Introduction

Human stem cell-based organoids are well established modeling systems widely used to investigate a range of different diseases. For the pancreas, especially endocrine stem cell-derived islets are used to model and decipher the complex development of diabetes. Although we and others also generated pancreatic duct-like organoids (PDLO) [1-4], engineering the main pancreatic compartment, the acinar cells, is underrepresented in literature [1, 4, 5]. Here, we used our advanced differentiation protocol to generate pancreatic progenitors (PPs) [1]. We enriched for the glycoprotein-2 (GP2) expressing multipotent pancreatic progenitors and generated a finetuned acinar differentiation protocol based on pathway and transcriptome analysis of PPs on single cell level. The optimized protocol robustly produces pure mature and functionally active pancreatic acinar-like organoids which we extensively characterized and validated by high resolution imaging, electron microscopy, transplantation studies and functional assays.

Aims & Methods

Immunofluorescence staining of key acinar markers was performed to comprehensively characterize the stem cell-based acinar organoids. High resolution imaging and transmission electron microscopy revealed the presence of secretory vesicles and digestive enzymes. Functionality was proven by enzyme activity assays. Further maturation of the acinar organoids was monitored by transplantation on an ex vivo scaffold, the porcine urinary bladder [6, 7].

Results

In this study we succeeded in producing functional and mature pancreatic acinar organoids by differentiation of human embryonic stem cells. The enrichment for multipotent pancreatic progenitors was facilitated by a double magnetic activated cell sorting, a straightforward approach to sort for GP2 high expressing multipotent progenitors. Finetuning and modulating relevant pathways identified with a PP single-cell RNA sequencing dataset was based on building trajectories, cell-cell communication patterns and analyzing expression profiles. A comprehensive pipeline for characterization and analysis of the resulting acinar organoids validated their functionality and maturity.

Conclusion

The here generated stem-cell derived pancreatic acinar-like organoids provide a useful human model system to investigate pancreatic exocrine disorders, like pancreatic cancer or pancreatitis. It offers a valuable tool for understanding cancer initiation and progression in an animal-reduced human setup.

References

1. Merz, S., et al., Single-cell profiling of GP2-enriched pancreatic progenitors to simultaneously create acinar, ductal, and endocrine organoids. Theranostics, 2023. 13(6): p. 1949.
2. Breunig, M., et al., Modeling plasticity and dysplasia of pancreatic ductal organoids derived from human pluripotent stem cells. Cell Stem Cell, 2021. 28(6): p. 1105-1124. e19.
3. Breunig, M., et al., Differentiation of human pluripotent stem cells into pancreatic duct-like organoids.STAR protocols, 2021. 2(4): p. 100913.
4. Huang, L., et al., Commitment and oncogene-induced plasticity of human stem cell-derived pancreatic acinar and ductal organoids. Cell Stem Cell, 2021. 28(6): p. 1090-1104. e6.
5. Hohwieler, M., et al., Human pluripotent stem cell-derived acinar/ductal organoids generate human pancreas upon orthotopic transplantation and allow disease modelling. Gut, 2017. 66(3): p. 473-486.
6. Melzer, M.K., et al., Organoids at the PUB: the porcine urinary bladder serves as a pancreatic niche for advanced cancer modeling. Advanced Healthcare Materials, 2022. 11(11): p. 2102345.
7. Melzer, M.K., et al., Protocol to use de-epithelialized porcine urinary bladder as a tissue scaffold for propagation of pancreatic cells. STAR protocols, 2022. 3(4): p. 101869.

Introduction

Even though numerous risk factors and methods for prevention of post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (PEP) have been identified, it persists as the most prevalent complication. Administration of 100mg diclofenac prior to ERCP represents the most frequently used prophylaxis. Diclofenac is predominantly metabolized by various cytochrome P450 enzymes. The potential presence of single nucleotide polymorphisms (SNP) in these enzymes could cause interindividual variability in metabolization rates, which could affect serum plasma levels of diclofenac. Given the limited available research on this subject, the aim of this study was to investigate whether differences in presence of SNPs in the CYP-enzymes involved in diclofenac metabolism were associated with PEP.

Aims & Methods

This multicentre cohort study included patients above 18 years who underwent ERCP between 2012 and 2022. Upon inclusion, patients were divided into the PEP or control group. Real-time polymerase chain reactions were performed on buccal smear samples to identify gene variants involved in the diclofenac metabolism (e.g. CYP2C8*3). Secondary endpoints were validation of known and possibly yet unknown risk factors for PEP.

Results

In total, 103 patients were included in the PEP group and 245 in the control group. DNA amplification was possible in 91.5% of the genetic variants and revealed that heterozygous carriers of CYP2C8*3 variant were associated with a lower frequency of PEP (OR 0.2; 95% CI 0.0-0.8). Pancreatic duct cannulation (OR 3.0; 95% CI 1.4-6.6) and precut sphincterotomy (OR 3.1; 95% CI 1.2-8.0) were both found as independent risk factors for PEP.

Conclusion

This study showed that heterozygous carriers of the CYP2C8*3 variant have a lower risk of developing PEP. To investigate gene variants affecting real-time diclofenac plasma levels, we have started a study in which we prospectively collect blood samples to enhance DNA quality and to analyze the existence of a correlation between PEP and reduced diclofenac levels. If this correlation is confirmed, further research is needed to investigate the optimal dosage of diclofenac in wild type CYP2C8 patients.

Introduction

Small bowel fibrostenotic strictures are common in patients with Crohn’s disease (CD). No global consensus recommendations on definitions, diagnosis and clinical management are available.

Aims & Methods

Several systematic reviews followed by a RAND/University of California Los Angeles appropriateness study on the definitions, diagnosis and clinical management of fibrostenosing CD in clinical practice were performed. A panel of 27 global experts and a patient representative were convened. They assessed a total of 152 candidate items. The items were subsequently evaluated for appropriateness.

Results

No accurate predictive biomarkers are available for naïve or anastomotic fibrostenosing strictures. Accurate diagnosis of fibrostenosing CD requires cross-sectional imaging which should evaluate bowel wall thickness, luminal narrowing and prestenotic dilatation. A potential inflammatory component should be assessed. Abdominal cross-sectional imaging was considered necessary prior to any treatment decision. The panel proposed an approach to medical, endoscopic, and surgical therapies. Technical characteristics for endoscopic balloon dilation and follow up strategies after successful dilation therapy were identified. Appropriateness, types and performance of different surgical approaches in various settings were evaluated.

Conclusion

This global consensus provides clinical guidance for the diagnostic and therapeutic management of patients with fibrostenotic CD.

Introduction

​​​​​Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-associated mortality in developed countries with a 5-year survival rate of less than 10%. Despite extensive research, there is still a lack of reliable biomarkers for early disease detection and pathophysiology. Therefore, the minute delineation of intimate molecular mechanisms involved in the intratumoral interplay between different compartments of PDAC became of paramount importance. DKK3 (Dickkopf-3), one of the main modulators of Wnt signaling, is a rather unique protein: in PDAC, suppression of DKK3 in a Trp53-deficient context negatively impacts tumor progression and prolongs survival. We previously identified DKK3 as a novel factor for organ regeneration during pancreatitis and, through state-of-the-art investigations involving a in vivo models, delineated a direct, previously unknown link between DKK3 and the canonical-Wnt- and Hedgehog-signaling. This, and given that pancreatitis renders as a risk factor for pancreatic cancer, prompted us to investigate the potential role of DKK3 in PDAC development.

Aims & Methods

To investigate the role of DKK3 in PDAC progression, we employed cutting-edge in vivo, in vitro, and ex vivo experimental settings at different stages of KRAS-driven tumorigenesis (from early dysplasia to cancer stage). In line, a KRAS-driven PDAC mouse model (LSL-KrasG12D/+; Ptf1aCre/+) harboring wildtype Dkk3 allele (KC), heterozygous Dkk3 deletion (DKC) or homozygous Dkk3 deletion (DDKC) was involved. To specifically examine stage-specific roles of DKK3, we characterized the mice at different stages, from development of early dysplasia (10 weeks) until invasive PDAC (36 weeks). To dissect the DKK3 compartment-specific role at early and late stages within the epithelial compartment, functional assays were conducted on acinar and tumor cells respectively. Comprehensive co-culture systems employing stellate and tumor cells were utilized to characterize the intratumoral heterogeneity traits.

Results

The phenotyping of mice revealed that DKCs and DDKCs animals displayed significantly more acinar-to-ductal metaplasia (ADM) and precursor lesions from 10 weeks onward. These lesions translated in a substantial PDAC invasiveness at 36 weeks, and negatively impacted the survival and metastasis at end stage. Additionally, the loss of DKK3 positively correlated with a higher fibrotic content, a key characteristic of PDAC tumorigenesis. In line, ex vivo ADM assays demonstrated a significantly increased capacity of DKK3-null acinar cells to form ductal structures. Additionally, functional assays employing knockout tumor cells revealed higher mesenchymal marker expression such as Vimentin and N cadherin, increased invasion and migration compared to KC, in line with increased metastasis at the end stage. Furthermore, DDKC tumor cells were able to redraft the fibroblast compartment and thus, to give rise to more myofibroblasts that could likely contribute to the tumor aggressiveness at the end stages.

Conclusion

Overall, we show a multifaceted role of DKK3 in pancreatic cancer progression, and especially highlight its tumor-restraining capability within the epithelial compartment as a moderator of early tumor initiating events. This could potentially enable DKK3 to rise as a novel stage-specific biomarker for early PDAC detection.

Introduction

Ozanimod (OZA) is approved for the treatment of moderately to severely active ulcerative colitis and relapsing multiple sclerosis. A previous phase 1 study of single dose OZA 0.23 mg showed a 31%–33% reduction of CC112273, the predominant active metabolite (MET) of OZA, in mild or moderate hepatic impaired participants (ppts) vs healthy ppts. That study was designed prior to identification of the 2 major METs of OZA (CC112273; CC1084037). Due to their slow accumulation and elimination, a new multiple dose study was designed with a long sampling schedule.

Aims & Methods

This multicenter, open-label phase 1 study (NCT04639115) evaluated the effect of mild (Child-Pugh [C-P] score 5–6) or moderate (C-P score 7–9) hepatic impairment on the multiple-dose pharmacokinetics (PK) of OZA and its 2 major active METs. Eligible ppts with hepatic impairment without active hepatitis were enrolled into the mild or moderate hepatic impairment groups (8 ppts each) based on their C-P scores. Ppts with normal hepatic function were matched to hepatic-impaired ppts by sex, age, weight, and smoking status. All ppts received once-daily OZA for 8 d in accordance with dose titration: 0.23 mg on Days (D) 1–4, 0.46 mg on D5–7, and 0.92 mg thereafter. Serial PK samples were collected on D1, D5, and D8, and additionally on D10, D12, and D15, and up to D72. Analysis of variance (ANOVA) fitted to the natural log-transformed PK parameters with hepatic function group as the fixed effect and the matching pair as a random effect was used to compare PK parameters between groups. Safety was assessed.

Results

All dosed ppts (N=26) completed the study. Each hepatic impairment group had 8 ppts; 10 ppts were in the normal hepatic function group, of whom 6 matched both mild and moderate hepatic-impaired ppts. Of the 16 hepatic-impaired ppts, 13 had documented hepatic cirrhosis. No clinically meaningful differences in total and unbound PK parameters for OZA and its major METs were observed between healthy and hepatic-impaired ppts on D1, D5, or D8 (dose titration) (Table). The unbound area under the plasma concentration-time curve from D8, time 0 to the time of last quantifiable concentration (AUC_0-last) (up to 64 d after the D8 dose) of both METs increased ~2-fold in both mild and moderate hepatic-impaired ppts (Table). Although steady state for both METs was not achieved by D8, AUC_0-last is a reasonable predictor of exposure at steady state. No correlation was observed between PK parameters for OZA or its major METs and measures of hepatic function (C-P score, serum albumin, and prothrombin time). No laboratory results (ie, absolute lymphocyte count), vital signs, physical examination, or 12-lead ECG results were reported as treatment-emergent adverse events.

Ratio of geometric LS mean (90% CI) for unbound PK parameters in ppts with hepatic impairment vs matched healthy ppts	PK parameter of interest	OZA	CC112273	CC1084037
Group comparison: Mild hepatic impairment (test) vs normal matched (reference)	D1 Cmax	134.42 (108.51, 166.52)	118.03 (77.19, 180.47)	NA
	D5 Cmax	135.60 (106.51, 172.63)	135.50 (92.26, 198.99)	141.26 (95.86, 208.17)
	D8 Cmax	186.48 (103.75, 335.19)	147.45 (87.15, 249.47)	145.26 (90.38, 233.45)
	D8 AUC0-last	182.53 (81.97, 406.46)	221.43 (106.61, 459.88)	229.74 (93.24, 566.10)
Group comparison: Moderate hepatic impairment (test) vs normal matched (reference)	D1 Cmax	136.12 (110.49, 167.71)	113.96 (85.55, 151.81)	NA
	D5 Cmax	124.92 (93.42, 167.04)	111.73 (80.73, 154.61)	120.98 (96.42, 151.80)
	D8 Cmax	168.91 (95.72, 298.09)	133.71 (84.78, 210.88)	135.77 (94.20, 195.68)
	D8 AUC0-last	145.87 (68.28, 311.64)	199.64 (119.55, 333.37)	212.36 (118.15, 381.70)
Note: The log-transformed PK parameters were analyzed using an ANOVA model with hepatic function group as a fixed effect and matching pair as a random effect. Cmax, maximum observed plasma concentration; LS, least squares; NA, not applicable.

Conclusion

Due to the lack of OZA PK differences between hepatic-impaired and healthy ppts during dose titration and the 2-fold increase of AUC_0-last of both METs in hepatic-impaired ppts, a dose adjustment of OZA 0.92 mg once every other day is recommended in patients with mild or moderate hepatic impairment after completing the standard 1-week dose titration.

Disclosure

This study was funded by Bristol Myers Squibb, Princeton, NJ, USA.
PZ, FH, KY, SW, MS, GT, BM, and DT: employees and/or shareholders of Bristol Myers Squibb.

Introduction

The molecular pathways for the development of ulcerative colitis (UC) are unclear. The purpose of the work is to study the molecular interactions of the apoptotic pathway in patients with UC.

Aims & Methods

The study was prospective comparative cohort with parallel design and included 65 patients (range from 22 to 35 years, 24 men and 41 women) with left-sided UC, mild and moderate activity (Truelove - Witts’ criteria, Mayo score). The induction of UC remission assumed the acceptance of mesalazine 3 to 4 g/day p.o. and rectally 1 to 2 g/day, the duration of therapy was 4.6 years. The control group
included 30 healthy individuals. The separation of individual proteins of colon mucosa was based on technologies of IEF, SDS-PAGE, 2DPAGE, by standard sets (MB-HIC C8 Kit, MB-IMAC Cu, MB-Wax Kit, «Bruker», USA). Automated mass spectrometry imaging was performed by matrix-assisted laser desorption-ionization timeof-flight mass spectrometry (MALDI-TOF-MS/MS, Ultraflex II, «Bruker», USA). The partially identified sequences were then submitted to “BLAST protein-protein” and screened against the Homo sapiens Swissprot database to check if this identification matched the MASCOT-identification (Matrix Science, UK). The data of the molecular interactions and functional features of proteins were received with STRING 10.0 database. Based on the data of standard methods of identification of UC and molecular phenotyping of colon mucosa we conducted new prognostic markers, molecular pathways of UC and diagnostic tests in patients with UC. Statistical analysis of the survey data was performed using the software “Statistica 12.0” (Statsoft).

Results

Bioinformatics analysis revealed the presence of molecules that are the participants in the universal pathways of UC in the active stage, and the molecular interactions involved. We identified following functional groups of peptides and proteins in molecular patterns of bioptates of colon mucosa in UC patients: peptides and proteins regulating the barrier function of colon mucosa; proteins-participants of specific metabolism in epitheliocytes and endocrinocytes; proteins of the fibrosis in colon mucosa; proteins regulating cell cycle, oncogenesis, proteolysis in cell, hormones processing, angiogenesis, coagulation factors; proteins of free radical oxidation and antioxidant system; proteins regulating the receptor activity of epitheliocytes and immune cells; structural proteins of colon mucosa; transcription and translation factors regulating the activity of cell nucleus, regulators of protein folding; transport proteins; proteins-enzyms of detoxification. Significant decrease of the PPARγ expression promotes the activation of STAT and AP-1 signaling pathways, which promotes an increase in the synthesis of IL-2,6,8,12, TNFα, matrix metalloproteinases, the activity of immune and inflamation processes in colon mucosa. A significant increase in the NF-kB expression in colon mucosa is associated with the activation of TNFα and IL-1, which promotes the increase of immune processes in colon mucosa. The molecular interactions of β-defensin-1: the reduction of the β-defensin-1 expression in cells of colon mucosa is accompanied by increased expression of CCR6, which promotes the formation of inflammatory infiltrates in colon submucosa in UC.

Conclusion

Identification of potential new molecular markers for UC development may provide opportunities for the development of diagnostic tests to evaluate the disease.

Introduction

Mental health is an overlooked aspect of inflammatory bowel disease (IBD) patient care and the most common psychological conditions in patients with IBD are anxiety and depression¹. The objective of this study was to examine the possible factors that will impact mental health burden of CD patients in Europe.

Aims & Methods

A multi-centre online medical chart review study of patients with CD was conducted between Jul – Sep 2022 among UK, FR, DE, IT & ES gastroenterologists practicing across hospital and private practices. Physicians were screened for practice duration and patient volume. Charts of patients prescribed with advanced therapy were included in the analysis.

Results

206 sampled physicians collectively reported 1178 CD patients. From the reported CD patients, 190 suffered mental health burden (depression or anxiety) and 988 CD patients did not suffered any mental health burden. Among CD patients suffered mental health burden, a higher proportion of CD patients are female vs those who did not suffered any (55% vs 46%). ‘Family history of IBD’ was the co-morbidity more likely to experience in reported CD patients suffered mental health burden vs those who did not (11% vs 7%).
When looking at employment status between these two patient groups, reported CD patients suffer mental health burden was less likely to be in full-time employment vs those not suffered mental health burden. A higher proportion of reported CD patients suffered mental health burden were in part-time employment or housewife/househusband.

Table 1: Employment status in reported CD patients (% patients)

CD patients	Suffered mental health burden	NOT suffered mental health burden
% full-time employment	47%	62%
% part-time employment	22%	14%
% housewife/ husband	8%	4%

CD patients with mental health burden had greater CRP level than patients without mental health burden (CRP mean: 16 vs 12). This aligns with reported CD patients with mental health burden had frequent surgery times vs without mental health burden (Mean surgery times: 2 vs 1) and had greater steroid usage (% reported patients currently on steroids – 18% vs 13%).

Conclusion

Comparisons in this study cohort highlight there are other factors could impact CD patients’ mental health burden other than IBD itself. This coincided with low full-time employment rate that attributed to financial stress may increase CD patients’ mental health burden. Also, higher rate of surgery and steroid usage in CD patients experienced mental health burden might suggest they had more severe or complications with their disease that will impact overall mental health. Further investigation using comparator cohort is warranted.

References

1. Shurong Hu, Yiping Chen, Yan Chen & Caihua Wang (2021): Depression and Anxiety Disorders in Patients With Inflammatory Bowel Disease, NCBI, DOI: 10.3389/fpsyt.2021.714057