by reintroducing faecal material in the gut have been shown in human IBD.26 Lactobacillus reuteri ATCC55730 is a heterofermentative probiotic bacterium widely administered as a dietary supplement. Its clinical usefulness and safety have been documented in different clinical trials showing efficacy in reducing incidence and severity of diarrhoea of different cause as well as in improving the course of gastrointestinal functional disorders in childhood.27-29 Previous experimental studies have documented the ability of L. reuteri ATCC55730 and other L. reuteri strains to colonise upper and lower intestinal mucosa as well as to modulate intestinal immunological response.30 These properties are widely thought to be critical and mandatory for successful biological activity of probiotic organisms. An anti‐inflammatory intestinal activity by different L. reuteri strains has been documented by inhibition of colitis in transgenic IL‐10 deficient mice and by reduction of the TNFα expression levels in a mouse model of colitis.31 It is also remarkable that L. reuteri exhibits a potent inhibitory effect on TNFα induced IL‐8 expression in human intestinal epithelial cells and that L. reuteri 100‐23 strain elicits an increased number of regulatory T‐cell in a murine gut model.32 Nevertheless, the anti‐inflammatory effects of L. reuteri strains on the intestinal mucosa are very rarely investigated in human ex vivo studies. We showed a striking reduction in the rectal mucosa expression levels of TNFα and IL1β, widely viewed as potent inflammatory mediators underlying IBD and previously shown by our group to be over‐expressed in the gut mucosa of IBD paediatric patients.33 It was also remarkable that rectal administration of L. reuteri ATCC55730 was accompanied by decreased expression of IL‐8, a chemokine involved in chemoattraction of polymorphonuclear cells and thought to be a main mediator in the innate immune system response.34 Indeed, an overexpression of chemokine axis components, including IL‐8, has been described in inflamed colonic tissue of paediatric and adult IBD patients.35 Our results are in agreement with observations in cultured epithelial cells that different L. reuteri strains and other Lactobacillus species can reduce over‐expression of IL‐8 due to pro‐inflammatory triggers.31 The increased mucosal expression of IL‐10 in our patients following L. reuteri administration should be highlighted. IL‐10 modulates both innate and adaptive immune responses and knowledge about its role in the pathogenesis of CD and UC is growing.36 There is evidence that IL‐10 regulates mucosal inflammation by inhibiting T cell/macrophage activation and pro‐inflammatory cytokine synthesis.37 However, recombinant IL‐10 has been tried in several human inflammatory conditions with disappointing results, attributed to insufficient local tissue concentrations.38 Administration of probiotics in patients with IBD has yielded mixed results with considerable variations in the selection of strains and disease. Efficacy and safety of a probiotic preparation combining eight different microorganisms (VSL#3) has been evaluated in 18 children with mild to moderate acute UC for 8 weeks, whereas concomitant mesalazine and low dose corticosteroids were maintained. This treatment gave a remission rate of 56% and a combined remission/response rate of 61%; however, five of the recruited patients had been withdrawn due to lack of response.17 In a 1‐year placebo‐controlled double‐blind study, the efficacy of the same probiotic mixture was evaluated on induction and maintenance of remission in 29 children with active UC, in addition to steroid induction and mesalazine maintenance treatment. Remission and maintenance rates were markedly higher in patients on VSL#3 than in those on placebo. Interestingly, at 6 and 12 months or at time of relapse, endoscopic and histological scores were significantly lower in the VSL#3 group than in the placebo.18 The use of probiotics in IBD is considerably increased in the recent years: two of five patients with IBD use regularly probiotics either alone or in combination with conventional drugs.39 However, the studies on the use of probiotics in IBD are heterogeneous for the microorganism species or strains given as well as for methodology and patient populations.15, 16 It should be also considered that benefits described with one species are not necessarily attributed to others. On the other hand, probiotics used in complex disorders such as IBD, with the aim to modulate the complex interactions between intestinal mucosa and gut microbiota, should possess some critical properties, such as the ability to adhere to the intestinal mucosa and to interact with immunological cells harbouring the gut wall.40 Several experimental observations document that the strain used in this study is able to modulate different steps of the intestinal mucosa immunity. In conclusion, our data document the efficacy of rectal infusion of L. reuteri ATCC 55730 in improving mucosal inflammation in children with distal active UC as well as in changing the mucosal expression levels of some cytokines involved in the mechanisms of IBD. It remains to be proved if this strain given on a long‐term basis is able to maintain such effectiveness and if its oral administration is more comfortable for patients, is as effective as rectal infusion. Other unsettled issues concern the ability of L. reuteri ATCC 55730 to reduce inflammation in proximal colonic areas of UC and if its administration alone is comparable in terms of efficacy to conventional anti‐inflammatory drugs.

Acknowledgement Declaration of personal and funding interests: None.