Introduction

Crohn’s disease (CD), a variant of inflammatory bowel disease, is a complex, debilitating, chronic and relapsing disorder of the gastrointestinal tract. If poorly controlled, patients are at risk of intestinal obstruction and infection due to the formation of strictures, fistulae and narrowing of the intestinal lumen, often requiring surgical intervention. As the precise aetiology remains unclear, current treatments for CD are limited. An altered composition of native gut microbiota may play a role in CD pathogenesis, with recent faecal microbiota transplantation (FMT) studies revealing variable therapeutic efficacy in CD management. Orally administered Emu Oil, derived from Emu adipose tissue, has demonstrated anti-inflammatory and reparative properties in models of intestinal disease.

Aims & Methods

The current study aimed to determine whether Emu Oil-modulated FMT could reduce disease severity and modulate the gut microbiota in a mouse model of trinitrobenzene sulfonic acid (TNBS)-induced Crohn’s-like colitis. Female ARC(s) mice were assigned to FMT donor (n=5/group) and recipient (n=10/group) groups. Donor mice were orally administered either Water (80μl), Olive Oil (OO) or Emu Oil (160μl) daily (days -7 to 0); fresh faeces were collected (days -1 to 3) and pooled per group. Recipient mice were administered TNBS (3mg; day 0) and donor faecal supernatant (120μl; day 3) intrarectally and euthanised on day 6. Daily bodyweight and disease activity index (DAI), colonoscopically-assessed disease severity, organ weights, intestinal permeability (fluorescein isothiocyanate [FITC]-dextran uptake), histologically-assessed colonic damage (disease severity, crypt depth), intestinal barrier function (mucin-secreting cells), acute inflammation (myeloperoxidase [MPO]) and faecal microbiome (16S rRNA amplicon sequencing) were assessed. p<0.05 was considered statistically significant.

Results

TNBS induced bodyweight loss in all groups, which normalised to day 0 values on the final day of the experimental period. Daily DAI remained elevated throughout the trial in all groups; only Emu Oil FMT normalised DAI on day 6 compared with day 0 (p<0.05). TNBS increased colonoscopically-assessed disease severity scores (p<0.05), with no significant Emu Oil nor OO FMT effect. Emu Oil FMT decreased liver weights and increased small intestinal weights relative to bodyweight, reduced histologically assessed disease severity and increased colonic mucin counts compared with Water and OO FMT (p<0.05). The most abundant genera across all groups were Muribaculaceae, Eisenbergiella and Bacteroides; while alpha and beta diversity, taxonomic profiles, FITC-dextran, crypt depth and MPO activity remained unchanged in all treatment groups (p>0.05).

Conclusion

Emu Oil-modulated FMT improved histologically-assessed indicators of intestinal disease and barrier function, compared with Water FMT and OO FMT. Interestingly, Emu Oil-modulated FMT affected liver and small intestinal organs weights, suggesting a systemic effect in this setting of disease. Importantly, key genera across taxonomic profiles were identified, however, the microbiome and remaining disease indicators were largely unchanged between treatments. Overall, Emu Oil-modulated FMT demonstrates therapeutic potential in CD management. Future studies would benefit from a deeper assessment of the gastrointestinal microbiome through long-read whole genome sequencing to further determine the role of the microbiome in EO FMT efficacy compared with conventional FMT.

Introduction

Patients suffering from inflammatory bowel diseases (IBD) manifest an increased risk of developing colitis-associated colon cancer (CAC). Interleukin 22 (IL22) is a cytokine involved in the proliferation and survival of epithelial cells. However, it also correlates with the development of tumoral lesions. IL22 is secreted by different immune cell types, including iNKT cells, tissue resident cells possessing cytotoxic properties in colorectal cancer (CRC). In IBD patients, though, iNKT cells perform both pro-inflammatory or tolerogenic functions in a microbiota-dependent fashion, opening questions on their functional role in CAC development.

Aims & Methods

Immune cell composition and iNKT cell phenotype of inflamed, non-tumor and cancerous tissue lesions of CAC (n=49), IBD (n=20) and sporadic CRC (n=38) patients were analysed in samples obtained from Policlinico Hospital, Milan, and IOV-IRCCS, Padua. High-dimensional single-cell flow cytometry, metagenomics, RNAseq, spatial immunophenotyping and trascriptomic (Cosmx, Nanostring), ex vivo and in vitro experiments were performed to evaluate the phenotype and function of human iNKT cells. Metabolomics was performed to identify microbial-derived molecules involved in iNKT cells activation.Mechanisms were dissected in CAC murine models, either iNKT proficient or deficient.

Results

An increased expression of IL22 and a higher NKT22 infiltration was observed in CAC tissues, as well as an enrichment of Odoribacter, a gram-negative bacteria implicated in tryptophan metabolism. Stimulation of iNKT cells with Odoribacter-derived metabolites induced Ahr-dependent IL22 production by iNKT cells. Metabolomic analyses revealed an increased presence of tryptophan metabolites in the Odoribacter supernatant, promoting NKT22 differentiation both in vitro and in vivo models. Moreover, the administration of Odoribacter Supernatant or of the single purified metabolites were sufficient to increase tumorigenesis in CAC mouse models, but not in iNKT-deficient mice.
Spatial proteomics and trascriptomic analyses of human CAC tissues confirms the microbiota-dependent functional skewing of iNKT cells residing closely to cancer cells.

Conclusion

iNKT cells in CAC patients are the major IL22 producing cells and are induced by the recognition of microbiota-derived metabolites implicated in tryptophan metabolism and Aryl hydrocarbon receptor (AhR) stimulation. NKT22-microbiota interaction in CAC patients plays a potential crucial role in tumor development.

Clinical Case Summary

A 13-year-old boy presented with abdominal distention, recurrent hypoglycemia, and self-resolving jaundice. Clinical examination revealed growth retardation and hepatosplenomegaly. Blood work revealed anemia, thrombocytopenia, elevated aspartate and alanine transaminase levels, and raised plasma cholesterol and triglycerides. Ultrasound showed an enlarged liver with surface irregularity, splenomegaly, and a dilated portal vein. Etiological work-up for cirrhosis was positive for hepatitis B (HBV) surface antigen (HbsAg) and antibodies for HBV core and envelope antigens (anti Hbc, antiHbe) but an undetectable HBV DNA level. HBV was considered to be an unlikely cause of cirrhosis due to the young age of the patient, hence further metabolic and genetic work-up was done. Tests for Wilson’s and autoimmune liver disease were negative. Liver biopsy revealed a distorted lobular architecture with nodules, thin septae, and portal fibrosis. Hepatocytes showed marked swelling and pyknotic nuclei, and negative HbsAg immunostaining. Molecular analysis revealed a novel mutation in the exon 3 of the PHKG2 gene (c.98G>T, p.Gly33Val). The patient was diagnosed with cirrhosis due to GSD IXc with inactive chronic hepatitis B. His symptoms resolved with an optimized diet of corn starch, high-protein and vitamin supplementation. The importance of liver transplantation was also discussed with the family.

Introduction

Potential celiac disease (PCD) is a clinical condition characterized by the presence of a positive CD-specific serology and a normal intestinal architecture. Asymptomatic PCD patients are generally addressed to continue on a gluten containing diet (GCD), but long-term risks of this approach have never been explored.

Aims & Methods

In the present study we aim at investigating nutritional and autoimmune complications possibly developing overtime in a cohort of asymptomatic PCD children left on a GCD. We compared parameters of growth, nutritional status, and autoimmunity between time of diagnosis and in occasion of last medical check, after a long-term gluten containing diet.

Results

Altogether we collected data from 171 PCD children with a mean follow-up time of 3 years (range 0.35-15.3 years). During follow-up, despite patients did not reduce the amount of daily gluten intake , anti-tissue transglutaminase (anti-TG2) antibodies spontaneously and significantly decreased. Most of parameters analysed did not change over follow-up (height centile, ferritin, albumin., cholesterol, calcium, alkaline phosphatase , parathormone, vitamin D, p>0.05) or even improved (weight and BMI centile, haemoglobin, blood iron, HDL, glycaemia and HbA1C, p<0.05), always remaining in the limit of normality. Equally autoantibodies for other concomitant autoimmune disorders did not increase overtime. Similar results were obtained excluding from the analysis patients that stopped producing anti-TG2 and those with a follow up < 3 years.

Conclusion

Our pilot study provides reassuring results on the maintenance of a gluten containing diet in asymptomatic PCD children, even when a long term follow-up is considered.

Disclosure

no conflict of interest to declare

Introduction

Aims & Methods

This retrospective cohort study encompasses all elective NRLAR, RLAR, and APR procedures performed for non-metastatic primary rectal carcinoma across 11 Dutch centers between 2013 and 2020. The primary outcome measured was 3-year disease-free survival (DFS), while secondary outcomes included 3-year overall survival (OS) and local recurrence (LR). Data analysis employed Kaplan-Meier survival analysis, and results were juxtaposed using log-rank testing. The association between resection type (NRLAR, RLAR, and APR) was further explored using multivariate Cox regression analysis.

Results

Conclusion

Disclosure

Introduction

The depth of early gastric cancer is an important factor in determining the treatment plan such as endoscopic resection or surgery. We developed an artificial intelligence (AI) program using deep neural networks to diagnose mucosal and submucosal cancers using endoscopic images. Performance evaluation tests were conducted for clinical applications.

Aims & Methods

A validation dataset was established comprising 204 patients with early gastric cancer who underwent endoscopic resection or surgery at our hospital during 2018–2021. The lesion sites were the upper part in 16 (8%), middle part in 106 (52%), and lower part in 82 (40%) patients. The median lesion size (range) was 14 (2–90) mm; the lesion depth was mucosal cancer in 136 (67%) and submucosal cancer in 68 (33%) patients. Five white-light images per case at a minimum were used (median: 8, range: 5–22). The primary endpoint was sensitivity of mucosal cancer, defined as the lower limit of the 95% confidence interval (CI) > 75%, based on previous reports on the depth diagnosis of early gastric cancer. The secondary endpoints were specificity and accuracy.

Results

Sensitivity of this program for the diagnosis of mucosal cancer was 91.2% (95% CI: 85.2%–94.9%), indicating the usefulness of this program. Specificity was 66.2% (95% CI: 54.3%–76.3%), and accuracy was 82.8% (95% CI: 77.1%–87.4%).

Conclusion

The AI program developed for diagnosing the depth of early gastric cancer is useful and expected to be applied clinically in the near future.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with the lowest 5-year survival rate of all cancers due to late diagnosis and lack of effective treatment. One of the challenges of developing molecular diagnosis and molecular targeted therapy for PDAC is the “mismatch” between research models and patients.

Aims & Methods

This study aimed i) to identify common proteins among different research models and patients and accordingly ii) to identify potential hub proteins and signaling pathways involved in the PDAC pathogenesis by using proteomics-based system modeling of multimodal datasets derived from multiple research models and patients. Mouse PDAC cells (derived from a genetically engineered mouse model with KrasG12D/Pdx1-Cre), PDAC orthotopically tumors in C57B/L6 mice (n=5), mouse PDAC spheroids, mouse pancreatic organoids, mouse PDAC organoids, normal mouse pancreatic tissues (n=5), human PDAC tumor tissue samples from treatment-naїve (n=20), neoadjuvant chemotherapy (n=22), and human PDAC organoids were included. Mass spectrometry-based proteomics, Rstudio, Cytoscape 3.10 and pathway enrichment analysis (using ingenuity pathways analysis, including human PDAC transcriptomic in addition) were used.

Results

In the comparison of PDAC models and patients, shared proteins were identified as follows: 60% between the mouse PDAC cells and the mouse PDAC spheroids, 78% between the mouse PDAC cells and the mouse PDAC tissue, 56% between the mouse PDAC spheroids and the mouse PDAC tissue, 66% between the mouse PDAC cells and the mouse PDAC organoids, 54% between the mouse PDAC spheroids and the mouse PDAC organoids, 38% between the mouse PDAC and the human PDAC tissues, 31% between the human PDAC tumor and the human PDAC organoids, and 60% between the mouse and the human PDAC organoids. A total of 1,367 common proteins were identified within all the PDAC models. Among these proteins ACTB, HSP90AA1, HSPA8, HSP90AB1, HSPA4, EEF2, RPS3, RPS20 and GAPDH were the top 10 hub proteins (with >300 node connections). Upon comparison of all eight models, eight proteins were identified as unique to PDAC, namely FAHD1, ARSA, NEK9, SERPINB9, LAMTOR2, SNRPG, TMED3, and RAB1A. There was a correlation in terms of z-score regarding signaling pathways between the mouse PDAC and human PDAC (p<0.001), particularly in activations of nerve growth factor signaling, integrin signaling, and neuregulin signaling. Central hub proteins commonly found in the mouse models included vinculin (membrane-cytoskeletal protein) as one of the high connectivity nodes and β-catenin and probable ubiquitin carboxyl-terminal hydrolase FAF-X (involved in axonal growth and neuronal cell migration) as high centrality nodes. In patients, central hub proteins that were dysregulated after chemotherapy included 14-3-3 protein zeta/delta (encoded by YWHAZ gene), β-catenin and SRC. Among the 47 proteins that were associated with altered metabolism after the chemotherapy in patients, CD44, CTNND1, APOA 1, PARVA, and SCRBS1 were nodes with low degrees, and desmosomes (DSP, KP3, PPL), PRTN3, thrombospondin-2 (THBS2), and DMBT1 were the most down-regulated proteins without nodes, and APOA1, caveolin-1 (encoded by CAV1), LRP1 were the most up-regulated proteins with nodes.

Conclusion

The system modeling identified the common proteins among the research models and patients and the hub proteins and signaling pathways, suggesting the potential translational targets of PDAC.