Gut Microbiota and Behcet's Syndrome: a Dietary Intervention Trial (MAMBA Study)

Study Overview
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Brief Summary
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Behçet's syndrome (BS) is an idiopathic, chronic, multi-systemic inflammatory disorder characterized by ocular disease, skin lesions, vascular, neurological and gastrointestinal involvement. A recent study showed a peculiar dysbiosis of gut microbiota (GM) in BS patients, with specific changes in the profiles of short-chain fatty acids, especially butyrate. Over the last few years, a growing interest on the role of GM in metabolic disturbances has been manifested. Diet is one of the major factors driving the GM composition and functionality. In this context, the influence of diets generally recognized healthy on GM has been explored, but consistent data on autoimmune and inflammatory diseases are not available. The aim of this intervention study is to investigate whether a lacto-ovo-vegetarian diet enriched in substrates with potential for butyrate production or a Mediterranean diet supplemented with oral butyrate could be beneficial for GM and metabolic risk profile in BS.

Detailed Description
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Background / State of Art: Behçet's syndrome (BS) is a systemic inflammatory disorder characterized by a wide range of potential clinical manifestations with no gold-standard therapy. Although BS is usually not a life-threatening condition, mortality can be associated with vascular-thrombotic and neurological affections. A recent study by our group, for the first time, provided evidence of a peculiar gut microbiota (GM) dysbiosis in BS patients, with reduced biodiversity, and decrease in short-chain fatty acid (SCFA)-producers and butyrate production. In light of this, controlled dietary interventions specifically designed to favor the increase of butyrate-producing members of the GM may support the recovery of a healthy GM ecosystem. Lacto-ovo-vegetarian diet is characterized by abstention from consuming meat and meat products, poultry, seafood and flesh from any other animal and by a large amount of plant- derived foods. This dietary pattern has been largely demonstrated to be beneficial for both patients with an established disease and for subjects with traditional risk factors for chronic diseases. Indeed, dietary patterns rich in plant-based food have been found to promote a more favorable GM profile, according to the high amount of dietary fiber and fermentable substrate, which are sources of metabolic fuel for GM fermentation that, in turn, result in end products - mainly SCFA - that are key microbial metabolites with a multifactorial role on the host health. Hypothesis and Specific aims: Although the pathogenesis of BS is currently unknown, it has been recently classified at the crossroad between autoimmune and autoinflammatory syndromes. GM has been found to deeply influence our metabolic and immunological health, and specific perturbed GM configurations have been indicating a fascinating link between intestinal microbes and health status. A recent study from our group showed that a peculiar dysbiosis of the GM ecosystem is present also in patients with BS, corresponding to specific changes in the profiles of SCFA production. In particular, the GM ecosystem in BS showed a low biodiversity, in line with several other chronic disorders. Moreover, a significant depletion of well- known butyrate producers, Roseburia and Subdoligranulum, and a corresponding decrease of butyrate production in BS patients was demonstrated. Butyrate - which is the preferred fuel for colonocytes - is able to induce T regulatory cell differentiation via several mechanisms, so the butyrate impairment in BS patients could favor a reduced T regulatory cell- mediated control, thus promoting a powerful immuno-pathological T cell responses. In this context, over the last years, growing evidence suggested that high-fiber dietary patterns are able to promote a more favorable GM profile, and are key mediators of microbial diversity. In particular, it has recently been demonstrated that high adherence to a lacto-ovo-vegetarian diet - including high intake of non-refined cereals, fruit, vegetables and legumes - is associated with a beneficial GM profile, with enrichment in fiber-degrading bacteria and increase of fecal SCFA. In a similar way, other dietary patterns have been shown to modulate GM dysbiosis, by supporting the recovery of a balanced microbial community of health-promoting SCFA-producing members with the decrease of pro-inflammatory groups. Furthermore, current evidence indicates that the consumption of certain fibers - such as inulin and resistant starch - leads to specific GM rearrangements with the production of more butyrate than others in humans. All these findings let hypothesize that the adherence to a controlled dietary profile such as lacto-ovo-vegetarian diet, possibly enriched in substrates with potential for butyrate production may select butyrate-producing bacteria - especially Roseburia spp. (Clostridium cluster XIVa) and Faecalibacterium prausnitzii (Clostridium cluster IV)- so reversing the pro- inflammatory dysbiosis observed in BS. Preliminary Data: A recent study by our group showed that a peculiar dysbiosis of the GM ecosystem is present in patients with BS, corresponding to specific changes in the profiles of SCFA production. By strengthening this dysbiotic GM structure, a significant decrease of fecal butyrate production was found in BS patients. More recently, a high percentage of Th1/Th17 cells at gut mucosal level in BS patients was observed, thus suggesting a reduced T regulatory activity, probably mediated by reduced levels of butyrate (unpublished data). Specific Aim 1: To conduct a dietary intervention randomized controlled trial in order to investigate whether a lacto-ovo-vegetarian diet enriched in substrates with potential for butyrate production or a Mediterranean diet supplemented with 2 g/day of butyrate could be beneficial for GM and for the amelioration of the clinical manifestations and disease severity of patients with BS. Specific Aim 2: To evaluate the effects of these interventions on: inflammatory parameters, circulating biomarkers of disease, endogenous butyrate production, and oxidative stress markers. Specific Aim 3: To validate and extend our preliminary results on GM ecosystem dysbiosis in BS patients

Official Title
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ModulAtion of Gut Microbiota Through Nutritional Interventions in Behcet's Syndrome pAtients: the MAMBA Study

Conditions
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Behcet Syndrome

Intervention / Treatment
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* Behavioral: Vegetarian diet
* Behavioral: Mediterranean diet with butyrate
* Behavioral: Mediterranean diet


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Behcet syndrome Age 18-65 years Willing to give informed consent Willing to participate in a study where one of the proposed dietary profile is a vegetarian pattern Exclusion Criteria: Pregnancy or lactation Concomitant presence of serious illness or unstable condition (autoimmune diseases; chronic viral infections; malignancies, recent myocardial infarction, chronic liver disease, inflammatory bowel diseases) Current or recent (past 6 months) participation in weight loss treatment program or use of weight loss medication Adoption of a vegetarian diet for the past 3 months

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 65 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Treatment
Allocation: Randomized
Intervention Model: Crossover Assignment
Interventional Model Description: Randomized crossover open dietary intervention study with 3 arms of intervention
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: VD group<br>Group that starts with Vegetarian diet (VD) | Behavioral: Vegetarian diet<br>* 7-days dietary profile with a Vegetarian diet (VD), containing inulin and resistant starch-rich foods and including no meat and fish, but containing eggs and dairy, for 3 months<br>* Other names: VD;Behavioral: Mediterranean diet with butyrate<br>* 7-days dietary profile with Mediterranean diet with oral supplementation with butyrate (MD+Bt), 2 g/day, for 3 months<br>* Other names: MD+Bt;Behavioral: Mediterranean diet<br>* 7-days dietary profile with Mediterranean diet (MD), including 2 portions per week of fish and 3 portions per week of fresh and processed meat (2 of which fresh or processed red meat), for 3 months<br>* Other names: MD;|
| Experimental: MD+Bt group<br>Group that starts with Mediterranean diet with oral supplementation with butyrate (MD+Bt) | Behavioral: Vegetarian diet<br>* 7-days dietary profile with a Vegetarian diet (VD), containing inulin and resistant starch-rich foods and including no meat and fish, but containing eggs and dairy, for 3 months<br>* Other names: VD;Behavioral: Mediterranean diet with butyrate<br>* 7-days dietary profile with Mediterranean diet with oral supplementation with butyrate (MD+Bt), 2 g/day, for 3 months<br>* Other names: MD+Bt;Behavioral: Mediterranean diet<br>* 7-days dietary profile with Mediterranean diet (MD), including 2 portions per week of fish and 3 portions per week of fresh and processed meat (2 of which fresh or processed red meat), for 3 months<br>* Other names: MD;|
| Active Comparator: MD group<br>Group that starts with Mediterranean diet (MD) | Behavioral: Vegetarian diet<br>* 7-days dietary profile with a Vegetarian diet (VD), containing inulin and resistant starch-rich foods and including no meat and fish, but containing eggs and dairy, for 3 months<br>* Other names: VD;Behavioral: Mediterranean diet with butyrate<br>* 7-days dietary profile with Mediterranean diet with oral supplementation with butyrate (MD+Bt), 2 g/day, for 3 months<br>* Other names: MD+Bt;Behavioral: Mediterranean diet<br>* 7-days dietary profile with Mediterranean diet (MD), including 2 portions per week of fish and 3 portions per week of fresh and processed meat (2 of which fresh or processed red meat), for 3 months<br>* Other names: MD;|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Disease severity of Behcet syndrome assessed by Behçet Disease Current Activity Form | The disease activity will be assessed by the use of the validated Behçet Disease Current Activity Form (BCDAF), at the baseline and after the dietary intervention. The BCDAF will assess the presence of oral and genital ulceration, skin, joint and gastrointestinal involvement, presence of fatigue and headache with a 5-point scale according to the duration of symptoms, with 0 meaning no symptoms and 4 meaning symptoms for 4 weeks. The presence of eyes, large vessels or central nervous system (CNS) involvement are document with yes/no answers. In addition, patients will be asked to rate on a 7-point scale how active they felt. Similarly, the clinicians will complete a 7-rating scale to assess their opinion of overall activity of the disease, with lower scores representing better outcomes. | 1 year | 
| Behcet disease's improvement of symptoms assessed by the Global Assessment of Improvement Scale (GAI) modified form | The Global Assessment of Improvement Scale (GAI) modified form will assess Behcet disease's improvement of symptoms using a 7-point scale, with higher scores meaning an improvement of the symptoms. The severity of abdominal pain, severity of abdominal distention, satisfaction with bowel habits, severity of headache, severity of exhaustion, severity of nausea, attention disorder, muscle/joint pain, and quality of life will be investigated in response to the following question: Compared to the way you felt before you entered the study, have your symptoms over the past 7 days been: 1) Substantially Worse, 2) Moderately Worse, 3) Slightly Worse, 4) No Change, 5) Slightly Improved, 6) Moderately Improved or 7) Substantially Improved. | 1 year | 
| Behcet disease's severity of gastrointestinal symptoms assessed by the Symptom Severity Scale (SSS) modified form | The Symptom Severity Scale (SSS) modified form is a multidimensional rating scale assessing overall symptoms' severity on a Visual Analogue Scale (VAS). An overall score will be calculated from six items: pain severity, pain frequency, abdominal bloating, bowel habit dissatisfaction, abdominal heaviness, and life interference. The modified SSS ranges from 0 to 600, with higher scores meaning more severe symptoms. | 1 year | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Gut microbiota assessed by Illumina MiSeq platform | Changes of gut microbiota profiles from baseline. The V3 and V4 hypervariable regions of the 16S rRNA gene will be sequenced on Illumina MiSeq platform, following the Illumina protocol for 16S Metagenomic Sequencing Library Preparation | 1 year | 
| Fecal SCFA assessed by Gas Chromatography - Mass Spectrometry system | Fecal SCFA's change from baseline, especially butyrate, will be assessed by Gas Chromatography - Mass Spectrometry system | 1 year | 
| Inflammatory profile assessed by cytofluorimetric approach | Inflammatory cytokines' change from baseline, will be assessed with the Bio-Plex cytokine assay, according to the manufacturer's instructions. In particular, Interleukin (IL)-1ra (pg/mL), IL-4 (pg/mL), IL-6 (pg/mL), IL-8 (pg/mL), IL-10 (pg/mL), IL-12 (pg/mL), IL-17 (pg/mL), monocyte chemoattractant protein (MCP)-1 (pg/mL), macrophage inflammatory protein (MIP)-1beta (pg/mL), vascular endothelial growth factor (VEGF) (pg/mL), tumor necrosis factor (TNF)-alpha (pg/mL), interferon (IFN)-gamma (pg/mL), interferon-gamma-induced protein (IP)-10 (pg/mL), will be measured. | 1 year | 
| Lipid peroxidation assessed by flow cytometry | Lipid peroxidation markers' change from baseline will be estimated using the Thiobarbituric Acid Reactive Substances (TBARS, pg/mL) assay kit through the flow cytometry. | 1 year | 
| Plasma total antioxidant capacity assessed by the oxygen radical absorbance capacity | Plasma total antioxidant capacity's (TAC, μmol/mL) change from baseline, will be measured using the oxygen radical absorbance capacity. | 1 year | 
| Reactive oxygen species (ROS) assessed by flow cytometry | Leukocyte (lymphocyte, monocyte, and granulocyte) reactive oxygen species' (ROS, RFU) change from baseline will be assessed through flow cytometry | 1 year | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Gut microbiota, Mediterranean diet, Vegetarian diet, Short chain fatty acids