Upregulated Matrisomal Proteins and Extracellular Matrix Mechanosignaling Underlie Obesity-Associated Promotion of Pancreatic Ductal Adenocarcinoma.

Diet-induced obesity (DIO) promotes pancreatic ductal adenocarcinoma (PDAC) in mice expressing KRasG12D in the pancreas (KC mice), but the precise mechanisms remain unclear. Here, we performed multiplex quantitative proteomic and phosphoproteomic analysis by liquid chromatography-tandem mass spectrometry and further bioinformatic and spatial analysis of pancreas tissues from control-fed versus DIO KC mice after 3, 6, and 9 months. Normal pancreatic parenchyma and associated proteins were steadily eliminated and the novel proteins, phosphoproteins, and signaling pathways associated with PDAC tumorigenesis increased until 6 months, when most males exhibited cancer, but females did not. Differentially expressed proteins and phosphoproteins induced by DIO revealed the crucial functional role of matrisomal proteins, which implies the roles of upstream regulation by TGFß, extracellular matrix-receptor signaling to downstream PI3K-Akt-mTOR-, MAPK-, and Yap/Taz activation, and crucial effects in the tumor microenvironment such as metabolic alterations and signaling crosstalk between immune cells, cancer-associated fibroblasts (CAFs), and tumor cells. Staining tissues from KC mice localized the expression of several prognostic PDAC biomarkers and elucidated tumorigenic features, such as robust macrophage infiltration, acinar-ductal metaplasia, mucinous PanIN, distinct nonmucinous atypical flat lesions (AFLs) surrounded by smooth muscle actin-positive CAFs, invasive tumors with epithelial-mesenchymal transition arising close to AFLs, and expanding deserted areas by 9 months. We next used Nanostring GeoMX to characterize the early spatial distribution of specific immune cell subtypes in distinct normal, stromal, and PanIN areas. Taken together, these data richly contextualize DIO promotion of Kras-driven PDAC tumorigenesis and provide many novel insights into the signaling pathways and processes involved.

Bladder cancer risk stratification with the Oncuria 10-plex bead-based urinalysis assay using three different Luminex xMAP instrumentation platforms.

BACKGROUND: No single marker of bladder cancer (BC) exists in urine samples with sufficient accuracy for disease diagnosis and treatment monitoring. The multiplex Oncuria BC assay noninvasively quantifies the concentration of 10 protein analytes in voided urine samples to quickly generate a unique molecular profile with proven BC diagnostic and treatment-tracking utility. Test adoption by diagnostic and research laboratories mandates reliably reproducible assay performance across a variety of instrumentation platforms used in different laboratories. METHODS: We compared the performance of the clinically validated Oncuria BC multiplex immunoassay when data output was generated on three different analyzer systems. Voided urine samples from 36 subjects (18 with BC and 18 Controls) were reacted with Oncuria test reagents in three 96-well microtiter plates on Day 1, and consecutively evaluated on the LED/image-based MagPix, and laser/flow-based Luminex 200 and FlexMap 3D (all xMAP instruments from Luminex Corp., Austin, TX) on Day 2. The BC assay uses magnetic bead-based fluorescence technology (xMAP, Multi-analyte profiling; Luminex) to simultaneously quantify 10 protein analytes in urine specimens [i.e., angiogenin (ANG), apolipoprotein E (ApoE), carbonic anhydrase IX (CA9), CXCL8/interleukin-8 (IL-8), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-10 (MMP-10), serpin A1/alpha-1 anti-trypsin (A1AT), serpin E1/plasminogen activator inhibitor-1 (PAI-1), CD138/syndecan-1 (SDC1), and vascular endothelial growth factor-A (VEGF-A)]. All three analyzers quantify fluorescence signals generated by the Oncuria assay. RESULTS: All three platforms categorized all 10 analytes in identical samples at nearly identical concentrations, with variance across systems typically < 5%. While the most contemporary instrument, the FlexMap 3D, output higher raw fluorescence values than the two comparator systems, standard curve slopes and analyte concentrations determined in urine samples were concordant across all three units. Forty-four percent of BC samples registered ≥ 1 analyte above the highest standard concentration, i.e., A1AT (n = 7/18), IL-8 (n = 5), and/or ANG (n = 2), while only one control sample registered an analyte (A1AT) above the highest standard concentration. CONCLUSION: Multiplex BC assays generate detailed molecular signatures useful for identifying BC, predicting treatment responsiveness, and tracking disease progression and recurrence. The similar performance of the Oncuria assay across three different analyzer systems supports test adaptation by clinical and research laboratories using existing xMAP platforms. TRIAL REGISTRATION: This study was registered at ClinicalTrials.gov as NCT04564781, NCT03193528, NCT03193541, and NCT03193515.

Bladder cancer risk stratification with the Oncuria 10-plex bead-based urinalysis assay using three different Luminex xMAP instrumentation platforms.

Background: No single marker of bladder cancer (BC) exists in urine samples with sufficient accuracy for disease diagnosis and treatment monitoring. The multiplex Oncuria BC assay noninvasively quantifies the concentration of 10 protein analytes in voided urine samples to quickly generate a unique molecular profile with proven BC diagnostic and treatment-tracking utility. Test adoption by diagnostic and research laboratories mandates reliably reproducible assay performance across a variety of instrumentation platforms used in different laboratories. Methods: We compared the performance of the clinically validated Oncuria BC multiplex immunoassay when data output was generated on three different analyzer systems. Voided urine samples from 36 subjects (18 with BC and 18 Controls) were reacted with Oncuria test reagents in three 96-well microtiter plates on Day 1, and consecutively evaluated on the LED/image-based MagPix, and laser/flow based Luminex 200 and FlexMap 3D (all xMAP instruments from Luminex Corp., Austin, TX) on Day 2. The BC assay uses magnetic bead-based fluorescence technology (xMAP, Multi-analyte profiling; Luminex) to simultaneously quantify 10 protein analytes in urine specimens [i.e., angiogenin (ANG), apolipoprotein E (ApoE), carbonic anhydrase IX (CA9), CXCL8/interleukin-8 (IL-8), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-10 (MMP-10), serpin A1/alpha-1 anti-trypsin (A1AT), serpin E1/plasminogen activator inhibitor-1 (PAI-1), CD138/syndecan-1 (SDC1), and vascular endothelial growth factor-A (VEGF-A)]. Results: All three platforms categorized all 10 analytes in identical samples at nearly identical concentrations, with variance across systems typically <5%. While the most contemporary instrument, the FlexMap 3D, output higher raw fluorescence values than the two comparator systems, standard curve slopes and analyte concentrations determined in urine samples were concordant across all three units. Forty-four percent of BC samples registered ≥1 analyte above the highest standard concentration, i.e., A1AT (n=7/18), IL-8 (n=5), and/or ANG (n=2). In Controls, A1AT was higher in one sample. Conclusion: Multiplex BC assays generate detailed molecular signatures useful for identifying BC, predicting treatment esponsiveness, and tracking disease progression and recurrence. The similar performance of the Oncuria assay across three different analyzer systems supports test adaptation by clinical and research laboratories using existing xMAP platforms. Trial Registration: This study was registered at ClinicalTrials.gov as NCT04564781, NCT03193528, NCT03193541, and NCT03193515.

Low dosage combination treatment with metformin and simvastatin inhibits obesity-promoted pancreatic cancer development in male KrasG12D mice.

Pancreatic ductal adenocarcinoma (PDAC), a highly lethal disease with limited therapeutic options, may benefit from repurposing of FDA-approved drugs in preventive or interceptive strategies in high-risk populations. Previous animal studies demonstrated that the use of metformin and statins as single agents at relatively high doses restrained PDAC development. Here, four-week-old mice expressing KrasG12D in all pancreatic lineages (KC mice) and fed an obesogenic high fat, high calorie diet that promotes early PDAC development were randomized onto low dosage metformin, simvastatin, or both drugs in combination administered orally. Dual treatment attenuated weight gain, fibro-inflammation, and development of advanced PDAC precursor lesions (pancreatic intraepithelial neoplasia [PanIN]-3) in male KC mice, without significant effect in females or when administered individually. Dual-treated KC mice had reduced proliferation of PanIN cells and decreased transcriptional activity of the Hippo effectors, YAP and TAZ, which are important regulators of PDAC development. Metformin and simvastatin also synergistically inhibited colony formation of pancreatic cancer cells in vitro. Together, our data demonstrated that a combination of low doses of metformin and simvastatin inhibits PDAC development and imply that both drugs are promising agents for being tested in clinical trials for preventing pancreatic cancer progression.

Basal Cell-derived WNT7A Promotes Fibrogenesis at the Fibrotic Niche in Idiopathic Pulmonary Fibrosis.

Loss of epithelial integrity, bronchiolarization, and fibroblast activation are key characteristics of idiopathic pulmonary fibrosis (IPF). Prolonged accumulation of basal-like cells in IPF may impact the fibrotic niche to promote fibrogenesis. To investigate their role in IPF, basal cells were isolated from IPF explant and healthy donor lung tissues. Single-cell RNA sequencing was used to assess differentially expressed genes in basal cells. Basal cell and niche interaction was demonstrated with the sLP-mCherry niche labeling system. Luminex assays were used to assess cytokines secreted by basal cells. The role of basal cells in fibroblast activation was studied. Three-dimensional organoid culture assays were used to interrogate basal cell effects on AEC2 (type 2 alveolar epithelial cell) renewal capacity. Perturbation was used to investigate WNT7A function in vitro and in a repetitive bleomycin model in vivo. We found that WNT7A is highly and specifically expressed in basal-like cells. Proteins secreted by basal cells can be captured by neighboring fibroblasts and AEC2s. Basal cells or basal cell-conditioned media activate fibroblasts through WNT7A. Basal cell-derived WNT7A inhibits AEC2 progenitor cell renewal in three-dimensional organoid cultures. Neutralizing antibodies against WNT7A or a small molecule inhibitor of Frizzled signaling abolished basal cell-induced fibroblast activation and attenuated lung fibrosis in mice. In summary, basal cells and basal cell-derived WNT7A are key components of the fibrotic niche, providing a unique non-stem cell function of basal cells in IPF progression and a novel targeting strategy for IPF.

Salivary Biomarker Evaluation of Chronic Pancreatitis Patients Reveals Alterations in Human Proteins, Cytokines, Prostaglandin E2 Levels, and Bacterial Diversity.

OBJECTIVES: Chronic pancreatitis (CP) is a chronic fibroinflammatory condition of the pancreas difficult to diagnose in early stages. Novel biomarkers useful to facilitate early diagnosis or treatment responses may be found in biofluids. Although saliva can be easily and noninvasively collected from patients, useful salivary biomarkers from CP patients have not yet been identified. METHODS: Here, we analyzed the proteome by quantitative proteomics, cytokine/chemokine levels by Luminex analysis, prostaglandin E2 (PGE2) levels by a mass spectrometry-based assay, and bacterial species diversity by 16S ribosomal ribonucleic acid sequencing in saliva samples from confirmed CP patients and healthy controls. RESULTS: Our results indicate the presence of various differentially expressed proteins, cytokines/chemokines, and a loss of oral bacterial diversity in the saliva of CP patients. The PGE2 levels trend toward elevation in CP patients. Area under the receiver operating characteristic curve models for proteomic, cytokine, and PGE2 assays ranged from 0.59 to 0.90. CONCLUSIONS: Collectively, our studies identify a range of putative CP biomarkers and alterations in human saliva requiring further validation. The biomarker discovery approaches we used might lead to identification of biomarkers useful for CP diagnosis and monitoring.

The unique pancreatic stellate cell gene expression signatures are associated with the progression from acute to chronic pancreatitis.

Chronic pancreatitis (CP) is characterized by irreversible fibro-inflammatory changes induced by pancreatic stellate cell (PSC). Unresolved or recurrent injury causes dysregulation of biological process following AP, which would cause CP. Here, we systematically identify genes whose expressions are unique to PSC by comparing transcriptome profiles among total pancreas, pancreatic stellate, acinar, islet and immune cells. We then identified candidate genes and correlated them with the pancreatic disease continuum by performing intersection analysis among total PSC and activated PSC genes, and genes persistently differentially expressed during acute pancreatitis (AP) recovery. Last, we examined the association between candidate genes and AP, and substantiated their potential as biomarkers in experimental AP and recurrent AP (RAP) models. A total of 68 genes were identified as highly and uniquely expressed in PSC. The PSC signatures were highly enriched with extracellular matrix remodeling genes and were significantly enriched in AP pancreas compared to healthy control tissues. Among PSC signature genes that comprised a fibrotic phenotype, 10 were persistently differentially expressed during AP recovery. SPARC was determined as a candidate marker for the pancreatic disease continuum, which was not only persistently differentially expressed even five days after AP injury, but also highly expressed in two clinical datasets of CP. Sparc was also validated as highly elevated in RAP compared to AP mice. This work highlights the unique transcriptional profiles of PSC. These PSC signatures' expression may help to identify patients with high risk of AP progression to CP.

Biomarkers of Chronic Pancreatitis: A systematic literature review.

BACKGROUND: Chronic pancreatitis (CP) does not have diagnostic or prognostic biomarkers. CP is the end stage of a progressive inflammatory syndrome that is diagnosed at late stages by morphologic features. To diagnose earlier stages of the disease, a new mechanistic definition was established based on identifying underlying pathogenic processes and biomarker evidence of disease activity and stage. Although multiple risk factors are known, the corresponding biomarkers needed to make a highly accurate diagnosis of earlier disease stages have not been established. The goal of this study is to systematically analyze the literature to identify the most likely candidates for development into biomarkers of CP. METHODS: We conducted a systematic review of candidate analytes from easily accessible biological fluids and identified 67 studies that compared CP to nonpancreatic-disease controls. We then ranked candidate biomarkers for sensitivity and specificity by area under the receiver operator curves (AUROCs). RESULTS: Five biomarkers had a large effect size (an AUROC > 0.96), whereas 30 biomarkers had a moderate effect size (an AUROC between 0.96 and 0.83) for distinguishing CP cases from controls or other diseases. However, the studies reviewed had marked variability in design, enrollment criteria, and biospecimen sample handling and collection. CONCLUSIONS: Several biomarkers have the potential for evaluation in prospective cohort studies and should be correlated with risk factors, clinical features, imaging studies and outcomes. The Consortium for the Study of Chronic Pancreatitis, Diabetes and Pancreas Cancer provides recommendations for avoiding design biases and heterogeneity in sample collection and handling in future studies.

Pathological Mechanisms in Diabetes of the Exocrine Pancreas: What's Known and What's to Know.

The clinical significance of diabetes arising in the setting of pancreatic disease (also known as diabetes of the exocrine pancreas, DEP) has drawn more attention in recent years. However, significant improvements still need to be made in the recognition, diagnosis and treatment of the disorder, and in the knowledge of the pathological mechanisms. The clinical course of DEP is different from type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). DEP develops in patients with previous existing exocrine pancreatic disorders which damage both exocrine and endocrine parts of pancreas, and lead to pancreas exocrine insufficiency (PEI) and malnutrition. Therefore, damage in various exocrine and endocrine cell types participating in glucose metabolism regulation likely contribute to the development of DEP. Due to the limited amount of clinical and experimental studies, the pathological mechanism of DEP is poorly defined. In fact, it still not entirely clear whether DEP represents a distinct pathologic entity or is a form of T2DM arising when ß cell failure is accelerated by pancreatic disease. In this review, we include findings from related studies in T1DM and T2DM to highlight potential pathological mechanisms involved in initiation and progression of DEP, and to provide directions for future research studies.

Targeting the CBP/ß-Catenin Interaction to Suppress Activation of Cancer-Promoting Pancreatic Stellate Cells.

BACKGROUND: Although cyclic AMP-response element binding protein-binding protein (CBP)/ß-catenin signaling is known to promote proliferation and fibrosis in various organ systems, its role in the activation of pancreatic stellate cells (PSCs), the key effector cells of desmoplasia in pancreatic cancer and fibrosis in chronic pancreatitis, is largely unknown. METHODS: To investigate the role of the CBP/ß-catenin signaling pathway in the activation of PSCs, we have treated mouse and human PSCs with the small molecule specific CBP/ß-catenin antagonist ICG-001 and examined the effects of treatment on parameters of activation. RESULTS: We report for the first time that CBP/ß-catenin antagonism suppresses activation of PSCs as evidenced by their decreased proliferation, down-regulation of "activation" markers, e.g., α-smooth muscle actin (α-SMA/Acta2), collagen type I alpha 1 (Col1a1), Prolyl 4-hydroxylase, and Survivin, up-regulation of peroxisome proliferator activated receptor gamma (Ppar-γ) which is associated with quiescence, and reduced migration; additionally, CBP/ß-catenin antagonism also suppresses PSC-induced migration of cancer cells. CONCLUSION: CBP/ß-catenin antagonism represents a novel therapeutic strategy for suppressing PSC activation and may be effective at countering PSC promotion of pancreatic cancer.

Pathogenic Carboxyl Ester Lipase (CEL) Variants Interact with the Normal CEL Protein in Pancreatic Cells.

Mutations in the gene encoding the digestive enzyme carboxyl ester lipase (CEL) are linked to pancreatic disease. The CEL variant denoted CEL-HYB predisposes to chronic pancreatitis, whereas the CEL-MODY variant causes MODY8, an inherited disorder of endocrine and exocrine pancreatic dysfunction. Both pathogenic variants exhibit altered biochemical and cellular properties compared with the normal CEL protein (CEL-WT, wild type). We here aimed to investigate effects of CEL variants on pancreatic acinar and ductal cell lines. Following extracellular exposure, CEL-HYB, CEL-MODY, and CEL-WT were endocytosed. The two pathogenic CEL proteins significantly reduced cell viability compared with CEL-WT. We also found evidence of CEL uptake in primary human pancreatic acinar cells and in native ductal tissue. Moreover, coexpression of CEL-HYB or CEL-MODY with CEL-WT affected secretion of the latter, as CEL-WT was observed to accumulate intracellularly to a higher degree in the presence of either pathogenic variant. Notably, in coendocytosis experiments, both pathogenic variants displayed a modest effect on cell viability when CEL-WT was present, indicating that the normal protein might diminish toxic effects conferred by CEL-HYB and CEL-MODY. Taken together, our findings provide valuable insight into how the pathogenic CEL variants predispose to pancreatic disease and why these disorders develop slowly over time.

Yes-Associated Protein 1 Plays Major Roles in Pancreatic Stellate Cell Activation and Fibroinflammatory Responses.

Background: Yes-associated protein 1 (YAP), a transcriptional co-activator and major effector of the Hippo pathway, regulates cell differentiation and morphology in many cell types and supports aberrant tumor growth. Recent studies showed that YAP is expressed in pancreas tissues in pancreatic ductal adenocarcinoma (PDAC) patients and experimental models of PDAC, with YAP largely found in cancer cells and pancreatic stellate cells (PaSC) in the stroma. Methods and Results: We studied here the role of YAP in the activated phenotype of PaSC. We found that YAP is expressed at low levels in normal mouse pancreas, but protein levels significantly increased after pancreas inflammatory damage induced by repeated cerulein administration in wild-type mice or upon initiation of neoplastic transformation of the pancreas parenchyma in Ptf1-Cre;LSL-KrasG12D/+ (KC) mice. In these animal models, YAP upregulation occurred in parallel with activation and proliferation of PaSC. Consistent with these findings, we found robust YAP expression in culture-activated mouse and human PaSC but not in quiescent, freshly isolated cells. Fully activated PaSC isolated from KC mice or PDAC patient tissues exhibited robust nuclear YAP suggesting YAP transcriptional activity. Agents that induce quiescence such as the Bromodomain and Extra-Terminal (BET) inhibitor iBET151 and the p38 MAPK inhibitor SB203580 reduced YAP levels in PaSC. Stimulation of PaSC with the potent mitogen PDGF elicited marked YAP Ser127 phosphorylation. However, unexpectedly, this effect did not diminish YAP nuclear localization, suggesting that YAP phosphorylation at this site does not govern YAP cellular localization in PaSC. siRNA-mediated knockdown of YAP reduced PDGF-induced PaSC expansion in culture and blunted the persistent activation of Akt and ERK elicited by PDGF stimulation, supporting a role for YAP in PDGF-induced cell growth. YAP knockdown also blunted fibroinflammatory gene expression responses both in unstimulated and transforming growth factor beta 1 (TGFß1)-stimulated PaSC. Conclusion: Our data suggest a central role for YAP in sustaining the activated phenotype and fibroinflammatory responses in PaSC. Moreover, our findings indicate that a complex crosstalk between YAP, TGFß1, and PDGF pathways regulates PaSC activity and growth.

Simvastatin induces autophagic flux to restore cerulein-impaired phagosome-lysosome fusion in acute pancreatitis.

BACKGROUND: During pancreatitis, autophagy is activated, but lysosomal degradation of dysfunctional organelles including mitochondria is impaired, resulting in acinar cell death. Retrospective cohort analyses demonstrated an association between simvastatin use and decreased acute pancreatitis incidence. METHODS: We examined whether simvastatin can protect cell death induced by cerulein and the mechanisms involved during acute pancreatitis. Mice were pretreated with DMSO or simvastatin (20 mg/kg) for 24 h followed by 7 hourly cerulein injections and sacrificed 1 h after last injection to harvest blood and tissue for analysis. RESULTS: Pancreatic histopathology revealed that simvastatin reduced necrotic cell death, inflammatory cell infiltration and edema. We found that cerulein triggered mitophagy with autophagosome formation in acinar cells. However, autophagosome-lysosome fusion was impaired due to altered levels of LAMP-1, AMPK and ULK-1, resulting in autophagosome accumulation (incomplete autophagy). Simvastatin abrogated these effects by upregulating LAMP-1 and activating AMPK which phosphorylated ULK-1, resulting in increased formation of functional autolysosomes. In contrast, autophagosomes accumulated in control group during pancreatitis. The effects of simvastatin to promote autophagic flux were inhibited by chloroquine. Mitochondria from simvastatin-treated mice were resistant to calcium overload compared to control, suggesting that simvastatin induced mitochondrial quality control to eliminate susceptible mitochondria. Clinical specimens showed a significant increase in cell-free mtDNA in plasma during pancreatitis compared to normal controls. Furthermore, genetic deletion of parkin abrogated the benefits of simvastatin. CONCLUSION: Our findings reveal the novel role of simvastatin in enhancing autophagic flux to prevent pancreatic cell injury and pancreatitis.

New-Onset Diabetes Mellitus After Chronic Pancreatitis Diagnosis: A Systematic Review and Meta-analysis.

OBJECTIVES: The aim of this study was to assess the occurrence of new-onset diabetes mellitus (DM) after chronic pancreatitis (CP) diagnosis via systematic review and meta-analysis. METHODS: A systematic review of literature and meta-analysis of relevant reports were performed. The primary outcome measures studied were newly diagnosed DM and DM treated with insulin. For the binary outcomes, pooled prevalence and 95% confidence interval (CI) were calculated. METHODS: Fifteen studies involving 8970 patients were eligible. The incidence of new-onset DM after CP diagnosis was 30% (95% CI, 27%-33%). Among all patients, 17% (95% CI, 13%-22%) developed insulin-dependent new-onset DM. The prevalence of newly diagnosed DM after CP diagnosis increased from 15% within 36 months to 33% after 60 months. The proportion of alcoholic CP, sex, age, and body mass index had minimal effect on the studied outcomes. CONCLUSIONS: This systematic review identified a clinically relevant risk of new-onset DM after CP diagnosis. Therefore, patients should be informed of the risk of DM and monitored.

Incidence of New Onset Diabetes Mellitus Secondary to Acute Pancreatitis: A Systematic Review and Meta-Analysis.

Background and Aims: Patients who have an episode of acute pancreatitis (AP) frequently develop diabetes mellitus (DM) over time. The reported incidence of DM after AP varies depending on the severity, etiology and the extent of pancreatic necrosis during AP. We performed a systematic review to determine the incidence of new-onset DM after AP episode (s), and compared the rate of DM in AP patients based upon different disease characteristics. Methods: A total of 31 relevant studies with 13894 subjects were collected from Medline, Embase, and Web of Science. Stata 15 software was used for data analyses in the meta-analysis. Results: The random-effects pooled incidence was 23.0% for DM (95% CI 16.0-31.0%) and 15.0% (95% CI 9.0-23.0%) for DM treated with insulin. We noted substantial heterogeneity in incidence estimates for DM and DM treated with insulin (I 2 = 95.61 and 71.78%; both p < 0·001). The DM incidence was higher in the populations that had a severe AP (SAP) episode than in those with mild acute pancreatitis (MAP) (39 vs. 14%). Patients that displayed pancreatic necrosis during the AP attack(s) had a higher frequency of DM than those without necrosis (37 vs. 11%). In addition, the pooled incidence of DM was higher after alcoholic compared to biliary AP (28 vs. 12%). The incidence of insulin use after SAP and alcoholic AP was 21 and 18%, respectively, with very low heterogeneities. According to duration of follow-up, the pooled rate of DM and insulin use within 5 years after AP was 20 and 14%, while the rate associated with follow-up duration of more than 5 years was elevated to 37 and 25%, respectively. On meta-regression, year of publication, male proportion, age at DM test, and duration of follow-up were neither positively nor negatively associated with the incidence of DM and DM treated with insulin in patients who had a prior AP attack. Conclusion: Patients with AP developed DM after discharge from hospital with a frequency of about 23%. SAP, alcoholic AP and acute necrotizing pancreatitis (ANP) were associated with increased incidence of DM. Assessments of severity, etiology, and pancreatic necrosis are critical for predicting DM development after AP.

The Orai Ca2+ channel inhibitor CM4620 targets both parenchymal and immune cells to reduce inflammation in experimental acute pancreatitis.

KEY POINTS: This work confirms previous reports that CM4620, a small molecule inhibitor of Ca2+ entry via store operated Ca2+ entry (SOCE) channels formed by stromal interaction molecule 1 (STIM1)/Orai complexes, attenuates acinar cell pathology and acute pancreatitis in mouse experimental models. Here we report that intravenous administration of CM4620 reduces the severity of acute pancreatitis in the rat, a hitherto untested species. Using CM4620, we probe further the mechanisms whereby SOCE via STIM1/Orai complexes contributes to the disease in pancreatic acinar cells, supporting a role for endoplasmic reticulum stress/cell death pathways in these cells. Using CM4620, we show that SOCE via STIM1/Orai complexes promotes neutrophil oxidative burst and inflammatory gene expression during acute pancreatitis, including in immune cells which may be either circulating or invading the pancreas. Using CM4620, we show that SOCE via STIM1/Orai complexes promotes activation and fibroinflammatory gene expression within pancreatic stellate cells. ABSTRACT: Key features of acute pancreatitis include excess cellular Ca2+ entry driven by Ca2+ depletion from the endoplasmic reticulum (ER) and subsequent activation of store-operated Ca2+ entry (SOCE) channels in the plasma membrane. In several cell types, including pancreatic acinar, stellate cells (PaSCs) and immune cells, SOCE is mediated via channels composed primarily of Orai1 and stromal interaction molecule 1 (STIM1). CM4620, a selective Orai1 inhibitor, prevents Ca2+ entry in acinar cells. This study investigates the effects of CM4620 in preventing or reducing acute pancreatitis features and severity. We tested the effects of CM4620 on SOCE, trypsinogen activation, acinar cell death, activation of NFAT and NF-κB, and inflammatory responses in ex vivo and in vivo rodent models of acute pancreatitis and human pancreatic acini. We also examined whether CM4620 inhibited cytokine release in immune cells, fibro-inflammatory responses in PaSCs, and oxidative burst in neutrophils, all cell types participating in pancreatitis. CM4620 administration to rats by i.v. infusion starting 30 min after induction of pancreatitis significantly diminished pancreatitis features including pancreatic oedema, acinar cell vacuolization, intrapancreatic trypsin activity, cell death signalling and acinar cell death. CM4620 also decreased myeloperoxidase activity and inflammatory cytokine expression in pancreas and lung tissues, fMLF peptide-induced oxidative burst in human neutrophils, and cytokine production in human peripheral blood mononuclear cells (PBMCs) and rodent PaSCs, indicating that Orai1/STIM1 channels participate in the inflammatory responses of these cell types during acute pancreatitis. These findings support pathological Ca2+ entry-mediated cell death and proinflammatory signalling as central mechanisms in acute pancreatitis pathobiology.

Recent Insights Into the Pathogenic Mechanism of Pancreatitis: Role of Acinar Cell Organelle Disorders.

Acute pancreatitis (AP) is a potentially lethal inflammatory disease that lacks specific therapy. Damaged pancreatic acinar cells are believed to be the site of AP initiation. The primary function of these cells is the synthesis, storage, and export of digestive enzymes. Beginning in the endoplasmic reticulum and ending with secretion of proteins stored in zymogen granules, distinct pancreatic organelles use ATP produced by mitochondria to move and modify nascent proteins through sequential vesicular compartments. Compartment-specific accessory proteins concentrate cargo and promote vesicular budding, targeting, and fusion. The autophagy-lysosomal-endosomal pathways maintain acinar cell homeostasis by removing damaged/dysfunctional organelles and recycling cell constituents for substrate and energy. Here, we discuss studies in experimental and genetic AP models, primarily from our groups, which show that acinar cell injury is mediated by distinct mechanisms of organelle dysfunction involved in protein synthesis and trafficking, secretion, energy generation, and autophagy. These early AP events (often first manifest by abnormal cytosolic Ca signaling) in the acinar cell trigger the inflammatory and cell death responses of pancreatitis. Manifestations of acinar cell organelle disorders are also prominent in human pancreatitis. Our findings suggest that targeting specific mediators of organelle dysfunction could reduce disease severity.

Proteomic Identification of Novel Plasma Biomarkers and Pathobiologic Pathways in Alcoholic Acute Pancreatitis.

Acute pancreatitis (AP) is a painful and potentially life-threatening disorder with the potential for therapeutic interventions. Biomarkers that characterize cases by severity and pathogenic mechanisms involved are not yet available but needed for the implementation of rational therapies. Here, we used shotgun proteomics to obtain information from plasma samples about local and systemic pathologies taking place during cases of alcoholic AP. Plasma was obtained at Kaunas University of Medicine Hospital (Lithuania) from 12 AP patients of alcohol related etiology (median age of 40) within 24 h of presentation, and 12 age-matched, healthy controls. Patients entered into the study had moderately severe AP with the following characteristics: mean blood lactate dehydrogenase level of 1127 mg/dl; median APACHEII score of 5.5 and mean IMRIE score of 3.5. For proteomic analysis, less-abundant proteins in plasma samples were enriched using Top 12 abundant protein depletion columns. Further processing was performed by a modified filter-assisted sample preparation combined with tandem mass tag labeling for quantitation. Samples were analyzed using an Orbitrap Elite mass spectrometer for high resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our analysis revealed 31 proteins that exhibited significant 1.5-fold or higher increases in the AP compared to control patients, and six that were significantly decreased. Gene ontology analysis indicated a strong correlation with exosomal origin in the elevated proteins, with 29/31 (93.5%) associated with this extracellularly-secreted compartment. Elevated proteins included established and proposed biomarkers of AP including C-reactive protein, LPS-binding protein, intercellular adhesion molecule-1, and von Willebrand factor, as well as several novel potential biomarkers. These results provide the methodology for proteomic analysis of plasma samples to discover novel biomarkers that characterize pancreatitis cases by pathogenic mechanism as well as disease activity at an early stage that is highly informative for routine clinical practice and clinical trials.