Journal of Crohn's and Colitis

Inflammatory bowel disease (IBD) is a heterogeneous chronic disease driven by dysregulated mucosal immunity and impaired epithelial barrier function. Although biologics have improved disease management, they are frequently associated with systemic immunosuppression and adverse effects, highlighting the need for localized therapeutic strategies that both control inflammation and promote tissue repair. Here, we developed a protein bispecific termed 7A2-IgG4-IL22, composed of a human IgG4-Fc domain displaying an antagonistic anti-human MAdCAM-1 single chain (sc)-Fv and a human interleukin (IL-)22. The anti-MAdCAM-1 scFv retained the functional activity of the parental monoclonal antibody, inhibiting T cell activation, expansion and differentiation from naive precursors. Blockade of the MAdCAM-1 signaling axis also reduced production of pro-inflammatory cytokines relevant to IBD pathogenesis, including IFN{gamma} and TNF. On the epithelial side, the IL-22 cargo induces robust signaling in epithelial cells, promoting the expression of IL-22 response genes associated with antimicrobial defense, mucosal homeostasis, as well as IL-10 and CXCL1 expression. This effect contributes to immune cell trafficking to the intestinal mucosa. Together, this bispecific provides a localized dual-mechanism strategy for restoring intestinal immune homeostasis.

Ulcerative colitis (UC) is characterised by chronic colonic inflammation with marked heterogeneity in disease severity and therapeutic outcomes. Here, we define a spatially organised, polyfunctional cytotoxic CD4 T-cell state associated with mucosal inflammation and adverse therapeutic outcomes in UC. Integrating ex vivo T-cell receptor stimulation with multi-cohort bulk and single-cell transcriptomics and multiparameter flow cytometry, we show that GZMB CD4 T cells are preferentially enriched in inflamed UC mucosa, but not peripheral blood, and co-express cytotoxic molecules, Th1- and Th17-associated cytokines and chemokines, and immunoregulatory receptors. Single-cell analyses implicate inflammatory cytokine and antigen-presentation signals in the acquisition or maintenance of this state. High-resolution spatial profiling localised this programme predominantly to Th17 cells, which were preferentially enriched within multicellular inflammatory and tertiary lymphoid structure-associated niches. Across independent patient cohorts, a transcriptional signature derived from this state increased with endoscopic disease severity and was associated with reduced response to anti-TNF and anti-IL-12/23p40 therapies. Adoptive transfer of Gzma/Gzmb-deficient rather than wild-type CD4 T cells into Rag2-deficient recipient mice markedly attenuated experimental colitis and abrogated the polyfunctional cytokine phenotype, demonstrating that granzyme-dependent effector activity is a key mechanism driving CD4+ T-cell-mediated intestinal inflammation. Finally, human host-microbiome analysis linked this programme to intestinal dysbiosis, while transfer of dysbiotic microbiota promoted the emergence of a corresponding state in vivo. Collectively, these findings define a microbiota-responsive, spatially organised polyfunctional cytotoxic CD4 T-cell programme that contributes to intestinal inflammation and is associated with disease severity and treatment resistance in UC.

CD28 costimulation contributes to pathogenic T cell responses in inflammatory bowel disease (IBD), but current B7-directed blockade also limits CTLA-4 signaling. Using a sensitive NanoBiT split-luciferase screening platform, we identified and optimized CA-23, a small molecule antagonist that directly binds human and mouse CD28 without measurable binding to CD80, CD86, or CTLA-4. CA-23 inhibited CD28-B7 engagement and CD28-dependent T cell activation without agonist activity in human whole blood and peripheral blood mononuclear cells. CA-23 achieved exposure in the colon and mesenteric lymph nodes and reduced disease severity, histologic injury, and pathogenic Th1 and Th17 responses in a T cell transfer model of colitis. In PBMCs from donors with ulcerative colitis or Crohns disease, CA-23 suppressed inflammatory cytokine production and T cell activation to a degree matching or exceeding Abatacept. In human intestinal epithelial-PBMC co-cultures, CA-23 preserved Treg suppressive activity and epithelial barrier integrity, whereas Abatacept reduced Treg function. CA-23 did not alter CD80 or CD86 expression on autologous antigen-presenting cells and showed no substantial off-target activity in the tested selectivity panel. These findings support direct CD28 antagonism as a mechanistically differentiated alternative to B7-directed co-stimulation blockade for suppressing pathogenic T cell responses in preclinical models of IBD. One Sentence SummaryA CD28-selective small molecule blocks pathogenic T cell activation and preserves Treg function unlike Abatacept in IBD models.

Prior anti-tumour necrosis factor (TNF) failure is associated with reduced efficacy of subsequent advanced therapies in ulcerative colitis (UC), but the biological basis of this treatment-refractory state remains unclear. We integrated clinical outcomes and baseline colonic transcriptomic data from UC patients in the UNIFI phase III trial programme with regulatory and signalling network inference, connectivity mapping, and single-cell-resolution spatial transcriptomics. Colonic transcriptomic analyses identified coordinated enrichment of extracellular matrix organisation, collagen remodelling and integrin-associated programmes, increased stromal cell representation and elevated inferred MAPK/EGFR activity in UC patients with prior anti-TNF failure. Causal network inference prioritised MAPK3 as a candidate regulator of this state, while connectivity mapping identified MEK/EGFR inhibitors as candidate perturbagens. MEK inhibition suppressed stromal pathways and reduced inferred MAPK/EGFR activity ex vivo. Spatial profiling of active UC and non-IBD colonic tissues localised these programmes to UC-enriched stromal niches. Ligand-receptor inference further identified reciprocal stromal-myeloid communication within these niches. Collectively, these findings define a stromal remodelling programme associated with prior anti-TNF failure and nominate MAPK/EGFR signalling as a potentially tractable component of treatment-refractory UC.

Environmental enteropathy (EE) is a chronic, subclinical disorder of the small intestine common in low- and middle-income countries (LMICs), where access to sanitation and exposure to enteric pathogens vary greatly by socioeconomic status (SES). Systemic immune cell activation by enteric microbial exposure is a suspected but poorly characterized driver of EE severity. We hypothesised that adults from Low-SES communities would have more severe EE than adults from High-SES communities and that this would be associated with distinct circulating immune cell phenotypes. We enrolled clinically healthy adults from High- (n=26) and Low-SES (n=76) communities in Lusaka, Zambia. Duodenal biopsies from these adults were used for microscopic morphometry assessments, while plasma and stool biomarkers of epithelial damage, intestinal inflammation, microbial translocation, and systemic inflammation were measured by ELISA. Circulating monocyte, neutrophil and T cell phenotypes were characterised in buffy coat cells by flow cytometry. Compared with the High-SES group, adults from Low-SES communities had higher duodenal villus width and crypt depth and lower epithelial surface area, indicative of more severe EE pathology, and higher levels of plasma biomarkers associated with microbial translocation and systemic inflammation. The Low-SES group also had higher expression of activation markers (CD86 and TLR4) and lower expression of HLA-DR on circulating classical monocytes and neutrophils, higher percentages of gut-homing (4{beta}7+) and activated/exhausted (PD-1+) T cells, including gut-homing (4{beta}7+) regulatory T cells. Principal Component Analysis identified key patterns of immune cell phenotypes across SES groups. Confounder-adjusted linear regression models showed that Principal Component 1 (monocyte/neutrophil activation) was inversely associated with duodenal villus height and epithelial surface area across SES groups. These findings indicate that EE severity varies by SES within LMIC and suggest that monocyte and neutrophil activation is linked to greater duodenal remodelling in adults with EE.

CTLA-4 (haplo)insufficiency displays incomplete penetrance and phenotypic heterogeneity, indicating the involvement of additional disease modifiers beyond the genetic defect. Microbiome analyses reveal a positive association between disease severity and intestinal dysbiosis, highlighting the microbiome as a critical contributor. To investigate this relationship mechanistically, we generated Ctla4/- wildlings harboring a natural microbiota. Unlike specific pathogen free (SPF) counterparts, which remain healthy, Ctla4/- wildlings spontaneously develop disease phenotypes resembling human CTLA-4 haploinsufficiency. Disease onset is followed by reduced microbial diversity and expansion of pathobionts. Integrative immunophenotyping shows that the natural microbiota synergizes with Ctla4 haploinsufficiency to reshape innate and adaptive immune compartments, generating a sustained pro-inflammatory milieu and reduced CTLA-4 expression in the cecum. Furthermore, microbiota-derived metabolites promote inflammatory cytokine production in both murine and human primary T cells via NF-{kappa}B activation. Collectively, Ctla4/- wildlings constitute an effective model for dissecting microbiome-immune crosstalk in CTLA-4 (haplo)insufficiency and for exploring therapeutic strategies.

Crohns disease (CD) is a chronic inflammatory condition of the gastrointestinal tract for which anti-tumour necrosis factor (anti-TNF) agents remain a first-line biologic therapy. However, remission rates are modest, and the mechanistic basis of non-response is poorly characterised. A common resistance mechanism is thought to emerge when alternative inflammatory cascades compensate for TNF inhibition, but the interactions underlying this rewiring have not been systematically characterised. We applied CytokineLink, our previously developed systems immunology framework, to single-cell RNA sequencing data from CD patients sampled before and after anti-TNF therapy. We reconstructed networks of interacting cytokines across samples stratified by treatment phase, response, and inflammation status, and identified condition-specific cytokine interactions and feedback loops, statistically validated against degree-matched random networks. We clustered the generated networks based on their inflammation, response, and treatment status. The pre-treatment inflamed non-responder network contained the largest set of unique interactions, organised around a connected module driven by IL17C targeting downstream TNF, IL6, IL1B, CXCL1/2/3/8, and CCL20. IL17C was produced by a population of non-ileal enteroendocrine cells, differentially abundant at baseline in non-responders. Gene set variation analysis in an independent cohort confirmed elevated non-responder module activity in colonic tissues of non-responders. Feedback loop analysis revealed that responder networks were characterised by persistent IL10 circuits sustained by macrophage populations and acquired tissue-remodelling interactions after therapy, whereas non-responders lost IL10 feedback loops post-treatment and gained TNF-containing motifs, including circuits signalling through the upstream activator TL1A. Our findings characterise the mechanism of anti-TNF non-response as a cytokine network, in which pre-existing epithelial-driven inflammatory modules and the failure to preserve regulatory feedback sustain TNF-independent inflammation in CD. By characterising cytokine interactions at the systems level, our approach moves beyond single-cytokine models of anti-TNF resistance to provide a mechanistic framework for understanding the biological basis of treatment failure in immune mediated diseases.

Regulatory T (Treg) cells expressing ROR{gamma}t accumulate in the intestinal mucosa, yet the signals that determine whether they remain suppressive or acquire inflammatory features are incompletely defined. We first reanalyzed human ileal single-cell data and identified Crohns disease-enriched FOXP3 states in which RORC, HIF1A, hypoxia-responsive, inflammatory, and metabolic programs converged. We then deleted Hif1a in ROR{gamma}t-expressing cells and tested acute DSS colitis, T cell transfer colitis, and azoxymethane/DSS-induced colitis-associated colorectal cancer (CAC). {Delta}Hif1a mice were protected in all three settings. In lymphopenic recipients given the same pathogenic naive T cells, changing only the genotype of the cotransferred Treg population enhanced protection, linking the phenotype to regulatory-cell function in vivo. Reanalysis of mouse colonic Treg single-cell ATAC-seq nominated suppressive and mitochondrial programs for cell-intrinsic testing during low HIF1- expression. {Delta}Hif1a ROR{gamma}t Treg produced more IL-10 and less IL-17A and IFN-{gamma}, limited responder-cell proliferation, contained fewer dysfunctional and mitochondrial-reactive-oxygen-species-high mitochondria, favored fusion-associated transcription, and displayed greater basal and maximal oxygen consumption and reserve capacity. During CAC, HIF-1 loss blunted inflammatory ROR{gamma}t Treg accumulation and reduced tumor burden. Human trajectory and gene-regulatory-network analyses further predicted that HIF1A perturbation would oppose selected disease-associated branches. Together, these findings identify HIF-1 as a context-dependent checkpoint that connects hypoxia-responsive transcription to mitochondrial fitness and inflammatory plasticity in intestinal ROR{gamma}t Treg.

Inflammatory bowel disease (IBD) often involves disrupted intestinal epithelial barrier, but therapies specifically targeting this barrier are limited. We found that downregulated AUF1 (HNRNPD) contributes to defective barrier integrity in ulcerative colitis (UC). Compared to controls, its expression level was decreased and inversely correlated with clinical severity. Knocking down AUF1 in human and mouse colonic organoids led to impaired barrier function, with reduced Occludin and upregulated Claudin-2, mimicking characteristic IBD-associated mucosal alterations. Distinct RNA-binding activity of AUF1 protein isoforms contributed to these changes: p37 stabilized Occludin mRNA and blocked microRNA-122/Ago2-mediated repression, whereas p40 promoted Claudin-2 mRNA degradation via ubiquitin-proteasome pathway. Restoring AUF1 expression in organoids enhanced epithelial properties and, when transplanted into mice with established colitis, accelerated mucosal healing and epithelial regeneration in recipient mice and decreased fibrosis. Our study unravelled a post-transcriptional mechanism important for intestinal homeostasis and demonstrated a concept of using engineered organoids for treating IBD.

Background: Faecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridioides difficile infections, yet the diversity of FMT formulations and delivery routes hampers comparisons across studies. Oral frozen capsules are widely used and current guidelines recommend storage at -80C for up to two years. Despite extensive use of FMT, data on the long-term persistence and maintenance of their microbial composition remains limited. Method: In this prospective study, we assessed the temporal stability of bacterial profiles in frozen FMT capsules derived from 48 donations of 10 healthy donors. Using metabarcoding, we longitudinally profiled one capsule per donation thawed within a month of production and after 3, 6, 12, and 24 months of storage. We used linear mixed effect models to evaluate changes in alpha diversity and community composition over time. Results: Species richness remained stable across all timepoints, whilst species evenness decreased slightly. Although changes in community composition were detectable, they were small and mostly affected low-abundance genera. Clinical efficacy, assessed in a subset of recipients, was not associated with storage duration. Conclusion: Our findings demonstrate that frozen FMT capsules preserve their bacterial community structure for at least two years of storage at -80C, supporting their suitability for long-term biobanking and standardised clinical or research use.

Importance: Understanding population differences in epidemiology, clinical presentation, and genetic architecture remains a major challenge for all rare genetic disorders. Hirschsprung disease (HSCR), despite being the commonest cause of neonatal intestinal obstruction, has been poorly studied with respect to its significant heterogeneity across U.S. populations. Objective: To characterize self-identified race and ethnicity differences in HSCR incidence, clinical presentation, and genetic architecture in the United States from diverse data sources. Design, Setting, and Participants: We used retrospective, population-based surveillance data from 3 independent US wide sources - (1) The National Birth Defects Prevention Network (NBDPN; 1996-2010), (2) aggregated electronic health record data from Epic COSMOS (1997-2025), and (3) individual level clinical and genomic data from the Hirschsprung Disease Research Collaborative (HDRC; 2011-2025). Statistical analyses of incident HSCR cases identified at birth, across time and geography, in conjunction with clinical phenotypes and genome sequences from unrelated HDRC probands were performed to characterize epidemiologic, phenotypic and genetic heterogeneity in HSCR. Exposures: HSCR cases were identified based on standardized clinical diagnostic criteria, primarily rectal biopsy with histopathologic confirmation of aganglionosis. The disease was defined using ICD-9-CM code 751.3, CDC/BPA code 751.30-751.34. and ICD-10-CM code Q43.1. Patients were classified by self-identified race and ethnicity (SIRE), with primary comparisons conducted between non-Hispanic Blacks/African Americans (Blacks) and non-Hispanic Whites (Whites). Main Outcomes and Measures: HSCR incidence and the frequency of clinical features were estimated overall and by SIRE. We also estimated the individual and total genetic burden of rare pathogenic coding variants and common noncoding regulatory variants at established HSCR genes by population. Results: Overall HSCR incidence in the U.S. was 2.04 per 10,000 live births (95% CI, 1.99-2.09) as previously estimated. We show, Blacks have the highest HSCR incidence (2.83-3.13 per 10 000 live births), in comparison to Whites (1.89-2.02) and Asians (1.54-1.98), a difference not previously ascertained from previous smaller cohorts from limited geographical regions. This difference persists across surveillance times and geography. This incidence difference from NBDPN is consistent with Epic COSMOS, a nation-wide, independent hospital-based data source. Clinically, Blacks are more likely to present with isolated HSCR and with milder manifestations at birth, including chronic severe constipation (CSC). Genetically, the burden of pathogenic coding variants did not differ between Blacks and Whites. However, Blacks had a significantly higher enrichment of two non-coding regulatory variants (rs199582499 and rs28735659) at the SOX10 gene locus, as compared with Whites. Conclusions and Relevance: This study demonstrates, for the first time, that Black HSCR patients in the U.S. have a higher incidence accompanied by milder clinical presentation and distinct noncoding regulatory SOX10 variants as compared to White patients. Nevertheless, Blacks are severely under-represented in U.S. studies of HSCR leading to significant health disparities in their care and management.

Interactions between intraepithelial lymphocytes (IELs) and the intestinal epithelium are central to mucosal homeostasis and disease. However, mechanistic in vitro studies describing their crosstalk in humans are limited by scarceness of primary material and insufficient knowledge about co-culture requirements. Here, we establish an autologous human duodenal IEL-organoid co-culture system encompassing expandable and bankable IEL and organoid protocols, with co-culture conditions that allow viability of both cell types. This system enables successive interrogation of lympho-epithelial interactions starting from minimal biopsy material. Under baseline conditions, CD45CD8CD103TCR{beta} IELs retain tissue-residency and effector features and induce an epithelial interferon response and chemokine production, without overt epithelial apoptosis. IL-15 and IL-21, essential cytokines involved in IEL-activation in intestinal enteropathies like celiac disease, increases granzyme B expression and interferon-{gamma} secretion but do not trigger epithelial cell death. However, enforcing IEL-epithelial contact using an anti-CD3-anti-Ep-CAM bispecific antibody induces epithelial apoptosis accompanied by increased tumor necrosis factor (TNF) and FAS-ligand (FASLG) secretion. These findings validate the platforms ability to resolve non-destructive and cytotoxic lympho-epithelial interaction and provide a tractable system for studying intestinal inflammation and immune-mediated epithelial cell death.

The gut microbiota regulates intestinal immunity through metabolite production, yet most disease-associated metabolites remain functionally uncharacterized. In inflammatory bowel diseases (IBD), where the microbial metabolome is profoundly altered, we identify N-acyl putrescines as microbiome-associated metabolites enriched across two independent cohorts. N-oleoylputrescine (NOP) emerges as the primary immunomodulatory candidate, inducing robust transcriptional responses in dendritic cells and colonic organoids. Enterocloster species harboring nonribosomal peptide synthetase gene clusters synthesize NOP, confirmed by isotope-tracing in vitro and germ-free mouse colonization in vivo. NOP suppresses core IBD inflammatory pathways in mouse dendritic cells and human monocytes, reducing signatures of histologic inflammation and therapy non-response. NOP dampens inflammation in four colitis models, decreases myeloid cell NF-{kappa}B activation, and suppresses type 1 immune responses through a T cell-intrinsic mechanism. That NOP accumulates in IBD despite its anti-inflammatory properties reveals a holobiont defense strategy: the gut microbiota deploys immunomodulatory metabolites as a compensatory response to restore homeostasis.

How ecological architectures within the gut microbiome convert complex inputs into specific host physiological outcomes remains poorly understood. We used CDD-2101, a multi-component botanical drug operating under an FDA (U.S. Food and Drug Administration) Investigational New Drug program, as a defined ecological perturbation in functional constipation (FC). Integrating a randomized, double-blind, placebo-controlled clinical trial with genome-resolved metagenomics, targeted metabolomics, staged prediction modeling, and receptor-level validation, we show that clinical efficacy of CDD-2101 depends on remodeling a function-specific substructure of the stable Two Competing Guilds (TCG) architecture. We term this substructure the FC-TCG, demonstrate its role along the gut-motility axis, and confirm its effect in three independent gut hypomotility cohorts. The two guilds responded asymmetrically: the intervention selectively suppressed the C1B guild (the pathobiont guild) while largely sparing the C1A guild, the foundation guild that anchors the core gut community, restoring its ecological dominance, producing a coordinated metabolic shift that elevates lithocholic acid and propionic acid. Through gnotobiotic transplantation and receptor antagonism, we demonstrate that lithocholic acid and propionic acid restore gut motility via concurrent engagement of Takeda G protein-coupled receptor 5 (TGR5) and G-protein coupled receptor 43 (GPR43). These findings identify microbial guild architecture as a function-resolved signal-transducing layer that converts multi-component botanical intervention into multi-receptor-mediated gut motility restoration, reframing the gut microbiome from a compositional system into a structural transducer between complex environmental inputs and host physiology.

Introduction Patients undergoing polypectomy at colonoscopy remain at risk of metachronous neoplasia despite surveillance guided by histopathological features. Mutational profiling of adenomas, including canonical driver mutations in APC, KRAS, and TP53, may offer additional predictive value. This study aimed to determine whether mutational status in index adenomas was associated with metachronous lesion risk. Methods The INCISE cohort included patients aged 50 to 74 years who underwent polypectomy within the Scottish Bowel Screening Programme and subsequent surveillance colonoscopy within 6 years. Targeted next-generation sequencing was performed on formalin-fixed paraffin-embedded polyps. Driver mutation frequency, tumour mutational burden (TMB), and variant allele frequency (VAF) were analysed and correlated with histopathological features and metachronous outcomes using appropriate statistical models. Results A total of 895 adenomas from 723 patients were analysed. In conventional adenomas, as the number of high-risk histopathological features (size >=10mm, villous architecture, and high-grade dysplasia) increased there was a stepwise increase in the proportion of samples with a mutation in KRAS from 13% to 51% (padj<0.001) and TP53 from 8% to 35% (padj<0.001). However, neither mutation frequency (p=0.901), nor median tumour mutation burden (TMB) (2.27 vs 2.15 mut/Mb, p=0.242), in index adenomas was associated with the development of metachronous lesions. Conclusions While classical driver mutations reflect histopathological progression within adenomas, they do not predict metachronous lesion risk post-polypectomy. Targeted mutation profiling alone is insufficient for surveillance risk stratification, highlighting the need for integrated molecular approaches in this setting.

Inflammatory bowel disease (IBD) is associated with chronic intestinal inflammation and gut microbial dysbiosis, yet effective microbiome-targeted therapeutics remain limited. Here, we investigated the anti-inflammatory and microbiome-modulating activities of metabolites isolated from Garcinia brassii, an endemic species of the Australian Wet Tropics. Five compounds, including a new natural product named garcitine, were isolated and structurally characterised. In human immune cells, garcinol and garcinia biflavonoid 1 significantly suppressed lipopolysaccharide-induced production of IL-1{beta}, IL-6, and TNF without detectable cytotoxicity, while parvifoliol F selectively inhibited IL-1{beta} release. Therapeutic efficacy was further evaluated in a TNBS-induced murine colitis model, where garcinia biflavonoid 1 and parvifoliol F significantly reduced colonic inflammation and improved histopathological outcomes. 16S rRNA sequencing demonstrated that both compounds restored gut microbial homeostasis by reversing colitis-associated dysbiosis and reducing inflammation-associated microbial signatures. Functional pathway prediction further suggested suppression of pro-inflammatory microbial metabolic pathways following treatment. Together, these findings demonstrate that Garcinia-derived metabolites alleviate experimental colitis through coordinated immunomodulatory and microbiome-reprogramming mechanisms and identify garcinia biflavonoid 1 and parvifoliol F as promising candidates for microbiome-targeted IBD therapeutics.

Esophageal adenocarcinoma (EAC) has a poor five-year survival rate and one of the fastest-rising incidences of any cancer. The presence of dysplasia in Barrett's esophagus (BE) is the main risk factor for EAC development and guides clinical management. Unfortunately, the current histopathological diagnosis of dysplasia is unreliable, with poor inter-observer agreement, highlighting the need for novel biomarkers that can improve diagnostic accuracy. Here, we performed transcriptome profiling across the full spectrum of BE-related neoplasia in 85 samples to delineate gene expression alterations in progressively worse disease stages and identify biomarkers that could complement histopathology to improve the detection of dysplasia and EAC in endoscopic biopsy specimens. Differential gene expression and pathway analyses revealed that the most extensive transcriptional changes occurred during the transition from normal squamous (NSq) to non-dysplastic BE (NDBE), consistent with metaplastic transformation. Compared to NDBE, dysplasia was characterized by enhanced cellular growth and proliferation; upregulation of immune processes and oncogenic signaling pathways were present in EAC. Using machine learning approaches, we identified a novel five-gene panel suitable for a potential RNAseq-based diagnostic test (SLC11A1, IL36A, LUCAT1, MIR215, RNU6-954P) and performed an initial validation of this signature in an additional 51 samples. We also identified several potential novel immunohistochemical markers that may warrant further evaluation, including TREM1, CXCL5, OSM, and motilin. In summary, by delineating transcriptional changes across the full disease spectrum, this study identifies several candidate biomarkers for improving current diagnostic methods for Barrett's dysplasia and EAC.

Early- and average-onset colorectal cancer (CRC) are separated at age 50, but whether this defines a biological threshold remains unclear. To clarify this, we identified molecular profiles in nine harmonised cBioPortal CRC cohorts (4,609 patients) by fitting Bernoulli mixture models to 31 repair-state, genomic-burden and gene-alteration features, excluding age, sex and tumour site, and compared their prevalence using <50/[&ge;]50 and decade-resolved groups. Four profiles captured conventional/CIN-like (P1), intermediate MSS (P2), KRAS/PI3K/APC-rich (P3) and hypermutated/MSI-high (P4) states along a left-to-right gradient. Although molecular identities remained stable, profile prevalence followed non-linear P1/P4 and opposing linear P2/P3 age trajectories. Profile-prevalence patterns did not track chronological proximity: profile composition at 30-39 differed from 50-59 but not clearly from 60-69. The age-50 threshold captured only 17.8% of decade-resolved deviance, whereas the optimal age-70 cut-off retained only 51.3%. Validation in 2,579 non-overlapping MSK-IMPACT patients (2,476 age-evaluable) reproduced molecular-feature patterns (r=0.97-0.98), age trajectories (r=0.92) and limited binary-threshold performance: age 50 and the optimal age-66 cut-off retained 12.7% and 45.1%, respectively. Thus, age reorganizes the prevalence of shared CRC states rather than defining a biological threshold at age 50.

Modern lifestyle factors have altered gut microbiota composition and function. Bacteria in the Clostridia class modulate mucosal immune responses through various mechanisms including production of secondary bile acids (SBA). Here, we present a novel system to study how the SBA isodeoxycholic acid (isoDCA) regulates host immunity. Through targeted mutagenesis of bile acid epimerization genes, we engineered Ruminococcus gnavus to ablate isoDCA production. Combining R. gnavus (WT or KO) with Peptacetobacter hiranonis created a two-member consortium that toggles isoDCA production on or off while keeping all other variables constant. Using this system, we demonstrate that isoDCA induces colonic lamina propria ROR{gamma}t{square} Foxp3{square} regulatory T cells (pTregs) through a mechanism requiring both the Takeda G protein-coupled receptor 5 (TGR5) and the Farnesoid X receptor (FXR). Engraftment of this isoDCA+ consortium protected against colitis in an adoptive T cell transfer model by reshaping the microbiota and suppressing host inflammation.

Juvenile systemic sclerosis (jSSc) is a rare autoimmune disease marked by skin fibrosis and multi-organ involvement. Autologous stem cell transplantation (ASCT) is an emerging therapy for severe, treatment-refractory jSSc, but its effects on immune cell dynamics remain poorly understood. PBMCs were collected from three patients with jSSc before ASCT and at 6, 12, and 24 months post-ASCT. Patient and healthy control samples were profiled using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq). We focused on monocytes, given their role in fibrosis-promoting inflammation. To detect longitudinal trends, pseudobulked gene expression (log scale) was regressed against time since ASCT. This approach identified widespread changes in jSSc monocytes, including decreased expression of systemic sclerosis-linked genes, such as SERPINE1. On the pathway level, NF-{kappa}B-associated inflammatory signaling was elevated in jSSc monocytes at baseline relative to healthy controls and decreased progressively post-ASCT. Genes related to mitochondrial function and oxidative phosphorylation progressively increased in expression after ASCT, suggesting a shift in metabolic state. Compositional changes in monocyte subpopulations were also identified and may have contributed to longitudinal gene expression patterns. Together, these findings characterize the dynamic immune changes in jSSc following ASCT and highlight a widely applicable longitudinal modeling framework for single-cell data.