Probiotic Mixture VSL Research Articles

Corrigendum to: Combined Therapy with Probiotic VSL#3 and Omega-3 Fatty Acids Attenuates Colonic Injury and Inflammation in Chronic DNBS-induced Colitis in Mice

We published the abovementioned article about probiotic mixture VSL#3 on October 6, 2021. Since the product was named VSL#3 in our purchasing process in early 2016, it is referred to as VSL#3 in our article. In addition, the product currently known as VSL#3 is not the same as De Simone Formulation. De Simone Formulation is now available as Visbiome® in the United States and Vivomixx® in Europe. The authors would like to apologise for any inconvenience caused

Probiotic mixture VSL#3: An overview of basic and clinical studies in chronic diseases.

Probiotics are known as “live microorganisms” and have been proven to have a health effect on hosts at the proper dose. Recently, a kind of probiotic mixture including eight live bacterial strains, VSL#3, has attracted considerable attention for its combined effect. VSL#3 is the only probiotic considered as a kind of medical food; it mainly participates in the regulation of the intestinal barrier function, including improving tight junction protein function, balancing intestinal microbial composition, regulating immune-related cytokine expression and so on. The objective of this review is to discuss the treatment action and mechanism for the administration of VSL#3 in chronic diseases of animals and humans (including children). We found that VSL#3 has a therapeutic or preventive effect in various systemic diseases per a large number of studies, including digestive systemic diseases (gastrointestinal diseases and hepatic diseases), obesity and diabetes, allergic diseases, nervous systemic diseases, atherosclerosis, bone diseases, and female reproductive systemic diseases.

Probiotics Upregulate Trefoil Factors and Downregulate Pepsinogen in the Mouse Stomach.

Probiotics are used in the management of some gastrointestinal diseases. However, little is known about their effects on normal gastric epithelial biology. The aim of this study was to explore how the probiotic mixture VSL#3 affects gastric cell lineages in mice with a special focus on protective and aggressive factors. Weight-matching littermate male mice (n = 14) were divided into treated and control pairs. The treated mice received VSL#3 (5 mg/day/mouse) by gastric gavage for 10 days. Control mice received only the vehicle. Food consumption and bodyweight were monitored. All mice were injected intraperitoneally with bromodeoxyuridine (120 mg/Kg bodyweight) two hours before sacrificed to label S-phase cells. Stomach tissues were processed for lectin- and immunohistochemical examination. ImageJ software was used to quantify immunolabeled gastric epithelial cells. Real-time quantitative polymerase chain reaction was used to provide relative changes in expression of gastric cell lineages specific genes. Results revealed that treated mice acquired (i) increased production of mucus, trefoil factor (TFF) 1 and TFF2, (ii) decreased production of pepsinogen, and (iii) increased ghrelin-secreting cells. No significant changes were observed in bodyweight, food consumption, cell proliferation, or parietal cells. Therefore, VSL#3 administration amplifies specific cell types specialized in the protection of the gastric epithelium.

Probiotic Mixture VSL#3 Alleviates Dextran Sulfate Sodium-induced Colitis in Mice by Downregulating T Follicular Helper Cells.

Clinical trials have shown beneficial effects of probiotics on inflammatory bowel diseases (IBD), although the exact mechanism remains unknown. VSL#3, a mixture of 8 probiotic bacteria, has been confirmed to have adjunctive therapeutic effects on colitis. T follicular helper (Tfh) cells, a new separate subset of CD4+ T helper cells, have been proved to play a vital role in autoimmunity. The present study aimed to identify the beneficial effect of the probiotic mixture VSL#3 on the mouse model of colitis by regulating Tfh cells. Dextran sulfate sodium (DSS) was used to induce chronic colitis in C57BL/6 mice. VSL#3 (3×109 live bacteria) was given to C57BL/6 mice every other day for 60 days by gavage. The disease activity index (DAI), histological activity index (HAI), colon length and myeloperoxidase (MPO) activity were detected. Immunofluorescence was used to visualize the location of Tfh cells. Immunoglobulins, Tfh cells and plasma cells were quantified by enzyme-linked immunosorbent assay (ELISA), flow cytometry, real-time PCR or Western blotting. The results showed that after DSS treatment, the humoral immunity was disordered in C57BL/6 mice, with increased IgM, IgG and IgA levels in colonic mucus and increased Tfh cells in mesenteric lymph nodes (MLN). VSL#3 treatment showed anti-inflammatory effects as evidenced by reduced DAI score, HAI score and MPO activity. IgM, IgG and IgA levels were significantly reduced in colon mucus, and the number of Tfh cells was markedly decreased in MLN after VSL#3 treatment. It was concluded that VSL#3 alleviates DSS-induced colitis by downregulating Tfh cells, and Tfh cells may become a potential therapeutic target for IBD.

Stress‐induced Effects on Endometriosis and Tight Junction Protein Expression are Counteracted by VSL#3 Administration in an Animal Model

IntroductionEndometriosis is a gynecological disorder in which tissue that normally lines the endometrium grows outside the uterus and commonly results in peritoneal inflammation and infertility. Our previous studies show that stress exacerbates the development of endometriosis symptoms with worsening of colonic damage, and probiotic administration can decrease lesion size. Tight junction proteins are important to maintain the integrity of the intestinal barrier, and their disturbance can contribute to a variety of pathological conditions. It is possible that in our model stress contributes to the intestinal effects via modulation of tight junction proteins, but it is unclear whether probiotic use could counteract any of these effects. In this project we focused on tight junction protein expression intensity using the specific integral membrane proteins occludin and claudin‐2 to investigate how probiotics might alleviate endometriotic damage in the colon. Downregulation of occludin can enhance paracellular permeability, while claudin‐2 overexpression has been linked to intestinal pathophysiology by decreasing barrier function of other claudins.HypothesisProbiotic administration will reverse the effects of stress on colonic tight junction protein expression in an animal model of endometriosis.MethodsEndometriosis was induced by suturing uterine horn tissue next to the intestinal mesenteries in female Sprague‐Dawley rats (11–12/group) on day 0. One week later, rats were given the probiotic mixture VSL#3 or placebo in their drinking water before subjecting to 7 days of water avoidance stress (60 mins/day; days 14–20). Non‐stressed controls also received VSL#3 or placebo. On day 60 the rats were sacrificed, the vesicles were retrieved and measured, and the tissues collected before paraffin embedding. Colonic damage was measured and immunofluorescence for occludin and claudin‐2 was performed.ResultsThe vesicle area from rats exposed to stress were significantly larger in size than non‐stressed controls (p<0.05), while after probiotic administration the size was reduced (p<0.01). Colonic macroscopic damage had a trend to increase in endometriosis animals and was reduced after probiotic administration (p<0.05). Colonic occludin expression showed a trend to increase when probiotic was administered after stress, while claudin‐2 expression was downregulated after stress (p<0.01) and probiotic administration (p<0.01).ConclusionStress aggravates endometriosis symptoms causing larger vesicles and increased colonic damage. After probiotic administration, these symptoms are reversed and integrity of the tight junctions in colonic tissue is increased. This tendency provides further support for the potential use of probiotics as a complementary treatment to reduce the effects of inflammation in this condition.Support or Funding InformationSupported in part by P20 GM103475‐15, R25GM096955 & R25GM082406This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Probiotic Mixture VSL#3 Reverses Olanzapine-Induced Metabolic Dysfunction in Mice.

Atypical antipsychotic drugs (AAPDs) such as olanzapine (OLZ) are associated with serious metabolic adverse effects such as weight gain, visceral fat accretion, glucose intolerance, and lipid abnormalities. Compelling evidence suggests that the gut microbiota is known to regulate metabolic homeostasis and therefore microbiota-modulating strategies such as probiotics may serve as an excellent approach for treating metabolic adverse effects associated with atypical antipsychotic drugs (AAPDs). The therapeutic potential of VSL#3 (20×109CFU/day), in reversing olanzapine-induced metabolic dysfunction, was assessed. VSL#3 administration led to attenuation of OLZ-induced body weight gain, uterine fat deposition, impaired glucose tolerance, and insulin resistance. Moreover olanzapine treatment also decreased inflammatory markers, abolished oxidative stress in the vWAT, and prevented shifts in gut microbiota abundance levels. These results indicate that VSL#3 via its ability to manipulate the gut microbiome confers beneficial metabolic effects and may, therefore, represent a novel therapeutic approach for reversing antipsychotic-induced metabolic dysfunction.

The effects of exhaustive swimming and probiotic administration in trained rats: Oxidative balance of selected organs, colon morphology, and contractility.

The duration and intensity of exercise are significant factors in oxidative, morphological, and functional changes of the gastrointestinal tract. This study aimed to investigate the effects of both exhaustive swimming and probiotic VSL#3 on rats that had been previously trained with moderate swimming. The rats were divided into four groups labeled: control (C), probiotic (P), exercise (E), and probiotic-exercise (PE). Groups P and PE were fed with probiotic mixture VSL#3. Groups E and PE had a 5-week moderate swimming program (1h/day for 5 days/week), followed by a 1-week exhaustive swimming program (trained like in moderate program but 3 times with 150min resting sessions, for 5days/week). At the end of the program, the rats were euthanized. Malondialdehyde, superoxide dismutase, catalase, and reduced glutathione levels were measured in tissue samples from the gastrocnemius muscle, heart, liver, kidney, and colon. In vitro contractile activity and histomorphology of the colon were also determined. Exercise and/or probiotic decreased the oxidative stress and also increased the level of one or more of the antioxidant enzymes in some of the organs. Probiotics had more pronounced effects on colon morphology than exercise but unexpectedly this effect was non-trophic. In the colon, the thickness of the tunica muscularis and the number of goblet cells were not affected; however, probiotic administration decreased the crypt depth and tunica mucosa thickness. Exercise increased the Emax value of acetylcholine (ACh), while decreased its sensitivity. These findings suggest that exhaustive swimming does not cause oxidative stress and that probiotic consumption improves oxidative balance in trained rats. The probiotic intake does not alter the effect of exercise on the contractile activity of the colon. Colon mucosal changes induced by probiotics are independent of exercise.

The Probiotic VSL#3 Modulates Colonic Macrophages, Inflammation, and Microflora in Acute Trinitrobenzene Sulfonic Acid Colitis.

The probiotic mixture VSL#3 attenuates colitis in patients with Inflammatory Bowel Disease (IBD) and in animal models of this condition, but the mechanisms involved are incompletely understood. VSL#3 alters macrophage morphology and secretory profile in vitro in a polarization-dependent manner. We examined the effect of VSL#3 on macrophages in acute trinitrobenzene sulfonic acid-induced colitis. Rats were randomized to normal, colitis, or colitis+VSL#3 groups. After sacrifice, the colons were evaluated for macroscopic and microscopic damage. Serum cytokine levels were measured, and microbiome analysis undertaken. Total and M1 colonic macrophages, and total and proliferating hepatic macrophages were assessed by double immunofluorescence staining. Colitis+VSL#3 rats had lower macroscopic damage, with less microscopic damage in the proximal colon, compared with colitis alone. Colitis significantly increased colonic macrophage infiltration, which was significantly reduced by VSL#3 treatment. VSL#3 did not decrease the colitis-induced surge of colonic M1 macrophages or hepatic macrophages. VSL#3 reduced colitis-induced serum cytokine levels, and induced restoration of colonic transcript levels for pro-inflammatory, anti-inflammatory, and barrier proteins to, or past, normal levels. Fecal bacteria distribution changed between groups. In summary, the probiotic VSL#3 reduces colitis severity, colonic macrophage infiltration, and serum cytokine levels, but does not dampen the pro-inflammatory phenotype of M1 macrophages.

P478 The current place of probiotics in treatment of pouchitis: systematic review

Abstract Background: Pouchitis is a common complication in patients undergoing restorative proctocolectomy for ulcerative colitis. Therapeutic attempts include manipulations of pouch flora composition. In this systematic review, we aimed to score the evidence supporting the use of probiotics and prebiotics in pouchitis patients, to clarify the place of these treatments in current therapeutic regimens. Methods: We conducted extensive electronic searches of the PubMed and SCOPUS databases, from their earliest records through Nov 2016, for MESH terms “probiotics” and “pouchitis”. Results: The electronic search retrieved 20 citations [1–20]. Six published RCTs [2,3,9,10,13,14] and a RCT presented as an abstract [12] evaluated clinical, endoscopic end/or histological effect of probiotics as a primary outcome; other reports ranged in level of evidence between meta-analyses [16–19] (4), open-labeled trials [1,4–8,11,15] (8) and letters [20] (1). Conclusions: Prevention of onset of pouchitis/Primary prevention. Three studies examined the ability of various probiotic regimens to prevent the onset of pouchitis after the restorative proctocolectomy [1–3]. Primary preventive effect of VSL#3 was indicated by a single RCT, with calculated effect ratio of 1.50 [1.02, 2.21] [16]. Table 1. Use of probiotics for prevention of onset of pouchitis Study No. of patients Duration (months) probiotic strain Strain Control Pouchitis-free survival Gosselink (2004) 117 36 LGG No treatment (historical control) 93% vs. 71% (p=0.011) Gionchetti (2003) [2] 40 12 VSL#3 Placebo 90% vs. 60% (p<0.05) Yasueda (2015) [3] 17 24 Clostridium butyricum Placebo 89% vs. 50% (NS) Treatment of acute episode. Seven – mostly open-labeled and uncontrolled – trials examined the use of probiotics for treatment of acute pouchitis episode [4–10]. Efficacy of probiotics in acute episodes of pouchitis needs to be proved in randomized controlled trials. Table 2. Use of probiotics for treatment of active pouchitis Study No. of patients Duration (months) Strain Control Outcome Gionchetti et al. (2007) [4] 23 1 VSL#3 Open-labeled; uncontrolled 69% remission rate Laake et al. (2005) [5] 51 (10 with active disease) 1 Cultura® fermented milk product Open-labeled; uncontrolled Symptomatic and endoscopic improvement in patients with active disease Laake et al. (1999) [21] Cultura® fermented milk product Open-labeled; uncontrolled Symptomatic improvement Laake et al. (2003) [7] 10 ½ month Cultura® fermented milk product Open-labeled; uncontrolled Endoscopic improvement Laake et al. (2004) [8] 41 UC + 10 FAP 1 Cultura® fermented milk product Open-labeled; uncontrolled Symptomatic remission and endoscopic improvement Tomasz et al. (2014) [9] 43 (14 with active disease) 9 Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. Bulgaricus, Bifidobacterium bifidus Placebo 43% remission rate vs 0% on placebo Kuisma (2003) [10] 20 3 LGG Placebo No benefit Seven studies aimed to determine the efficacy of probiotic strains in preventing recurrences in patients with previous episode/s of pouchitis [4,9,11–15]. Probiotic mixture VSL#3 effectively prevents relapses after successful antibiotic treatment of active inflammation, with calculated effect ratio of 20.24 [4.28, 95.81] [16]. Side effects may affect the adherence of the patients with the long-term treatment [15,16]. Table 3. Use of probiotics for prevention of recurrences of pouchitis Study No. of patients Duration (months) Probiotic strain Control Outcome Pronio (2008) [11] 31 12 VSL#3 No treatment Small reduction of PDAI scores Brown (2004) [12] (abstract) 17 6 Bifidobacterium longum BB-536 Placebo Pouchitis-free survival 86% vs. 60% on placebo (NS); small reduction of PDAI scores Tomasz et al. (2014) [9] 43 9 Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. Bulgaricus, Bifidobacterium bifidus Placebo 43% in remission vs 0% on placebo Gionchetti et al. (2007) [4] 16 6 VSL#3 Open-labeled; uncontrolled 69% remission rate Gionchetti (2000) [13] 40 9 VSL#3 Placebo Pouchitis-free survival 85% vs. 0% on placebo (P<0.001) Mimura (2004) [14] 36 12 VSL#3 Placebo Pouchitis-free survival 85% vs. 6% on placebo (P<0.0001) Shen (2005) [15] 31 8 VLS#3 (self administration) Open-labeled compliance trial 80% non-adherence; 74% self-reported recurrence of symptoms

Probiotic mixture VSL#3 reduces colonic inflammation and improves intestinal barrier function in Muc2 mucin-deficient mice.

It is unclear whether probiotics require an intact mucin barrier to first colonize and/or exert their protective functions. In this study we used mucin-deficient (Muc2-/-) mice to interrogate if the multispecies probiotic mixture VSL#3 could enhance epithelial barrier function. In the absence of a mucus bilayer, VSL#3 dampened proinflammatory and chemokine production, accelerated restitution, and markedly improved gut permeability mediated by the short-chain fatty acid acetate in the colon.

Probiotic mixture VSL#3 reduce high fat diet induced vascular inflammation and atherosclerosis in ApoE(-/-) mice.

Atherosclerosis results from chronic inflammation potentially caused by translocation of bacterial components from the oro-gastrointestinal tract to circulation. Specific probiotics have anti-inflammatory effects and may reduce bacterial translocation. We thereby tested whether a probiotic mixture with documented anti-inflammatory potential could reduce atherosclerosis. ApoE−/− mice were fed high fat diet alone or with VSL#3 or a positive control treatment, telmisartan or both for 12 weeks. All treatments reduced atherosclerotic plaques significantly compared to high fat diet alone. VSL#3 significantly reduced proinflammatory adhesion molecules and risk factors of plaque rupture, reduced vascular inflammation and atherosclerosis to a comparable extent to telmisartan; and VSL#3 treated mice had the most distinctly different intestinal microbiota composition from the control groups. Combining the VSL#3 and telmisartan brought no further benefits. Our findings showed the therapeutic potential of VSL#3 in reducing atherosclerosis and vascular inflammation.

The Probiotic Mixture VSL#3 Attenuates Colitis, Decreases Macrophage Infiltration, and Reduces Serum Cytokine Levels in an Acute Colitis Model

BackgroundOur previous work has shown that the probiotic mixture VSL#3 alters the morphology and secretory profile of proinflammatory (M1), anti‐inflammatory (M2), and unpolarized macrophages in vitro. The effect of VSL#3 on macrophages in vivo is unclear. This knowledge may prove useful for developing and improving treatments for colitis in patients with Inflammatory Bowel Disease.AimExamine the effect of VSL#3 administration on disease parameters, macrophage phenotype and infiltration, and serum cytokine levels in an acute colitis model.MethodsMale rats were randomized to one of the following groups: a normal group (n=5), a colitis group (n=9), or a colitis+VSL#3 group (n=12). Rats in the colitis+VSL#3 group received 10 billion CFU of the probiotic in drinking water daily from a week prior to colitis induction until the time of sacrifice, whereas the control rats received water during this period. Rats underwent colitis induction by intracolonic administration of 0.5 mL of trinitrobenzene sulfonic acid (60 mg/mL) and were sacrificed 72 hours later. The colon of each rat was resected and evaluated for macroscopic and microscopic damage. Double immunofluorescence staining of colonic sections for CD68 and inducible nitric oxide synthase (iNOS) was used to assess total macrophage (CD68+) and M1 macrophage (CD68+iNOS+) numbers. Serum cytokine levels at the time of sacrifice were measured with a multiplex bead array.ResultsAll of the colitis rats significantly lost weight when compared to normal rats (p<0.001), which gained weight. The probiotic did not have a significant effect on weight loss. All of the colitis rats had significant macroscopic (p<0.001) and microscopic damage (p<0.01) in the colon compared to normal rats as expected. Rats in the colitis+VSL#3 group had lower macroscopic damage scores overall (p<0.01), and lower microscopic damage scores in the proximal colon (p<0.01), when compared to rats in the colitis group. This improvement was not found in the mid or distal colon. M1 macrophages were mainly limited to areas of mucosa and submucosa that stained for iNOS. Within these areas, rats with colitis contained significantly higher percentages of M1 macrophages than normal rats (p<0.05), treatment with VSL#3 did not decrease the percentage of M1 macrophages in rats with colitis, and total macrophage numbers did not differ significantly between the three groups. Overall, colitis induction led to a significant increase in submucosal macrophage infiltration (p<0.01), and this infiltration was significantly reduced by treatment with VSL#3 (p<0.01). Mucosal macrophage infiltration was significantly higher in the colitis group when compared to normal controls (p<0.001) and the colitis+VSL#3 group (p<0.01). Colitis induction significantly increased serum levels of GRO/KC, MCP‐1, and VEGF. Serum levels of EGF, IL‐5, IL‐12p70, IL‐13, IL‐17A, IL‐18, MIP‐1α, and TNF‐α were significantly increased in the colitis group, but not the colitis+VSL#3 group, when compared to normal controls. Treatment with VSL#3 significantly reduced serum levels of EGF, eotaxin, IL‐2, and VEGF.ConclusionIn this model of acute colitis, the probiotic VSL#3 reduces colitis severity, colonic macrophage infiltration, and serum cytokine and chemokine levels. Our results suggest that VSL#3 treatment does not dampen the proinflammatory phenotype of M1 macrophages, consistent with our previously reported in vitro results for human monocyte‐derived M1 macrophages.Support or Funding InformationR25GM082406 & William Townsend Porter Predoctoral Fellowship from the American Physiological Society

VSL#3 induces and maintains short-term clinical response in patients with active microscopic colitis: a two-phase randomised clinical trial

BackgroundThe probiotic mixture VSL#3 has proven efficacious in inflammatory bowel diseases and irritable bowel syndrome; however, its efficacy in microscopic colitis (MC) is being investigated.ObjectiveTo evaluate the safety and efficacy...

Role of Probiotics in Crohn's Disease and in Pouchitis.

The alterations in the gut microbiota observed in patients with inflammatory bowel disease and in particular in Crohn's disease and in ulcerative colitis patients with pouchitis, provide the rationale for administering probiotic agents in the medical treatment of those conditions. In the maintenance treatment of inactive Crohn's disease probiotics, when administered alone, were found ineffective in preventing clinical and/or endoscopic recurrence. By contrast, a combination of a probiotic agent (eg, Saccharomyces boulardii) with standard pharmacological therapy can promote clinical benefit. In patients with pouchitis, so far only the probiotic mixture VSL #3 proved to effectively prevent relapses after successful antibiotic treatment of active inflammation. Further controlled studies, enrolling higher numbers of patients, are needed to better identify the exact role of probiotics in this area.

Probiotics Improve Inflammation-Associated Sickness Behavior by Altering Communication between the Peripheral Immune System and the Brain.

This research shows that probiotics, when eaten, can improve the abnormal behaviors (including social withdrawal and immobility) that are commonly associated with inflammation. Probiotics are able to cause this effect within the body by changing how the immune system signals the brain to alter brain function. These findings broaden our understanding of how probiotics may beneficially affect brain function in the context of inflammation occurring within the body and may open potential new therapeutic alternatives for the treatment of these alterations in behavior that can greatly affect patient quality of life.

The Probiotic Mixture VSL#3 Has Differential Effects on Intestinal Immune Parameters in Healthy Female BALB/c and C57BL/6 Mice1–3

Background: Probiotic bacteria may render mice resistant to the development of various inflammatory and infectious diseases.Objective: This study aimed to identify mechanisms by which probiotic bacteria may influence intestinal immune homeostasis in noninflammatory conditions.Methods: The effect of VSL#3, a mixture of 8 probiotic bacteria, on intestinal gene expression was studied in healthy female BALB/c and C57BL/6 mice after prolonged oral treatment (28 d, triweekly) with 3 × 108 colony-forming units of VSL#3. In a separate experiment in BALB/c mice, the effects of prolonged administration of VSL#3 and of phosphate-buffered saline (PBS), followed by 1 single dose of VSL#3, on innate and adaptive immune cells were evaluated.Results: Microarray analysis of the intestines of mice treated with PBS confirmed well-established differences in the expression of immune-related genes between C57BL/6 and BALB/c mice. Prolonged administration of VSL#3 was associated with downregulation of Il13[fold change (FC) = 0.46] and Eosinophil peroxidase (Epx) (FC = 0.44) and upregulation of Il12rb1 (FC = 2.1), C-C chemokine receptor type 5 (Ccr5) (FC = 2.6), chemokine (C-X-C motif) receptor 3 (Cxcr3) (FC = 1.6), and C-X-C motif chemokine 10 (Cxcl10) (FC = 2.8) in BALB/c mice but not in C57BL/6 mice. In BALB/c mice, it was shown that 28 d of treatment with VSL#3 affected the Peyer's patches (PPs) and mesenteric lymph nodes (MLNs), which was evident from an increase in B cells (26% and 8%, respectively), a decrease in T cells (21% and 8%, respectively), and an increase in cluster of differentiation (CD) 11c+ cells (57% in PPs) compared with PBS-treated mice. This treatment was also associated with increased frequencies of T helper 17 (13%) and regulatory T cells (11%) in the MLNs. Treatment with PBS followed by 1 single dose of VSL#3, 18 h before killing, was associated with a 2-fold increase in CD103+CD11c+ dendritic cells in MLNs and PPs.Conclusion: VSL#3 treatment mediates mouse strain–specific alterations in immunologic phenotype in conditions of homeostasis, suggesting that the effects of probiotic bacteria depend on the genetic background of the host.

Effects of Probiotic VSL#3 on Cytokine and Tight Junction Protein Expression in Intestinal Epithelial Cells

In the gut, benefits of probiotic bacteria may be mediated through multiple mechanisms, including alteration of the composition or metabolism of the gut microbiota, direct action on host intestinal cells, or modulation of the immune system. The commercially available probiotic VSL#3 includes a mixture of eight species, mostly lactobacilli and bifidobacteria. VSL#3 has been shown to have clinical benefits in ulcerative colitis patients, and has also been demonstrated to alter expression of mucins and other genes in cultured intestinal epithelial cells. This study aimed to assess effects of VSL#3 on pro‐ and anti‐inflammatory cytokine and tight junction protein expression in Caco‐2 colonic epithelial cells. Cells were treated for 4 hours with VSL#3 (1x108 CFU/ml) and expression of the anti‐inflammatory cytoking transforming growth factor‐beta‐1 (TGFβ‐1), pro‐inflammatory interleukin (IL)‐6 and the housekeeping gene GAPDH was analyzed by semi‐quantitative PCR. Expression of the tight junction protein claudin‐2 was detected by immunofluorescent staining. VSL#3 treated Caco‐2 cells expressed increased levels of TGFβ‐1 mRNA compared to untreated controls. Preliminary experiments suggested a similar increase in TGFβ‐1 in murine CMT‐93 cells. In contrast, IL‐6 mRNA expression was not increased by VSL#3. Immunofluorescent staining showed increased claudin‐2 staining in cells treated with VSL#3 for 24 hours compared to untreated controls. In conclusion, the probiotic mixture VSL#3 stimulates expression of TGFβ‐1 and claudin‐2 in Caco‐2 cells, providing a potential mechanism for protective effects of VSL#3 on the intestinal epithelium. This project was supported by the Missouri Western State University PORTAL program.

Prebiotics and probiotics in irritable bowel syndrome and inflammatory bowel disease in children.

Underlying pathophysiological mechanisms of irritable bowel syndrome (IBS), a common disorder characterized by abdominal pain associated to a change in stool consistency or frequency, include low-grade inflammation and intestinal microbiota changes. Few and disappointing data are available for prebiotics. A few controlled trials (RCTs) of probiotics are instead available with favourable effects, although most are limited by suboptimal design and small sample size. A recent report from the Rome foundation group included 32 RCTs of probiotics, most of which showed an overall modest improvement in symptoms, with the patients most benefitting from probiotics being those with predominant diarrhoea and those having a post-infectious IBS. A review focusing only on children with functional gastrointestinal disorders concluded that probiotics are more effective than placebo in the treatment of patients with abdominal pain-related functional gastrointestinal disorders, although no effect on constipation was evident. The role for probiotics in inflammatory bowel disease (IBD) appears logical: the endogenous intestinal microbiota plays a central role in their development, and various probiotics have been found effective in animal models of IBD. However, research in humans has been overall quite limited, and it would seem that after a phase of intense research in the first decade of this century, the pace has slowed down, with fewer clinical trials been published in the past 2-3 years. To summarize current evidence: no probiotic has proven successful in Crohn's disease. In ulcerative colitis, on the other hand, data are more promising, and a very recent meta-analysis, that included 23 randomized controlled trials, concluded that there is evidence of efficacy for the probiotic mixture VSL#3 in helping inducing and maintaining remission, as well as in maintaining remission in patients with pouchitis. It is fair to state that for both IBD and IBS, more well-designed, rigorous, randomized clinical trials must be performed.

The probiotic mixture VSL#3 dampens LPS-induced chemokine expression in human dendritic cells by inhibition of STAT-1 phosphorylation.

VSL#3, a mixture of 8 different probiotic bacteria, has successfully been used in the clinic to treat Ulcerative Colitis. We previously identified the modulation of chemokines as a major mechanism in the protective effect of the VSL#3 in a mouse model of colitis. This was supported by in vitro studies that implicated a role for VSL#3 in the suppression of LPS-induced chemokine production by mouse bone marrow-derived dendritic cells (DC). Herein, we validated these findings employing human monocyte-derived DC. Stimulation of human DC with LPS, VSL#3, or a combination of both resulted in their maturation, evident from enhanced expression of activation markers on the cell-surface, as well as the induction of various chemokines and cytokines. Interestingly, a set of LPS-induced chemokines was identified that were suppressed by VSL#3. These included CXCL9, CXCL10, CCL2, CCL7, and CCL8. In silico approaches identified STAT-1 as a dominant regulator of these chemokines, and this was confirmed by demonstrating that LPS-induced phosphorylation of this transcription factor was inhibited by VSL#3. This indicates that VSL#3 may contribute to the control of inflammation by selective suppression of STAT-1 induced chemokines.

The probiotic mixture VSL#3 mediates both pro- and anti-inflammatory responses in bone marrow-derived dendritic cells from C57BL/6 and BALB/c mice.

Probiotic bacteria express a wide range of molecular structures that bind to receptors on innate immune cells and mediate health-promoting effects in the host. We have recently demonstrated in a colitis model that favourable effects of the probiotic mixture VSL#3 may in part be due to the suppression of intestinal chemokine expression. To obtain more insights into the underlying mechanisms, in the present study, we analysed the modulation of bone marrow-derived dendritic cells (BM-DC) from BALB/c (T helper (Th)2 biased) v. C57BL/6 (Th1 biased) mice. Our findings showed that VSL#3 differed from pure Toll-like receptor (TLR) ligands by inducing the production of various cytokines, including IL-12 p70 subunit (IL-12p70), IL-23 and IL-10. Dedicated TLR arrays were employed to profile mRNA from BM-DC cultured with lipopolysaccharide (LPS), VSL#3, or a combination of both. This approach led to the identification of (1) a cluster of genes that were up- or down-regulated, irrespective of the stimulus, (2) a cluster of genes that were synergistically up-regulated by LPS and VSL#3 in BM-DC from C57BL/6 mice, but not in those from BALB/c mice, and (3) a cluster of LPS-induced genes that were suppressed by VSL#3, in particular chemokine genes. These data show that this probiotic mixture has both pro- and anti-inflammatory effects on BM-DC and suggest that their immune-modulating properties in vivo may depend on the genetic background of the host.