Chymotrypsin activity signals to intestinal epithelium by protease-activated receptor-dependent mechanisms.

BACKGROUND AND PURPOSE: Chymotrypsin is a pancreatic protease secreted into the lumen of the small intestine to digest food proteins. We hypothesized that chymotrypsin activity may be found close to epithelial cells and that chymotrypsin signals to them via protease-activated receptors (PARs). We deciphered molecular pharmacological mechanisms and gene expression regulation for chymotrypsin signalling in intestinal epithelial cells. EXPERIMENTAL APPROACH: The presence and activity of chymotrypsin were evaluated by Western blot and enzymatic activity tests in the luminal and mucosal compartments of murine and human gut samples. The ability of chymotrypsin to cleave the extracellular domain of PAR1 or PAR2 was assessed using cell lines expressing N-terminally tagged receptors. The cleavage site of chymotrypsin on PAR1 and PAR2 was determined by HPLC-MS analysis. The chymotrypsin signalling mechanism was investigated in CMT93 intestinal epithelial cells by calcium mobilization assays and Western blot analyses of (ERK1/2) phosphorylation. The transcriptional consequences of chymotrypsin signalling were analysed on colonic organoids. KEY RESULTS: We found that chymotrypsin was present and active in the vicinity of the colonic epithelium. Molecular pharmacological studies have shown that chymotrypsin cleaves both PAR1 and PAR2 receptors. Chymotrypsin activated calcium and ERK1/2 signalling pathways through PAR2, and this pathway promoted interleukin-10 (IL-10) up-regulation in colonic organoids. In contrast, chymotrypsin disarmed PAR1, preventing further activation by its canonical agonist, thrombin. CONCLUSION AND IMPLICATIONS: Our results highlight the ability of chymotrypsin to signal to intestinal epithelial cells via PARs, which may have important physiological consequences in gut homeostasis.

Minute virus of mice shows oncolytic activity against pancreatic cancer cells exhibiting a mesenchymal phenotype.

Pancreatic cancer will soon become the second cause of death by cancer in Western countries. The main barrier to increase the survival of patients with this disease requires the development of novel and efficient therapeutic strategies that better consider tumor biology. In this context, oncolytic viruses emerge as promising therapeutics. Among them, the fibrotropic minute virus of mice prototype (MVMp) preferentially infects migrating and undifferentiated cells that highly resemble poorly differentiated, basal-like pancreatic tumors showing the worst clinical outcome. We report here that MVMp specifically infects, replicates in, and kills pancreatic cancer cells from murine and human origin with a mesenchymal, basal-like profile, while sparing cancer cells with an epithelial phenotype. Remarkably, MVMp infection, at a dose that does not provoke tumor growth inhibition in athymic mice, shows significant antitumoral effect in immune-competent models; extended mouse survival; and promoted the massive infiltration of tumors by innate, myeloid, and cytotoxic T cells that exhibit a less terminally exhausted phenotype. Collectively, we demonstrate herein for the first time that MVMp is specific and oncolytic for pancreatic tumors with mesenchymal, basal-like profile, paving the way for precision-medicine opportunities for the management of the most aggressive and lethal form of this disease.

Curative surgical treatment of common bile duct stones: Retrospective cohort study.

BACKGROUND: In the event of symptomatic common bile duct (CBD) stones with dilated CBD, one possible curative treatment option is stone extraction through choledocotomy associated with cholecystectomy. Endoscopic treatment is only reserved for residual stones at 6 weeks. The aim of this study was to evaluate the results from laparoscopic curative surgical treatment of CBD stones with dilated CBD. METHODS: This is a retrospective single-centered cohort study. All consecutive patients admitted for laparoscopic cholecystectomy with evidence of CBD stones with dilated CBD from January 2010 to December 2020 at our center were included. Success was defined by CBD clearance at 6 weeks. Need for additional procedures, such as endoscopic sphincterotomy, immediate, and end-of-procedure morbi-mortality as well as factors associated with procedure failure, were also studied. RESULTS: A total of 246 patients who received curative treatment were included in the study. The success rate for the curative treatment was 93.1% (229 patients). Immediate postoperative morbidity was 24.4% with a 5.3% reintervention rate. Immediate and 6-week postoperative mortality rates were zero and 0.4%, respectively. The mean length of stay was 11.3 days. Factors associated with procedure failure appeared to be the occurrence of an early postoperative complication and the need for readmission during the period between surgery and drain removal. CONCLUSION: This study indicates that laparoscopic curative surgical treatment for symptomatic CBD stones may be performed with acceptable results without routine need for additional procedures.

Cytidine Deaminase Resolves Replicative Stress and Protects Pancreatic Cancer from DNA-Targeting Drugs.

Cytidine deaminase (CDA) functions in the pyrimidine salvage pathway for DNA and RNA syntheses and has been shown to protect cancer cells from deoxycytidine-based chemotherapies. In this study, we observed that CDA was overexpressed in pancreatic adenocarcinoma from patients at baseline and was essential for experimental tumor growth. Mechanistic investigations revealed that CDA localized to replication forks where it increased replication speed, improved replication fork restart efficiency, reduced endogenous replication stress, minimized DNA breaks, and regulated genetic stability during DNA replication. In cellular pancreatic cancer models, high CDA expression correlated with resistance to DNA-damaging agents. Silencing CDA in patient-derived primary cultures in vitro and in orthotopic xenografts in vivo increased replication stress and sensitized pancreatic adenocarcinoma cells to oxaliplatin. This study sheds light on the role of CDA in pancreatic adenocarcinoma, offering insights into how this tumor type modulates replication stress. These findings suggest that CDA expression could potentially predict therapeutic efficacy and that targeting CDA induces intolerable levels of replication stress in cancer cells, particularly when combined with DNA-targeted therapies. SIGNIFICANCE: Cytidine deaminase reduces replication stress and regulates DNA replication to confer resistance to DNA-damaging drugs in pancreatic cancer, unveiling a molecular vulnerability that could enhance treatment response.

Preclinical development of non-viral gene therapy for patients with advanced pancreatic cancer.

Pancreatic cancer remains one of the greatest challenges in oncology for which therapeutic intervention is urgently needed. We previously demonstrated that the intra-tumoral gene transfer of somatostatin receptor 2, to combat tumor aggressiveness, or of deoxycytidine kinase and uridylate monophosphate kinase, to sensitize to gemcitabine chemotherapy, has anti-tumoral potential in experimental models of cancer. Here, we describe the development of the CYL-02 non-viral gene therapy product that comprises a DNA-plasmid encoding for the three aforementioned genes, which expression is targeted to tumor cells, and complexed with polyethyleneimine non-viral vector. We performed pre-clinical toxicology, bio-distribution, and therapeutic activity studies of CYL-02 in two rodent models of pancreatic cancer. We found that CYL-02 is safe, does not increase gemcitabine toxicity, is rapidly cleared from blood following intravenous administration, and sequestered in tumors following intra-tumoral injection. CYL-02 drives the expression of therapeutic genes in cancer cells and strongly sensitizes tumor cells to gemcitabine, both in vitro and in vivo, with significant inhibition of tumor cells dissemination. This study was instrumental for the later use of CYL-02 in patients with advanced pancreatic cancer, demonstrating that rigorous and thorough preclinical investigations are informative for the clinical transfer of gene therapies against this disease.

Pancreatic Cancer in Chronic Pancreatitis: Pathogenesis and Diagnostic Approach.

Chronic pancreatitis is one of the main risk factors for pancreatic cancer, but it is a rare event. Inflammation and oncogenes work hand in hand as key promoters of this disease. Tobacco is another co-factor. During alcoholic chronic pancreatitis, the cumulative risk of cancer is estimated at 4% after 15 to 20 years. This cumulative risk is higher in hereditary pancreatitis: 19 and 12% in the case of PRSS1 and SPINK1 mutations, respectively, at an age of 60 years. The diagnosis is difficult due to: (i) clinical symptoms of cancer shared with those of chronic pancreatitis; (ii) the parenchymal and ductal remodeling of chronic pancreatitis rendering imaging analysis difficult; and (iii) differential diagnoses, such as pseudo-tumorous chronic pancreatitis and paraduodenal pancreatitis. Nevertheless, the occurrence of cancer during chronic pancreatitis must be suspected in the case of back pain, weight loss, unbalanced diabetes, and jaundice, despite alcohol withdrawal. Imaging must be systematically reviewed. Endoscopic ultrasound-guided fine-needle biopsy can contribute by targeting suspicious tissue areas with the help of molecular biology (search for KRAS, TP53, CDKN2A, DPC4 mutations). Short-term follow-up of patients is necessary at the clinical and paraclinical levels to try to diagnose cancer at a surgically curable stage. Pancreatic surgery is sometimes necessary if there is any doubt.

The PRSS3P2 and TRY7 deletion copy number variant modifies risk for chronic pancreatitis.

BACKGROUND: PRSS1 and PRSS2 constitute the only functional copies of a tandemly-arranged five-trypsinogen-gene cluster (i.e., PRSS1, PRSS3P1, PRSS3P2, TRY7 and PRSS2) on chromosome 7q35. Variants in PRSS1 and PRSS2, including missense and copy number variants (CNVs), have been reported to predispose to or protect against chronic pancreatitis (CP). We wondered whether a common trypsinogen pseudogene deletion CNV (that removes two of the three trypsinogen pseudogenes, PRSS3P2 and TRY7) might be associated with CP causation/predisposition. METHODS: We analyzed the common PRSS3P2 and TRY7 deletion CNV in a total of 1536 CP patients and 3506 controls from France, Germany, India and Japan by means of quantitative fluorescent multiplex polymerase chain reaction. RESULTS: We demonstrated that the deletion CNV variant was associated with a protective effect against CP in the French, German and Japanese cohorts whilst a trend toward the same association was noted in the Indian cohort. Meta-analysis under a dominant model yielded a pooled odds ratio (OR) of 0.68 (95% confidence interval (CI) 0.52-0.89; p = 0.005) whereas an allele-based meta-analysis yielded a pooled OR of 0.84 (95% CI 0.77-0.92; p = 0.0001). This protective effect is explicable by reference to the recent finding that the still functional PRSS3P2/TRY7 pseudogene enhancers upregulate pancreatic PRSS2 expression. CONCLUSIONS: The common PRSS3P2 and TRY7 deletion CNV was associated with a reduced risk for CP. This finding provides additional support for the emerging view that dysregulated PRSS2 expression represents a discrete mechanism underlying CP predisposition or protection.

Pancreatic Cancer Cell and Gene Biotherapies: Past, Present, and Future.

Solid cancers remain a major health challenge in terms of research, not only due to their structure and organization but also in the molecular and genetic variations present between tumors as well as within the same tumor. When adding on the tumor microenvironment with cancer-associated cells, vasculature, and the body's immune response (or lack of), the weapons used to tackle this disease must also be diverse and intricate. Developing gene-based therapies against tumors contributes to the diverse lines of attack already established for cancers and can potentially overcome certain obstacles encountered with these strategies, the lack of tumor selectivity with chemotherapies, for example. Given the high mortality and relapse rate associated with pancreatic cancer, novel treatments, including gene therapy, are actively being investigated. Even though no gene therapy for pancreatic cancer is currently on the market, a significant amount of clinical trials are underway, especially in active and recruiting or recently completed phases.

Prevention of post-operative recurrence of Crohn's disease among patients with prior anti-TNFα failure: A retrospective multicenter study.

BACKGROUND: Anti-TNFα are recommended for preventing Crohn's disease (CD) postoperative recurrence (POR) in patients with risk factors. However, few data exploring anti-TNFα efficacy in patients with preoperative anti-TNFα failure are available so far. AIMS: The aim of the present study was to compare the efficacy of anti-TNFα with other biologics and immunosuppressants to prevent POR in this setting. METHODS: Consecutive CD patients who underwent bowel resection between January 2010 and December 2019 after failure of at least one anti-TNFα were retrospectively included among three tertiary centers if they started a postoperative medical prophylaxis within the three months after index surgery. The main outcome was to compare rates of objective recurrence (endoscopic or radiological recurrence in absence of colonoscopy) between patients treated with an anti-TNFα agent or another treatment as prevention of POR. RESULTS: Among the 119 patients included, 71 patients received an anti-TNFα (26 infliximab, 45 adalimumab) and 48 another treatment (18 ustekinumab, 7 vedolizumab, 20 azathioprine and 3 methotrexate) to prevent POR. Rates of objective recurrence at two years were 23.9% in patients treated with anti-TNFα and 44.9% in the others (p = 0.011). CONCLUSION: Anti-TNFα remained an effective option to prevent POR for patients operated upon with previous anti-TNFα failure.

Long-Term Follow-Up and Immunomonitoring of Relapsing Type 1 Autoimmune Pancreatitis Treated With Rituximab.

OBJECTIVES: To evaluate the efficacy and safety of rituximab in relapsing type 1 autoimmune pancreatitis especially the long-term clinical and immunologic impacts. METHODS: All consecutive patients with type 1 autoimmune pancreatitis were retrospectively included. The rituximab protocol was induction therapy of 375 mg·m -2 intravenous weekly for 4 weeks, followed by 500 mg intravenous every 6 months for 2 years. The follow-up included clinical examinations, biological tests, positron emission tomography scan, and immunomonitoring of lymphocyte CD 19+. RESULTS: Among the 43 patients included, 15 received rituximab induction therapy, followed by maintenance in 10 cases because of 1 or more relapses after steroids (whether or not followed by immunosuppressants) and multiple organ involvement. All patients had a clinical, biological and morphological response, a deep and persistent drop in serum immunoglobulin G4 levels, an extinction of both pancreatic and extra pancreatic hypermetabolic positron emission tomography scan signals, and a depletion of B lymphocyte CD19+. No relapse occurred during the follow-up (62.8 ± standard error of the mean of 11.1 months). CONCLUSIONS: Rituximab is an effective treatment for type 1 autoimmune pancreatitis that provides a rapid strong clinical, biological, and morphological response, which persists after discontinuation without any safety issues.

Endoscopic ultrasound as a reliable tool for assessment of pancreatic adenocarcinoma treatment: Example of in situ gene therapy.

Background and study aims In pancreatic cancer, the antitumor effect can only be assessed by means of a computed tomography (CT) scan using RECIST (Response Evaluation Criteria in Solid Tumours) criteria. The aim of this study was to assess the intra-observer and interobserver agreement of endoscopic ultrasound (EUS) imaging in assessing tumor volume in primary pancreatic cancer. Patients and methods During a Phase 1 gene therapy trial, 21 patients had EUS before the first and second EUS-guided in situ gene therapy injections. All anonymized EUS files were then randomly distributed to three gastroenterologists/endosonographers and three radiologists (blind status). The largest tumor diameter was measured and the intraclass correlation coefficient (ICC) was determined. Results Intra-observer and interobserver agreements were good to excellent, regardless of operator experience (junior versus senior member of staff) (ICC: 0.65 to 0.84). A comparison of pretreatment and post-treatment measurements by the investigators highlighted a significant antitumor effect (-11 %; P  = 0.0098), similar to that obtained during the generic protocol (-10 %; P  = 0.0045). Conclusions Interobserver agreement regarding primary pancreatic adenocarcinoma measurements appears good to excellent, thus paving the way for the future inclusion of EUS assessments, particularly in trials assessing local therapies for pancreatic tumors.

Guidelines for the management of patients with severe acute pancreatitis, 2021.

OBJECTIVE: To provide guidelines for the management of the intensive care patient with severe acute pancreatitis. DESIGN: A consensus committee of 22 experts was convened. A formal conflict-of-interest (COI) policy was developed at the beginning of the process and enforced throughout. The entire guideline construction process was conducted independently of any industrial funding (i.e. pharmaceutical, medical devices). The authors were required to follow the rules of the Grading of Recommendations Assessment, Development and Evaluation (GRADE®) system to guide assessment of quality of evidence. The potential drawbacks of making strong recommendations in the presence of low-quality evidence were emphasised. METHODS: The most recent SFAR and SNFGE guidelines on the management of the patient with severe pancreatitis were published in 2001. The literature now is sufficient for an update. The committee studied 14 questions within 3 fields. Each question was formulated in a PICO (Patients Intervention Comparison Outcome) format and the relevant evidence profiles were produced. The literature review and recommendations were made according to the GRADE® methodology. RESULTS: The experts' synthesis work and their application of the GRADE® method resulted in 24 recommendations. Among the formalised recommendations, 8 have high levels of evidence (GRADE 1+/-) and 12 have moderate levels of evidence (GRADE 2+/-). For 4 recommendations, the GRADE method could not be applied, resulting in expert opinions. Four questions did not find any response in the literature. After one round of scoring, strong agreement was reached for all the recommendations. CONCLUSIONS: There was strong agreement among experts for 24 recommendations to improve practices for the management of intensive care patients with severe acute pancreatitis.

Variants in the pancreatic CUB and zona pellucida-like domains 1 (CUZD1) gene in early-onset chronic pancreatitis - A possible new susceptibility gene.

OBJECTIVE: Non-alcoholic chronic pancreatitis (NACP) frequently develops in the setting of genetic susceptibility associated with alterations in genes that are highly expressed in the pancreas. However, the genetic basis of NACP remains unresolved in a significant number of patients warranting a search for further risk genes. DESIGN: We analyzed CUZD1, which encodes the CUB and zona pellucida-like domains 1 protein that is found in high levels in pancreatic acinar cells. We sequenced the coding region in 1163 European patients and 2018 European controls. In addition, we analyzed 297 patients and 1070 controls from Japan. We analyzed secretion of wild-type and mutant CUZD1 from transfected cells using Western blotting. RESULTS: In the European cohort, we detected 30 non-synonymous variants. Using different prediction tools (SIFT, CADD, PROVEAN, PredictSNP) or the combination of these tools, we found accumulation of predicted deleterious variants in patients (p-value range 0.002-0.013; OR range 3.1-5.2). No association was found in the Japanese cohort, in which 13 non-synonymous variants were detected. Functional studies revealed >50% reduced secretion of 7 variants, however, these variants were not significantly enriched in European CP patients. CONCLUSION: Our data indicate that CUZD1 might be a novel susceptibility gene for NACP. How these variants predispose to pancreatitis remains to be elucidated.

Severe Vulvar and Perianal Crohn's Disease: A Case Report and Review of the Literature.

Association of vulvar Crohn's disease (CD) with pyoderma gangrenosum (PG) has been described, yet due to its low prevalence, data on therapeutic management of this association are still limited. We here present a 22-year-old woman with severe vulvar and perianal CD with a major inguinal and perineal ulceration. In the hypothesis of an associated PG, oral corticosteroids were prescribed at a dose of 40 mg with limited efficacy for a duration of 3 months. Afterward, a combination treatment of cyclosporine and infliximab followed by negative pressure wound therapy and split-thickness skin graft resulted in complete healing.

Incidence and Risk Factors of Cancer in the Anal Transitional Zone and Ileal Pouch following Surgery for Ulcerative Colitis and Familial Adenomatous Polyposis.

Proctocolectomy with ileal pouch-anal anastomosis is the intervention of choice for ulcerative colitis and familial adenomatous polyposis requiring surgery. One of the long-term complications is pouch cancer, having a poor prognosis. The risk of high-grade dysplasia and cancer in the anal transitional zone and ileal pouch after 20 years is estimated to be 2 to 4.5% and 3 to 10% in ulcerative colitis and familial polyposis, respectively. The risk factors for ulcerative colitis are the presence of pre-operative dysplasia or cancer, disease duration > 10 years and severe villous atrophy. For familial polyposis, the risk factors are the number of pre-operative polyps > 1000, surgery with stapled anastomosis and the duration of follow-up. In the case of ulcerative colitis, a pouchoscopy should be performed annually if one of the following is present: dysplasia and cancer at surgery, primary sclerosing cholangitis, villous atrophy and active pouchitis (every 5 years without any of these factors). In the case of familial polyposis, endoscopy is recommended every year including chromoendoscopy. Even if anal transitional zone and ileal pouch cancers seldom occur following proctectomy for ulcerative colitis and familial adenomatous polyposis, the high mortality rate associated with this complication warrants endoscopic monitoring.

Cannabinoid-Related Acute Pancreatitis: An Update from International Literature and Individual Case Safety Reports.

INTRODUCTION: In addition to the growing interest of different cannabinoids for therapeutic purposes, the safety profile of these substances has changed, with the recent identification of new events such as acute pancreatitis. OBJECTIVE: The aim of this study was to characterize cannabinoid-related acute pancreatitis, based on the recent literature and the analysis of pharmacovigilance data available worldwide. METHODS: Nine national and international pharmacovigilance databases were requested for individual case safety reports of acute pancreatitis related to cannabinoid exposure. A systematic review was performed searching in PubMed®, Web of Science®, and Google Scholar® for any publication dealing with acute pancreatitis and cannabinoid exposure (cannabis, cannabinoid, cannabidiol, tetrahydrocannabinol, nabilone, dronabinol), to identify case reports, observational studies, clinical trials, or reviews. All queries were updated on 1 January, 2021. RESULTS: Twenty-two individual case safety reports were identified in the pharmacovigilance databases and 51 in the literature, corresponding to a predominantly young male population (74% of men, median age 28 interquartile range [21-39]) using recreational Cannabis sativa with high intensity. A therapeutic purpose was identified in 13 cases (including tetrahydrocannabinol, cannabidiol, and dronabinol). The outcome was often favorable after dechallenge (except three deaths), and frequent recurrences were observed in the case of rechallenge or sustained consumption. Eleven cross-sectional studies and one ecological study showed an increasing trend of cannabis use in in-patients with acute pancreatitis, with a significantly lower in-hospital mortality. CONCLUSIONS: This review underlines that acute pancreatitis is a potential adverse effect of cannabinoid use. It remains often unrecognized and can occur during recreational or therapeutic use. The development of the therapeutic use of cannabinoids in frail patients deserves a better investigation of the benefit-risk ratio of these different products.

The antitumoral activity of TLR7 ligands is corrupted by the microenvironment of pancreatic tumors.

Toll-like receptors (TLRs) are key players in the innate immune system. Recent studies have suggested that they may affect the growth of pancreatic cancer, a disease with no cure. Among them, TLR7 shows promise for therapy but may also promotes tumor growth. Thus, we aimed to clarify the therapeutic potential of TLR7 ligands in experimental pancreatic cancer models, to open the door for clinical applications. In vitro, we found that TLR7 ligands strongly inhibit the proliferation of both human and murine pancreatic cancer cells, compared with TLR2 agonists. Hence, TLR7 treatment alters cancer cells' cell cycle and induces cell death by apoptosis. In vivo, TLR7 agonist therapy significantly delays the growth of murine pancreatic tumors engrafted in immunodeficient mice. Remarkably, TLR7 ligands administration instead increases tumor growth and accelerates animal death when tumors are engrafted in immunocompetent models. Further investigations revealed that TLR7 agonists modulate the intratumoral content and phenotype of macrophages and that depleting such tumor-associated macrophages strongly hampers TLR7 agonist-induced tumor growth. Collectively, our findings shine a light on the duality of action of TLR7 agonists in experimental cancer models and call into question their use for pancreatic cancer therapy.