bluuebunny/crossref_metadata_2025_split huggingface dataset:
P037 Enhancing the efficacy of cannabidiol using protein nanoparticles to treat inflammatory bowel disease
Authors: Moniruzzaman M , Rewatkar P , Begun J , Popat A 
Published: January 2023

Abstract

Background
Inflammatory bowel disease (IBD) is a global health burden and approximately 30% of the patients with severe IBD require surgery following treatments, leaving an unmet need of new and safe therapies. Cannabidiol (CBD) is one of the major non-psychoactive and water-insoluble constituents of medicinal cannabis, known for its anti-inflammatory properties in epilepsy. The compound is safe at high doses and doesn’t have the psychotropic effects of delta-9-tetrahydrocannabinol. Therefore, this study investigated the therapeutic potential of CBD in controlling intestinal inflammation.

Methods
CBD was loaded in the protein nanoparticles (PNP-CBD) and tested for its water solubility. Primary intestinal epithelial cells (IECs; organoids) and bone marrow-derived macrophages (BMDM) were isolated from C57BL/6 mice and treated with IL-1β and LPS, respectively. Stimulated cells were treated with ±CBD and checked for downstream signalling through inflammatory gene expression using qRT PCR. Conditioned media from LPS-treated BMDM was also harvested to treat mIECs to check link between macrophage activation and intestinal inflammation. Accordingly, we assessed the efficacy of CBD in suppressing intestinal inflammation using dextran sulfate sodium (DSS)-induced acute experimental colitis in the C57BL/6J mice. At the end, blood samples were collected for hematology and serum analyses, mesenteric lymph nodes for immune cell signatures, colon samples for histological analysis and qRT PCR analysis for inflammatory genes.

Results
CBD loading in the protein nanoparticles (PNP-CBD) increased its water solubility up to 60 times. In the mIECs, CBD did not have any effects on cytokine-induced inflammation; however, it significantly dampened LPS-induced BMDM activation and inflammatory cytokine production. The results also showed that inhibition of CB2 but not CB1 receptor can completely abolish the anti-inflammatory potential of CBD in the BMDM. Transferring conditioned media from BMDM to mIECs showed macrophage-specific targeting alleviated inflammation in the mIECs. These have also been observed in the acute DSS colitis model where not CBD alone but PNP-CBD improved disease severity index including body weight loss, diarrhoea, shorter colon length, and inflammatory gene expression in the colon. PNP-CBD also reduced histological colitis and increased goblet cell mucin production in the DSS-treated colon.

Conclusion
Encapsulation of CBD increased water solubility and efficacy in alleviating intestinal inflammation in ulcerative colitis. These data also demonstrated that CB2 receptor targeting of macrophage activation is the primary mechanism of CBD’s effect.
P041 The upregulation of tissue factor in colonic mesenchymal cells in active ulcerative colitis is associated with severe and resistant to treatment disease, but tofacitinib has not effect on it
Authors: Drygiannakis I , Tzenaki N , Archontoulaki E , Filidou E , Kandilogiannakis L , Valatas V , Kolios G , Koutroubakis I E 
Published: January 2023

Abstract

Background
Tissue factor (TF) plays an important role on blood clotting and the risk of thromboembolic events is increased in ulcerative colitis (UC). Tofacitinib, recently introduced in UC therapeutics, may further increase the risk.
To delineate thrombosis pathophysiology in this context, TF expression in primary human colonic mesenchymal cells (PHCMC) of patients with active UC was correlated with clinical parameters, serum markers of inflammation and endoscopy. We then treated those PHCMC with tofacitinib with or without cytokines to better mimic the in vivo milieu they are exposed to.

Methods
PHCMC from endoscopic biopsies of the inflamed mucosa of 10 UC patients with an endoscopic Mayo score ≥2 were treated with all major T helper (Th)1 (TNF-α, IFN-γ), Th2 (IL-4, IL-13) or T regulatory (Treg; TGF-β, IL-10) cytokines with or without tofacitinib. Cells were lysed, RNA was isolated and reverse-transcribed to cDNA. TF and RPL4 (housekeeping) cDNAs were quantified with real-time PCR. Wilcoxon and Mann-Whitney U tests were used to compare TF ΔΔCT for paired or unpaired values, respectively, and Spearman’s rho to correlate TF ΔΔCT with scale clinical variables and continuous laboratory values.

Results
Increased TF mRNA abundance in PHCMC was correlated to increased partial Mayo score (Spearman’s rho 0.661, p &lt; 0.044), reduced serum albumin (Spearman’s rho -0.723, p &lt; 0.05) and to more severe endoscopic lesions: cells originating from UC patients of an endoscopic Mayo score 3 expressed 20 times more TF than those from an endoscopic Mayo score 2 (p &lt; 0.017; Figure 1). Moreover, PHCMC from difficult-to-treat patients, defined as requiring &gt;1 biologics, expressed 9 times more TF (Figure 2).
On the other hand, treatment with tofacitinib did not upregulate TF (Figure 3). PHCMC expressed receptors and responded to treatment with all major Th1 (TNF-α, IFN-γ), Th2 (IL-4, IL-13) or Treg (TGF-β, IL-10) cytokines by further upregulating TF by 5.5-7.5 times (p 0.001-0.035). IL-13 had the maximal effect (Figure 4). Even when tofacitinib was added together with the aforementioned cytokines, it did not further upregulate TF; instead, it tended to partially inhibit their effects. For example, it decreased upregulation of TF by IL-13 by 40%.

Conclusion
The expression of TF mRNA in active UC is significantly associated with clinically and endoscopically severe disease and resistance to treatment. Tofacitinib per se does not increase TF. Instead, it may limit the upregulating effect of pro-inflammatory cytokines.
P042 Distinct gene expression profile associated to inflammation and neoplastic progression in longstanding ulcerative colitis
Authors: Alsøe L , Lefol Y , Wennerström A B , Liu Y , Andersen S N , Klepp P , Brackmann S , Nilsen H 
Published: January 2023

Abstract

Background
One of the main drivers of colitis associated colorectal cancer (CA-CRC) is suspected to be long-standing mucosal inflammation, but far from all colitis patients develop cancer. We analyzed mucosal gene expression profiles in ulcerative colitis (UC) patients who have progressed to neoplasia (progressors) compared to those who have not (non-progressors) in order to identify underlying mechanisms for neoplastic progression in UC patients.

Methods
We performed transcriptome profiling on 143 mucosal samples from non-neoplastic colon segments of 14 UC progressors and 30 UC non-progressors from the prospective CA-CRC cohorts from Lovisenberg Diakonale- and Akershus University Hospitals in Norway. Differentially expressed genes were determined using SurePrint G3 human gene expression 60K microarray (Agilent Tech Inc.).

Results
609 genes were differentially expressed between non-progressor- and progressor inflamed tissue, 24 genes between non-progressor- and progressor non-inflamed tissue, 404 genes between non-progressor non-inflamed- and -inflamed tissue and 26 genes between progressor non-inflamed- and inflamed tissue.
In inflamed mucosa, genes related to metabolic processes such as xenobiotic- as well as carbohydrate and lipid metabolism were significantly higher expressed in progressors compared to non-progressors. Genes related to innate and adaptive immune responses were expressed at significantly lower levels. These main traits were not influenced by type of inflammation but apparent in both, chronic- and chronic active inflammation. However, chronic inflamed mucosa of progressors were also characterised by dysregulation of processes related to chromatin organization. Treatment with immunomodulators and biologics were equally distributed in the groups. Restriction of the analysis to patients without treatment of these drugs did not significantly influence the main results.
In non-inflamed mucosa, the gene expression profile of progressors and non-progressors were remarkably similar. In fact, within the positively and negatively enriched gene ontologies, only the gene S100P was significantly deregulated with a higher expression in non-inflamed mucosa of the progressors.

Conclusion
Upregulated detoxification as well as dysregulated immune response and chromatin organization may be underlying processes for neoplastic progression in ulcerative colitis. Our findings warrant further evaluation.
P043 ALI culture derived from human organoids recapitulates cell composition during intestinal crypt formation
Authors: Dotti I , Álvarez I , Esteller M , Ricart E , Salas A 
Published: January 2023

Abstract

Background
The ALI (air liquid-interface) culture derived from human primary intestinal epithelium is generating considerable interest for closely reproducing the mature cell features of the differentiated epithelium in a 2D configuration. However, the phenotypic changes occurring in the human ALI culture during cell differentiation have thus far not been explored in depth. Here, we aimed to characterize the cell composition of human ALI culture over time.

Methods
3D epithelial organoids (n=3) were expanded from human colonic samples and organoid-derived single cells were seeded on a Transwell. After reaching confluence, the submerged 2D was switched to ALI and maintained for 7 days. The ALI culture was analysed every 24h by qPCR, membrane staining (H&E and immunofluorescence) and trans-epithelial resistance (TER) (Figure 1).

Results
The confluent pre-ALI (TER&gt;200 Ω*cm2) showed high mRNA expression of stem cell markers (i.e., LGR5, AXIN2), while several differentiation markers (i.e., ZG16, CLCA1, ANPEP, AQP8, BEST4, CHGA) were almost undetectable, thus resembling the stem cell niche compartment. Air exposure of the 2D culture induced an early and transient (limited to ALI-day 1) transcriptional up-regulation of proliferation markers (i.e., MKI67, CDCA7) and OLFM4, followed by a concomitant marked downregulation of stem cell markers, thus mimicking the Transit Amplifying compartment. OLFM4 protein, as observed by immunofluorescence, showed an early cytoplasmic accumulation followed by secretion at later time points (from ALI-day 5). Differentiation towards absorptive and secretory lineages increased gradually from ALI-day 5 and ALI-day 7 onward, respectively, at both the transcriptional and protein level. Expression changes were accompanied by the progressive transition of the ALI monolayer from a squamous to a columnar morphology. In parallel, TER promptly increased upon ALI switch, thus indicating the early establishment of cell-cell junctions.

Conclusion
By recapitulating the cell composition identified along the crypt, the ALI culture holds promise to be a versatile in vitro model for studying how the different epithelial cell compartments respond to specific environmental stimuli, like those present during an inflammatory flare.
P048 Deep phenotyping of the peripheral immune cell compartment in Crohn‘s Disease
Authors: Haag L M , Walling S , Kunkel D , Hecker J , Letizia M , Huck A , Weidinger C , Glauben R , Kuehl A , Siegmund B 
Published: January 2023

Abstract

Background
Crohn’s disease (CD) is characterized by chronic inflammation that can discontinuously affect any segment of the gastrointestinal tract. New findings concerning clinical behaviour, epidemiology, genetics, and gut microbiota suggest that CD in the ileum (iCD) and the colon (cCD) should be considered as two distinct subtypes of IBD. As systemic immune cell signatures differ between CD and UC, we propose a similar strategy for the differentiation between iCD and cCD. The aim of this study is to analyse and compare peripheral blood mononuclear cell (PBMC) subsets of iCD and cCD patients. For a robust analysis of the peripheral immune compartment in inflammatory bowel diseases (IBD), PBMC subsets of ileocolonic CD (icCD), ulcerative colitis (UC) and healthy controls (HC) were included as well.

Methods
In this study a total of 50 adults, 10 subjects per subgroup – iCD, cCD, icCD, UC and HC – were included. From each individual, both naïve and PMA/Ionomycin stimulated samples were stained for phenotypic and functional characterization using mass cytometry. For data analysis pre-gating was performed in Cytobank followed by debarcoding, compensation, normalization, clustering, and analysis on differential abundances edge R (DA_edgeR) and differential states limma (DS_limma) with CATALYST in R.

Results
FlowSOM allowed us to cluster CD45+ PBMCs of all samples into 16 major cell subtypes as visualized via UMAP (Fig. 1). DA testing showed significant differences between iCD and cCD, iCD and icCD but not cCD and icCD (Fig 2). Significantly different abundances among CD subgroups and HC included cluster 1 CD33+ myeloid cells, 7 CD45RO+ CD16+ cells, 8 CD11b+ cells, 9 CD4+ IL7R+ T cells and 12 CD27+ CD38+ CD45RA+ B cells (Fig. 3).
When focussing on differences between iCD, cCD and icCD, DS testing revealed significantly higher IFNgamma and TNFalpha production in cluster 9 (CD4+ IL7R+ T cells), 15 (CD8+ T cells) and 16 (CD8+ CD45RO+ T cells) in iCD compared to icCD. Moreover, cluster 9 showed significantly higher levels of IFNgamma in iCD compared to cCD, and for TNFalpha cluster 16 revealed significantly higher levels in cCD compared to icCD (Fig. 4).
Fig. 1 UMAP clustering of stimulated cells.

Fig. 2 Differential analysis of stimulated samples with DA_edgeR and DS_limma.

Fig. 3 Relative population abundances with DA_edge R testing.

Fig. 4 Relative state marker abundance within clusters with DS_limma testing.

Conclusion
Deep phenotyping of the peripheral immune cell compartment in CD might serve as a confirmatory marker for disease location and therefore as predictor of clinical behaviour.
P050 Intraperitoneally injected 3-dimensional cultured tonsil-derived mesenchymal stem cells in murine colitis model form aggregates with mouse immune cells: A preliminary study
Authors: Byeon J R , Song E M , Joo Y H , Park Y , Shim K N , Choe A R , Tae C H , Kim S E , Moon C M , Jung H K , Hong J T , Jung S A 
Published: January 2023

Abstract

Background
3-dimensional (3D) cultured tonsil-derived mesenchymal stem cells (TMSCs) showed higher therapeutic efficacy in murine colitis model than conventional 2D-cultured TMSCs via increased anti-inflammatory cytokine expression. Here, we evaluated the in-vivo localization and formation of 3D-cultured TMSCs using electron microscopy.

Methods
Chronic mouse colitis was induced by oral administration of 1.5% dextran sulfate sodium for 30 days. 3D-cultured TMSCs and 2D-cultured TMSCs was injected intraperitoneally on day 6 and day 16 after inducing colitis in mice. TMSC was 3D-cultured as a spheroid for 24 hours before intraperitoneal injection. On day 31, mice were euthanized and the intraperitoneal localization of 3D-cultured TMSCs was evaluated using immunofluorescence analysis. Moreover, the in-vivo characterization of 3D-cultured TMSCs was evaluated using electron microscopy.

Results
In vitro 3D-cultured TMSC spheroids were measured to have an average size of 182.4 μm on day 1, 169.4 μm on day 2, and 154.1 μm on day 3; however, the size of spheroids gradually decreased after 2 days of culture. In mice treated with 2D-cultured TMSCs, there were no detectable TMSCs at the time of sacrifice. In mice treated with 3D-cultured TMSCs, white variable sized spherical aggregates were observed. These aggregates were attached to peritoneal cavity including omentum and mesentery. In fluorescence microscopy, these aggregates were stained with human nuclear antigen antibody, indicating that the transplanted 3D-cultured TMSCs formed a cluster in the peritoneum even 14 days after intraperitoneal injection. In electron microscopy, these aggregates were covered with mouse lymphocytes. In the inside of spheroids, suspected mesenchymal cell and mesothelial cell were distributed.

Conclusion
Intraperitoneally injected 3D-cultured TMSCs were found as aggregates in the peritoneal cavity of mouse even 14 days after injection. In electron microscopy, these aggregates were covered with mouse lymphocytes. Based on the results of current study, functional studies evaluating the role of TMSC as a cell therapy, including cell-to-cell communication between TMSC and immune cells, are needed.
P054 Secreted inflammatory protein profiles following treatment of ex-vivo human inflammatory bowel disease explants with licenced biologic therapies
Authors: Corcoran R , O'Connell F , O'Sullivan J , Kevans D 
Published: January 2023

Abstract

Background
Patient-derived inflammatory bowel disease (IBD) ex-plants have potential for biomarker and therapy discovery. Infliximab (IFX), ustekinumab (USTK) and vedolizumab (VDZ) are biologic therapies licenced for the induction and maintenance of remission in ulcerative colitis (UC) and Crohn’s disease (CD). All are efficacious in IBD despite differing mechanisms of action. Treatment of IBD explants ex-vivo with licensed biologic therapies may result in further insights into their mechanism of action. We aimed to evaluate the effect of IFX, USTK and VDZ on inflammatory protein secretion profiles in ex-vivo human IBD ex-plants.

Methods
Patients with IBD, undergoing endoscopy, were prospectively recruited. Biopsies were collected from the sigmoid and IBD ex-plants generated as per previously described methods. IBD explants were then co-cultured for 24 hours with an IgG control vehicle, IFX, USTK and VDZ. After 24 hours, tissue conditioned media (TCM) from IBD explants was collected. TCM secreted inflammatory protein profiles were quantified using 54 V-plex ELISA (Meso Scale Diagnostics, USA). Secreted inflammatory protein profiles were compared between IgG vehicle (control) and USTK, IFX, VDZ treated ex-plants. All continuous variables are presented as median [interquartile range (IQR)]. P values &lt; 0.05 were considered significant in analyses.

Results
37 patients with IBD were included (51% CD, 49% UC); age (median, [IQR]) 41[33-53] years, 49% male; disease duration (median, [IQR]) 9 [5-14] years; 51% of patients were anti-TNF naïve. TNF-a secretion was significantly lower in IBD explants treated with IFX compared with IgG control, p=0.006. IL-23 secretion was significantly lower in IBD explants treated with USTK compared with IgG control, p=0.043. Biologic therapy ex-vivo resulted in significant differences in angiogenesis and vascular injury panel biomarkers compared to control; CRP, VEGF-A and SAA. Pro-inflammatory cytokines and chemokines were also significantly decreased with ex-vivo biologic therapy compared to control; IL-2, IL-10, IL-6, MCP-1, IL-1a, TNF-a and MDC. Three Th17 panel proteins were significantly decreased with ex-vivo biologic therapy compared to control; IL-22, IL-17A, IL17A Gen B.

Conclusion
The statistically significant decrease in both TNF-a and IL-23 secretion with ex vivo IFX and USTK treatment respectively demonstrates that the IBD explant model recapitulates expected IBD disease biology and therapy responses. These data demonstrate that treatment with licenced biologic therapies ex-vivo reduces multiple pro-inflammatory cytokines, chemokines and secreted proteins in IBD explants. Further study is required to completely understand the effects of licenced biologic therapies on the IBD tissue microenvironment.
P060 Restoration of occludin and claudin-1 expression in patients with Crohn’s disease receiving anti-TNF treatment.
Authors: Tsounis E , Geramoutsou C , Aggeletopoulou I , Lourida T , Pastras P , Theocharis G , Diamantopoulou G , Zolota V , Assimakopoulos S F , Bravou V , Thomopoulos K , Triantos C 
Published: January 2023

Abstract

Background
Occludin and claudin-1 are integral components of tight junctions (TJs) that contribute to intestinal barrier integrity. The aim of this study was to prospectively evaluate the expression of occludin and claudin-1 in the intestinal mucosa of patients with Crohn’s disease (CD) undergoing treatment with tumor necrosis factor (TNF) inhibitors.

Methods
Clinical assessment, laboratory testing, and colonoscopy were performed on all participants prior to receiving anti-TNF therapy. Colonic or ileal biopsies were obtained from the affected intestinal areas. A follow-up exam and colonoscopy were performed at least 6 months after the start of anti-TNF therapy, and paired biopsies were obtained from the initially affected segments. The expression of the TJ proteins was evaluated by immunohistochemistry (IHC).

Results

In this prospective observational study, 21 consecutive patients with active CD [median age 32.5 years (IQR: 18.5-51), 66.7% male] and 10 healthy controls [36 years (IQR: 29-58), 60% male] were enrolled. Patients with CD received treatment with TNF inhibitors (infliximab, adalimumab, and certolizumab: 61.9%, 33.3%, and 4.8%, respectively), according to current guidelines, and a follow-up examination was conducted after a median period of 25 months (IQR: 9.5-66). Before treatment administration, patients with active CD exhibited an increased expression of both occludin [2 (IQR: 1.75-2.75) vs. 1.5 (IQR: 0-1.75); p = 0.032] and claudin-1 [2.5 (IQR: 2-3) vs. 1 (IQR: 0.75-2); p = 0.01] in comparison to healthy controls. In control mucosa, IHC staining for occludin and claudin-1 was weak to moderate, cytoplasmic, and membranous, confined to the surface epithelium, while staining in intestinal crypts was negative. On the other hand, an enhanced expression of both proteins in the enteric crypts and surface epithelium was demonstrated in CD patients upon initial examination. Treatment with TNF inhibitors resulted in a significant reduction of claudin-1 expression (p = 0.018) as well as a notable decline of occludin expression (p = 0.08). Occludin and claudin-1 immunostaining was restricted to the surface epithelium in post-treatment biopsies. In addition, the activity of the disease, as estimated by the Crohn’s Disease Activity Index (r = 0.476; p = 0.014) and C-reactive protein (r = 0.411; p = 0.039), was positively correlated with the expression of claudin-1 in the mucosa.

Conclusion
Active CD is plagued with a disruption of the expression and distribution of claudin-1 and occludin throughout the affected intestinal mucosa. Successful treatment with biological agents induces the restoration of intestinal barrier function, which is associated with an improvement in the patients' clinical condition.