To Örebro University

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.

Background: Mental health (MH) has been reported to be poorer among patients with inflammatory bowel disease (IBD) than general population. However, it is not known whether MH is more driven by inflammation itself or related to gastrointestinal (GI) symptoms. Also, the dynamics of MH following IBD diagnosis is not well understood.

Methods: In the Swedish Inception Cohort of IBD (SIC-IBD), patients with Crohn’s disease (CD), ulcerative colitis (UC) and unclassified IBD (IBD-U) as well as symptomatic controls (SC) and healthy controls (HC) filled in Patient Health Questionnaire-9 (PHQ-9), a validated screening tool for depression. Patients completed PHQ-9 at diagnosis and at one year follow-up while the controls completed it once. Disease outcome was defined at one year based on requirement of advanced treatments/ surgery.

Results: In total, 286 individuals (16 HC, 89 SC, 62 CD, 104 UC, 15 IBD-U) completed the questionnaire at baseline. HC had significantly lower PHQ-9 score, (fewer depressive symptoms), at baseline compared to all the other groups (p<0.01). The baseline PHQ-9 score was not significantly different between SC and CD, UC and IBD-U patients (p=0.06).

At one year follow-up, 38 CD and 53 UC patients completed the PHQ-9. Between baseline and follow-up, UC patients had a significant drop in their PHQ-9 score (p<0.0000001), whereas CD patients did not have any significant change in their PHQ-9 score (p=0.06). Furthermore, UC patients had significantly lower PHQ-9 score compared with CD patients at follow-up (p=0.04, Figure 1).

Baseline PHQ-9 score was not correlated with calprotectin at baseline neither in UC nor CD patients (p=0.7 and 0.5 respectively). Also, there was no positive correlation between PHQ-9 score change and calprotectin change in either UC or CD patients, and neither baseline nor follow-up PHQ-9 scores were significantly different in patients with poor or good outcome (p>0.05). When analysed separately by sex, there was still no correlation between baseline PHQ-9 score and outcome in neither CD nor UC patients (p>0.05)

Conclusion: IBD patients have at the time of diagnosis more depressive symptoms than HC, but not different from SC. Depressive symptoms are more related to GI symptoms than IBD specific inflammation or disease outcome. UC patients show more of an improvement in their depressive symptoms one year after diagnosis than CD patients.

BACKGROUND: Sepsis is defined as a dysfunctional host response to infection. The diverse clinical presentations of sepsis pose diagnostic challenges and there is a demand for enhanced diagnostic markers for sepsis as well as an understanding of the underlying pathological mechanisms involved in sepsis. From this perspective, metabolomics has emerged as a potentially valuable tool for aiding in the early identification of sepsis that could highlight key metabolic pathways and underlying pathological mechanisms.

OBJECTIVE: The aim of this investigation is to explore the early metabolomic and lipidomic profiles in a prospective cohort where plasma samples (n = 138) were obtained during ambulance transport among patients with infection according to clinical judgement who subsequently developed sepsis, patients who developed non-septic infection, and symptomatic controls without an infection.

METHODS: Multiplatform metabolomics and lipidomics were performed using UHPLC-MS/MS and UHPLC-QTOFMS. Uni- and multivariable analysis were used to identify metabolite profiles in sepsis vs symptomatic control and sepsis vs non-septic infection.

RESULTS: Univariable analysis disclosed that out of the 457 annotated metabolites measured across three different platforms, 23 polar, 27 semipolar metabolites and 133 molecular lipids exhibited significant differences between patients who developed sepsis and symptomatic controls following correction for multiple testing. Furthermore, 84 metabolites remained significantly different between sepsis and symptomatic controls following adjustment for age, sex, and Charlson comorbidity score. Notably, no significant differences were identified in metabolites levels when comparing patients with sepsis and non-septic infection in univariable and multivariable analyses.

CONCLUSION: Overall, we found that the metabolome, including the lipidome, was decreased in patients experiencing infection and sepsis, with no significant differences between the two conditions. This finding indicates that the observed metabolic profiles are shared between both infection and sepsis, rather than being exclusive to sepsis alone.

Background: Faecal biomarkers can be used to assess inflammatory bowel disease (IBD).

Aim: To explore the performance of some promising biomarkers in diagnosing and predicting disease course in IBD.

Methods: We included 65 patients with treatment-na & iuml;ve, new-onset Crohn's disease (CD), 90 with ulcerative colitis (UC), 67 symptomatic controls (SC) and 41 healthy controls (HC) in this prospective observational study. We analysed faecal samples for calprotectin (FC), myeloperoxidase (MPO), human neutrophil lipocalin (HNL), eosinophil cationic protein ECP and eosinophil-derived neurotoxin (EDN) and compared markers among groups. We assessed the diagnostic capability of biomarkers with receiver operating characteristic curves. Clinical disease course was determined for each patient with IBD and analysed the association with biomarkers by logistic regression.

Results: All markers were elevated at inclusion in patients with IBD compared with HC (p < 0.001) and SC (p < 0.001). FC (AUC 0.85, 95% CI: 0.79-0.89) and MPO (AUC 0.85, 95% CI: 0.80-0.89) showed the highest diagnostic accuracy in distinguishing IBD from SC. The diagnostic ability of biomarkers differed between IBD subtypes with the highest performance for FC and MPO in CD. The diagnostic accuracy was further improved by combining FC and MPO (p = 0.02). Levels of FC, MPO and HNL at inclusion were predictive of an aggressive disease course with MPO showing the strongest association (p = 0.006).

Conclusions: This study provides new insight into the diagnostic and prognostic capability of neutrophil and eosinophil biomarkers in IBD and suggests that MPO, alone or in combination with FC, may add to the diagnostic power of faecal biomarkers.

Improved biomarkers are needed for pediatric inflammatory bowel disease. Here we identify a diagnostic lipidomic signature for pediatric inflammatory bowel disease by analyzing blood samples from a discovery cohort of incident treatment-naïve pediatric patients and validating findings in an independent inception cohort. The lipidomic signature comprising of only lactosyl ceramide (d18:1/16:0) and phosphatidylcholine (18:0p/22:6) improves the diagnostic prediction compared with high-sensitivity C-reactive protein. Adding high-sensitivity C-reactive protein to the signature does not improve its performance. In patients providing a stool sample, the diagnostic performance of the lipidomic signature and fecal calprotectin, a marker of gastrointestinal inflammation, does not substantially differ. Upon investigation in a third pediatric cohort, the findings of increased lactosyl ceramide (d18:1/16:0) and decreased phosphatidylcholine (18:0p/22:6) absolute concentrations are confirmed. Translation of the lipidomic signature into a scalable diagnostic blood test for pediatric inflammatory bowel disease has the potential to support clinical decision making.

INTRODUCTION: Maternal SARS-CoV-2 infection can affect pregnancy outcome, but the placental response to and the effect of timing of infection is not well studied. The aim of this study was to investigate the placental levels of inflammatory and cardiovascular markers in pregnancies complicated by SARS-CoV-2 infection compared to non-infected pregnancies, and to investigate whether there was an association between time point of infection during pregnancy and placental inflammatory and cardiovascular protein levels.

METHODS: Placental samples from a prospectively recruited pregnancy cohort of SARS-CoV-2-infected (n = 53) and non-infected (n = 50) women were analysed for 177 inflammatory and cardiovascular proteins, using an antibody-based proximity extension assay. In the SARS-CoV-2-infected group, half of the women were infected before 20 weeks of gestation, and five women were hospitalised for severe SARS-CoV-2 infection. Single-protein analyses were performed with linear mixed effects models, followed by Benjamini-Hochberg correction for multiple testing. Multi-protein analyses were performed using principal component analysis and machine learning algorithms.

RESULTS: The perinatal outcomes and the placental levels of inflammatory or cardiovascular proteins in women with SARS-CoV-2 infection were similar to those in non-infected women. There were no differences in inflammatory or cardiovascular protein levels between early and late pregnancy SARS-CoV-2 infection, nor any linear correlations between protein levels and gestational age at time of infection.

DISCUSSION: Women with SARS-CoV-2 infection during pregnancy without clinical signs of placental insufficiency have no changes in inflammatory or cardiovascular protein patterns in placenta at time of birth regardless of the timing of the infection.

Background: We aimed to identify protein signatures predictive of a future diagnosis of inflammatory bowel disease (IBD).

Methods: We conducted a case-control study, nested within large population-based cohorts with biorepositories. Samples were obtained from individuals who later in life were diagnosed with IBD (preclinical cases) and compared with age and sex-matched individuals who remained free from IBD during follow-up (controls). Using proximity extension assays (Olink, Uppsala), we measured 176 proteins. We applied regularized logistic regression to identify protein signatures of preclinical disease in serum from the discovery cohort (n=312). Their performance was validated in an external preclinical cohort (n=222). The biological relevance of identified proteins was further assessed in an inception cohort (n=144). Finally, we used an IBD twin cohort (n=327) to examine the impact of genetic and shared environmental factors on identified proteins.

Results: We identified 34 proteins associated with preclinical Crohn’s disease (CD) in the discovery cohort (Pfalse discovery rate <0.10), with 9 confirmed in the validation cohort (Pfalse discovery rate <0.05). For preclinical ulcerative colitis (UC), 45 proteins were identified and 12 validated (Fig. 1A-B). In the discovery cohort, a signature of 29 proteins differentiated preclinical CD cases from controls with an AUC of 0.85 (Fig. 1G). Its performance was confirmed when applied to the preclinical validation cohort (AUC=0.84, Fig. 1H). Moreover, the signature had excellent capacity to differentiate newly diagnosed CD from healthy controls in the inception cohort (AUC = 0.99, Fig. 1I). The preclinical UC signature had a significant, but albeit lower, predictive capacity in the discovery (AUC=0.77), validation (AUC=0.67) and inception cohort (AUC=0.90, Fig. 1G-I).15 of 17 proteins associated with preclinical IBD demonstrated significantly higher intra-pair correlation coefficients in healthy monozygotic- compared to dizygotic twin pairs, indicating an influence from genetic factors on the regulation of these protein markers. The preclinical signature for CD demonstrated an AUC of 0.87 when comparing twins with preclinical CD (n=10) to matched external healthy twins. However, its predictive capacity was lower when comparing preclinical CD twins with their healthy twin siblings (AUC=0.58), i.e., when accounting for genetic and shared environmental factors. The difference in AUC estimates in the twin cohort was not significant (P=0.07).

Background: Better prognostic measures for ulcerative colitis (UC) could significantly advance patient care. While the prognostic capacity of circulating proteins in UC has been explored, the role of mucosal proteins remains largely unknown. We examined mucosal protein markers in patients with incident ulcerative colitis and evaluated their prognostic value.

Methods: Biopsies from macroscopically inflamed colonic/rectal mucosa of adult patients in the Swedish inception cohort of IBD (SIC IBD) were obtained at diagnosis of UC. Patients were followed prospectively, and clinical data were recorded after 3 and 12 months. Disease course was categorised as indolent or aggressive at 12 months, based on a composite outcome of colectomy, hospital admission for active disease, treatment refractoriness towards ≥2 biological agents; the use of >2 courses of corticosteroids, or a cumulative dose of >2.5 g. Relative estimates of 162 protein markers were assessed in homogenised tissue supernatants, using proximity extension assay technology (Olink Proteomics, Uppsala, Inflammation and Oncology II panel). Mann-Whitney U test, with Benjamini-Hochberg correction was used to identify differentially regulated mucosal proteins in aggressive vs indolent disease course, with a 5% false discovery rate (FDR). Smoothly clipped absolute deviation regularised logistic regression models were used to identify prognostic signatures distinguishing aggressive from indolent disease course. Performance was estimated in a leave-one-out cross-validation and reported as the area under the receiver operating characteristic (ROC) curve (AUC).

Results: 117 patients provided a macroscopically inflamed colonic/rectal biopsy at diagnosis of UC. Basic demographics and clinical characteristics are presented in Table 1. Relative protein levels of WFdc2 and CCL20 were significantly lower in lysates from patients developing an aggressive course vs patients developing an indolent course, while estimates of MMP1, CCL11, WISP-1, OPG, RSPO3 and VEGFR2 were higher (Figure 1A). Regularized logistic regression identified signatures restricted to 28 proteins, distinguishing aggressive from indolent UC courses, yielding an AUC of 0.68 (95% confidence interval (CI): 0.56-0.80) for left-out samples (Figure 1B). Incorporating extent of inflammation at diagnosis in the model improved the AUC to 0.71 (95% CI: 0.60-0.83).

Conclusion: We identified prognostic mucosal protein signatures associated with future course of ulcerative colitis by analysing inflamed mucosal biopsies that were obtained at diagnosis. These protein markers may highlight pathways of relevance for ulcerative colitis outcomes.