Introduction

Xanthine oxidase (XO) is involved in the last steps of purine metabolism; indeed, it catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid (UA) with the production of superoxide anion. The accumulation of UA has been shown to initiate the inflammatory process through NLRP3 inflammasome and the production of reactive oxygen species (ROS) contributes to inflammation-related tissue damage. In murine models of colitis, XO inhibitors, allopurinol or febuxostat, reduced the expression of proinflammatory cytokines supporting the involvement of XO in the intestinal inflammatory process.

Aims & Methods

This study aims to conduct a thorough investigation of the role XO plays in IBD pathogenesis and to explore the potential of XO inhibition as a therapeutic strategy.
Biopsies from the sigmoid colon of 15 Healthy Controls (HC), 20 Ulcerative Colitis (UC), and 15 Crohn’s Disease (CD) patients, as well as from the ileum of 10 HC and 10 CD patients, were collected at the University Hospital of Cagliari. Disease activity was assessed using Mayo and SES-CD scores. XO expression levels were evaluated using Real-Time PCR and validated by GEO database (GSE11223, GSE117993). XO enzymatic activity was quantified spectrophotometrically in protein extracts from the biopsies. XO and NLRP3 inflammasome activation pathway, including NLRP3, ASC, and CASPASE1, was investigated using immunohistochemistry (IHC). Biopsies samples were lysed in RIPA buffer and Capillary Western analyses were performed with ProteinSimple Jess System utilizing 1:10 dilution for XO, NLRP3, ASC, and Caspase 1; 1:100 dilution for Beta-actin as primary antibodies. Ex-vivo organ culture were assayed with Intestinal biopsies from UC and CD patients. Briefly, the biopsies were cultivated with or without XO inhibitors, allopurinol and febuxostat, for 24 hours in a 24-well plate with 250 µL RPMI at 37°C with 95% O 2 /5% CO 2 atmosphere. After 24h the biopsies were used for IHC analysis of NLRP3 pathways.

Results

Increased XO mRNA expression was found in UC and CD sigmoid colon mucosa compared to HC in the GEO database. This finding was further confirmed by Real-Time PCR in our patients, with a Relative Quantification (RQ) of 1.93 vs. 0.99 (p=0.004) for UC and CD sigmoid colon, and RQ of 2.72 vs. 1.26 (p=0.004) for CD ileum. XO activity was higher in UC and CD sigmoid colon (3.86 nmol UA/min/mg in UC, 4.82 in CD vs. 1.03 in HC, p<0.001) and CD ileum (8.41 vs. 1.83 in HC, p<0.001). IHC and Western analysis confirmed that XO and the NLRP3 inflammasome pathway are more expressed in IBD patients than in HC, predominantly within epithelial cells. The ex-vivo organ culture of CD patients, and UC patients, showed a reduced UA levels and reduced NLRP3 pathways activation after treatment with XO inhibitors.

Conclusion

XO expression was higher in UC and CD patients than in HC individuals. Alongside this increase in XO activity, there was a concurrent hyperactivation of the NLRP3 inflammasome. The inhibition of XO activity in ex-vivo organ culture resulted in reduced activation of the NLRP3 inflammasome pathway. These findings support the hypothesis that XO plays a critical role in the pathogenesis of IBD and highlight its potential as a therapeutic target.

Disclosure

I declare that there are no conflicts of interest.

Introduction

Xanthine oxidase (XO) catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid (UA) with the production of superoxide anion. The accumulation of UA has been shown to initiate the inflammatory process through NLRP3 inflammasome and the production of reactive oxygen species (ROS), which contributes to inflammation-related tissue damage observed in inflammatory bowel disease (IBD). In murine models of colitis, XO inhibitors, allopurinol or febuxostat, reduced the expression of proinflammatory cytokines supporting the involvement of XO in the intestinal inflammatory process.

Aims & Methods

The aim of this study was to evaluate the expression level and enzymatic activity of XO in the intestinal mucosa of IBD patients (pts), both ulcerative colitis (UC) and Crohn’s disease (CD), compared to healthy controls (HC).
Sigmoid-colon biopsies from consecutive UC and CD pts, with a confirmed diagnosis (by ECCO guidelines) in regular follow-up at University Hospital of Cagliari with an indication to endoscopy according to standard clinical practice, were collected. Site-matched biopsies from individuals undergoing screening colonoscopy for colorectal cancer were used as controls.
Demographic data and endoscopic grade for disease activity scored by endoscopic Mayo and SES-CD score were collected. Real-Time PCR was used to evaluate XO relative expression levels (RQ) and data was confirmed by analyzing GEO database (study code GSE11223). XO activity was measured in whole protein extracts from biopsies by spectrophotometric quantification of UA production and expressed as nmol of UA/min/mg of total protein. The relative abundance of XO was further investigated by Western Blot. Finally, XO expression was assessed in sections of paraffin-embedded intestinal biopsies by immunohistochemistry.

Results

Biopsies of 15 HC, 20 UC, and 15 CD pts were collected. Mean age was 50 for UC, 49 for CD and 54 for HC. Mayo endoscopic subscore was 0 in 25% of UC pts, 1 in 18,75%, 2 in 21,2% and 3 in 25%. SES-CD was between 0-2 in 28,6% of CD pts, 3-6 in 21,4%, 7-14 in 42,8% and >14 in 7,1%. Real-time PCR showed that XO expression values were increased in the sigmoid mucosa of pts with UC compared with HC individuals (RQ 1,93 vs 0,99. p=0,004). This data was confirmed by analysis of XO expression in the GEO database study code GSE11223. The difference in XO expression between HC and CD was not statistically significant (p=0,13). Western blot analysis of XO protein expression in CD and UC pts compared with HC showed a significant increase in IBD pts (p<0,001).
XO activity was significantly higher in UC and CD pts as compared to HC. XO activity mean was 3,75 in UC pts and 5,10 in CD pts vs 1,11 nmol of UA/min/mg of protein in HC individuals (p<0.001). Moreover, in UC pts XO activity was correlated with Mayo endoscopic sub-score (r= 0.346; p=0.05). Immunohistochemistry confirmed higher expression of XO in IBD pts than HC with a prevalent expression observed in epithelial cells.

Conclusion

XO expression was higher in UC pts than in HC individuals. XO activity was higher in both CD and UC pts than HC and in UC pts XO activity correlated with endoscopic disease suggesting its potential role as therapeutic target.

Introduction

Periodontitis (gum disease) and inflammatory bowel disease (IBD) are both chronic progressive inflammatory diseases with many similarities in their aetiopathogenesis. In addition, patients with IBD are reported to be at increased risk of developing periodontitis, which in turn seems to impact IBD activity and disability as shown in recent studies. However, these findings need to be validated. Thus, we aimed to assess whether the presence of periodontitis is associated with increased IBD activity, disability, and severity, and/or reduced IBD specific quality of life, in a Swedish patient cohort.

Aims & Methods

An on-line questionnaire was distributed to the members of the Swedish patient organisation for gastrointestinal diseases. The survey included questions on social demographics, oral health, as well as IBD-related characteristics. Oral health was assessed by the number of missing teeth, self-reported state of teeth and gums, and the Periodontal Screening Score (PESS), which is a validated instrument to screen for severe periodontitis. Disease activity in patients with Ulcerative Colitis (UC) and Crohn’s disease (CD) was assessed by the Simple Clinical Colitis Index (SCCAI) and the Harvey & Bradshaw’s Activity Index (HBI), respectively. Disease related disability was assessed by the Inflammatory bowel disease disability index (IBD-DI). IBD-specific health-related quality of life measured by the short inflammatory bowel disease questionnaire (sIBDQ).

Results

A total of 786 IBD patients responded to the invitation, including 371 patients with CD and 415 patients with UC. For both CD and UC, self-reported severe periodontitis was significantly associated with a higher level of current disease activity [OR 1.72 (1.86;2.49); p=0.004] based on the HBI (for CD) and the SCCAI (for UC), an increased IBD disability score [Coef. 5.07 (1.86;8.27); p=0.002], and an increased self-reported disease activity in the last 12 months [OR 1.44 (1.05;1.98); p=0.025]. Likewise, number of missing teeth (tooth loss is the ultimate outcome of untreated periodontitis and as such an indirect indicator of its severity), was associated with an increased IBD disability score [Coef. 4.47 (0.71;8.22); p=0.020]. IBD severity (analysed as composite parameter of history of IBD-related surgery and/or treatment with biological therapy) was neither associated with tooth loss [OR 1.57 (0.97;2.52); p=0.065], nor with self-reported severe periodontitis [OR 1.20 (0.80;1.80); p=0.367]. IBD-specific health-related quality of life measured by sIBDQ score showed a tendency to be decreased in the presence of self-reported severe periodontitis [OR -1.45 (-3.01;0.10); p=0.067] but was not associated with tooth loss [Coef. -1.20 (-3.01;0.62); p=0.194].

Conclusion

The present study provides further evidence that the presence of periodontitis is associated with an increased disease activity and disability in IBD patients. These results underline that increased attention should be given to the oral health of IBD patients.

Introduction

Chronic abdominal pain is prevalent in individuals with inflammatory bowel diseases (IBD). However, the determinants and phenotypes of IBD-associated pain are not well-characterized, frustrating effective pain management and mitigation strategies

Aims & Methods

Aims: This is an ongoing descriptive, cross-sectional study aimed at phenotyping the types of pain experienced by patients with IBD, the impact on health-related quality of life (HRQoL) and psychological health, and relationship to disease activity assessed by clinical metrics and endoscopy. Methods: Fifty-eight adult patients diagnosed with IBD were prospectively enrolled at a single academic medical center between October 17, 2022 and November 7, 2023. Patients completed a set of online patientreported outcome measures (PROM)-based standardized and validated questionnaires (Table 2) just prior to a scheduled colonoscopy. Endoscopically active disease was defined as Mayo Endoscopic Score ≥ 1 for ulcerative colitis (UC) and SES-CD ≥ 3 for Crohn’s Disease.

Results

A total of 58 patients (28 UC/30 Crohn’s, 24 male/34 female, median age 44.5±14.5 years) were included. An initial analysis examined relationships based on endoscopic disease activity. Interestingly, there was no significant difference between those with endoscopically active disease and those without on metrics of pain, HRQoL, or impact on psychological health (p>0.05). However, those with endoscopically inactive disease had significantly higher scores on central sensitization (CS) (31.1±11.5 vs. 23±12.1, p<0.05). Using a validated score threshold of 40 on the central sensitization inventory (CSI), the cohort was then stratified into two new groups (those with and without evidence of CS). Overall, 12/58 patients met diagnostic criteria for CS. Table 1 summarizes the baseline characteristics of the two groups. As detailed in Table 2, those with CS had higher scores on abdominal pain (severity and interference, including catastrophizing), visceral hypersensitivity, lower scores on HRQoL questionnaires, and greater scores on depression and anxiety scales (p<0.05). There was no significant difference in neuropathic pain (p=0.85).

Conclusion

Central sensitization affected at least 20% of this cohort of IBD patients, which is consistent with prior studies. Interestingly, there was a trend towards more central sensitization in those with quiescent disease. CS was associated with worse pain, HRQoL, and psychosocial distress across a broad range of outcomes measured. These data support enhanced attention to contribution of CS to pain in IBD patients, particularly those with persistent pain in remission, as therapies emerge for the treatment of CS.

Introduction

Inflammatory bowel disease (IBD) is a chronic multi-factorial disease characterized by inflammation of the gastrointestinal tract. Currently, the mechanisms underlying the disease are far from being elucidated. Thus, the study of proteins involved in its pathogenesis would allow further insights into the molecular mechanisms underlying IBD.

Aims & Methods

The serum and intestinal mucosa (ileum and left colon) proteomic profiles of patients with Crohn´s disease (CD) and ulcerative colitis (UC) and healthy controls (HC) were analyzed through label-free relative quantitative proteomic profiling using nLC MS/MS. Data mining and pattern recognition techniques were performed to identify hidden relationships that are not detectable using classical linear classifiers, and thus identify potential markers able to discriminate between the different study groups. Six candidate biomarkers were selected for further validation using enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) in serum and intestinal tissue samples, respectively.

Results

Comparison of serum proteins between UC patients and HC revealed statistically lower levels of protein 1 and 2 and higher levels of protein 3. The comparison of potential biomarkers between CD and HC group showed lower levels of protein 1 and higher levels of protein 3. In addition, protein 4 was differentially upregulated in CD patients compared to UC. Regarding the intestinal biopsies, protein 5 showed higher intensity in the IHC staining of ileum from CD patients compared to HC, whereas protein 6 was significantly decreased in left colon and ileum from CD and UC patients compared to HC. These proteins are involved in thyroxine transport, regulation of chemotactic chemokine activity, inflammatory processes, triglyceride homeostasis, oxidative stress and regulation of NF-kappa-B activation in the cytosol.

Conclusion

In this study, we have identified a panel of six potential biomarkers that could help to elucidate mechanisms involved in IBD pathogenesis. Their roles in the pathophysiology need to be clarified in further experiments.