Crohn Disease (CD)
Conditions
Keywords
Crohn Disease, Fibrosis, Dysbiosis, Microbiota, Fibrostenosis
Brief summary
The goal of this study is to find out if there is a direct connection between an imbalance of gut bacteria and the development of scar tissue in the gut by identifying important bacterial proteins found in scarred gut tissue. Our aim is to identify which types of cells and biological processes are affected by these bacterial proteins in people with Crohn's Disease. We will also study how these bacterial proteins cause changes in 3D models of gut fibrosis.
Detailed description
More than 50% of CD patients develop a penetrating disease or stenosis due to fibrostenosis, which in most cases requires surgery, as no effective therapies have yet been found. The disease leads to both structural and functional alterations of the intestinal mucosa. Although the functional alteration of the mucosa is mainly caused by the continuous tissue damage that occurs during the chronic inflammation associated with CD, recent studies have suggested that the fibrosis associated with CD may be driven by triggering factors independent of inflammation, such as dysbiosis of the microbiota. Our proposal aims to establish the causal link between gut dysbiosis and fibrosis by studying the role of key bacterial proteins present in fibrotic gut tissue. This project will ultimately offer new molecular targets for the development of possible tailor-made antifibrotic treatments, with likely benefits for healthcare, as it will facilitate the management of severe CD, avoiding surgery and reducing SSN costs.
Interventions
PROCEDURECollection of surgical specimen
Specimens of CD patients and non-IBD related patients will be collected during the surgery performed for clinical practice, without other risks for the patients, since we will use only leftover material after pathologist analysis. All of the collected surgical samples will be used for this project and, for this reason, they will not be conserved after the research period. Any residual samples will be destroyed.
PROCEDURECollection of stool sample
One week before the surgery to remove the stricture, fecal samples from CD patients belonging to B1, B2, B3 groups will be collected. They will be used for the exploratory objective. All of the collected fecal samples will be used for this project and, for this reason, they will not be conserved after the research period. Any residual samples will be destroyed.
Sponsors
University of Florence
Humanitas Research Hospital IRCCS, Rozzano-Milan
Ministero dell'Istruzione, dell'Università e della Ricerca
Università Vita-Salute San Raffaele (MI)
IRCCS San Raffaele
Study design
Observational model
CASE_CONTROL
Time perspective
CROSS_SECTIONAL
Eligibility
Sex/Gender
ALL
Age
18 Years to 69 Years
Healthy volunteers
Yes
Inclusion criteria
* Adult patients ≥18 and \<70 years * Patients able to autonomously sign the informed consent * Non pregnant or breastfeeding patients * For CD patients: CD patients with established diagnosis and additionally classified according to 3 stages: B1 (more inflammatory, non-stricturing), B2 (stricturing, non-penetrating) and B3 (stricturing and penetrating). Patients will be stratified on the basis of CT, MRI or ultrasound analysis (stratification already known before surgery) * For non-IBD patients: patients with other pathologies but not affected by IBD according to the previously reported clinical and endoscopic evaluation criteria (ex. diverticulitis)
Exclusion criteria
* Patients \<18 years or \> 70 years * Pregnant or breastfeeding patients * For CD Patients: Subjects with CD who do not meet evaluation criteria described above * For non-IBD patients: Patients undergoing anti-inflammatory and/or immunosuppressive treatments for IBD-related diseases
Design outcomes
Primary
| Measure | Time frame | Description |
|---|---|---|
| To identify the cellular subtypes and molecular pathways impacted by the specific bacterial factors during CD-associated fibrosis. | Experiments and analysis: 11th to 36th month | Control subjects' and CD-derived surgical specimens will be processed to obtain a cell suspension, that will be frozen and stored for the subsequent cell sorting. Frozen CD and healthy cell suspensions will be then thawed and undergo FACS for specific cell markers (CD31 for endothelial cells, EpCam for epithelial cells, CD90 for fibroblasts, CD45 for leukocytes, including CD4 and CD8 for T cells, CD20 for B cells, CD14 and CD163 for macrophages (MΦ), CD11b and c for dendritic cells (DCs). Single-cell populations will undergo library preparation and will be analyzed by ribo-minus RNAseq at 30M reads of depth. Metatranscriptomics for profiling the microbial composition, as well as the transcriptomics to determine both the differential gene expression (DGE) and the Gene Set Enrichment Analysis (GSEA), will be performed. |
Secondary
| Measure | Time frame | Description |
|---|---|---|
| To unravel the cellular and molecular mechanisms and dynamics induced by the bacterial factors in 3D models of intestinal fibrosis | Experiments and analysis: 11th to 36th month | We will isolate and sequence RNA of specific cellular subtypes from the phenotype B1, B2, and B3 of fibrotic CD tissues and healthy non-IBD tissues. We will profile the microbial composition by Metatranscriptomics as well as we will determine both the differential gene expression (DGE) and the Gene Set Enrichment Analysis (GSEA) through transcriptomics. Moreover, to identify the cellular type(s) where bacterial proteins will be expected to exert the most prominent pro-fibrotic effects, we will set up the transwell-based experimental platform by plating epithelial cells (organoids) and/or endothelial cells, and/or fibroblasts in the upper chamber of the transwell, while lamina propria mononuclear cells (LPMCs) will be plated in the lower chamber |
Other
| Measure | Time frame | Description |
|---|---|---|
| To validate in vitro results in an animal model by assessing the role of microbial factors in inducing fibrosis in an entire organism. Moreover, we will provide a possible therapeutic approach with strong clinical implications in the future | Experiments on in-vivo models: 13th to 24th month | To evaluate the efficacy of nanomedicine therapy in inhibiting the deleterious processes induced by bacterial factors by exploiting in vivo models that recapitulate CD-associated fibrosis. We will evaluate the pro-fibrotic potential of specific bacterial factors during experimental intestinal fibrosis, testing a possible treatment through the liposome-mediated inhibition of bacterial factor-induced mechanisms. For this. purpose, fecal samples collected from patients will be handled and processed for analysis in our Lab and will then be shipped to UO2 for in vivo studies |
Countries
Italy
Contacts
Primary ContactSilvio Danese, PhD.-MD
Backup ContactFederica Ungaro, PhD.
Outcome results
None listed