To Örebro University

Health care practitioners (HCPs) strive to provide the best medical care for each individual patient. The question as to what constitutes 'the best' does, however, not have a single straightforward answer. Evidence-based Medicine (EBM) and Personalized Medicine (PM) are two paradigms that have emerged as means to improve intervention selection. Both paradigms have their own strengths and weaknesses that affect their use in clinical decision-making. In this review we discuss the strengths and weaknesses from the patient's and HCP perspective: how to find the best intervention for a particular patient. We review methodological and practical aspects, and zoom out from the scientific level to the epistemological level to integrate EBM and PM. Both EBM and PM are based on a realist worldview and by adopting a pragmatist worldview the strengths of both paradigms can be combined. We apply this pragmatic approach, called Evidence-based Personalized Medicine (EBPM), to microbiome-targeting interventions. The example EBPM implementation uses four steps. First, it allows HCPs to provide information (clinical diagnosis, complaints, patient needs, laboratory measures) about an individual patient. Second, it uses a GRADE-based system to grade evidence of specific intervention components. Next, it combines the patient profile data and preferences with the graded evidence, to come to a suggestion for a personalized intervention. Finally, this method enables gathering of treatment effects providing feedback into the system and further improve suggestions for future patients.

Intestinal luminal microbial metabolites affect tryptophan and serotonin metabolism, and cross or modify the blood-brain barrier (BBB). Understanding those mechanisms further necessitates integrated gut-brain axis model systems. Using an ex vivo-in vitro approach, H2O2-stressed or non-stressed human dermal fibroblasts - representing the BBB - are cultured with serosal fluids of healthy or irritable bowel syndrome human colonic biopsies collected from Ussing chamber experiments, after participant's colon was exposed to butyrate in vivo, fecal fiber fermentation or control supernatant ex vivo. Culturing fibroblasts with serosal fluids does not compromise viability or have cytotoxic effects. Serosal fluids alone do not alter expression of tryptophan-related large amino acid membrane transporter genes and proteins, nor their activity (i.e., tryptophan uptake). However, adding serosal fluids to fibroblasts prior to oxidative stress indicate a protective role. This new model allows investigation of direct effects of serosal content on BBB-representing fibroblasts and is highly promising for more personalized applications.

Background: Pectic rhamnogalacturonan-I (RG-I) is a dietary fiber that modulates the gut-immune axis. This study evaluates a novel variant of RG-I from chicory root (chRG-I).

Methods: In a randomized, double-blind, placebo-controlled trial, 55 healthy adults were stratified by habitual fiber intake and baseline Bifidobacterium levels before receiving 500 mg/day of chRG-I or placebo for four weeks. Primary endpoints included fecal Bifidobacterium counts. Secondary outcomes assessed fecal metabolites, systemic immune cell activation markers, and gastrointestinal symptoms. To provide mechanistic insights, donor-matched fecal samples were used in in vitro fermentation and Caco-2/peripheral blood mononuclear cell co-culture gut barrier models.

Results: Supplementation with chRG-I induced a statistically significant bifidogenic effect, with absolute levels peaking at week three, and lower levels of some fecal short-chain fatty acids (SCFA) compared to placebo. However, donor-matched in vitro fermentations with chRG-I confirmed robust production of SCFA and reduction of branched-chain fatty acids levels (BCFA). Systemically, chRG-I upregulated HLA-DR expression on myeloid dendritic cells. Clinically, chRG-I was well-tolerated and slightly improved stool consistency compared to placebo. In an intestinal barrier challenge model, chRG-I fermentates (a pool of metabolites including SCFA and fragments of chRG-I) protected barrier integrity, modulated the cytokine milieu away from a predominantly pro-inflammatory response, as characterized by increased IL4 and IL22 and reduced IL9, IL17A, and IL21.

Conclusion: Supplementation with a low dose of chRG-I is well-tolerated, beneficially modulates the gut microbiome - which can protect the intestinal barrier, and subtly enhances systemic immune readiness, suggesting that chRG-I may have benefits as a functional food ingredient.

Shifting to a plant-based diet naturally alters protein source choices. In many countries, protein from yellow pea is widely used as a main ingredient in meat alternatives. Still, its biological effects, especially regarding gastrointestinal health, remain incompletely understood. The aim of our study was to investigate how a weekly increase in the intake of a well-characterized pea protein isolate affects surrogate markers of health, fecal short-chain fatty acids and gut microbiota composition in healthy individuals. Male and female adults (N = 29) participated in this exploratory intervention study. A 4-week pre-intervention period for questionnaires and fecal samples collection was followed by a 4-week supplementation. Participants consumed isolated pea protein in weekly increasing amounts, starting from 0.25 g per kg body mass per day in week 5 to 1.00 g per kg body mass per day in week 8. Questionnaire data, fecal samples as well as fasting blood and 24 h urine samples were collected weekly. Data from biological samples and questionnaires confirmed a healthy study population and compliance. Fecal calprotectin levels significantly increased only in a subset of participants, which was accompanied by higher fecal water cytotoxicity in vitro. Short-chain fatty acids mainly rose in those subjects with stable calprotectin levels. Relative abundances of Limosilactobacillus frumenti, Odoribacter splanchnicus and Lactobacillus crispatus increased significantly in the total population during the intervention while the relative abundance of Bifidobacterium longum and Bifidobacterium catenulatum decreased. Our results indicate that an increased intake of pea protein isolate affects the growth of certain beneficial bacterial strains and differentially influences markers related to gut inflammation in healthy individuals.

BACKGROUND: Modulating the gut-brain axis via probiotic supplementation has emerged as attractive strategy to promote brain health, but its long-term effects are largely unknown.

METHODS: This study with 32 community-dwelling self-reported healthy older adults (68.0 ± 5.4 years, 21 f/11 m, without contraindications for study examinations) assesses whether probiotic effects persist after intake cessation using brain imaging, targeting function (exploratory outcome of main study, primary outcome of report) and structure, and questionnaires for psychological assessments (exploratory). The 4-6 weeks discontinuation follow-up was performed after assessing immediate effects of a six-week randomised, blinded (investigators, nurses, participants), placebo-controlled parallel trial with two different formulations of Lacticaseibacillus rhamnosus HN001 of identical appearance/taste, in autumn 2023 in Örebro, Sweden.

RESULTS: Trial status: completed; participants analysed (of randomised) per group: n = 8(29) encapsulated probiotic, n = 12(31) non-encapsulated probiotic, n = 12(30) placebo; no related adverse events at discontinuation follow-up. Previously reported immediate intervention effects evoked alteration in anxiety symptom scores and distinct resting state functional connectivity patterns. While some changes persisted, others were not detectable any longer at discontinuation follow-up. Comparing both probiotic formulations, connectivity between left superior parietal lobule and right occipital/cuneal/intracalcarine/supracalcarine cortex differed significantly (seed-to-voxel analysis, T = + 7.38, cluster-size p = 0.0001 FDR-corrected, additionally correct for number of tests); levels for Hospital Anxiety and Depression Scale Total Score and its Anxiety Subscore differed significantly (time*group interaction effects); both between weeks 10-12 and week 6, but not baseline.

CONCLUSIONS: These findings suggest that some brain-related probiotic effects may indeed persist 4-6 weeks post-intervention cessation, especially on the level of brain function as indicated by resting state functional connectivity, suggesting longer-lasting gut-brain axis effects than previously presumed.

REGISTRATION: ClinicalTrials.gov NCT05801042.

Irritable bowel syndrome (IBS) is a prevalent gastrointestinal disorder for which effective treatment strategies are insufficient. Butyrate, a microbiota-derived short-chain fatty acid believed to strengthen the intestinal barrier function, might be a potential new treatment option. This study aimed to investigate potential protective effects of acute in vivo butyrate exposure on intestinal barrier function in healthy subjects and patients with IBS. For this, we used an experimental colonoscopy-perfusion model for colon-specific butyrate delivery and adequate tissue sampling. Seventeen IBS and 17healthy subjects underwent a colonoscopy procedure exposing a predefined colonic area to 100mmol/L butyrate for 90 min in vivo. Mucosal biopsies collected pre- and post-butyrate exposure were stimulated in Ussing chambers with/without sodium deoxycholate (DC) to induce intestinal hyperpermeability. Intestinal permeability was measured by fluorescein isothiocyanate-dextran and horseradish peroxidase passage. DC-stimulation significantly increased para- and transcellular permeability in biopsies collected pre-butyrate exposure. DC-induced transcellular hyperpermeability was significantly alleviated in biopsies collected post-butyrate exposure compared to pre-exposure in patients with IBS (p = 0.034). In conclusion, we established a colonoscopy research model for colon-specific delivery and sampling and demonstrated acute protective effects of butyrate on transcellular intestinal permeability in patients with IBS. The results support butyrate's potential role in novel treatment strategies in IBS. Clinicaltrials.gov number: NCT05249023.

Stress induction tests such as carbon dioxide (CO2) inhalation challenges, are often used in research of anxiety and panic disorders. Physiological parameters of the autonomic response, e.g., heart rate variability (HRV) and skin conductance (SC), are often measured alongside questionnaires for evaluation. Previous studies have shown varied results on CO2 inhalation-induced physiological reactivity and further knowledge is of interest. The aim of this study was to explore the effects of a 35 % CO2 inhalation test on HRV frequencies and SC, in healthy subjects; and to set those into relation with subjective psychological ratings. In this single-blinded, repeated measures study, healthy subjects underwent a 35 % CO2 inhalation challenge, whereof the first and third double vital capacity inhalations were with normal air and the second with 35 % CO2. HRV (low and high frequency) and SC (as electrodermal activity (EDA)) were measured throughout. CO2 inhalation resulted in a significant increase of HRV's high and very high frequencies compared to the first air inhalation (median difference + 0.0001633; + 0.0000348) and of HRV's very high and very low frequencies compared to the last air inhalation (+ 0.0000321; + 0.0000154). Mean and maximum SC increased significantly during the CO2 inhalation compared to both air inhalations (mean difference to first air inhalation + 1.151; + 1.964; to last air inhalation + 0.5751; +1.484), but also between the separate air inhalations (+ 0.5754; + 0.4799). The HRV results indicate increased parasympathetic activity, while the SC results indicate increased sympathetic activity during CO2 inhalation. SC only minorly correlated with provoked panic symptoms (EDA minimum to VAS minimum r = -0.559, and not between any other EDA and VAS/PSL measure). While those results seem contradictory, this study confirms that a 35 % CO2 inhalation challenge in young healthy adults, provokes a physiological as well as psychological reaction.

Dietary fibers (DF) from plant-based foods promote health benefits through their physicochemical properties and fermentation by the gut microbiota, often studied in relation to changes in gut microbiota profile and production of gut microbiota-derived metabolites. Here, we characterized structural motifs (i.e., oligomers) produced during DF breakdown upon colonic fermentation and explored their interaction with toll-like receptors (TLRs) present on the surface of human intestinal and immune system cells. Wheat arabinoxylan (WAX) was subjected to in vitro colonic fermentation, with its structural motifs identified and tracked throughout the fermentation process. Using carbohydrate-active enzymes, six well-defined fractions of arabinoxylans and linear xylans identified during colonic fermentation were produced and tested for interaction with tool-like receptors (TLR)2 and TLR4 via reporter cell assay. The results showed structure-dependent effects, with TLR2 inhibition and TLR4 activation varying based on the degree of polymerization and branching. Molecular docking confirmed that minor structural changes in oligomers structure significantly influenced these interactions. The study supports the hypothesis that oligomers and polysaccharides affect cell receptors through complex, multi-receptor interactions, and highlights the potential for enzymatic tailoring of DF to create functional ingredients with targeted effects on human health.

Background: Rhamnogalacturonan-I (RG-I) is a pectic polysaccharide with emerging prebiotic and immunomodulatory potential. This randomised, double-blind, placebo-controlled trial (ID: NCT06081972) evaluated the effects of carrot-derived RG-I (cRG-I) supplementation, compared to placebo (maltodextrin), on gut microbiota composition and immune cell activation in healthy adults.

Methods: A total of 54 participants (18-70 years old) were randomised in a double-blind manner to receive either 500 mg/day of cRG-I or placebo for four weeks. Pre-screening ensured balanced randomisation based on habitual fibre intake and faecal Bifidobacterium counts. Questionnaires assessed potential gut health and well-being effects, while in vitro and ex vivo models were used to evaluate effects on intestinal permeability.

Results: cRG-I was well tolerated with excellent compliance. Faecal Bifidobacterium counts increased significantly, peaking at week 3. Isobutyric acid levels rose, though no other SCFAs differed. Immunologically, cRG-I enhanced the percentage of circulating myeloid dendritic cells expressing activation markers (CD86, HLA-DR) on. Stool consistency improved slightly. Preclinical models further showed that cRG-I and its fermentation products protected intestinal barrier integrity under stress.

Conclusion: These results support cRG-I as a safe, low-dose dietary intervention capable of beneficially modulating gut microbiota, immune responses, and barrier function in healthy adults within a short supplementation period.

Background: This study investigates the impact of fermentation supernatants (FSs) from Pleurotus eryngii whole mushrooms (PEWS), as well as its subcomponents, digested (PEWSD) and extracted (PEWSE) forms, on intestinal barrier function and immune modulation in lipopolysaccharide (LPS) -stimulated Caco-2 cells.

Methods: Gene expression of tight junction (TJs) genes, cytokines, and key immune/metabolic receptors was assessed via qRT-PCR, while cytokine protein levels were measured using ELISA to explore post-transcriptional regulation.

Results: LPS challenge significantly downregulated TJs zonula occludens-1 (ZO-1,) occludin, and claudin-1, compromising epithelial integrity. Treatment with FS-PEWS notably restored ZO-1 and occludin expression, outperforming FS-PEWSD and FS-PEWSE, which only partially mitigated the LPS-induced damage. FS-PEWS further demonstrated potent immunomodulatory effects, upregulating anti-inflammatory IL-10 and pro-inflammatory cytokines such as IL-8 and TNF-α. The activation of key receptors like TLR-2 and mTOR suggests that FS-PEWS modulates critical immune and metabolic pathways, such as NF-kB signaling, to maintain immune homeostasis. Although mRNA expression of pro-inflammatory cytokines was altered, no corresponding protein release was detected, suggesting potential post-transcriptional regulation.

Conclusions: FS-PEWS preserves intestinal barrier integrity and modulates immune responses, particularly in low-grade inflammation, highlighting the whole food matrix's role in enhancing its bioactivity and functional food potential.