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 Keratins, a major subgroup of intermediate filament proteins, play a critical role in maintaining epithelial barrier and intracellular epithelial integrity. Studies have demonstrated possible links between inflammatory signaling and colonic keratins type II K8, and type I K18, K19 and K20, in animal models of colitis, and in patients with Inflammatory Bowel Disease (IBD). K7 is de novo expressed in patients with the IBD subtypes Ulcerative Colitis (UC) and Crohn's Disease (CD). However, the histopathological roles of colonocyte keratins across IBD, microscopic colitis (MC), and Irritable Bowel Syndrome (IBS) remain poorly understood. Given the established utility as biomarkers in cancer diagnostics, we investigated whether keratin expression patterns could be used to distinguish inflammatory and functional colonic disorders. Methods Biobank samples from patients with IBD (n=27), MC (n=18), IBS (n=32) and healthy controls (n=31), were collected and immunohistochemically stained for K7, K8, K18, K19, and K20. Digital image analysis quantified staining intensities, which were correlated with histopathological severity scores and clinical parameters. Results Colonic keratin expression was significantly elevated in IBD, particularly in UC, while they were decreased in MC, and unaltered in IBS. Notably, K8 and K19 expression were strongly associated with areas of severe epithelial damage in IBD. Keratin expression was most pronounced in patients who had undergone colectomy due to treatment-resistant IBD. Discussion Keratin changes in IBD and MC but not in IBS highlight their importance in maintaining barrier homeostasis. Whether these changes are causes or consequences for these diseases will warrant further research. ### Competing Interest Statement The authors have declared no competing interest. Academy of Finland, 315139/332582 InFLAMES Flagship Programme, 337531 Novo Nordisk Foundation, NNF23OC0087039 Sigrid Juselius Foundation ÅAU Center of Excellence of Cellular Mechanostasis Medicinska Understödsföreningen Liv och Hälsa Foundation Business Finland Tor, Joe, and Pentti Borg Memorial Fund Satakunta Hospital District, Pori, Finland Swedish Cultural Foundation Satakunta Regional Fund of the Finnish Cultural Foundation

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. Proof-of-concept of a humanised gut-brain axis ex vivo model with physiological coupling of intestinal and blood-brain barrier for mechanistical assessment of butyrate- or fiber-rich diets on the serotonergic system in healthy or diseased states.

Abstract 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.

Pleurotus eryngii (PE), an edible mushroom rich in bioactive compounds, has been shown to exert immunomodulatory, anti-inflammatory, antioxidant, anti-carcinogenic, anti-microbial, antihepatotoxic and hypolipidemic activities, all important for the well-being of the ageing population. This study assessed in vitro the prebiotic-like effects of multiple forms of this mushroom. An in vitro static batch fermentation was performed for 24 h with faecal inocula from five apparently healthy older adults in the presence of the following PE forms: whole food matrix (PEWS), in vitro digested (PEWSD) and rich in beta-glucans extract (PEWSE). The changes in bacterial communities upon fermentation at family, genera and species level were detected via 16S rRNA Next Generation Sequencing and Quantitative real-time PCR. Short-chain fatty acids (SCFAs) were quantified using gas chromatography (GC), whereas other metabolites were analysed through ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). DEseq2 analysis indicated that PEWS presence exhibited the largest impact on faecal microbial families' and genera's abundance compared to negative (noncarbon source) and positive (inulin) controls. Only PEWS significantly increased Bifidobacterium spp. and F. prausnitzii populations, while all three forms robustly increased Bacteroides spp. levels and levels of butyrate, acetate and propionate acids. Statistical significance was set at p < 0.05. Overall, the findings highlight the beneficial effect of PE on intestinal health of older adults supporting its potential incorporation into innovative functional foods. However, additional in vivo studies are required to substantiate these findings before translating them into dietary guidelines or clinical applications.

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.

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.

Increasing evidence suggests that modulations of the gut-brain axis with probiotics impact healthy ageing. This double-blinded, randomised, placebo-controlled study compared effects of micro-encapsulated and non-encapsulated Lacticaseibacillus rhamnosus HN001 in 87 community-dwelling elderly (60-80 years). Resting state functional connectivity differed significantly in regions involved in visual processing and perception between the two probiotic groups (p < 0.0001). Brain morphometry was not altered. Significant time*group effects (p < 0.05) were observed for processing speed, non-significant effects were observed for short-term memory and anxiety symptoms, while other cognitive domains, depression, perceived stress, and sleep quality were unaffected. Distribution of available and stored peripheral serotonin was significantly affected (p < 0.05), while levels of γ-aminobutyric acid and glutamate in striatum and circulating brain-derived neurotrophic factor did not show significant time*group effects. Micro-encapsulated probiotics target the gut differently, which impacts the effects on brain health assessed by (functional) magnetic resonance imaging in older adults. The trial is registered at ClinicalTrials.gov under ID: NCT05801042.

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: This study explores the potential of the Pleurotus eryngii mushroom fermentation supernatant (FS-PEWS) as an intervention for mitigating sodium deoxycholate (SDC)-induced intestinal barrier dysfunction and inflammation. Methods: FS-PEWS was assessed for its protective effects against SDC-induced barrier dysfunction and inflammation using an in vitro Caco-2 cell model and ex vivo colonic biopsies from healthy adult donors, where barrier integrity, permeability, immunomodulation and receptor-mediated pathways were evaluated. Results: In Caco-2 cells, SDC exposure downregulated ZO-1, occludin, and claudin-1 expression, with FS-PEWS restoring ZO-1 and claudin-1 levels while maintaining cell viability. In colonic biopsies from healthy adults, FS-PEWS maintained tissue integrity and selectively mitigated transcellular permeability without affecting paracellular permeability when combined with the stressor. Additionally, FS-PEWS exhibited potent anti-inflammatory effects, reducing pro-inflammatory cytokines, e.g., TNF-α, IL-6, and IL-1β and modulating receptor-mediated pathways, i.e., TLR-4, dectin-1. Conclusions: These results demonstrate the potential of FS-PEWS to sustain intestinal barrier function and modulate immune responses under stress, highlighting its therapeutic potential for managing gut barrier dysfunction and inflammation associated with microbial metabolite-induced disruptions.

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 17 healthy subjects underwent a colonoscopy procedure exposing a predefined colonic area to 100 mmol/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

Aim: The cell matrix of plant foods has received little attention in prebiotic fiber research. We aimed to understand the impact of the plant cell matrix in dried chicory root on its breakdown in the human gut to explain its reported beneficial effects on gut and metabolic health. Methods: We applied in vitro digestion and fermentation models together with an ex vivo gut barrier integrity model. Plant cell matrix intactness in the upper gastrointestinal tract was investigated by scanning electron microscopy. Colonic breakdown of inulin, and chicory root cubes and powder was assessed by gut microbiota analysis using 16S rRNA gene amplicon sequencing and determining the kinetics of changes in pH, gas, and short- chain fatty acid (SCFA) production. Finally, effects on gut barrier integrity were explored by exposing colonic biopsies to fermentation supernatants in an Ussing chamber model. Results: The plant cell matrix of dried chicory root cubes remained intact throughout upper gastrointestinal transit. Dried chicory root fermentation resulted in higher final relative abundances of pectin-degrading Monoglobus and butyrate-producing Roseburia spp. compared to inulin and a seven-fold increase in Bifidobacterium spp. in donors where these species were present. Dried chicory root cubes yielded similar total SCFAs but higher final butyrate levels than chicory root powder or isolated inulin with less gas produced. No uniform but donor-specific effects of fermentation supernatants on the maintenance of gut barrier integrity were detected. Conclusion: The intact plant cell matrix of dried chicory root affected its colonic breakdown kinetics and microbiota, underpinning its beneficial effect in vivo.

AbstractStrenuous exercise can result in disruption of intestinal barrier function and occurrence of gastrointestinal symptoms. The aim of this exploratory study was to elucidate systemic effects of increased intestinal permeability after high‐intensity exercise. Forty‐one endurance‐trained subjects performed a 60‐min treadmill run at 80% VO2max. Small intestinal permeability was measured as urinary excretion ratio of lactulose/rhamnose (L/R). Blood, saliva and feces were analyzed for gut barrier and immune‐related biomarkers. The exercise challenge increased several markers of intestinal barrier disruption, immune function and oxidative stress. We found a negative correlation between L/R ratio and uric acid (r = −0.480), as well as a positive correlation between the L/R ratio and fecal chromogranin A in male participants (r = 0.555). No significant correlations were found between any of the markers and gastrointestinal symptoms, however, perceived exertion correlated with the combination of IL‐6, IL‐10 and salivary cortisol (r = 0.492). The lack of correlation between intestinal permeability and gastrointestinal symptoms could be due to minor symptoms experienced in lab settings compared to real‐life competitions. The correlation between L/R ratio and uric acid might imply a barrier‐protective effect of uric acid, and inflammatory processes due to strenuous exercise seem to play an important role regarding physical exhaustion.

It is well known that dietary fibers (DF) from plant-source foods can induce beneficial health effects through their physicochemical properties and utilization by the gut microbiota during fermentation, which is mainly explored with a focus on changes in the gut microbiota profile and the production of microbial-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. Firstly, a source of wheat arabinoxylan (WAX) was subjected to in vitro simulation of human colonic fermentation, followed by characterization and quantification of WAX structural motifs to explore their dynamics throughout fermentation. The identified structural motifs were further produced through enzymatic catalysis of WAX using carbohydrate-active enzymes and fractionated into six well-defined fractions of arabinoxylans and linear xylans. These fractions of structural motifs were then tested for interaction with TLR2 and TLR4 using a reporter cell assay. Results revealed structure-dependent effects, primarily with inhibition of TLR2 and activation of TLR4 depending on the degree of polymerization and branching of WAX structural motifs. The role of the fine structure of WAX structural motifs was confirmed by molecular docking, which revealed that minor structural changes substantially influence the interaction between structural motifs and TLRs. The results from in vitro and in silico studies also support the hypothesis that the direct effects of oligomers and polysaccharides on cell receptors are likely the result of complex interactions involving multiple cell surface receptors. Finally, in addition to highlighting that direct effects of structural motifs might play an important role in the overall effects of DF, this work suggests that enzymatic-tailoring design of DF can be a potential tool for producing functional ingredients with specific effects on human health. ### Competing Interest Statement The authors have declared no competing interest.

Serious infections may result in greater risk of Parkinson's disease. However, high-quality cohort studies focusing on a potential causal role of different types and sites of infection are lacking. Gastrointestinal infections are of a particular interest due to growing evidence implicating gut dysbiosis in Parkinson's disease aetiology. This population-based cohort study used the Swedish Total Population Register to identify individuals born during 1944-77 and resident in Sweden between 1990 and 2018 (N = 3 698 319). Hospital-treated infections at ages 21-30 and 31-40 years were identified from the National Patient Register. Participants were followed to identify Parkinson's disease diagnoses from age 41 years up to December 31, 2018, when the oldest individual reached 75 years. Cox regression with a sibling comparison design to tackle familial genetic and environmental confounding was used to derive hazard ratios and 95% confidence intervals for each infection site, type, or any infections at ages 21-30 and 31-40 years. During a median follow-up of 15.4 years, 8815 unique Parkinson's disease diagnoses were accrued, with a crude rate of 17.3 (95% confidence interval 17.0, 17.7) per 100 000 person-years. After controlling for shared familial factors, hospital-treated gastrointestinal and respiratory infections between 21 and 30 years of age were associated with a greater risk of Parkinson's disease [hazard ratios 1.35 (95% confidence interval: 1.05, 1.75) and 1.45 (95% confidence interval: 1.08, 1.95), respectively]; no association was found for any infections at age 31-40 [hazard ratio 1.05 (95% confidence interval: 0.93, 1.19)]. After adjustment, no statistically significant associations were observed for other sites including genitourinary and skin. These findings suggest that hospital-treated infections of the gastrointestinal tract and lungs, both of which may have an influence on the gut microbiome, by age 30 years may be risk factors for Parkinson's disease. Vingeliene et al. report that hospital-treated gastrointestinal and respiratory infections at age 21-30 are a risk factor for Parkinson's disease using sibling comparison design to control for shared familial factors. It is consistent with a role of gastrointestinal microbiome dysbiosis caused by infections in the aetiology of Parkinson's disease. Graphical Abstract