Proteomic Profiling of Colorectal Adenomas Identifies a Predictive Risk Signature for Development of Metachronous Advanced Colorectal Neoplasia.

BACKGROUND & AIMS: Colonic adenomatous polyps, or adenomas, are frequent precancerous lesions and the origin of most cases of colorectal adenocarcinoma. However, we know from epidemiologic studies that although most colorectal cancers (CRCs) originate from adenomas, only a small fraction of adenomas (3%-5%) ever progress to cancer. At present, there are no molecular markers to guide follow-up surveillance programs. METHODS: We profiled, by mass spectrometry-based proteomics combined with machine learning analysis, a selected cohort of formalin-fixed, paraffin-embedded high-grade (HG) adenomas with long clinical follow-up, collected as part of the Danish national screening program. We grouped subjects in the cohort according to their subsequent history of findings: a nonmetachronous advanced neoplasia group (G0), with no new HG adenomas or CRCs up to 10 years after polypectomy, and a metachronous advanced neoplasia group (G1) where individuals developed a new HG adenoma or CRC within 5 years of diagnosis. RESULTS: We generated a proteome dataset from 98 selected HG adenoma samples, including 20 technical replicates, of which 45 samples belonged to the nonmetachronous advanced neoplasia group and 53 to the metachronous advanced neoplasia group. The clear distinction of these 2 groups seen in a uniform manifold approximation and projection plot indicated that the information contained within the abundance of the ∼5000 proteins was sufficient to predict the future occurrence of HG adenomas or development of CRC. CONCLUSIONS: We performed an in-depth analysis of quantitative proteomic data from 98 resected adenoma samples using various novel algorithms and statistical packages and found that their proteome can predict development of metachronous advanced lesions and progression several years in advance.

Remote ischemic postconditioning has a detrimental effect and remote ischemic preconditioning seems to have no effect on small intestinal anastomotic strength.

BACKGROUND: The effect of remote pre- and postconditioning on anastomotic healing has been sparsely studied. The aim of our study was to investigate whether remote ischemic conditioning (RIC) applied before and after the creation of a small bowel anastomosis had an effect on anastomotic healing on postoperative day five evaluated by a tensile strength test and histological analysis. MATERIALS AND METHODS: Twenty-two female piglets were randomized into two groups. The intervention group (n = 12) received RIC on the forelimbs consisting of 15 min of ischemia followed by 30 min of reperfusion before the first end-to-end ileal anastomosis was created. The RIC procedure was repeated and the second and more distal anastomosis was performed. The control group (n = 10) had two similar anastomoses with similar time intervals but without RIC. On postoperative day five, the anastomoses were subjected to macroscopic evaluation, tensile strength test and histological examination. RESULTS: Mean tensile strength when the first transmural rupture appeared (MATS-2) was significantly lower in the first anastomosis in the intervention group compared to the control group (11.4 N vs 14.7 N, p < .05). Similar result was found by the maximal strength (MATS-3) as defined by a drop in the load curve (12.3 N vs 15.9 N, p < .05). Histologically, a significantly higher necrosis score was found in the anastomosis in the intervention group (1.4 vs 0.8, p < .05). No other significant differences were found. CONCLUSIONS: In conclusion, post-anastomotic remote ischemic conditioning had a detrimental effect and pre-anastomotic conditioning seems to have no effect on small intestinal anastomotic strength.

Segmental reversal of the distal small intestine in a short bowel syndrome model in piglets showed detrimental effect on weight gain.

BACKGROUND: To investigate the effects of a reversed segment of the distal small intestine to improve weight gain in an experimental short bowel syndrome (SBS) model in piglets. METHODS: Twenty-four piglets underwent resection of 70% of the distal small intestine. In half of the animals a conventional anastomosis was performed, and in the other half, the distal 25 cm of the remnant jejunum was reversed before the intestinal continuity was recreated. Weight was measured daily until day 28, where the animals were euthanized. Glucagon-Like Peptide-2 (GLP-2) and Glucose-dependent Insulinotropic Peptide (GIP) was measured pre- and postoperatively at day 28. RESULTS: The group with reversal of small intestine had a significant lower weight gain at 5.26 ± 3.39 kg (mean ± SD) compared to the control group with 11.14 ± 3.83 kg (p < 0.05). In the control group greater villus height and crypt depth was found distally, and greater muscular thickness was found proximally in the intervention group. GLP-2 and GIP levels increased significantly in the control group. CONCLUSIONS: Treatment of short bowel syndrome with a reversed jejunal segment of 25 cm had a detrimental effect on the weight gain.

A streamlined mass spectrometry-based proteomics workflow for large-scale FFPE tissue analysis.

Formalin fixation and paraffin-embedding (FFPE) is the most common method to preserve human tissue for clinical diagnosis, and FFPE archives represent an invaluable resource for biomedical research. Proteins in FFPE material are stable over decades but their efficient extraction and streamlined analysis by mass spectrometry (MS)-based proteomics has so far proven challenging. Herein we describe a MS-based proteomic workflow for quantitative profiling of large FFPE tissue cohorts directly from histopathology glass slides. We demonstrate broad applicability of the workflow to clinical pathology specimens and variable sample amounts, including low-input cancer tissue isolated by laser microdissection. Using state-of-the-art data dependent acquisition (DDA) and data independent acquisition (DIA) MS workflows, we consistently quantify a large part of the proteome in 100 min single-run analyses. In an adenoma cohort comprising more than 100 samples, total workup took less than a day. We observed a moderate trend towards lower protein identification in long-term stored samples (>15 years), but clustering into distinct proteomic subtypes was independent of archival time. Our results underscore the great promise of FFPE tissues for patient phenotyping using unbiased proteomics and they prove the feasibility of analyzing large tissue cohorts in a robust, timely, and streamlined manner. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Extracellular Matrix Fragments of the Basement Membrane and the Interstitial Matrix Are Serological Markers of Intestinal Tissue Remodeling and Disease Activity in Dextran Sulfate Sodium Colitis.

BACKGROUND: Chronic intestinal inflammation results in tissue damage partly caused by an increase in matrix metalloproteinases (MMP) activity causing degradation of extracellular matrix (ECM) proteins. We studied intestinal tissue remodeling by quantifying ECM protein fragments in serum in dextran sulfate sodium (DSS)-induced colitis, to investigate ECM protein fragments as serological biomarkers of intestinal tissue remodeling and disease activity. METHODS: Male Sprague-Dawley rats received 5% DSS in drinking water for 5 days followed by 11 days with regular water. Disease activity index (DAI) was scored daily. Serum was collected on day 0, 6, 7, and 16. ELISAs were used to quantify MMP-derived remodeling fragments of basement membrane type IV collagen (C4M and PRO-C4) and interstitial matrix type III collagen (C3M and rPRO-C3). RESULTS: In DSS rats, serum levels relative to baseline of C4M, PRO-C4, and C3M were elevated (P < 0.01; P < 0.001; P < 0.001) at day 7, which declined at day 16. Levels of rPRO-C3 were lower in DSS rats at day 7 and increased to normal levels at day 16. The ratio between C3M and rPRO-C3 showed an overall degradation (P < 0.0001) of collagen type III in DSS rats at day 7, which correlated to the DAI (r2 = 0.5588, P < 0.0001). CONCLUSION: Our data suggest that remodeling of the basement membrane (C4M and PRO-C4) and the interstitial matrix (C3M and rPRO-C3) increased during DSS-induced colitis and declined with reversal of the disease. Thus, serological biochemical biomarkers of the ECM reflect tissue remodeling and could be studied as markers of disease activity in IBD.

Segmental reversal of distal small intestine in short bowel syndrome: a study on the influence on postoperative weight and intestinal topography in piglets.

PURPOSE: The objective of the present study was to investigate the effect of segmental reversal of the distal 20 cm of the small intestine in piglets with induced SBS compared to controls with SBS alone. Primary endpoint was postoperative weight change over a period of 1 month. Secondary endpoints were the influence on cell proliferation and mucosal architecture shown by histological analysis. METHODS: Sixteen piglets underwent a 60% resection of the distal small intestine and were randomized into two groups. Group 1 short bowel syndrome alone (SBS) (n = 8) and group 2 with reversal of a distal small intestinal segment (SBS-RS) (n = 8). Body weight was measured daily and the pigs were euthanized after 1 month. Crypt depths, villus heights and muscle layers thicknesses were measured. For the evaluation of microvilli of the brush border of the epithelium and cell proliferation, immunohistochemical staining with Villin and Ki-67 was performed. RESULTS: No statistically significant differences were observed concerning weight gain. Mean ± SD weight gain was 2.31 ± 2.85 kg for SBS-RS and 2.03 ± 1.27 kg for SBS (p = 0.8). In the proximal jejunal segment a significant increase in villus heights was found in the SBS group and increase in the thickness of the circular and longitudinal muscle layers in the SBS-RS group. In the distal ileal segment the longitudinal muscle layer thicknesses were increased in the SBS group. Otherwise, no significant changes were found. CONCLUSION: Reversal of a 20-cm distal segment showed no effect on weight gain, but there were some significant histological changes of unknown clinical significance.

Impact of sex and socioeconomic status on the likelihood of surgery, hospitalization, and use of medications in inflammatory bowel disease: a systematic review and meta-analysis.

BACKGROUND: Inflammatory bowel diseases (IBDs) are associated with high healthcare utilization. This systematic review aimed to summarize what is known about the impact of sex, income, and education on the likelihood of bowel surgery, hospitalization, and use of corticosteroids and biologics among patients with IBD. METHODS: We used EMBASE, MEDLINE, CINAHL, and Web of Science to perform a systematic literature search. Pooled hazard ratios (HRs) and odds ratios (ORs) with 95% confidence intervals (CIs) were calculated using random effects meta-analysis for the impact of sex on the likelihood of surgery and hospitalization. In addition, we performed subgroup analyses of the effect of IBD type (Crohn's disease or ulcerative colitis) and age. Finally, meta-regression was undertaken for the year of publication. RESULTS: In total, 67 studies were included, of which 23 studies were eligible for meta-analysis. In the main meta-analysis, male sex was associated with an increased likelihood of bowel surgery (HR 1.42 (95% CI 1.13;1.78), which was consistent with the subgroup analysis for UC only (HR 1.78, 95% CI 1.16; 2.72). Sex did not impact the likelihood of hospitalization (OR 1.05 (95% CI 0.86;1.30), although the subgroup analysis revealed an increased likelihood of hospitalization in CD patients (OR 1.42, 95% CI 1.28;1.58). In 9 of 10 studies, no significant sex-based differences in the use of biologics were reported, although in 6 of 6 studies, female patients had lower adherence to biologics. In 11 of 13 studies, no significant sex-based difference in the use of corticosteroids was reported. The evidence of the impact of income and education on healthcare utilization was sparse and pointed in different directions. The substantial heterogeneity between studies was explained, in part, by differences in IBD type and age. CONCLUSIONS: The results of this systematic review indicate that male patients with IBD are significantly more likely to have surgery than female patients with IBD but are not, overall, more likely to be hospitalized, whereas female patients appear to have statistically significantly lower adherence to biologics compared to male patients. Thus, clinicians should not underestimate the impact of sex on healthcare utilization. Evidence for income- and education-based differences remains sparse. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42022315788.

Clinical procedures used to diagnose inflammatory bowel disease: real-world evidence from a Danish nationwide population-based study.

OBJECTIVE: Although clinical guidelines exist, the diagnostic work-up for diagnosing inflammatory bowel disease (IBD) is complex and varies in clinical practice. This study used real-life data to characterise the current diagnostic procedures used to establish IBD diagnoses in a Danish nationwide setting. DESIGN: Person-level data on patients diagnosed with IBD between 1 January 2014 and 30 June 2018 were linked between Danish health registers. Information on age, sex, registration of other gastrointestinal diseases, and diagnostic procedures (endoscopies, biopsies, and imaging) performed in relation to the first IBD hospital admission was analysed for the total study population and was stratified by IBD type, sex, and age. RESULTS: The majority of the 12 871 patients with IBD included underwent endoscopy (84%), had a biopsy taken (84%), and/or underwent imaging procedures (44%). In total, 7.5% of the population (6% for Crohn's disease and 8% for ulcerative colitis) were diagnosed with IBD despite not undergoing any of these diagnostic procedures. Patients with Crohn's disease underwent more procedures than patients with ulcerative colitis (94% vs 92%, p<0.001). Children underwent slightly fewer diagnostic procedures than adults (92% vs 93%, p=0.004). Slightly more men underwent at least one procedure than women (92% vs 94%, p<0.001). CONCLUSION: For 7.5% of patients with IBD, this study did not detect any registrations of the recommended diagnostic procedures for establishing an IBD diagnosis. Further research is needed to examine whether these findings are mainly explained by limitations of the register data or also indicate shortcomings of the general approach to IBD.

Peptidoglycan Recognition Peptide 2 Aggravates Weight Loss in a Murine Model of Chemotherapy-Induced Gastrointestinal Toxicity.

Introduction: Chemotherapy-induced gastrointestinal toxicity (CIGT) is a frequent, severe and dose-limiting side effect. Few treatments have proven effective for CIGT. CIGT is characterized by activation of the nuclear factor kappa B pathway which, leads to upregulation of proinflammatory cytokines. The innate immune protein peptidoglycan recognition peptide 2 (PGLYRP2) binds to and hydrolyzes microbial peptidoglycan. Expression of PGLYRP2 is upregulated in the intestine of chemotherapy-treated piglets. In this experimental study, we investigated the role of Pglyrp2 in the development and severity of murine CIGT. Methods: Pglyrp2 wildtype and Pglyrp2 knockout mice received intraperitoneal injections of chemotherapy (Doxorubicin 20 mg/kg) to induce CIGT. Weight was monitored daily, and animals were euthanized after 2 or 7 days. Expression of proinflammatory cytokines in the jejunum was measured by quantitative real-time polymerase-chain reaction and enzyme-linked immunosorbent assay. Villus height, crypt depth, and histologic inflammation were evaluated on haematoxylin and eosin stained tissue specimens. Results: Chemotherapeutic treatment induced weight loss (p < 0.05), shortening of the small intestine (p < 0.05), elongation of villus height (p < 0.05), increased crypt depth (p < 0.05), and led to elevated mRNA levels of II1ß (p < 0.05), II6 (p < 0.05), and Tnf (p < 0.001) at day 2. Protein levels of IL1ß, IL6, and TNFα did not change after exposure to chemotherapy. Doxorubicin treated wildtype mice had a more pronounced weight loss compared to knockout mice from day 3 to day 7 (D3-D6: p < 0.05 and D7: p < 0.01). No other phenotypic differences were detected. Conclusion: Pglyrp2 aggravates chemotherapy-induced weight loss but does not induce a specific pattern of inflammation and morphological changes in the small intestine.