Henning Glerup

Neutrophil Extracellular Traps in Ulcerative Colitis: A Proteome Analysis of Intestinal Biopsies

Background:The etiology of the inflammatory bowel diseases, including ulcerative colitis (UC), remains incompletely explained. We hypothesized that an analysis of the UC colon proteome could reveal novel insights into the disease etiology. Methods:Mucosal colon biopsies were taken by endoscopy from noninflamed tissue of 10 patients with UC and 10 controls. The biopsies were either snap-frozen for protein analysis or prepared for histology. The protein content of the biopsies was characterized by high-throughput gel-free quantitative proteomics, and biopsy histology was analyzed by light microscopy and confocal microscopy. Results:We identified and quantified 5711 different proteins with proteomics. The abundance of the proteins calprotectin and lactotransferrin in the tissue correlated with the degree of tissue inflammation as determined by histology. However, fecal calprotectin did not correlate. Forty-six proteins were measured with a statistically significant differences in abundances between the UC colon tissue and controls. Eleven of the proteins with increased abundances in the UC biopsies were associated with neutrophils and neutrophil extracellular traps. The findings were validated by microscopy, where an increased abundance of neutrophils and the presence of neutrophil extracellular traps by extracellular DNA present in the UC colon tissue were confirmed. Conclusions:Neutrophils, induced neutrophil extracellular traps, and several proteins that play a part in innate immunity are all increased in abundance in the morphologically normal colon mucosa from patients with UC. The increased abundance of these antimicrobial compounds points to the stimulation of the innate immune system in the etiology of UC.

Gastric emptying: a comparison of three methods

Objective. A better understanding of the clinical relevance of delayed gastric emptying (e.g. in diabetes) requires a simple, easily accessible and inexpensive method for measuring it. Two “new” methods for measuring gastric emptying of liquids (the paracetamol absorption test and the 13C-acetate breath test) are compared with the gold standard (gastric emptying scintigraphy (GES)). Material and methods. The three techniques were used simultaneously in 10 healthy subjects. A gastric emptying time-retention curve was drawn for each technique and the results were compared at the 75%, 50% and 25% retention quartiles. Results. Agreement was found between the paracetamol absorption test and GES (p=0.95; Hotellings T2 test). Using the Wagner-Nelson one compartment correction produced a retention curve for the 13C-acetate breath test statistically significantly below GES (p<0.01). Conclusion. In healthy subjects, the paracetamol absorption test produced results comparable to those of liquid GES, but not to the results of the 13C-acetate breath test.

The effect of a single oral megadose of vitamin D provided as either ergocalciferol (d2) or cholecalciferol (d3) in alcoholic liver cirrhosis

Objective The goal of this study was to examine the effects of a single oral dose of 300 000 international units of either ergocalciferol (D2) or cholecalciferol (D3) on the plasma levels of 25-hydroxyvitamin D in patients with alcoholic liver cirrhosis. Methods Inclusion criteria for this study were diagnosis of alcoholic liver cirrhosis and plasma levels of 25-hydroxyvitamin D less than 25 nmol/l. At baseline, patients were divided into Child–Pugh groups A, B, or C and were given one oral dose of 300 000 international units of ergocalciferol (D2 group, N=23) or cholecalciferol (D3 group, N=13). Plasma concentrations of 25(OH) vitamin D and vitamin D-binding protein were measured on days 0, 7, 30, and 90. Results On days 7 and 30, patients from the D3 group had higher vitamin D levels than patients from the D2 group (P<0.05). On day 7, vitamin D levels were found to correlate with Child–Pugh scores from patients in the D3 group. For patients in the D2 group, there was a positive correlation between vitamin D and vitamin D-binding protein as indicated by the area under the concentration versus time curves (Spearmen’s &rgr;=0.64 P<0.001). Conclusion In patients with alcoholic liver cirrhosis, a single oral megadose of cholecalciferol was more effective than ergocalciferol in the treatment of vitamin D deficiency. Severe liver disease and low levels of vitamin D-binding protein were predictors for poor treatment outcomes.

Proteome analysis of rheumatoid arthritis gut mucosa

Rheumatoid arthritis (RA) is an inflammatory joint disease leading to cartilage damage and ultimately impaired joint function. To gain new insight into the systemic immune manifestations of RA, we characterized the colon mucosa proteome from 11 RA-patients and 10 healthy controls. The biopsies were extracted by colonoscopy and analyzed by label-free quantitative proteomics, enabling the quantitation of 5366 proteins. The abundance of dihydrofolate reductase (DHFR) was statistically significantly increased in RA-patient biopsies compared with controls and correlated with the administered dosage of methotrexate (MTX), the most frequently prescribed immunosuppressive drug for RA. Additionally, our data suggest that treatment with Leflunomide, a common alternative to MTX, increases DHFR. The findings were supported by immunohistochemistry with confocal microscopy, which furthermore demonstrated that DHFR was located in the cytosol of the intestinal epithelial and interstitial cells. Finally, we identified 223 citrullinated peptides from 121 proteins. Three of the peptides were unique to RA. The list of citrullinated proteins was enriched in extracellular and membrane proteins and included known targets of anticitrullinated protein antibodies (ACPAs). Our findings support that the colon mucosa could trigger the production of ACPAs, which could contribute to the onset of RA. The MS data have been deposited to ProteomeXchange with identifiers PXD001608 and PXD003082.

A proposal for a study on treatment selection and lifestyle recommendations in chronic inflammatory diseases: A Danish multidisciplinary collaboration on prognostic factors and personalised medicine

Chronic inflammatory diseases (CIDs), including Crohn’s disease and ulcerative colitis (inflammatory bowel diseases, IBD), rheumatoid arthritis, psoriasis, psoriatic arthritis, spondyloarthritides, hidradenitis suppurativa, and immune-mediated uveitis, are treated with biologics targeting the pro-inflammatory molecule tumour necrosis factor-α (TNF) (i.e., TNF inhibitors). Approximately one-third of the patients do not respond to the treatment. Genetics and lifestyle may affect the treatment results. The aims of this multidisciplinary collaboration are to identify (1) molecular signatures of prognostic value to help tailor treatment decisions to an individual likely to initiate TNF inhibitor therapy, followed by (2) lifestyle factors that support achievement of optimised treatment outcome. This report describes the establishment of a cohort that aims to obtain this information. Clinical data including lifestyle and treatment response and biological specimens (blood, faeces, urine, and, in IBD patients, intestinal biopsies) are sampled prior to and while on TNF inhibitor therapy. Both hypothesis-driven and data-driven analyses will be performed according to pre-specified protocols including pathway analyses resulting from candidate gene expression analyses and global approaches (e.g., metabolomics, metagenomics, proteomics). The final purpose is to improve the lives of patients suffering from CIDs, by providing tools facilitating treatment selection and dietary recommendations likely to improve the clinical outcome.

Efficacy and safety of faecal microbiota transplantation in patients with psoriatic arthritis: protocol for a 6-month, double-blind, randomised, placebo-controlled trial

Introduction An unbalanced intestinal microbiota may mediate activation of the inflammatory pathways seen in psoriatic arthritis (PsA). A randomised, placebo-controlled trial of faecal microbiota transplantation (FMT) infused into the small intestine of patients with PsA with active peripheral disease who are non-responsive to methotrexate (MTX) treatment will be conducted. The objective is to explore clinical aspects associated with FMT performed in patients with PsA. Methods and analysis This trial is a randomised, two-centre stratified, double-blind (patient, care provider and outcome assessor), placebo-controlled, parallel-group study. Eighty patients will be included and randomised (1:1) to either placebo (saline) or FMT provided from an anonymous healthy donor. Throughout the study, both groups will continue the weekly self-administered subcutaneous MTX treatment, remaining on the preinclusion dosage (15–25u2009mg/week). The clinical measures of psoriasis and PsA disease activity used include the Short (2-page) Health Assessment Questionnaire, the Dermatology Quality of Life Index, the Spondyloarthritis Research Consortium of Canada Enthesitis Index, the Psoriasis Area Severity Index, a dactylitis digit count, a swollen/tender joint count (66/68), plasma C reactive protein as well as visual analogue scales for pain, fatigue and patient and physician global assessments. The primary end point is the proportion of patients who experience treatment failure during the 6-month trial period. The number of adverse events will be registered throughout the study. Ethics and dissemination This is a proof-of-concept clinical trial and will be performed in agreement with Good Clinical Practice standards. Approvals have been obtained from the local Ethics Committee (DK-S-20150080) and the Danish Data Protection Agency (15/41684). The study has commenced in May 2017. Dissemination will be through presentations at national and international conferences and through publications in international peer-reviewed journal(s). Trial registration number NCT03058900; Pre-results.

Impact of red and processed meat and fibre intake on treatment outcomes among patients with chronic inflammatory diseases: protocol for a prospective cohort study of prognostic factors and personalised medicine

Introduction Chronic inflammatory diseases (CIDs) are frequently treated with biological medications, specifically tumour necrosis factor inhibitors (TNFi)). These medications inhibit the pro-inflammatory molecule TNF alpha, which has been strongly implicated in the aetiology of these diseases. Up to one-third of patients do not, however, respond to biologics, and lifestyle factors are assumed to affect treatment outcomes. Little is known about the effects of dietary lifestyle as a prognostic factor that may enable personalised medicine. The primary outcome of this multidisciplinary collaborative study will be to identify dietary lifestyle factors that support optimal treatment outcomes. Methods and analysis This prospective cohort study will enrol 320 patients with CID who are prescribed a TNFi between June 2017 and March 2019. Included among the patients with CID will be patients with inflammatory bowel disease (Crohn’s disease and ulcerative colitis), rheumatic disorders (rheumatoid arthritis, axial spondyloarthritis, psoriatic arthritis), inflammatory skin diseases (psoriasis, hidradenitis suppurativa) and non-infectious uveitis. At baseline (pretreatment), patient characteristics will be assessed using patient-reported outcome measures, clinical assessments of disease activity, quality of life and lifestyle, in addition to registry data on comorbidity and concomitant medication(s). In accordance with current Danish standards, follow-up will be conducted 14–16 weeks after treatment initiation. For each disease, evaluation of successful treatment response will be based on established primary and secondary endpoints, including disease-specific core outcome sets. The major outcome of the analyses will be to detect variability in treatment effectiveness between patients with different lifestyle characteristics. Ethics and dissemination The principle goal of this project is to improve the quality of life of patients suffering from CID by providing evidence to support dietary and other lifestyle recommendations that may improve clinical outcomes. The study is approved by the Ethics Committee (S-20160124) and the Danish Data Protecting Agency (2008-58-035). Study findings will be disseminated through peer-reviewed journals, patient associations and presentations at international conferences. Trial registration number NCT03173144; Pre-results.

Proteomics dataset: The colon mucosa from inflammatory bowel disease patients, gastrointestinal asymptomic rheumatoid arthritis patients, and controls

The datasets presented in this article are related to the research articles entitled “Neutrophil Extracellular Traps in Ulcerative Colitis: A Proteome Analysis of Intestinal Biopsies” (Bennike et al., 2015 [1]), and “Proteome Analysis of Rheumatoid Arthritis Gut Mucosa” (Bennike et al., 2017 [2]). The colon mucosa represents the main interacting surface of the gut microbiota and the immune system. Studies have found an altered composition of the gut microbiota in rheumatoid arthritis patients (Zhang et al., 2015; Vaahtovuo et al., 2008; Hazenberg et al., 1992) [5], [6], [7] and inflammatory bowel disease patients (Morgan et al., 2012; Abraham and Medzhitov, 2011; Bennike, 2014) [8], [9], [10]. Therefore, we characterized the proteome of colon mucosa biopsies from 10 inflammatory bowel disease ulcerative colitis (UC) patients, 11 gastrointestinal healthy rheumatoid arthritis (RA) patients, and 10 controls. We conducted the sample preparation and liquid chromatography mass spectrometry (LC-MS/MS) analysis of all samples in one batch, enabling label-free comparison between all biopsies. The datasets are made publicly available to enable critical or extended analyses. The proteomics data and search results, have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001608 for ulcerative colitis and control samples, and PXD003082 for rheumatoid arthritis samples.

P204 Inflammatory joint- and bowel diseases – a clinical proteomics study seeking to identify the underlying biological triggers

content of n-3 fatty acids, antioxidants and fiber, and a low content in sugars and saturated fats. The Mediterranean Diet (MD) comprises all the former nutrients, and thus it has been hypothesized that it could be beneficial for patients with Inflammatory Bowel Disease (IBD). The purpose of our study was to evaluate compliance of IBD patients with the MD and assess its impact on disease activity. Methods: A total of 59 patients [41 with Crohn’s disease (CD) and 18 with ulcerative colitis (UC)] with IBD were enrolled in this cross-sectional study. Clinical data and anthropometric measures were recorded. Dietary intake was assessed using a semi-quantitative food frequency questionnaire. Conversion of foodstuffs to nutrient intake was accomplished with the software Food Processor Plus (ESHA Research, Salem, Oregon). Compliance to MD, was analyzed using the Mediterranean Adequacy Index (MAI), which is calculated as a ratio between foodstuffs consumed that characterize MD and others that are not consistent with MD. MAI enables comparison between the estimated nutrient intake and Healthy Reference National Mediterranean Diet. Disease activity was measured using Harvey Bradshaw Index (HBI) for CD and Cinical Activity Index for UC (CAI). Data analysis was performed with SPSS 20 (IBM SPSS statistics). Results: 37.3% (n = 22) of the studied population were overweight, 10.2% (n = 6) obese, 47.5% (n = 28) normal weight and 5% (n = 3) had a low BMI. Patients with no relapse during the last year showed a higher intake of red meat (40.57±20.3 g/d vs 26.4±20.7 g/d; p = 0.022), canned food (12.9±12.9 g/d vs 8.1±11.9 g/d; p = 0.022), and cured meats (5.6±4.2 g/d vs 4.3±6.8 g/d, p = 0.063). Also, patients in remission had a lower intake of skim milk (6.6±40.3mL vs 55.1±151.2mL; p = 0.082) and a higher intake of canned food (12.4±13.2 g/d vs 5.7±9.9 g/d; p = 0.007), sugar (18.1±16.3 g/d vs 11.0±13.4 g/d; p = 0.098) and coffee (78.3±57.5 vs 51.19±60.9; p = 0.052). Only 14% (n = 8) of our population was classified as compliant with MD (MAI 4.6), whereas the mean MAI was 2.46±5.21. Moreover, no correlation was found between MAI and HBI (r = 0.242; p = 0.304) or CAI (r = 0.201; p = 0.44). Conclusions: The studied population with IBD showed a high prevalence of overweight/obesity and only a small number of patients were compliant with the MD. No association was found between MAI and disease activity. Future interventional nutritional studies aiming at investigating the role of MD in the maintenance of remission or relapse frequency are warranted.