Nutritional profile of rodent diets impacts experimental reproducibility in microbiome preclinical research.

The lack of reproducibility of animal experimental results between laboratories, particularly in studies investigating the microbiota, has raised concern among the scientific community. Factors such as environment, stress and sex have been identified as contributors, whereas dietary composition has received less attention. This study firstly evaluated the use of commercially available rodent diets across research institutions, with 28 different diets reported by 45 survey respondents. Secondly, highly variable ingredient, FODMAP (Fermentable Oligo-, Di-, Mono-saccharides And Polyols) and gluten content was found between different commercially available rodent diets. Finally, 40 mice were randomized to four groups, each receiving a different commercially available rodent diet, and the dietary impact on cecal microbiota, short- and branched-chain fatty acid profiles was evaluated. The gut microbiota composition differed significantly between diets and sexes, with significantly different clusters in ß-diversity. Total BCFA were highest (p = 0.01) and SCFA were lowest (p = 0.03) in mice fed a diet lower in FODMAPs and gluten. These results suggest that nutritional composition of commercially available rodent diets impact gut microbiota profiles and fermentation patterns, with major implications for the reproducibility of results across laboratories. However, further studies are required to elucidate the specific dietary factors driving these changes.

Gluten-free and low-FODMAP sourdoughs for patients with coeliac disease and irritable bowel syndrome: A clinical perspective.

Wheat- and gluten-containing products are often blamed for triggering a wide range of gastrointestinal symptoms, and this has fueled demand for gluten-free products worldwide. The best studied 'gluten intolerance' is coeliac disease, an auto-immune disease that affects the small intestine. Coeliac disease occurs in 1% of the population and requires strict, life-long avoidance of gluten-containing foods as the only medical treatment. There is a larger group of individuals (around 10-15% of the population) who report a wide-range of gastrointestinal symptoms that respond well to a 'gluten-free diet', but who do not have coeliac disease - so called 'non-coeliac gluten sensitivity (NCGS)'. The team at Monash University has identified other factors in gluten-containing foods that may be responsible for symptoms in this group of individuals with so-called, NCGS. We have evidence that certain poorly absorbed short chain carbohydrates (called FODMAPs) present in many gluten-containing food products, induce symptoms of abdominal pain, bloating, wind and altered bowel habit (associated with irritable bowel syndrome, IBS). Our research has shown that FODMAPs, and not gluten, triggered symptoms in NCGS. Going forward, there are great opportunities for the food industry to develop low FODMAP products for this group, as choice of grain variety and type of food processing technique can greatly reduce the FODMAP levels in foods. The use of sourdough cultures in bread making has been shown to reduce the quantities of FODMAPs (mostly fructan), resulting in bread products that are well tolerated by patients with IBS. Greater interaction between biomedical- and food-scientists will improve understanding about the clinical problems many consumers face, and lead to the development of food products that are better tolerated by this group.

Review article: short chain fatty acids as potential therapeutic agents in human gastrointestinal and inflammatory disorders.

BACKGROUND: Butyrate, propionate and acetate are short chain fatty acids (SCFA), important for maintaining a healthy colon and are considered as protective in colorectal carcinogenesis. However, they may also regulate immune responses and the composition of the intestinal microbiota. Consequently, their importance in a variety of chronic inflammatory diseases is emerging. AIMS: To review the physiology and metabolism of SCFA in humans, cellular and molecular mechanisms by which SCFA may act in health and disease, and approaches for therapeutic delivery of SCFA. METHODS: A PubMed literature search was conducted for clinical and pre-clinical studies using search terms: 'dietary fibre', short-chain fatty acids', 'acetate', 'propionate', 'butyrate', 'inflammation', 'immune', 'gastrointestinal', 'metabolism'. RESULTS: A wide range of pre-clinical evidence supports roles for SCFA as modulators of not only colonic function, but also multiple inflammatory and metabolic processes. SCFA are implicated in many autoimmune, allergic and metabolic diseases. However, translating effects of SCFA from animal studies to human disease is limited by physiological and dietary differences and by the challenge of delivering sufficient amounts of SCFA to the target sites that include the colon and the systemic circulation. Development of novel targeted approaches for colonic delivery, combined with postbiotic supplementation, may represent desirable strategies to achieve adequate targeted SCFA delivery. CONCLUSIONS: There is a large array of potential disease-modulating effects of SCFA. Adequate targeted delivery to the sites of action is the main limitation of such application. The ongoing development and evaluation of novel delivery techniques offer potential for translating promise to therapeutic benefit.

Randomised clinical trial: efficacy, safety and dosage of adjunctive allopurinol in azathioprine/mercaptopurine nonresponders (AAA Study).

BACKGROUND: Thiopurine hypermethylation towards 6-methylmercaptopurine (6MMP) instead of 6-thioguanine nucleotides (6TGN) is associated with inefficacy in patients with IBD. Allopurinol reverses such hypermethylation. AIMS: To prospectively determine efficacy of allopurinol-thiopurine combination and to compare 2 doses of allopurinol. DESIGN: In a multicentre, double-blind trial, patients with clinically active or steroid-dependent IBD and thiopurine shunting were randomised to 50 or 100 mg/d allopurinol and 25% of their screening thiopurine dose, which was subsequently optimised, aiming for 6TGN of 260-500 pmol/8x108 RBCs. The primary endpoint was steroid-free clinical remission at 24 weeks. RESULTS: Of 73 patients, 39 (53% [95% CI 42-65]) achieved steroid-free remission, (54% with 50 mg/d and 53% with 100 mg/d). 81% were able to discontinue steroids. Therapeutic 6TGN levels were achieved in both groups. Final thiopurine doses were lower with 100 mg/d allopurinol (P < 0.005). 6MMP: 6TGN ratio decreased from mean 64 to 4 (P < 0.001), being higher with 50 mg/d (6 ± 1.83) than for 100 mg/d ([1 ± 0.16], P = 0.003). Three patients on 50 mg/d failed to sustain low ratios at 24 weeks. Toxicity was minimal; three patients on 50 mg/d allopurinol developed transient leukopenia. Alanine aminotransferase concentrations decreased (P < 0.001) similarly in both arms. Faecal calprotectin levels at study end were lower in patients who achieved the primary endpoint (median 171 [85-541] vs 821[110-5892] ug/g, P = 0.03). CONCLUSIONS: Low-dose allopurinol-thiopurine combination safely reverses shunting and optimises 6TGN with associated improvement in disease activity. 100 mg/d allopurinol is preferable due to greater metabolite profile stability and lower thiopurine dose without additional toxicity.

Vedolizumab in patients with concurrent primary sclerosing cholangitis and inflammatory bowel disease does not improve liver biochemistry but is safe and effective for the bowel disease.

BACKGROUND: Blocking of lymphocyte trafficking to bile ducts is a potential mechanism to alter the disease course of patients with primary sclerosing cholangitis (PSC). AIM: To describe the effect of the α4 ß7 integrin antibody, vedolizumab, on liver biochemistry and disease activity in patients with PSC and inflammatory bowel disease (IBD). METHODS: This is a retrospective multi-centre study of adult patients with a diagnosis of both IBD and PSC. The primary outcome was change in serum alkaline phosphatase level at weeks 14 and 30. Secondary outcomes included changes in other liver biochemistries and in clinical outcomes for the bowel disease. A safety analysis for adverse events was performed. RESULTS: Thirty-four patients (16 Crohn's disease, 18 ulcerative colitis) were included. Nine (26%) had a history of liver transplant. Median follow-up on vedolizumab was 9 months (IQR: 7-16). There was no overall change in serum alkaline phosphatase level with vedolizumab therapy (median 268 [IQR: 105-551] IU/L at baseline versus 249 [IQR: 183-634] IU/L, P = 0.99 at week 30). No significant changes in other liver biochemistries or the Mayo PSC Risk Score were demonstrated at week 30. Clinical remission was achieved at week 30 in 55% of Crohn's disease and 29% of ulcerative colitis patients. Seven (21%) patients ceased vedolizumab; six patients stopped therapy due to persistent IBD activity and one for worsening of liver biochemistries. CONCLUSION: Vedolizumab treatment in patients with PSC and IBD did not improve liver biochemistry but was associated with improvement in bowel disease and a favourable safety profile.

Performance of an algorithm-based approach to the diagnosis and management of functional gastrointestinal disorders: A pilot trial.

BACKGROUND: Recent advances in the development of diagnostic criteria and effective management options for functional gastrointestinal disorders (FGIDs) have not yet been integrated into clinical practice. There is a clear need for the development and validation of a simple clinical pathway for the diagnosis and management of FGIDs which can be used in primary care. METHODS: In this controlled pilot study, we designed and evaluated a non-specialist-dependent, algorithm-based approach for the diagnosis and management of FGIDs (ADAM-FGID). Patients referred to 1 tertiary referral center with clinically suspected functional gastrointestinal disorders were allocated to waitlist control or algorithm group. The algorithm group was screened for organic disease, and those without clinical alarms received a written FGID diagnosis and management options. All participants were followed up for 1 year. KEY RESULTS: The ADAM-FGID was found to be feasible and acceptable to both patients and primary healthcare providers. The diagnostic component identified that 39% of referrals required more urgent gastroenterological review than original triage category, with organic disease subsequently diagnosed in 31% of these. The majority of patients (82%) diagnosed with a FGID did not receive a relevant alternative diagnosis during follow-up. Patient buy-in to the model was good, with all reading the diagnostic/management letter, 80% entering management, and 61% reporting symptom improvement at 6 weeks. Moreover, 68% of patients and all referring doctors found the approach to be at least moderately acceptable. Patients reported being reassured by the approach and found the management options useful. Primary healthcare providers acknowledged the potential of this approach to reduce waiting times for endoscopic procedures and to provide reassurance to both patients and themselves. CONCLUSIONS & INFERENCES: This pilot study provides preliminary evidence to support a clinical pathway for the diagnosis and management of FGIDs which does not depend upon specialist review. Further rigorous testing within primary care is needed to conclusively establish safety and efficacy. However, this approach is safer than current management and has potential to build capacity by reducing specialist burden and expediting effective care.

Reducing the maternal dietary intake of indigestible and slowly absorbed short-chain carbohydrates is associated with improved infantile colic: a proof-of-concept study.

BACKGROUND: To investigate if a low fermentable oligo-, di- and mono-saccharides and polyols (FODMAP) diet consumed by breastfeeding mothers may be associated with reduced symptoms of infantile colic. METHODS: Exclusively breastfeeding mothers and their typically-developing healthy infants who met the Wessel Criteria for infantile colic were recruited from the community, to this single-blind, open-label, interventional study. After a 3-day qualifying period, mothers were provided a low FODMAP 7-day diet. On days 5, 6 and 7 mothers completed a Baby Day Diary. At baseline and at the end of the 7-day dietary intervention, breast milk was analysed for FODMAP content and infant faecal samples for pH. RESULTS: Eighteen breastfeeding mothers (aged 27-40 years) adhered (100%) to the low FODMAP diet. Infants were of gestational age 37-40.3 weeks and aged 2-17 weeks. At entry, crying durations were a mean [95% CI] of 142 [106-61] min and fell by 52 [178-120] min (P = 0.005; ancova). Combined crying-fussing durations fell by 73 [301-223] min (n = 13; P = 0.007), as did crying episodes (P = 0.01) and fussing durations (P = 0.011). Infant sleeping, feeding, or awake-and-content durations did not change. Infant faecal pH did not change. Breast milk lactose content was stable and other known FODMAPs were not detected. At end of study, mothers reported their baby 'is much more content' and 'can be put down without crying'. CONCLUSIONS: Maternal low FODMAP diet may be associated with a reduction in infant colic symptoms. A randomized controlled study is warranted to determine if a maternal low FODMAP diet is effective in reducing symptoms.

Two weeks of repetitive gut-challenge reduce exercise-associated gastrointestinal symptoms and malabsorption.

Debilitating gastrointestinal symptoms is a common feature of endurance running and may be exacerbated by and/or limit the ability to tolerate carbohydrate intake during exercise. The study aimed to determine whether two weeks of repetitive gut-challenge during running can reduce exercise-associated gastrointestinal symptoms and carbohydrate malabsorption. Endurance runners (n=18) performed an initial gut-challenge trial (GC1) comprising 2-hour running exercise at 60% VO2max (steady state) while consuming a formulated gel-disk containing 30 g carbohydrates (2:1 glucose-fructose, 10% w/v) every 20 minutes, followed by a 1-hour running effort bout. Gastrointestinal symptoms, feeding tolerance, and breath hydrogen (H2 ) were determined along the gut-challenge trial. After GC1, participants were randomly assigned to a blinded carbohydrate (CHO, 90 gCHO hour-1 ) or placebo (PLA, 0 gCHO hour-1 ) gut-training group. This comprised of consuming the group-specific feeding intervention during 1-hour running exercise at 60% VO2max equivalent, daily over a period of two weeks. Participants then repeated the gut-challenge trial (GC2). In GC2, a reduced gut discomfort (P=.012), total (P=.009), upper- (P=.015), and lower-gastrointestinal (P=.008) symptoms, and nausea (P=.05) were observed on CHO, but not PLA. Feeding tolerance did not differ between GC1 and GC2 on CHO and PLA. H2 peak was attenuated in GC2 (6±3 ppm) compared to GC1 (13±6 ppm) on CHO (P=.004), but not on PLA (GC1 11±7 ppm, and GC2 10±10 ppm). The effort bout distance was greater in GC2 (12.3±1.3 km) compared with GC1 (11.7±1.5 km) on CHO (P=.035) only. Two weeks of repetitive gut-challenge improve gastrointestinal symptoms and reduce carbohydrate malabsorption during endurance running, which may have performance implications.

Inaccuracy of patient-reported descriptions of and satisfaction with bowel actions in irritable bowel syndrome.

BACKGROUND: Perception of diarrhea and constipation differs greatly. This study aimed to correlate subjective and objective assessment of fecal characteristics in irritable bowel syndrome (IBS) patients. METHODS: Data from two interventional dietary trials with varying FODMAP (Fermentable Oligosaccharides, Disaccharides, Monosaccharides And Polyols) or gluten content were interrogated. Subjects rated their dissatisfaction with stool consistency daily using a visual analog scale during the interventions. Subjects collected stools at the end of each intervention. Each stool was scored according to the King's Stool Chart (KSC). Fecal water content (FWC) was measured on pooled feces by freeze drying, with diarrhea defined as ≥78%. KEY RESULTS: Seventy IBS (Rome III) and eight healthy subjects were studied. Each subject's self-rating of stool consistency during the most symptomatic diet was approximately double that of their least. Degree of dissatisfaction with stool consistency correlated poorly with changes in FWC and KSC. IBS subtype related poorly to objective measures of stool consistency. Sixty percent of IBS-D subjects had diarrhea on objective measures. Eighty-five percent with IBS-C had hard and formed stools but three patients met the criteria for diarrhea. One healthy subject had diarrhea on FWC and KSC, and six had hard, formed stools. No differences in FWC was observed when subjects consumed differing amounts of FODMAPs or gluten (all P > .200). CONCLUSIONS AND INFERENCES: There are major disparities between patients' stool descriptions and objective features of constipation and diarrhea. Patient-reported bowel habits require more interrogation for accurate IBS subtyping. Varying FODMAP or gluten content of the diet is not associated with consistent change in FWC.

Increasing Symptoms in Irritable Bowel Symptoms With Ingestion of Galacto-Oligosaccharides Are Mitigated by α-Galactosidase Treatment.

OBJECTIVES: Galacto-oligosaccharides (GOS) are dietary FODMAPs (fermentable carbohydrates) associated with triggering gastrointestinal symptoms in patients with irritable bowel syndrome (IBS). This randomized, double-blind, placebo-controlled, cross-over trial aimed to assess whether oral α-galactosidase co-ingestion with foods high in GOS and low in other FODMAPs would reduce symptoms. METHODS: Patients meeting the Rome III criteria for IBS who were hydrogen-producers on breath testing were recruited. Participants were treated with full-dose (300 GALU (galactosidic units) α-galactosidase) and half-dose enzyme (150 GALU α-galactosidase), and placebo (glucose) in a random order with ≤14 days washout between arms. Following a 3-day low FODMAP run-in period, participants consumed provided diets high in GOS for a further 3-days. Gastrointestinal symptoms were measured daily using a 100 mm visual-analogue-scale, and breath samples taken hourly on the second last day with hydrogen content analysed as area-under-the-curve. RESULTS: Thirty-one patients with IBS (20 IBS-D, 4 IBS-C, 7 IBS-M) completed the study. The addition of high GOS foods resulted in a significant increase in overall symptoms with 21 patients exhibiting GOS-sensitivity (>10 mm increase for overall symptoms). Of those, full-dose enzyme reduced overall symptoms (median 24. 5(IQR 17.5-35.8) vs. 5.5(1.5-15.0) mm; P=0.006) and bloating (20.5(9.5-42.0) vs. 6.5(2.0-15.8); P=0.017). Breath hydrogen production was minimal with no differences seen between placebo and full-dose (P=0.597). CONCLUSIONS: Oral α-galactosidase taken with high GOS foods provides a clinically significant reduction in symptoms in GOS-sensitive individuals with IBS. This strategy can be translated into practice to improve tolerance to high GOS foods as an adjunct therapy to the low FODMAP diet.

Systematic review: exercise-induced gastrointestinal syndrome-implications for health and intestinal disease.

BACKGROUND: "Exercise-induced gastrointestinal syndrome" refers to disturbances of gastrointestinal integrity and function that are common features of strenuous exercise. AIM: To systematically review the literature to establish the impact of acute exercise on markers of gastrointestinal integrity and function in healthy populations and those with chronic gastrointestinal conditions. METHODS: Search literature using five databases (PubMed, EBSCO, Web of Science, SPORTSdiscus, and Ovid Medline) to review publications that focused on the impact of acute exercise on markers of gastrointestinal injury, permeability, endotoxaemia, motility and malabsorption in healthy populations and populations with gastrointestinal diseases/disorders. RESULTS: As exercise intensity and duration increases, there is considerable evidence for increases in indices of intestinal injury, permeability and endotoxaemia, together with impairment of gastric emptying, slowing of small intestinal transit and malabsorption. The addition of heat stress and running mode appears to exacerbate these markers of gastrointestinal disturbance. Exercise stress of ≥2 hours at 60% VO2max appears to be the threshold whereby significant gastrointestinal perturbations manifest, irrespective of fitness status. Gastrointestinal symptoms, referable to upper- and lower-gastrointestinal tract, are common and a limiting factor in prolonged strenuous exercise. While there is evidence for health benefits of moderate exercise in patients with inflammatory bowel disease or functional gastrointestinal disorders, the safety of more strenuous exercise has not been established. CONCLUSIONS: Strenuous exercise has a major reversible impact on gastrointestinal integrity and function of healthy populations. The safety and health implications of prolonged strenuous exercise in patients with chronic gastrointestinal diseases/disorders, while hypothetically worrying, has not been elucidated and requires further investigation.

Infliximab and adalimumab drug levels in Crohn's disease: contrasting associations with disease activity and influencing factors.

BACKGROUND: Discriminative drug level thresholds for disease activity endpoints in patients with Crohn's disease. have been consistently demonstrated with infliximab, but not adalimumab. AIMS: To identify threshold concentrations for infliximab and adalimumab in Crohn's disease according to different disease endpoints, and factors that influence drug levels. METHODS: We performed a cross-sectional service evaluation of patients receiving maintenance infliximab or adalimumab for Crohn's disease. Serum drug levels were at trough for infliximab and at any time point for adalimumab. Endpoints included Harvey-Bradshaw index, C-reactive protein and faecal calprotectin. 6-tioguanine nucleotide (TGN) concentrations were measured in patients treated with thiopurines. RESULTS: A total of 191 patients (96 infliximab, 95 adalimumab) were included. Differences in infliximab levels were observed for clinical (P=.081) and biochemical remission (P=.003) and faecal calprotectin normalisation (P<.0001) with corresponding thresholds identified on ROC analysis of 1.5, 3.4 and 5.7 µg/mL. Adalimumab levels were similar between active disease and remission regardless of the endpoint assessed. Modelling identified that higher infliximab dose, body mass index and colonic disease independently accounted for 31% of the variation in infliximab levels, and weekly dosing, albumin and weight accounted for 23% of variation in adalimumab levels. TGN levels did not correlate with drug levels. CONCLUSIONS: Infliximab drug levels are associated with the depth of response/remission in patients with Crohn's disease, but no such relationship was observed for adalimumab. More data are needed to explain the variation in drug levels.

Intra-patient variability in adalimumab drug levels within and between cycles in Crohn's disease.

BACKGROUND: Whether therapeutic drug monitoring for adalimumab needs to be performed at trough has not been defined. AIM: To determine intra-patient adalimumab drug-level variation and to identify modulating patient and disease factors. METHODS: In this prospective observational study, adult patients with Crohn's disease established on maintenance adalimumab had drug levels measured repeatedly according to pre-defined schedules (visit 1: day 4-6, visit 2: day 7-9, trough: day 13-14) across two consecutive fortnightly cycles. Disease activity was assessed using Harvey-Bradshaw Index, C-reactive protein and faecal calprotectin. For this analysis, trough levels ≥4.9 µg/mL were considered therapeutic. RESULTS: Nineteen patients underwent 111 evaluations. Mean intra-patient drug levels from paired visits between cycles did not differ (visit1 cycle1: 4.81, cycle2: 5.21 µg/mL, P = 0.24, visit2 cycle1: 4.86, cycle2: 4.82, P = 0.91 and trough cycle1: 3.95, cycle2: 3.95, P = 0.99), irrespective of disease activity. Drug levels were stable over the first 9 days (visit 1-2), but declined to trough by a mean 1.06 and 0.89 µg/mL between visit 1 or 2, respectively (P < 0.001). Models using nontemporal factors (smoking, syringe delivery device) and levels at earlier visits accounted for 66-80% of the variance in trough levels. On receiver-operating curve analysis, thresholds identified in the first 9 days that predicted a therapeutic trough level were similar to the trough threshold itself, with high sensitivity but modest specificity. CONCLUSION: While therapeutic drug monitoring should be performed at trough, a drug level ≥4.9 µg/mL obtained during the first 9 days predicts a therapeutic trough drug level with reasonable confidence.

Adding glucose to food and solutions to enhance fructose absorption is not effective in preventing fructose-induced functional gastrointestinal symptoms: randomised controlled trials in patients with fructose malabsorption.

BACKGROUND: In healthy individuals, the absorption of fructose in excess of glucose in solution is enhanced by the addition of glucose. The present study aimed to assess the effects of glucose addition to fructose or fructans on absorption patterns and genesis of gastrointestinal symptoms in patients with functional bowel disorders. METHODS: Randomised, blinded, cross-over studies were performed in healthy subjects and functional bowel disorder patients with fructose malabsorption. The area-under-the-curve (AUC) was determined for breath hydrogen and symptom responses to: (i) six sugar solutions (fructose in solution) (glucose; sucrose; fructose; fructose + glucose; fructan; fructan + glucose) and (ii) whole foods (fructose in foods) containing fructose in excess of glucose given with and without additional glucose. Intake of fermentable short chain carbohydrates (FODMAPs; fermentable, oligo-, di-, monosaccharides and polyols) was controlled. RESULTS: For the fructose in solution study, in 26 patients with functional bowel disorders, breath hydrogen was reduced after glucose was added to fructose compared to fructose alone [mean (SD) AUC 92 (107) versus 859 (980) ppm 4 h-1 , respectively; P = 0.034). Glucose had no effect on breath hydrogen response to fructans (P = 1.000). The six healthy controls showed breath hydrogen patterns similar to those with functional bowel disorders. No differences in symptoms were experienced with the addition of glucose, except more nausea when glucose was added to fructose (P = 0.049). In the fructose in foods study, glucose addition to whole foods containing fructose in excess of glucose in nine patients with functional bowel disorders and nine healthy controls had no significant effect on breath hydrogen production or symptom response. CONCLUSIONS: The absence of a favourable response on symptoms does not support the concomitant intake of glucose with foods high in either fructose or fructans in patients with functional bowel disorders.