Ulf Helwig

Prophylaxis of pouchitis onset with probiotic therapy: a double-blind, placebo-controlled trial

BACKGROUND & AIMS We have recently documented the efficacy of a highly concentrated probiotic preparation (VSL#3) in the prevention of flare-up in patients with chronic pouchitis. The aim of this study was to compare probiotic therapy with VSL#3 versus placebo in the ability to prevent the onset of acute pouchitis during the first year after ileal pouch-anal anastomosis. METHODS Forty consecutive patients who underwent ileal pouch-anal anastomosis for ulcerative colitis were randomized to receive either VSL#3 (1 packet containing 900 billion bacteria/day) (n = 20) or an identical placebo (n = 20) immediately after ileostomy closure for 1 year. The patients were assessed clinically, endoscopically, and histologically after 1, 3, 6, 9, and 12 months. Health-related quality of life was assessed using the Inflammatory Bowel Disease Questionnaire. RESULTS Two of the 20 patients (10%) treated with VSL#3 had an episode of acute pouchitis compared with 8 of the 20 patients (40%) treated with placebo (log-rank test, z = 2.273; P < 0.05). Treatment with VSL#3 determined a significant improvement in Inflammatory Bowel Disease Questionnaire score, whereas this was not the case with placebo. CONCLUSIONS Treatment with VSL#3 is effective in the prevention of the onset of acute pouchitis and improves quality of life of patients with ileal pouch-anal anastomosis.

Once daily high dose probiotic therapy (VSL#3) for maintaining remission in recurrent or refractory pouchitis

Background: Ten to 15% of patients with pouchitis experience refractory or recurrent disease. The aim of this study was to evaluate the effectiveness of a single daily high dose probiotic preparation (VSL#3) in maintaining antibiotic induced remission, and quality of life (QOL), for one year in such patients. Methods: Patients with pouchitis at least twice in the previous year or requiring continuous antibiotics, associated with a pouchitis disease activity index (PDAI) ⩾7 (0 = perfect; 18 = worst), in whom remission was induced by four weeks of combined metronidazole and ciprofloxacin, were randomised to receive VSL#3 6 g or placebo once daily for one year or until relapse. Symptomatic, endoscopic, and histological evaluations were made before, and two and 12 months after randomisation or at the time of relapse. Remission was defined as a clinical PDAI ⩽2 and endoscopic PDAI ⩽1. Relapse was defined as an increased clinical PDAI score ⩾2 and increased endoscopic PDAI score ⩾3. QOL was assessed using the inflammatory bowel disease questionnaire (IBDQ). Results: Thirty six patients were randomised: 20 to VSL#3 and 16 to placebo. Remission was maintained at one year in 17 patients (85%) on VSL#3 and in one patient (6%) on placebo (p<0.0001). The IBDQ score remained high in the VSL#3 group (p = 0.3) but deteriorated in the placebo group (p = 0.0005). Conclusion: The once daily high dose probiotic VSL#3 is effective in maintaining antibiotic introduced remission for at least a year in patients with recurrent or refractory pouchitis. This is associated with a high level of quality of life.

Bacterial and fungal microbiota in relation to probiotic therapy (VSL#3) in pouchitis

Background: The intestinal microbiota plays a critical role in the pathophysiology of pouchitis, a major complication after ileal pouch anal anastomosis in patients with ulcerative colitis. Recently, controlled trials have demonstrated that probiotics are effective in maintenance of remission in pouchitis patients. However, the mechanism by which therapy with probiotics works remains elusive. This study explores the role of the bacterial and fungal flora in a controlled trial for maintenance of remission in pouchitis patients with the probiotic VSL#3 compound. Methods: The mucosa associated pouch microbiota was investigated before and after therapy with VSL#3 by analysis of endoscopic biopsies using ribosomal DNA/RNA based community fingerprint analysis, clone libraries, real time polymerase chain reaction (PCR), and fluorescence in situ hybridisation. Patients were recruited from a placebo controlled remission maintenance trial with VSL#3. Results: Patients who developed pouchitis while treated with placebo had low bacterial and high fungal diversity. Bacterial diversity was increased and fungal diversity was reduced in patients in remission maintained with VSL#3 (p = 0.001). Real time PCR experiments demonstrated that VSL#3 increased the total number of bacterial cells (p = 0.002) and modified the spectrum of bacteria towards anaerobic species. Taxa specific clone libraries for Lactobacilli and Bifidobacteria showed that the richness and spectrum of these bacteria were altered under probiotic therapy. Conclusions: Probiotic therapy with VSL#3 increases the total number of intestinal bacterial cells as well as the richness and diversity of the bacterial microbiota, especially the anaerobic flora. The diversity of the fungal flora is repressed. Restoration of the integrity of a “protective” intestinal mucosa related microbiota could therefore be a potential mechanism of probiotic bacteria in inflammatory barrier diseases of the lower gastrointestinal tract.

Expression of cytokines, inducible nitric oxide synthase, and matrix metalloproteinases in pouchitis: effects of probiotic treatment

OBJECTIVE:The efficacy of probiotic organisms in the treatment of pouchitis has been reported. In the present study, we evaluated the tissue levels of pro- and anti-inflammatory cytokines, nitric oxide synthase, and matrix metalloproteinases in control and inflamed pouches before and after antibiotic and probiotic treatment of patients with acute pouchitis.METHODS:Pouch biopsy samples were obtained from seven patients with pouchitis before and after antibiotic and probiotic treatment. Tissue samples from five patients with normal pouches were used as controls. Cytokines were determined by ELISA, matrix metalloproteinase activity was evaluated by zymograms, and nitric oxide synthase activity was determined by measuring arginine to citrulline conversion.RESULTS:Tissue levels of tumor necrosis factor α increased (p 0.01) in pouchitis relative to uninflamed pouches and reduced after antibiotic and probiotic treatment. Also, interferon γ and interleukin 1α (IL-1α) augmented in pouchitis, but their increase did not reach statistical significance. The latter, however, were lower (p < 0.05) after treatment with the antibiotics and probiotics. Tissue levels of IL-4 and IL-10 were unchanged in inflamed pouches and unaffected by antibiotic treatment. However, IL-10 increased (p < 0.05) after probiotic treatment. Moreover, inflamed pouches had higher levels of inducible nitric oxide synthase and gelatinase activities, which decreased after treatment.CONCLUSIONS:The ability of antibiotic and probiotic treatments to increase tissue levels of IL-10, at a higher level than those observed in control pouches, and to decrease, to levels present in control pouches, proinflammatory cytokine, inducible nitric oxide synthase, and matrix metalloproteinase activity may suggest a mechanism of action to explain the efficacy of this therapeutic regime in pouchitis.

Four-week open-label trial of metronidazole and ciprofloxacin for the treatment of recurrent or refractory pouchitis

Preliminary data suggest that short‐term antibiotic therapy with a single drug is effective for the treatment of patients with pouchitis. However, some patients are resistant to treatment.

Effect of probiotic strains on interleukin 8 production by HT29/19A cells

OBJECTIVES:Promising results from clinical studies on the effect of probiotics as maintenance therapy in inflammatory bowel disease and in the prevention of onset of pouchitis ask for studies to unravel the still poorly understood mechanism of action of probiotics.METHODS:To evaluate whether the probiotic bacteria that were used in the clinical studies (VSL#3, Escherichia coli Nissle 1917, and Lactobacillus GG) are able to induce chemokine production in epithelial cells, HT29/19A monolayers were incubated with cell debris and cell extract fractions of single strains of the probiotic bacteria in doses ranging from 103 to 109 colony-forming units/ml for 32 h. Supernatants were measured for interleukin 8 by ELISA.RESULTS:Lactobacilli and bifidobacteria strains from VSL#3 and Lactobacillus GG did not induce interleukin 8, whereas both cell debris and cell extracts from E. coli Nissle 1917 induced interleukin 8 production in a dose-dependent way. Cell extracts from streptococcal strains induced interleukin 8 when applied at high concentrations.CONCLUSIONS:Probiotic Gram-positive bacteria did not induce interleukin 8, whereas the nonpathogenic, Gram-negative E. coli Nissle 1917 strain induced interleukin 8 in a dose-dependent way in this culture model. These results suggest that probiotic Gram-positive bacteria and E. coli Nissle 1917 may exert their beneficial effects on the host by a different mechanism of action.

CLA Does Not Impair Endothelial Function and Decreases Body Weight as Compared with Safflower Oil in Overweight and Obese Male Subjects

Objective: Conjugated linoleic acid (CLA) showed a wide range of beneficial biological effects with relevance for cardiovascular health in animal models and humans. Most human studies used olive oil as a reference. This study assessed the effect of CLA as compared with safflower oil on endothelial function and markers of cardiovascular risk in overweight and obese men. Heated safflower oil and olive oil were given for additional descriptive control. Methods: Eighty-five overweight men (aged 45–68 years, body mass index 25–35 kg/m2) were randomized to receive 4.5 g/d of the CLA isomeric mixture, safflower oil, heated safflower oil, or olive oil in a 4-week double-blind study. Endothelial function was assessed by peripheral arterial tonometry (PAT) index determination in the fasting and postprandial state (i.e., 4 hours after consumption of a fat- and sucrose-rich meal). Results: CLA as compared with safflower oil consumption did not impair fasting or postprandial PAT index but decreased body weight. CLA as compared with safflower oil did not change total, low-density lipoprotein (LDL), or high-density lipoprotein (HDL) cholesterol; triglycerides; insulin sensitivity indices; C-reactive protein; soluble adhesion molecules; oxidized LDL; lipoprotein a (Lp[a]); paraoxonase; or platelet-activating factor acetylhydrolase (PAF-AH) activity, but significantly reduced arylesterase activity and increased concentrations of the F2-isoprostane 8-iso-prostaglandin F (PGF)2α. Conclusion: CLA did not impair endothelial function. Other parameters associated with metabolic syndrome and oxidative stress were not changed or were slightly improved. Results suggest that CLA does not increase cardiovascular risk. Increased F2-isoprostane concentrations in this context may not indicate increased oxidative stress.

Epigenetic imprinting by commensal probiotics inhibits the IL-23/IL-17 axis in an in vitro model of the intestinal mucosal immune system

The pathophysiology of IBD is characterized by a complex interaction between genes and the environment. Genetic and environmental differences are attributed to the heterogeneity of the disease pathway and to the epigenetic modifications that lead to altered gene expression in the diseased tissues. The epigenetic machinery consists of short interfering RNA, histone modifications, and DNA methylation. We evaluated the effects of Bifidobacterium breve (DSMZ 20213) and LGG (ATCC 53103), as representatives of commensal probiotics on the expression of IL‐17 and IL‐23, which play an important role in IBD, and on the epigenetic machinery in a 3D coculture model composed of human intestinal HT‐29/B6 or T84 cells and PBMCs. The cells were treated with LPS in the presence or absence of bacteria for 48 h, and the expression of IL‐17, IL‐23, and CD40 at the mRNA and protein levels was assessed using TaqMan qRT‐PCR and ELISA, respectively. Western blotting was used to assess the expression of the MyD88, the degradation of IRAK‐1 and IκBα, the expression of the NF‐κB p50/p65 subunits, the p‐p38 MAPK and p‐MEK1, as well as histone modifications. NF‐κB activity was assessed by NF‐κB‐dependent luciferase reporter gene assays. The accumulation of Ac‐H4 and DNA methylation was quantitatively assessed using colorimetric assays. B. breve and LGG diminished the LPS‐induced expression of IL‐17, IL‐23, CD40, and histone acetylation, while slightly enhancing DNA methylation. These effects were paralleled by a decrease in the nuclear translocation of NF‐κB, as demonstrated by a decrease in the expression of MyD88, degradation of IRAK‐1 and IκBα expression of the nuclear NF‐κB p50/p65 subunits, p‐p38 MAPK and p‐MEK1, and NF‐κB‐dependent luciferase reporter gene activity in LPS‐stimulated cells. B. breve and LGG may exert their anti‐inflammatory effects in the gut by down‐regulating the expression of the IBD‐causing factors (IL‐23/IL‐17/CD40) associated with epigenetic processes involving the inhibition of histone acetylation and the optimal enhancement of DNA methylation, reflected in the limited access of NF‐κB to gene promoters and reduced NF‐κB‐mediated transcriptional activation. We describe a new regulatory mechanism in which commensal probiotics inhibit the NF‐κB‐mediated transcriptional activation of IBD‐causing factors (IL‐23/IL‐17/CD40), thereby simultaneously reducing histone acetylation and enhancing DNA methylation.

Isomer-specific effects of CLA on gene expression in human adipose tissue depending on PPARγ2 P12A polymorphism: a double blind, randomized, controlled cross-over study

BackgroundPeroxisome proliferator-activated receptor (PPAR)γ is a key regulator in adipose tissue. The rare variant Pro12Ala of PPARγ2 is associated with a decreased risk of insulin resistance. Being dietary PPARγ ligands, conjugated linoleic acids (CLAs) received considerable attention because of their effects on body composition, cancer, atherosclerosis, diabetes, obesity and inflammation, although some effects were only demonstrated in animal trials and the results in human studies were not always consistent. In the present study effects of CLA supplementation on genome wide gene expression in adipose tissue biopsies from 11 Ala12Ala and 23 Pro12Pro men were investigated. Subjects underwent four intervention periods (4 wk) in a randomized double blind cross-over design receiving 4.25 g/d of either cis-9, trans-11 CLA, trans-10,cis-12 CLA, 1:1 mixture of both isomers or a reference linoleic acid oil preparation. After each intervention biopsies were taken, whole genome expression microarrays were applied, and genes of interest were verified by realtime PCR.ResultsThe following genes of lipid metabolism were regulated by CLA: LDLR, FASN, SCD, FADS1 and UCP2 were induced, while ABCA1, CD36 and CA3 were repressed. Transcription factors PPARγ, NFAT5, CREB5 and EBF1, the adipokine NAMPT, members of the insulin signaling cascade SORBS1 and IGF1 and IL6ST were repressed, while the adipokine THBS1 and GLUT4 involved in insulin signaling were induced. Compared to trans-10,cis-12 CLA and the CLA mixture the cis-9, trans-11 CLA isomer exerted weaker effects. Only CD36 (-1.2 fold) and THBS1 (1.5 fold) were regulated. The CLA effect on expression of PPARγ and leptin genes depends on the PPARγ2 genotype.ConclusionThe data suggest that the isomer specific influence of CLA on glucose and lipid metabolism is genotype dependent and at least in part mediated by PPARγ.Trial registrationhttp://www.controlled-trials.com: ISRCTN91188075

Corticosteroids and immunosuppressive therapy influence the result of QuantiFERON TB Gold testing in inflammatory bowel disease patients

INTRODUCTION Latent tuberculosis infection is detected by the tuberculin skin test before treating with anti-Tumour-Necrosis factor alpha (anti TNFα) reagents. More accurate are Interferon gamma release assays (IFNγ release assays) to identify patients with latent tuberculosis. Because of a positive control in this assay, it is possible to identify those patients in which a result of tuberculosis testing is not available due to a lack of stimulation capacity of lymphocytes (indeterminate result). Patients suffering from IBD are often treated with immunosuppressive agents, which may influence the results of tuberculosis testing. AIM The aim is to investigate the influence of immunosuppressive agents on the outcome of IFNγ-release assay. METHODS 50 consecutive patients were documented before introducing anti-TNF-treatment in this single centre study between April 2009 and April 2010. Data of INFγ release assay for latent tuberculosis, skin test and laboratory data and current medication were enrolled. RESULTS For the period of one year data of 45 consecutive patients was available for statistical analysis. 24 patients out of 45 (corresponding to 53.3%) received at least low doses of corticoid treatment and 27 patients out of 45 (corresponding to 60.0%) received immunosuppressive agents. 13 patients out of 45 (corresponding to 28.9%) had an indeterminate result of the QuantiFERON test. A correlation between the indeterminate result and combination therapy of corticosteroids was found. The concomitant therapy of immunosuppressive agents lead to a lower IFN release but no significance was found. CONCLUSIONS Steroid treatment and further combination therapy with immunosuppressive agents lead to a high risk of indeterminate QuantiFERON test.