Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn's Disease.

OBJECTIVE: Epigenetic mechanisms, including DNA methylation (DNAm), have been proposed to play a key role in Crohn's disease (CD) pathogenesis. However, the specific cell types and pathways affected as well as their potential impact on disease phenotype and outcome remain unknown. We set out to investigate the role of intestinal epithelial DNAm in CD pathogenesis. DESIGN: We generated 312 intestinal epithelial organoids (IEOs) from mucosal biopsies of 168 patients with CD (n=72), UC (n=23) and healthy controls (n=73). We performed genome-wide molecular profiling including DNAm, bulk as well as single-cell RNA sequencing. Organoids were subjected to gene editing and the functional consequences of DNAm changes evaluated using an organoid-lymphocyte coculture and a nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 (NLRC5) dextran sulphate sodium (DSS) colitis knock-out mouse model. RESULTS: We identified highly stable, CD-associated loss of DNAm at major histocompatibility complex (MHC) class 1 loci including NLRC5 and cognate gene upregulation. Single-cell RNA sequencing of primary mucosal tissue and IEOs confirmed the role of NLRC5 as transcriptional transactivator in the intestinal epithelium. Increased mucosal MHC-I and NLRC5 expression in adult and paediatric patients with CD was validated in additional cohorts and the functional role of MHC-I highlighted by demonstrating a relative protection from DSS-mediated mucosal inflammation in NLRC5-deficient mice. MHC-I DNAm in IEOs showed a significant correlation with CD disease phenotype and outcomes. Application of machine learning approaches enabled the development of a disease prognostic epigenetic molecular signature. CONCLUSIONS: Our study has identified epigenetically regulated intestinal epithelial MHC-I as a novel mechanism in CD pathogenesis.

Precision nutrition in pediatric IBD: A position paper from the ESPGHAN special interest group for basic science and translational research, the IBD Porto group, and allied health professionals.

Stratified and precision nutrition refers to disease management or prevention of disease onset, based on dietary interventions tailored to a person's characteristics, biology, gut microbiome, and environmental exposures. Such treatment models may lead to more effective management of inflammatory bowel disease (IBD) and reduce risk of disease development. This societal position paper aimed to report advances made in stratified and precision nutritional therapy in IBD. Following a structured literature search, limited to human studies, we identified four relevant themes: (a) nutritional epidemiology for risk prediction of IBD development, (b) food-based dietary interventions in IBD, (c) exclusive enteral nutrition (EEN) for Crohn's disease (CD) management, and (d) pre- and probiotics for IBD management. There is scarce literature upon which we can make recommendations for precision or stratified dietary therapy for IBD, both for risk of disease development and disease management. Certain single-nucleotide polymorphisms related to polyunsaturated fatty acid (PUFA) metabolism may modify the effect dietary PUFA have in increasing the risk of IBD development. Non-colonic CD, mild-to-moderate CD, and high microbiota richness may predict success of EEN and may be used both for prediction of treatment continuation, but also for early cessation in nonresponders. There is currently insufficient evidence to make recommendations for precision or stratified dietary therapy for patients with established IBD. Despite the great interest in stratified and precision nutrition, we currently lack data to support conclusive recommendations. Replication of early findings by independent research groups and within structured clinical interventions is required.

Orai1 calcium channel inhibition prevents progression of chronic pancreatitis.

Patients with recurrent acute pancreatitis (RAP) are at significant risk of developing early chronic pancreatitis (CP), which progresses into irreversible, end-stage CP with severe symptoms. There is no specific therapy in RAP or in early CP that may hinder disease progression. The pathogenesis of CP is complex and involves interactions among multiple cell types, including pancreatic acinar, ductal, and stellate cells (PSC). Therefore, it is pivotal to identify common pathogenic pathways in these cells that could be targeted pharmacologically. The Orai1-mediated store-operated Ca2+ entry (SOCE) is a ubiquitous signaling mechanism that may become overactivated in pathological states resulting in intracellular Ca2+ overload. In this study, we used ex vivo and in vivo preclinical disease models to demonstrate that Orai1 inhibition prevents progression of RAP and early CP. The selective Orai1 inhibitor CM5480 restored the expression of SOCE-associated regulatory factor in acinar cells, prevented uncontrolled Ca2+ elevation, protected acinar and ductal functions, mitigated immune cell infiltration, and diminished PSC activation, proliferation, and migration. We suggest that the overactivation of Orai1 is a crucial pathogenetic event in the progression of early CP and that inhibition of Orai1 could prevent the development of end-stage CP.

Thiopurines impair the apical plasma membrane expression of CFTR in pancreatic ductal cells via RAC1 inhibition.

BACKGROUND AND AIMS: Thiopurine-induced acute pancreatitis (TIP) is one of the most common adverse events among inflammatory bowel disease patients treated with azathioprine (AZA), representing a significant clinical burden. Previous studies focused on immune-mediated processes, however, the exact pathomechanism of TIP is essentially unclear. METHODS: To model TIP in vivo, we triggered cerulein-induced experimental pancreatitis in mice receiving a daily oral dose of 1.5 mg/kg AZA. Also, freshly isolated mouse pancreatic cells were exposed to AZA ex vivo, and acinar cell viability, ductal and acinar Ca2+ signaling, ductal Cl- and HCO3- secretion, as well as cystic fibrosis transmembrane conductance regulator (CFTR) expression were assessed using microscopy techniques. Ras-related C3 botulinum toxin substrate (RAC1) activity was measured with a G-LISA assay. Super-resolution microscopy was used to determine protein colocalization. RESULTS: We demonstrated that AZA treatment increases tissue damage in the early phase of cerulein-induced pancreatitis in vivo. Also, both per os and ex vivo AZA exposure impaired pancreatic fluid and ductal HCO3- and Cl- secretion, but did not affect acinar cells. Furthermore, ex vivo AZA exposure also inhibited RAC1 activity in ductal cells leading to decreased co-localization of CFTR and the anchor protein ezrin, resulting in impaired plasma membrane localization of CFTR. CONCLUSIONS: AZA impaired the ductal HCO3- and Cl- secretion through the inhibition of RAC1 activity leading to diminished ezrin-CFTR interaction and disturbed apical plasma membrane expression of CFTR. We report a novel direct toxic effect of AZA on pancreatic ductal cells and suggest that the restoration of ductal function might help to prevent TIP in the future.

Clinical assessment of children with long COVID syndrome.

BACKGROUND: There is a need for further understanding pediatric long COVID syndrome (LCS) to be able to create specific case definitions and guidelines for providing good clinical care. METHODS: Medical records of all LCS patients who presented at our designated LC clinic were collected. We carried out descriptive analyses summarizing the history, clinical presentation, and findings of children, while doing a diagnosis of exclusion with multi-disciplinary medical examinations (physical, laboratory, and radiological examinations, specialist consultations, etc.) without a control group. RESULTS: Most children reported at least minor impairment to their quality of life, of which 17 (23%) had moderate or severe difficulties. Findings that could be directly connected to the linked complaint category were observed in an average of 18%, respiratory symptoms with objective alterations being the most frequent (37%). Despite our detecting mostly non-specific conditions, in a smaller number we identified well-described causes such as autoimmune thyroiditis (7%). CONCLUSIONS: The majority of children stated an impairment in their quality of life, while symptom-related conditions were detected only in a minority. Controlled studies are needed to separate the effect of the pandemic era from the infection itself. Evidence-based pediatric guidelines could aid to rationalize the list of recommended examinations. IMPACT: Long COVID syndrome is a complex entity with a great impact on children's everyday lives. Still, there is no clear guidance for pediatric clinical management. Systematic, detailed studies with medical assessment findings could aid the process of creating evidence-based guidelines. We present validated systematic information collected during in-person medical assessments with detailed medical findings and quality of life changes. While making a diagnosis of exclusion, we could confirm symptom-related conditions only in a minority of children; however, the majority reported at least minor impairment to their quality of life.

Geno- and phenotypic characteristics and clinical outcomes of CACNA1C gene mutation associated Timothy syndrome, "cardiac only" Timothy syndrome and isolated long QT syndrome 8: A systematic review.

Background: Mutations in the CACNA1C gene-encoding for the major Ca2+ channel of the heart-may exhibit a variety of clinical manifestations. These include typical or atypical Timothy syndromes (TS) which are associated with multiple organ manifestations, and cardiac involvement in form of malignant arrhythmias, QTc prolongation, or AV block. "Cardiac only" Timothy syndrome (COTS) shows no extracardiac manifestation, whereas some CACNA1C gene mutations are associated with QTc prolongation alone (isolated long QT syndrome 8, LQT8). Methods: A systematic search of the literature reporting cases of CACNA1C gene mutation associated syndromes, including TS, COTS and isolated LQT8 via major databases published from 2004 through 2019 was performed. Detailed patient-level phenotypic and genotypic characteristics, as well as long-term outcome measures were collected and compared between pre-specified patient groups, defined both on phenotype and genotype. Results: A total of 59 TS, 6 COTS, and 20 isolated LQT8 index cases were identified. Apart of syndactyly or baldness, there were no major differences regarding clinical manifestations or outcome measures between TS subtypes, either defining TS subtypes on the genotype or based on the phenotype. Both subtypes were characterized by an extreme degree of QTc prolongation (median ≥600 ms) which were reflected in high major adverse cardiac event rate. On the other hand, there were marked differences between TS, COTS, and isolated LQT8. Timothy syndrome was characterized by a much earlier disease onset, much more pronounced QTc prolongation and much higher mortality rate than COTS or isolated LQT8. Similar differences were observed comparing CACNA1C exon 8/8A vs. non-exon 8/8A mutation carriers. TS showed a high degree of genetic homogeneity, as the p.Gly406Arg mutation either in exon 8 or exon 8A alone was responsible for 70% of the cases. Conclusions: Clinical phenotypes associated with mutations in the CACNA1C gene show important clinical differences. Timothy syndrome is associated with the most severe clinical phenotype and with the highest risk of morbidity and mortality. However, distinguishing TS subtypes, in any form, are not supported by our data. Systematic review registration: [https://www.crd.york.ac.uk/prospero/], identifier [CRD42020184737].

Impaired regulation of PMCA activity by defective CFTR expression promotes epithelial cell damage in alcoholic pancreatitis and hepatitis.

Alcoholic pancreatitis and hepatitis are frequent, potentially lethal diseases with limited treatment options. Our previous study reported that the expression of CFTR Cl- channel is impaired by ethanol in pancreatic ductal cells leading to more severe alcohol-induced pancreatitis. In addition to determining epithelial ion secretion, CFTR has multiple interactions with other proteins, which may influence intracellular Ca2+ signaling. Thus, we aimed to investigate the impact of ethanol-mediated CFTR damage on intracellular Ca2+ homeostasis in pancreatic ductal epithelial cells and cholangiocytes. Human and mouse pancreas and liver samples and organoids were used to study ion secretion, intracellular signaling, protein expression and interaction. The effect of PMCA4 inhibition was analyzed in a mouse model of alcohol-induced pancreatitis. The decreased CFTR expression impaired PMCA function and resulted in sustained intracellular Ca2+ elevation in ethanol-treated and mouse and human pancreatic organoids. Liver samples derived from alcoholic hepatitis patients and ethanol-treated mouse liver organoids showed decreased CFTR expression and function, and impaired PMCA4 activity. PMCA4 co-localizes and physically interacts with CFTR on the apical membrane of polarized epithelial cells, where CFTR-dependent calmodulin recruitment determines PMCA4 activity. The sustained intracellular Ca2+ elevation in the absence of CFTR inhibited mitochondrial function and was accompanied with increased apoptosis in pancreatic epithelial cells and PMCA4 inhibition increased the severity of alcohol-induced AP in mice. Our results suggest that improving Ca2+ extrusion in epithelial cells may be a potential novel therapeutic approach to protect the exocrine pancreatic function in alcoholic pancreatitis and prevent the development of cholestasis in alcoholic hepatitis.

Making Research Flourish Through ESPGHAN: A Position Paper From the ESPGHAN Special Interest Group for Basic and Translational Research.

ABSTRACT: Recent research breakthroughs have emerged from applied basic research throughout all scientific areas, including adult and paediatric gastroenterology, hepatology and nutrition (PGHAN). The research landscape within the European Society of Paediatric Gastroenterology and Nutrition (ESPGHAN) is also inevitably changing from clinical research to studies involving applied laboratory research. This position paper aims to depict the current status quo of basic science and translational research within ESPGHAN, and to delineate how the society could invest in research in the present and future time. The paper also explores which research areas in the field of PGHAN represent the current and future priorities, and what type of support is needed across the ESPGHAN working groups (WGs) and special interest groups (SIGs) to fulfil their research goals.

Combined use of indomethacin and hydration is the best conservative approach for post-ERCP pancreatitis prevention: A network meta-analysis.

OBJECTIVES: Post-ERCP pancreatitis (PEP) is a life-threatening complication. Given the lack of a causative treatment for pancreatitis, it is of vital importance to minimize this risk of PEP. Multi-target preventive therapy may be the best choice for PEP prevention as disease development is multifactorial. AIM: We aimed to assess the efficacy of a combination of indomethacin and hydration - type and amount - for PEP prevention via a network meta-analysis. METHODS: Through a systematic search in three databases, we searched all randomized controlled trials involving hydration and indomethacin and ranked the PEP preventive efficacy with a Bayesian network meta-analysis using the PRISMA for Network Meta-Analyses (PRISMA-NMA) guideline. The RoB2 tool was used for risk of bias assessment, surface under the cumulative ranking curve (SUCRA) for ranking and PROSPERO for the study protocol [reg. no. CRD42018112698]. We used risk ratios (RR) for dichotomous data with 95% credible intervals (95% CrI). RESULTS: The quantitative analysis included 7559 patients from 24 randomized controlled trials. Based on the SUCRA values, a combination of lactated Ringer's and indomethacin is more effective than single therapy with a 94% certainty. The percent relative risk ratios estimate preventive efficacy 70-99% higher for combinations than single therapies. Aggressive hydration with indomethacin (SUCRA 100%) is also significantly more effective than all other interventions (percent relative effect 94.3-98.1%). CONCLUSIONS: A one-hit-on-each-target therapeutic approach is recommended in PEP prevention with an easily accessible combination of indomethacin and aggressive hydration for all average and high-risk patients without contraindication.

Caspase-9 acts as a regulator of necroptotic cell death.

Necroptosis is a regulated necrotic-like cell death modality which has come into the focus of attention since it is known to contribute to the pathogenesis of many inflammatory and degenerative diseases as well as to tumor regulation. Based on current data, necroptosis serves as a backup mechanism when death receptor-induced apoptosis is inhibited or absent. However, the necroptotic role of the proteins involved in mitochondrial apoptosis has not been investigated. Here, we demonstrated that the stimulation of several death and pattern recognition receptors induced necroptosis under caspase-compromised conditions in wild-type, but not in caspase-9-negative human Jurkat and murine MEF cells. Cerulein-induced pancreatitis was significantly reduced in mice with acinar cell-restricted caspase-9 gene knockout. The absence of caspase-9 led to impaired association of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3 and resulted in decreased phosphorylation of RIP kinases, but the overexpression of RIPK1 or RIPK3 rescued the effect of caspase-9 deficiency. Inhibition of either Aurora kinase A (AURKA) or its known substrate, glycogen synthase kinase 3ß (GSK3ß) restored necroptosis sensitivity of caspase-9-deficient cells, indicating an interplay between caspase-9 and AURKA-mediated pathways to regulate necroptosis. Our findings suggest that caspase-9 acts as a newly identified regulator of necroptosis, and thus, caspase-9 provides a promising therapeutic target to manipulate the immunological outcome of cell death.

Inflammatory Bowel Diseases Elevate the Risk of Developing Acute Pancreatitis: A Meta-analysis.

OBJECTIVES: Increasing data suggest that acute pancreatitis (AP) occurs more frequently among patients with inflammatory bowel diseases (IBDs) than in the non-IBD population; however, currently no comprehensive meta-analysis is available. METHODS: Systematic literature search was conducted in 4 major databases. We included observational studies sampling from the general population. Basic study characteristics and crude incidences of AP were extracted. Pooled odds ratios (ORs) with 95% confidence interval (CIs) were calculated using the random-effects model. Subgroups were set up by Crohn disease and ulcerative colitis. Heterogeneity was tested with I statistics. RESULTS: Eight studies were eligible for the analysis. The odds of AP were 3 times higher in IBD (OR, 3.11; 95% CI, 2.93-3.30; I, 0.0%), significantly higher in Crohn disease than in ulcerative colitis (P < 0.001; OR, 4.12 vs OR, 2.61; I, 0.0%). The pooled annual incidence of AP in IBD was 210/100,000 person-years (95% CI, 84-392/100,000 person-years; I, 98.66%). CONCLUSIONS: We confirmed that IBD elevates the risk of AP and of 100,000 IBD patients 210 AP cases are to be expected annually. Therefore, it is important to include pancreatic enzyme level measurements and radiological investigations in the workup of IBD patients with acute abdominal pain.

Eosinophil Counts in the Small Intestine and Colon of Children Without Apparent Gastrointestinal Disease: A Meta-analysis.

The aim of the current study was to review the available data regarding eosinophil density in healthy tissue specimen originating from lower gastrointestinal segments to support suggested diagnostic cutoffs widely used in clinical practice. A systematic search was performed in 3 different databases. Calculations were made with Comprehensive MetaAnalysis software using random-effects model. Cell number measurements were pooled using the random-effects model and displayed on forest plots. Summary point estimations, 95% confidence intervals (CIs), and 95% prediction intervals (PIs) were calculated. The cumulative mean cell numbers were 8.26 (95% CI 4.71-11.80) with PI of 0-25.32 for the duodenum, 11.52 (95% CI 7.21-15.83) with PI 0-60.64 for the terminal ileum, and 11.10/ high-power field (HPF) (95% CI 9.11-13.09) with PI of 0.96 to 21.23 in the large intestine and the rectum (HPF area = 0.2 mm). Previous studies included control patients with irritable bowel syndrome and functional gastrointestinal disorders. As mucosal eosinophils have a role in their pathomechanism, those patients should have been excluded. A critical point of interpreting reported data is that HPF is relative to the technical parameters of the microscopes; therefore, it is important to report findings in cell/mm. The present meta-analysis does not support the higher (>20) or lower (<10) cutoff values for healthy tissue eosinophil number. In contrast to the esophagus, there is no normal cutoff eosinophil density in the small intestine and the colon. A prospective, multicenter study to establish normal mucosal eosinophil density is clearly needed.

Specific MicroRNA Pattern in Colon Tissue of Young Children with Eosinophilic Colitis.

Eosinophilic colitis (EC) is a common cause of haematochezia in infants and young children. The exact pathomechanism is not understood, and the diagnosis is challenging. The role of microRNAs as key class of regulators of mRNA expression and translation in patients with EC has not been explored. Therefore, the aim of the present study was to explore the miRNA profile in EC with respect to eosinophilic inflammation. Patients enrolled in the study (n = 10) had persistent rectal bleeding, and did not respond to elimination dietary treatment. High-throughput microRNA sequencing was carried out on colonic biopsy specimens of children with EC (EC: n = 4) and controls (C: n = 4) as a preliminary screening of the miRNA profile. Based on the next-generation sequencing (NGS) results and literature data, a potentially relevant panel of miRNAs were selected for further measurements by real-time reverse transcription (RT)-PCR (EC: n = 14, C: n = 10). Validation by RT-PCR resulted in significantly altered expression of miR-21, -31, -99b, -125a, -146a, -184, -221, -223, and -559 compared to controls (p ≤ 0.05). Elevation in miR-21, -99b, -146a, -221, and -223 showed statistically significant correlation to the extent of tissue eosinophilia. Based on our results, we conclude that the dysregulated miRNAs have a potential role in the regulation of apoptosis by targeting Protein kinase B/Mechanistic target of rapamycin (AKT/mTOR)-related pathways in inflammation by modulating Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-related signalling and eosinophil cell recruitment and activation, mainly by regulating the expression of the chemoattractant eotaxin and the adhesion molecule CD44. Our results could serve as a basis for further extended research exploring the pathomechanism of EC.