The role of intracellular Ca2+signaling in the exocrine pancreatic damage duringacute pancreatitis

Acute biliary pancreatitis poses a significant clinical challenge as currently no specific pharmaceutical treatment exists. Disturbed intracellular Ca2+ signalling caused by bile acids is a hallmark of the disease, which induces increased reactive oxygen species (ROS) production, mitochondrial damage, intra-acinar digestive enzyme activation and cell death. Because of this mechanism of action, prevention of toxic cellular Ca2+ overload is a promising therapeutic target. Transient receptor potential melastatin 2 (TRPM2) is a non-selective cation channel that has recently emerged as an important contributor to oxidative-stress-induced cellular Ca2+ overload across different diseases. However, the expression and possible functions of TRPM2 in the exocrine pancreas remain unknown. Here we found that TRPM2 is expressed in the plasma membrane of mouse pancreatic acinar, which can be activated by increased oxidative stress induced by H2O2 treatment. TRPM2 activity was found to contribute to bile acid-induced extracellular Ca2+ influx in acinar cells. The generation of intracellular ROS in response to bile acids was remarkably higher in pancreatic acinar cells. This activity promoted acinar cell necrosis in vitro independently from mitochondrial damage or mitochondrial fragmentation. In addition, bile-acid-induced experimental pancreatitis was less severe in TRPM2 knockout mice, whereas the lack of TRPM2 had no protective effect in cerulein-induced acute pancreatitis. Our results suggest that the inhibition of TRPM2 may be a potential treatment option for biliary pancreatitis

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

Novel mitochondrial transition pore inhibitor N‐methyl‐4‐isoleucine cyclosporin is a new therapeutic option in acute pancreatitis

Bile acids, ethanol and fatty acids affect pancreatic ductal fluid and bicarbonate secretion via mitochondrial damage, ATP depletion and calcium overload. •Pancreatitis-inducing factors open the membrane transition pore (mPTP) channel via cyclophilin D activation in acinar cells, causing calcium overload and cell death; genetic or pharmacological inhibition of mPTP improves the outcome of acute pancreatitis in animal models. •Here we show that genetic and pharmacological inhibition of mPTP protects mitochondrial homeostasis and cell function evoked by pancreatitis-inducing factors in pancreatic ductal cells. •The results also show that the novel cyclosporin A derivative NIM811 protects mitochondrial function in acinar and ductal cells, and it preserves bicarbonate transport mechanisms in pancreatic ductal cells. •We found that NIM811 is highly effective in different experimental pancreatitis models and has no side-effects. NIM811 is a highly suitable compound to be tested in clinical trials

TRPM2 channel-mediated cell death: an important mechanism linking oxidative stress-inducing pathological factors to associated pathological conditions

Oxidative stress resulting from the accumulation of high levels of reactive oxygen species is a salient feature of, and a well-recognised pathological factor for, diverse pathologies. One common mechanism for oxidative stress damage is via the disruption of intracellular ion homeostasis to induce cell death. TRPM2 is a non-selective Ca2+-permeable cation channel with a wide distribution throughout the body and is highly sensitive to activation by oxidative stress. Recent studies have collected abundant evidence to show its important role in mediating cell death induced by miscellaneous oxidative stress-inducing pathological factors, both endogenous and exogenous, including ischemia/reperfusion and the neurotoxicants amyloid-β peptides and MPTP/MPP+ that cause neuronal demise in the brain, myocardial ischemia/reperfusion, proinflammatory mediators that disrupt endothelial function, diabetogenic agent streptozotocin and diabetes risk factor free fatty acids that induce loss of pancreatic β-cells, bile acids that damage pancreatic acinar cells, renal ischemia/reperfusion and albuminuria that are detrimental to kidney cells, acetaminophen that triggers hepatocyte death, and nanoparticles that injure pericytes. Studies have also shed light on the signalling mechanisms by which these pathological factors activate the TRPM2 channel to alter intracellular ion homeostasis leading to aberrant initiation of various cell death pathways. TRPM2-mediated cell death thus emerges as an important mechanism in the pathogenesis of conditions including ischemic stroke, neurodegenerative diseases, cardiovascular diseases, diabetes, pancreatitis, chronic kidney disease, liver damage and neurovascular injury. These findings raise the exciting perspective of targeting the TRPM2 channel as a novel therapeutic strategy to treat such oxidative stress-associated diseases

Mouse pancreatic ductal organoid culture as a relevant model to study exocrine pancreatic ion secretion

Pancreatic exocrine secretory processes are challenging to investigate on primary epithelial cells. Pancreatic organoid cultures may help to overcome shortcomings of the current models, however the ion secretory processes in pancreatic organoids-and therefore their physiological relevance or their utility in disease modeling-are not known. To answer these questions, we provide side-by-side comparison of gene expression, morphology, and function of epithelial cells in primary isolated pancreatic ducts and organoids. We used mouse pancreatic ductal fragments for experiments or were grown in Matrigel to obtain organoid cultures. Using PCR analysis we showed that gene expression of ion channels and transporters remarkably overlap in primary ductal cells and organoids. Morphological analysis with scanning electron microscopy revealed that pancreatic organoids form polarized monolayers with brush border on the apical membrane. Whereas the expression and localization of key proteins involved in ductal secretion (cystic fibrosis transmembrane conductance regulator, Na+/H+ exchanger 1 and electrogenic Na+/HCO3- cotransporter 1) are equivalent to the primary ductal fragments. Measurements of intracellular pH and Cl- levels revealed no significant difference in the activities of the apical Cl-/HCO3- exchange, or in the basolateral Na+ dependent HCO3- uptake. In summary we found that ion transport activities in the mouse pancreatic organoids are remarkably similar to those observed in freshly isolated primary ductal fragments. These results suggest that organoids can be suitable and robust model to study pancreatic ductal epithelial ion transport in health and diseases and facilitate drug development for secretory pancreatic disorders like cystic fibrosis, or chronic pancreatitis

TRPM2-mediated extracellular Ca2+ entry promotes acinar cell necrosis in biliary acute pancreatitis

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CD8+ tissue-resident memory T-cell development depends on infection-matching regulatory T-cell types

Abstract Immunological memory is critical for immune protection, particularly at epithelial sites, which are under constant risk of pathogen invasions. To counter invading pathogens, CD8+ memory T cells develop at the location of infection: tissue-resident memory T cells (TRM). CD8+ T-cell responses are associated with type-1 infections and type-1 regulatory T cells (TREG) are important for CD8+ T-cell development, however, if CD8+ TRM cells develop under other infection types and require immune type-specific TREG cells is unknown. We used three distinct lung infection models, to show that type-2 helminth infection does not establish CD8+ TRM cells. Intracellular (type-1) and extracellular (type-3) infections do and rely on the recruitment of response type-matching TREG population contributing transforming growth factor-β. Nevertheless, type-1 TREG cells remain the most important population for TRM cell development. Once established, TRM cells maintain their immune type profile. These results may have implications in the development of vaccines inducing CD8+ TRM cells

Implementing continuous auditing processes in Quality Management

The aim of this thesis work was to create an internal quality audit system for Sallila Energia Oy. The thesis work is based on the ISO standards, that the Sallila Energia follows. The logic behind the internal quality audit system also works for other standards and supports an organization in achieving compliance and performance through continuous development. Altogether, the standards were parsed into 441 requirements, consisting of four different standards. The internal auditing system contains each of these requirements with one row per requirement. The row contains information of whether it has been completed or not (marked with either “k” for compliance or “?” for need of further specifications or development before compliance), action to be taken and further information regarding the implementation of the potential change or changes. The implementation was supported by the PDSA (Plan-Do-Study-Act) theory, of which the plan and do-phases were part of this thesis work. Findings of this paper show that continuous internal auditing routines function as a base for the development of organizational process planning, and that the internal auditing can raise the organization from being standard complying to being a platform for organizational performance through continuous auditing principles. The internal audit system can be found in Appendix IV