To Örebro University

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.

BACKGROUND AND AIMS: The diagnostic and prognostic properties of anti-integrin αvβ6 IgG autoantibodies in ulcerative colitis (UC) are poorly understood. We aimed to assess the diagnostic performance of anti-integrin αvβ6 autoantibodies and examine their association with disease outcomes.

METHODS: Serum samples from a Swedish inception cohort of patients with suspected inflammatory bowel disease (IBD, n=473) were analysed using an in-house fluorescence enzyme immunoassay based on EliA™technology. Findings were validated in a Norwegian population-based inception cohort (n=570). Diagnostic performance was assessed by calculating the area under the curve (AUC) with 95% confidence intervals (CIs) and determining sensitivity and specificity. Reclassification was evaluated using the net reclassification index.

RESULTS: In the discovery cohort, patients with UC, IBD-unclassified, or colonic Crohn's disease exhibited higher median autoantibody levels compared to symptomatic and healthy controls. In the validation cohort, the autoantibody demonstrated 79% sensitivity and 94% specificity for UC vs symptomatic controls at a cut-off of 400 UA/l. Its diagnostic performance (AUC=0.92, 95%CI 0.89-0.95) was superior to hs-CRP (AUC=0.65, 95%CI 0.60-0.70, P<0.001) and faecal calprotectin (fcalpro) (AUC=0.88, 95%CI 0.84-0.92, P=0.09). Combining the autoantibody with fcalpro further improved diagnostic accuracy (AUC=0.97, 95%CI 0.95-0.98) and patient reclassification (P<0.001). Autoantibody positivity was associated with a severe phenotype of UC, characterised by increased inflammatory activity and higher IL-17A and granzyme B levels. Higher autoantibody levels were linked to an aggressive disease course, remaining stable in aggressive UC but decreasing in indolent disease (P=0.003).

CONCLUSIONS: Anti-integrin αvβ6 is a reliable diagnostic and prognostic marker for UC, with potential clinical implementation.

BACKGROUND: Recent genetic and transcriptomic data highlight the need for improved molecular characterisation of inflammatory bowel disease (IBD). Proteomics may advance the delineation of IBD phenotypes since it accounts for post-transcriptional modifications. AIM: We aimed to assess the IBD spectrum based on inflammatory serum proteins and identify discriminative patterns of underlying biological subtypes across multiple European cohorts.

METHODS: Using proximity extension methodology, we measured 86 inflammation-related serum proteins in 1551 IBD patients and 312 healthy controls (HC). We screened for proteins exhibiting significantly different levels among IBD subtypes and between IBD and HC. Classification models for differentiating between Crohn's disease (CD) and ulcerative colitis (UC) were employed to explore the IBD spectrum based on estimated probability scores.

RESULTS: Levels of multiple proteins, such as IL-17A, MMP-10, and FGF-19, differed (fold-change>1.2; FDR<0.05) between ileal vs colonic IBD. Using multivariable models, a protein signature reflecting the IBD spectrum was identified, positioning colonic CD between UC and ileal CD, which were at opposite ends of the spectrum. Based on area under the curve (AUC) estimates, classification models more accurately differentiated UC from ileal CD (median AUCs>0.73) than colonic CD (median AUCs<0.62). Models differentiating colonic CD from ileal CD demonstrated intermediate performance (median AUCs 0.67-0.69).

CONCLUSION: Our findings in serum proteins support the presence of a continuous IBD spectrum rather than a clear separation of CD and UC. Within the spectrum, disease location may reflect a more similar disease than CD vs UC, as colonic CD resembled UC more closely than ileal CD.

Purpose: There is a need for diagnostic and prognostic biosignatures to improve long-term outcomes in inflammatory bowel disease (IBD). Here, we describe the establishment of the Swedish Inception Cohort in IBD (SIC-IBD) and demonstrate its potential for the identification of such signatures.

Participants: Patients aged >= 18 years with gastrointestinal symptoms who were referred to the gastroenterology unit due to suspected IBD at eight Swedish hospitals between November 2011 and March 2021 were eligible for inclusion.

Findings to date: In total, 367 patients with IBD (Crohn's disease, n=142; ulcerative colitis, n=201; IBD-unclassified, n=24) and 168 symptomatic controls were included. In addition, 59 healthy controls without gastrointestinal symptoms were recruited as a second control group. Biospecimens and clinical data were collected at inclusion and in patients with IBD also during follow-up to 10 years. Levels of faecal calprotectin and high-sensitivity C-reactive protein were higher in patients with IBD compared with symptomatic controls and healthy controls. Preliminary results highlight the potential of serum protein signatures and autoantibodies, as well as results from faecal markers, to differentiate between IBD and symptomatic controls in the cohort. During the first year of follow-up, 37% (53/142) of the patients with Crohn's disease, 24% (48/201) with ulcerative colitis and 4% (1/24) with IBD-U experienced an aggressive disease course.

Future plans: We have established an inception cohort enabling ongoing initiatives to collect and generate clinical data and multi-omics datasets. The cohort will allow analyses for translation into candidate biosignatures to support clinical decision-making in IBD. Additionally, the data will provide insights into mechanisms of disease pathogenesis.

Background: Inter-individual differences in drug clearance are common in inflammatory bowel disease (IBD) patients treated with biologics. Associations between inflammatory markers, clinical features, and drug levels may indicate that the inflammatory immune response influences the pharmacokinetics of biologics. We assessed the impact of inflammatory and immunity-related proteins on drug levels in IBD.

Methods: Serum samples from 144 IBD patients initiating biologics in a prospective multicentre cohort were analysed using AFIAS-3 (Boditech Med Inc. Korea). The primary outcome was drug levels after the end of induction treatment (post-induction), analysed as continuous variables or categorised into high or low groups based on the median. Correlations were assessed using the Pearson’s correlation coefficients with false discovery rate (FDR) adjustment. Predictive capacity of clinical and protein data was evaluated with regularised linear regression models. Analyses were stratified by treatment (infliximab, adalimumab, vedolizumab) and diagnosis (Crohn’s disease (CD), ulcerative colitis (UC)).

Results: Patient demographics and clinical characteristics are provided in Table 1. Median (interquartile range, IQR) post-induction infliximab levels were 5.3 (2.2-9.5) μg/ml in patients with CD (n=34) and 3.8 (1.7-5.6) μg/ml in UC (n=33). The corresponding levels were 12.5 (7.0-16.1) μg/ml (n=36) and 8.8 (5.7-12.7) μg/ml (n=13) for adalimumab, 13.9 (6.4-24.4) μg/ml (n=19) and 17.7 (9.8-22.6) μg/ml (n=9) for vedolizumab. Principal component analyses revealed potential baseline protein profile differences between patients with high vs low post-induction levels for infliximab (p=0.09) and adalimumab (p=0.07) in CD, but no differences for vedolizumab (p=0.78) or UC. Individual protein analyses indicated that patients with higher drug levels post-induction seemed to display lower baseline estimates of inflammatory proteins compared to those with lower drug levels. However, statistical significance after correction for multiple comparisons was only observed for CDCP1 in adalimumab-treated CD patients (PFDR=0.04) (Figure 1). Additionally, baseline Flt31 (r=-0.57, PFDR=0.03) and Wisp1 (r=-0.61, PFDR=0.02) correlated with post-induction s-adali-mumab levels in CD, while Mcp3 (r=-0.89, PFDR=0.04) and Frgamma (r=-0.87, PFDR=0.05) correlated in UC. Integrating baseline protein data in prediction models for post-induction drug levels did not improve their accuracy compared to models based on only clinical and biochemical variables.

Conclusion: Inflammatory protein levels may influence post-induction drug levels in IBD, particularly in adalimumab treated CD patients. This insight could aid in optimising personalised treatment.

As the volume of plastic waste from electrical and electronic equipment (WEEE) continues to rise, a significant portion is disposed of in the environment, with only a small fraction being recycled. Both disposal and recycling pose unknown health risks that require immediate attention. Existing knowledge of WEEE plastic toxicity is limited and mostly relies on epidemiological data and association studies, with few insights into the underlying toxicity mechanisms. Therefore, this study aimed to perform comprehensive chemical screening and mechanistic toxicological assessment of WEEE plastic-associated chemicals. Chemical analysis, utilizing suspect screening based on high-resolution mass spectrometry, along with quantitative target chemical analysis, unveiled numerous hazardous compounds including polyaromatic compounds, organophosphate flame retardants, phthalates, benzotriazoles, etc. Toxicity endpoints included perturbation of morphological phenotypes using the Cell Painting approach, inflammatory response, oxidative stress, and endocrine disruption. Results demonstrated that WEEE plastic chemicals altered the phenotypes of the cytoskeleton, endoplasmic reticulum, and mitochondria in a dose-dependent manner. In addition, WEEE chemicals induced inflammatory responses in resting macrophages and altered inflammatory responses in lipopolysaccharide-primed macrophages. Furthermore, WEEE chemicals activated the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, indicating oxidative stress, and the aryl hydrocarbon receptor (AhR). Endocrine disruption was also observed through the activation of estrogenic receptor-α (ER-α) and the induction of anti-androgenic activity. The findings show that WEEE plastic-associated chemicals exert effects in multiple subcellular sites, via different receptors and mechanisms. Thus, an integrated approach employing both chemical and toxicological methods is essential for comprehensive assessment of the toxicity mechanisms and cumulative chemical burden of WEEE plastic-associated chemicals.

Background: Inflammatory bowel disease (IBD) is a chronic disease where the aetiology is suggested to involve genetic, microbial, and environmental factors as well as their interactions. Though the bacterial component of the gut microbiome has been of substantial interest in recent years, the mycobiome (fungal community) is less studied. Recent studies have, however, shown that the efficacy of faecal microbiota transplantation in ulcerative colitis (UC) is dependent on the pre-transplantation of host mycobiome. Furthermore, there is an association between anti-Saccharomyces cerevisiae antibodies (ASCA) for Crohn’s disease (CD), but not UC. We aimed to characterize the gut mycobiome of incident IBD patients.

Methods: In this study, we analysed samples from 216 adult treatment-naïve patients in the NORDTREAT cohort, who were referred on suspicion of IBD. Patients were recruited in 4 Nordic countries from 2022 to 20231. The diagnosis of IBD was based on internationally accepted criteria (UC n=72, CD n=48 and IBD-unclassified [IBD-U] n=8) or, if IBD was ruled out, categorized as symptomatic controls (SC, n=88). Stool samples and colonic biopsies were collected at inclusion. From these, DNA was extracted, and ITS2 amplicon sequencing was performed as previously described2. The primary end-point of subsequent bioinformatic analysis was relative abundance at the genus level. The secondary end-points were alpha and beta diversity, as well as a correlation analysis between faecal samples and biopsies.

Results: The relative abundance at genus level was dominated by Saccharomyces in stool samples whereas this genus was markedly reduced in biopsies and Malassezia was found to be increased (see figure 1, data not shown for IBD-U). We did not observe statistically significant differences in relative abundance between patient groups. Alpha diversity and beta diversity did not significantly differ for fungi across patient groups, neither in stool nor in biopsies. However, canonical correspondence analysis of the dominant genera between biopsies and faecal samples demonstrated a low constrained proportion in SC (0,2), increasing from UC (0,25) through CD (0,5) indicating that in CD, the mycobiome of stool and mucosa is more similar than in the UC and SC.

Conclusion: This study of the gut mycobiome in new onset and treatment naïve IBD patients is the largest so far and does not find evidence of disease specific differences. Previous reports with inconsistent findings regarding alterations of the mycobiome in IBD patients might rather reflect treatment effects than a role of the mycobiome in disease aetiology.

References:

1.Fejrskov A, Füchtbauer JD, Davíðsdóttir LG, et al. Novel biomarker profiles to improve individual diagnosis and prognosis in patients with suspected inflammatory bowel disease: protocol for the Nordic inception cohort study (NORDTREAT). BMJ Open. 2024;14(5):e083144. doi:10.1136/bmjopen-2023-083144

2.Rühlemann MC, Solovjeva MEL, Zenouzi R, et al. Gut mycobiome of primary sclerosing cholangitis patients is characterised by an increase of Trichocladium griseum and Candida species. Gut. Published online October 25, 2019. doi:10.1136/gutjnl-2019-320008

Metal additive manufacturing (AM), also known as industrial 3D printing, has revolutionized modern industry, enabling the creation of complex, high-performance components across sectors such as aerospace, automotive, and biomedicine. While the printing process itself is often well-contained, a critical and understudied phase – post-processing – has emerged as a source of potentially hazardous airborne particulate matter. These emissions may pose health risks to workers, particularly through interaction with the immune system, which serves as the body's first line of defense and a sentinel of environmental stressors. Yet, limited data exist on the physicochemical properties and immunotoxicological impact of these particles. This study aimed to assess the immunological consequences of particle emissions released during the post-processing of metallic AM alloys, using a human macrophage model and a multi-omics framework.

Airborne particles were collected directly from an operational AM facility using a cascade impactor, separating them into five size fractions, ranging from coarse (>2.5 μm) to nanoscale (<250 nm). A comprehensive physicochemical characterization was performed using scanning electron microscopy with energy-dispersive spectroscopy and X-ray photoelectron spectroscopy. The emitted particles were highly heterogeneous, with irregular, sharp morphologies, and exhibited increased surface oxidation compared to virgin feedstock powders. Functional toxicological assessments were performed in human macrophages, including transmission electron microscopy to evaluate particle uptake. Macrophages, both resting and lipopolysaccharide-primed, displayed potent and dose-dependent inflammatory responses, as seen by elevated secretion of several cytokines (e.g., IL-1β, IL-6). RNA sequencing revealed profound alterations in macrophage gene expression, including dysregulation of NF-κB signaling, cellular senescence, and lipid metabolism pathways. Gene set enrichment analysis indicated broader perturbations in immune regulation and macrophage homeostasis. Non-targeted metabolomics demonstrated significant changes in intracellular metabolic profiles. Specifically, there was an upregulation of numerous lipids and a suppression of several metabolites involved in immunomodulation and cellular energy homeostasis, including tryptophan, NAD, and phenylalanine. Integrated multi-omics analysis revealed a coordinated crosstalk between transcriptional and metabolic responses, pointing to an acute and multifaceted inflammatory reprogramming of macrophages in response to post-processing AM particles.

In conclusion, this study provides the first integrative multi-omics characterization of human immune cell responses to airborne particulate emissions from metal AM post-processing. These results not only advance the field of nanosafety in industrial AM environments but also underscore the urgent need for targeted risk mitigation strategies during post-processing.

BACKGROUND AND AIMS: Biomarkers are needed to identify individuals at elevated risk of inflammatory bowel disease (IBD). This study aims to identify protein signatures predictive of IBD.

METHODS: Using large population-based cohorts (n≥180,000), blood samples were obtained from individuals who later in life were diagnosed with IBD and compared to age and sex-matched controls, free from IBD during follow-up. 178 proteins were measured on Olink platforms. We used machine learning methods to identify protein signatures of preclinical disease in the discovery cohort (n=312). Their performance was validated in an external preclinical cohort (n=222) and assessed in an inception cohort (n=144) and a preclinical twin cohort (n=102).

RESULTS: In the discovery cohort, a signature of 29 proteins differentiated preclinical Crohn's disease (CD) cases from controls, with an area under the curve (AUC) of 0.85. Its performance was confirmed in the preclinical validation (AUC=0.87) and the inception cohort (AUC=1.0). In preclinical samples, downregulated (but not upregulated) proteins related to gut barrier integrity and macrophage functionality correlated with time to diagnosis of CD. The preclinical ulcerative colitis (UC) signature had a significant, albeit lower, predictive ability in the discovery (AUC=0.77), validation (AUC=0.67), and inception cohorts (AUC=0.95). The preclinical signature for CD demonstrated an AUC of 0.89 when comparing twins with preclinical CD to matched external healthy twins, but its predictive ability was lower (AUC=0.58; P=.04) when comparing them with their healthy twin siblings, i.e., when accounting for genetic and shared environmental factors.

CONCLUSION: We identified protein signatures for predicting a future diagnosis of CD and UC, validated across independent cohorts. In the context of CD, the signature offers potential for early prediction.

Cancer is the leading cause of death from disease in children. Survival depends not only on surgery, cytostatic drugs, and radiation but also on systemic immune responses. Factors influencing these immune responses in children of different ages and tumor types are unknown. Novel immunotherapies can enhance anti-tumor immune responses, but few children have benefited, and markers of effective responses are lacking. Here, we present a systems-level analysis of immune responses in 191 children within a population-based cohort with diverse tumors and reveal that age and tumor type shape immune responses differently. Systemic inflammation and cytotoxic T cell responses correlate with tumor mutation rates and immune cell infiltration. Clonally expanded T cell responses are rarely detected in blood or tumors at diagnosis but are sometimes elicited during treatment. Expanded T cells are similarly regulated in children and adults with more immunogenic cancers. This research aims to facilitate the development of precision immunotherapies for children with cancer.