Abstract Background and Aims Fatigue is a prevalent and disabling symptom in inflammatory bowel disease (IBD), yet its underlying biological mechanisms remain poorly understood. We aimed to characterize fatigue-associated molecular signatures in IBD patients by integrating DNA methylation and mRNA expression analyses. Methods Peripheral blood was collected from 40 patients with Crohn’s disease (CD), 29 with ulcerative colitis (UC), and 10 healthy controls. Fatigue severity was assessed continuously using the Multidimensional Fatigue Inventory (MFI). Epigenome-wide DNA methylation profiling and mRNA sequencing were performed, identifying differentially methylated regions (DMRs) and differentially expressed genes (DEGs) for active and quiescent CD and UC, adjusting for age, sex, and smoking status. Pathway enrichment analysis was performed on genes with differential methylation and expression. Results In active CD, more severe fatigue was associated with transcriptional suppression of immune and metabolic pathways (246 DMRs; 1,090 DEGs), versus upregulation of mitochondrial and metabolic processes in quiescent CD (200 DMRs; 1,619 DEGs). In active UC, fatigue was associated with anabolic pathway upregulation and epigenetic silencing of neuroactive pathways (6,927 DMRs; 343 DEGs; 56 concordant genes). Quiescent UC showed transcriptional changes without significant epigenetic pathway enrichment (1,710 DMRs; 3,224 DEGs). Healthy controls exhibited a distinct profile spanning metabolic, immune, and neuronal pathways (8,621 DMRs; 395 DEGs). Fatigue-associated signatures were largely non-overlapping across all five groups. Conclusions Fatigue-associated molecular profiles differed substantially by disease subtype and activity state, highlighting the biological heterogeneity of IBD-related fatigue and laying the foundation for multi-omics approaches to identify biomarkers and potential therapeutic targets.

BACKGROUND:The skin-to-blood route is traditionally considered the main pathway in Candida late-onset sepsis (LOS) development in preterm infants. However, emerging evidence suggests that the gut also serves as a source of infection. We aimed to characterize fecal mycobiota and microbiota profiles preceding onset of Candida LOS to assess the role of the preterm gut microbiome in disease development. METHODS:This multicenter case-control study included very preterm infants (<30 weeks of gestation) with Candida LOS. Each case was matched to nonaffected controls by gestational and postnatal age, hospital site, and/or cumulative antibiotic exposure prior to day of LOS onset (t = 0). Fecal samples collected at t = 0 and the 5 preceding days were analyzed by ITS1 and 16S RNA sequencing. Microbial amplicon yields, composition, and interkingdom correlations were assessed. RESULTS:Of 2397 screened infants, fecal samples were available for 8 of 19 infants with Candida LOS. In these 8 cases, the ITS/16S amplicon yield ratio was increased (P < .001), and the relative abundance of fecal Candida albicans correlated positively with fungal amplicon yield (ρ = 0.71, adjusted P = .005), suggesting increased absolute abundance up to 5 days before onset. Additionally, bacterial yields were significantly lower (P = .02) and α-diversity significantly decreased (P = .012) when compared to the controls. CONCLUSIONS:Increased fecal C albicans preceded Candida LOS onset, implicating the preterm gut as a potential source of infection. Reduced bacterial yields and diversity suggest ecological alterations that may facilitate Candida pathogenicity in the preterm gut. These findings support further research into gut-derived Candida LOS and the potential for microbiota-targeted prevention strategies.

BACKGROUND & AIMS:Colorectal cancer (CRC) often requires surgical resection of the tumor and an anastomosis. Anastomotic leakage (AL) occurs in 2.8% to 30% of patients, which increases postoperative morbidity and complications. The preoperative microbiome composition is implicated in AL. Recent studies have shown that microbial tryptophan (Trp) metabolism into aryl hydrocarbon receptor (AhR) indole-derivatives contributes to intestinal tissue healing. Here, we addressed the role of Trp and its metabolites in AL in a CRC patient cohort and a colon-anastomosis mouse model. METHODS:Targeted quantitative metabolomics was performed in preoperative fecal samples from patients with CRC recruited in the REVEAL cohort (n = 388), including 19 AL cases. Anastomotic healing (AH) was tested in a mouse model using wild-type (WT), AhR-/-, VillinCreAhrfl/fl, and interleukin (IL)-22 drug-targeting to evaluate the role of AhR and IL-22 in AL. RESULTS:Fifty-two of the 388 patients with available preoperative fecal samples were matched for AH/AL occurrence (AL, n = 19; AH, n = 33). Among Trp metabolites, indole-3-acetic acid was significantly reduced in AL compared with matched AH male patients. AhR-/- mice displayed more severe AL, reduced IL-22 expression, and a marked loss of IL-22-expressing type 3 ILCs compared with WT mice. Neutralizing IL-22 antibody augmented AL in WT mice, whereas IL-22Fc application ameliorated AL in AhR-/- mice. AhR agonism failed to rescue healing under IL-22 deficiency. Furthermore, low-Trp diet-fed WT mice exhibited reduced fecal concentration of AhR agonists, AhR-agonist producing bacteria, and augmented AL. This phenotype was prevented by dietary supplementation with the AhR agonist indole-3-carbinol. CONCLUSIONS:Stimulation of the AhR/IL-22 by synthetic agonists or dietary-derived Trp-metabolites can prevent AL.

Background:The Crohn's disease (CD) exclusion diet (CDED) is an emerging dietary therapy for inducing remission in CD. However, data on its effects on gut microbiome in adults remain limited. This study investigated microbial responses to CDED in adults with mild-to-moderate CD and compared them with pediatric patients and healthy pediatric controls. Methods:Microbiome data were analyzed from a randomized controlled trial (RCT) in adults (baseline, weeks 6, 12, 24) and a pediatric RCT (baseline, weeks 6, 12). Baseline microbial composition, diversity, and functional potential were compared between patients who achieved sustained clinical remission (SCR) at both weeks 12 and 24 and those who did-not. Functional profiling was performed using gene ortholog annotations, linear discriminant analysis, and metabolite inference. Results:Baseline microbial and functional profiles differed between patients with and without SCR. SCR was associated with lower alpha diversity, higher relative abundances of Alistipes and Faecalibacterium, and increased flagellin gene expression. SCR was associated with enrichment of genes for redox balance, fatty acid metabolism, and DNA repair, while non-SCR showed elevated NAD biosynthesis, bacterial adhesion, and pro-inflammatory pathways. Haemophilus and Prevotella were negatively linked to SCR. Compositional and functional microbiome analyses revealed a microbiome shift during CDED-induced remission toward a profile more similar to healthy pediatric controls. Conclusions:Before and during CDED, distinct baseline microbial and functional profiles were associated with SCR. These highlight the potential of the gut microbiome as a biomarker for identifying patients most likely to benefit from sustained effects of dietary therapy, supporting a more personalized approach to CD management.

Damage to the intestinal epithelial barrier is a hallmark of inflammatory diseases such as necrotizing enterocolitis. Specialized proresolving mediators (SPMs), such as lipoxin A4, resolvin D1, and resolvin E1, which are derived from essential fatty acids, have been shown to aid in resolving inflammation and promote mucosal healing. This study aimed to explore the effects of specific SPMs on intestinal inflammatory response in an early life in vitro model. We established 3-dimensional and 3-dimensional organoid cultures from fetal and pediatric intestines and investigated the effect of an SPM cocktail (lipoxin A4, resolvin D1, and resolvin E1) on gut epithelial maturation and barrier function. An inflammatory response of the gut barrier was provoked by lipopolysaccharide and flagellin stimulations combined with proinflammatory cytokines, tumor necrosis factor-α, and interferon gamma. Additionally, repetitive mechanical wounding was developed to test the effects of the SPM cocktail on 2-dimensional organoid monolayers. Under physiological conditions, we observed no effect of SPM cocktail treatment on gut epithelial maturation. Upon cytokine challenge, there was no modulation of the inflammatory tone of the gut barrier by the SPM cocktail. However, during the repetitive wounding and recovery assay, SPM cocktail treatment accelerated barrier recovery and maintained barrier integrity for 24 hours after repeated injuries. Our findings suggest that the SPM cocktail does not affect bacterial product- or cytokine-induced epithelial inflammation, although it may accelerate epithelial barrier recovery in mechanically wounded monolayers. These results provide valuable insights into the therapeutic potential of SPMs in neonatal intestinal inflammation. SIGNIFICANCE STATEMENT: Using early life intestinal organoid models, we found that although specialized proresolving mediators did not alter cytokine- or bacterial product-induced inflammation, they significantly enhanced epithelial barrier recovery following repeated mechanical injury.

Early-life microbial metabolism of tryptophan plays a critical role in immune modulation and may influence susceptibility to inflammatory disorders such as eczema. However, longitudinal human data linking microbial tryptophan metabolism to eczema onset are limited. We conducted a prospective cohort study of 40 term-born UK infants, followed from birth to 24 months, stratified by early-onset (infants developing eczema <12 months, n = 12) or late-onset (infants developing eczema ≥12 months, n = 7) doctor-diagnosed eczema. Fecal samples underwent targeted tryptophan LC-MS metabolomics and 16S rRNA sequencing. Tryptophan metabolomics revealed dynamic changes in tryptophan metabolites that were associated with disease status compared to healthy infants. Infants with early-onset eczema (<12 months) exhibited elevated tryptophan and tryptamine levels at 6 months, while those with late-onset eczema (≥12 months) showed reduced indole-3-lactic acid (ILA) levels prior to diagnosis at 9 months. 16S and metabolomics correlations highlighted a metabolic shift in Bifidobacterium from ILA, a metabolite that predominated fecal metabolome during early infancy but decreases by 12 months, coinciding with increased indole-3-aldehyde (I3AD) metabolism by Bifidobacterium. Receiver operating characteristic (ROC) curve analysis identified a small panel of early predictive indole-pathway markers, including tryptophan and the indole/indole-3-sulfate (I3S), I3S/ILA, and tryptophan/ILA ratios, discriminating both early- and late-onset eczema development. Our findings reveal distinct, time-dependent disruptions in microbial tryptophan cometabolism associated with eczema onset. ILA may serve as a biomarker for the diagnosis of infantile eczema. Further validation studies to establish its clinical utility are now required.

BACKGROUND:Previous studies have shown that low skeletal muscle mass and excess adiposity are predictors of mortality both in the general population and in colorectal cancer (CRC) patients. However, it remains unclear whether body composition is associated with disease recurrence. The aim of this study was to investigate the association between body composition and recurrence in patients with non-metastatic CRC, while explicitly accounting for the competing risk of non-CRC-related mortality. METHODS:This prospective multicenter study included stage I-III CRC patients undergoing curative-intent resection. Body composition was assessed on axial CT images at the third lumbar (L3) and supra-acetabular (SA) levels. Visceral, subcutaneous, and intermuscular adipose tissue and skeletal muscle mass and density were quantified. Recurrence, CRC-related mortality, and non-CRC-related mortality were analyzed using Fine and Gray competing risks models. RESULTS:Among 402 patients with a median age of 68 years, 70 (17%) developed recurrence and 67 (17%) died during a median follow-up of 5.0 years (IQR 4.4-5.3; range 0.1-10.0). Of the 67 deaths, 34 (51%) were due to non-CRC-related causes. None of the 11 body composition parameters were associated with recurrence. Higher skeletal muscle mass at the SA level was associated with lower all-cause mortality (HR 0.71, 95% CI 0.59-0.84, p = 0.003) and CRC-related mortality (HR 0.67, 95% CI 0.50-0.90, p = 0.01). Higher visceral fat mass was associated with higher non-CRC-related mortality (HR 1.07, 95% CI 1.01-1.14, p = 0.02). CONCLUSIONS:Preoperative body composition was not associated with recurrence in non-metastatic CRC and is therefore unlikely to represent a modifiable target for reducing recurrence risk. Nonetheless, skeletal muscle mass was associated with both CRC-related and non-CRC-related mortality, suggesting that its optimization may reduce these risks through mechanisms independent of recurrence.

Purpose:While gut microbiome dysbiosis is known to play a role in colorectal cancer (CRC) initiation and progression, its role in CRC recurrence remains unclear. This study investigates whether the gut microbiome is associated with CRC recurrence. Patients and Methods:In a prospective observational cohort, preoperative fecal samples from patients with stage I to III CRC undergoing surgical resection were analyzed using 16S rRNA gene sequencing. Alpha diversity and beta diversity were compared between patients with and without recurrence, and differential abundance analyses were conducted to identify bacterial genera associated with recurrence risk. Results:Among 294 patients, 61 (21%) patients developed recurrence during a median follow-up of 56 months, with a median time to recurrence of 19 months. Alpha diversity did not differ between groups, but beta diversity analysis revealed significantly distinct microbial clustering in patients with recurrence, particularly those with locoregional recurrence. Differential abundance analysis identified five bacterial genera associated with locoregional recurrence (Acidaminococcus, Alloprevotella, Butyrivibrio, Ruminococcaceae CAG-352, and Lachnospiraceae UCG-003), one with distant recurrence (Megamonas), and two with overall recurrence (Anaeroplasma, Porphyromonas). Stratifying patients into high- and low-abundance subgroups revealed that those with a high relative abundance of Porphyromonas had an increased risk of overall recurrence (hazard ratio, 2.80 [95% CI, 1.54 to 5.10]). Conclusion:Patients with CRC who develop locoregional recurrence exhibit a distinct preoperative fecal microbial composition compared with those without recurrence. Our findings provide novel insights into the role of the intestinal microenvironment in recurrence and identify Porphyromonas as a potential fecal biomarker for overall recurrence risk.

Introduction:Crohn's disease (CD) is commonly treated with biologic therapies, including anti-TNFα agents, vedolizumab (VDZ), and ustekinumab (USTE), yet only a subset of patients respond to these treatments. This study aimed to evaluate the potential of the gut microbiome to predict treatment response. Methods:Adult CD patients initiating anti-TNFα (infliximab or adalimumab), VDZ or USTE were enrolled. Pre-treatment ileal and/or colonic biopsies were collected endoscopically. Treatment response after 26-52 weeks was defined by ≥50% reduction in the simple endoscopic score for CD and either a corticosteroid-free clinical response (≥3-point HBI decrease or remission [HBI ≤4] without systemic steroids) or a biochemical response (≥50% or ≤5 mg/L CRP reduction and ≥50% or ≤250 μg/g faecal calprotectin reduction) versus baseline. Mucosal microbiota was profiled by 16S rRNA gene sequencing of biopsies. Machine learning models predicting treatment response were trained using ASV-level count data. The impact of heat-killed bacteria on anti-TNFα-induced CD14+CD206+ macrophages was tested in mixed lymphocyte reactions (MLRs). Results:A total of 125 patients were included: 39 on anti-TNFα, 47 on VDZ, and 39 on USTE. Clinical features were similar between responders and non-responders, aside from sex (USTE-colon) and CRP (USTE-ileum). No major microbial differences were observed in VDZ, USTE ileal or colon samples. However, in colonic biopsies, anti-TNFα responders had significantly higher pre-treatment α-diversity, and 3.9% of β-diversity variation associated with response. Among six models, the anti-TNFα colonic model performed significantly better than random (AUC = 0.90) to predict response. Mediterraneibacter gnavus ASVs associated with non-response, whereas Blautia ASVs associated with response, to anti-TNFα. When tested in MLRs, pretreatment with M. gnavus and B. luti led to a reduction in macrophage polarization, with a significantly stronger effect observed for M. gnavus compared with B. luti. Discussion:Taken together, this study demonstrates that the colonic mucosal microbiome prior to anti-TNFα treatment can distinguish responders from non-responders in CD, supporting its potential as a predictive biomarker.

Intestinal bacterial translocation to the bloodstream is a route of infection for late-onset sepsis (LOS) in preterm infants, highlighting the potential of fecal microbiota profiling for early risk stratification. We aimed to identify and validate LOS-specific gut microbiota signatures. Fifty-eight preterm infants (gestational age < 30 weeks) with blood culture-proven LOS (excluding coagulase-negative staphylococci) were matched to controls (1:1) across three cohorts (Discovery (DC) n = 18; Validation 1 and 2; VC1 n = 12, VC2 n = 28). Fecal samples collected up to 10 days before LOS onset underwent 16S rRNA gene sequencing. Microbial composition, diversity, and discriminatory taxa were compared across LOS subgroups. Random Forest (RF) models were trained in DC and validated in VC1/VC2. Microbiota variation was largely explained by LOS pathogen (R2 = 17%, P < 0.001). Infants with non-staphylococcal and E. coli-LOS showed a temporal increase in relative abundance of Escherichia/Shigella. The RF model distinguishing E. coli-LOS from controls displayed the highest discriminatory performance (AUC = 0.99/0.78/0.61 for DC/VC1/VC2) compared to non-staphylococcal LOS (AUC = 0.96/0.46/0.41). Our findings demonstrate profound microbiota shifts preceding E. coli-LOS, with higher discriminatory ability compared to non-staphylococcal-LOS. While pathogen-specific microbiota-based risk stratification may offer added clinical value, reduced validation performance highlights the limited generalizability and underscores the need for future research before clinical translation.

BACKGROUND:Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by relapsing and remitting mucosal inflammation of the colon. While active UC mucosa is characterized by dysregulated B cell responses and increased B cell and IgG plasma cell populations, targeting CD20-expressing B cells in UC has proven ineffective. METHODS:We conducted an exploratory single-cell transcriptomic analysis of colonic biopsies obtained from UC patients with (n = 5) or without (n = 5) active inflammation, and non-UC controls (n = 4). To explore whether B cells contribute to colitis severity, we transferred various ratios of spleen-derived naive B cells with CD45RBhigh T cells into severe combined immune deficient mice to induce colitis. RESULTS:Our analysis identified a distinct subset of naive (MS4A1+CD27-IGHD+TCL1A+) B cells that are significantly enriched and present a more matured phenotype in inflamed compared to non-inflamed biopsies from UC patients. Cell-cell communication analysis indicated that naive B cells interacted predominantly with CD4+ T cell subsets. In the mice transfer colitis model, co-transfer of naive B cells at a ratio of 1-2 T and B cells, respectively, showed an increased maturation and activity, which led to exacerbation of colitis as measured by weight loss, increased colon density, and histological inflammation. CONCLUSION:Our findings suggest that naive B cells expand in actively inflamed mucosa from UC patients and play a pathogenic role in experimental colitis.

Background The gut microbiome influences health by regulating metabolism, modulating the immune system, and protecting against pathogens. Surgical interventions, antibiotics, and dietary changes can disrupt this balance. In rectal cancer surgery, the perioperative microbiome's role in selective digestive decontamination and ileostomy formation remains underexplored. This manuscript aimed to investigate perioperative microbiome dynamics in patients undergoing rectal cancer surgery, focusing on the impact of selective digestive decontamination and ileostomy. Methods This study was part of the IMARI-trial. Faecal samples were collected before surgery and faecal or ileostomy samples on postoperative day 4. Microbial composition and diversity were assessed using 16S ribosomal RNA sequencing; alpha and beta diversity analyses were stratified by time point, ileostomy status, and selective digestive decontamination administration. Results Of the 246 enrolled in the IMARI-trial, 214 patients were analysed. Postoperative samples showed significantly lower alpha diversity than preoperative samples, indicating reduced microbial richness and evenness across groups. Beta diversity analyses revealed distinct clustering between pre- and postoperative samples, with surgery explaining the largest variance in microbial composition (R2 = 15.6%; P < 0.001). Ileostomy status (R2 = 9.6%; P < 0.001) and selective digestive decontamination administration (R2 = 3.1%; P = 0.002) also contributed significantly. Notable taxonomic shifts included increased postoperative abundances of Enterococcus and Klebsiella, alongside reduced Firmicutes genera, particularly in ileostomy samples. Conclusion Surgery and perioperative factors induce significant, early microbiome alterations, favouring facultative anaerobes over obligate anaerobes. Although selective digestive decontamination selectively modulates specific taxa, its overall impact on diversity appears less pronounced than surgery and ileostomy status. These findings underscore the need for studies linking perioperative microbiome trajectories to clinical endpoints and for evaluating microbiome-informed perioperative strategies, including antibiotic stewardship, diet, or microbial therapeutics, in rectal cancer care.