Transgenic Expression of PRSS1R122H Sensitizes Mice to Pancreatitis.

BACKGROUND & AIMS: Mutations in the trypsinogen gene (PRSS1) cause human hereditary pancreatitis. However, it is not clear how mutant forms of PRSS1 contribute to disease development. We studied the effects of expressing mutant forms of human PRSS1 in mice. METHODS: We expressed forms of PRSS1 with and without the mutation encoding R122H (PRSS1R122H) specifically in pancreatic acinar cells under control of a full-length pancreatic elastase gene promoter. Mice that did not express these transgenes were used as controls. Mice were given injections of caerulein to induce acute pancreatitis or injections of lipopolysaccharide to induce chronic pancreatitis. Other groups of mice were fed ethanol or placed on a high-fat diet to induce pancreatitis. Pancreata were collected and analyzed by histology, immunoblots, real-time polymerase chain reaction, and immunohistochemistry. Trypsin enzymatic activity and chymotrypsin enzymatic activity were measured in pancreatic homogenates. Blood was collected and serum amylase activity was measured. RESULTS: Pancreata from mice expressing transgenes encoding PRSS1 or PRSS1R122H had focal areas of inflammation; these lesions were more prominent in mice that express PRSS1R122H. Pancreata from mice that express PRSS1 or PRSS1R122H had increased levels of heat shock protein 70 and nuclear factor (erythroid-derived 2)-like 2, and reduced levels of chymotrypsin C compared with control mice. Increased expression of PRSS1 or PRSS1R122H increased focal damage in pancreatic tissues and increased the severity of acute pancreatitis after caerulein injection. Administration of lipopolysaccharide exacerbated inflammation in mice that express PRSS1R122H compared to mice that express PRSS1 or control mice. Mice that express PRSS1R122H developed more severe pancreatitis after ethanol feeding or a high-fat diet than mice that express PRSS1 or control mice. Pancreata from mice that express PRSS1R122H had more DNA damage, apoptosis, and collagen deposition and increased trypsin activity and infiltration by inflammatory cells than mice that express PRSS1 or control mice. CONCLUSIONS: Expression of a transgene encoding PRSS1R122H in mice promoted inflammation and increased the severity of pancreatitis compared with mice that express PRSS1 or control mice. These mice might be used as a model for human hereditary pancreatitis and can be studied to determine mechanisms of induction of pancreatitis by lipopolysaccharide, ethanol, or a high-fat diet.

Health Economic Impact of a Pulmonary Artery Pressure Sensor for Heart Failure Telemonitoring: A Dynamic Simulation.

AIMS: Recently, a permanently implantable wireless system, designed to monitor and manage pulmonary artery (PA) pressures remotely, demonstrated significant reductions in heart failure (HF) hospitalizations in high-risk symptomatic patients, regardless of ejection fraction. The objectives of this study were to simulate the estimated clinical and economic impact in Germany of generalized use of this PA pressure monitoring system considering reductions of HF hospitalizations and the improvement in Quality of Life. MATERIALS AND METHODS: Based on the Prospective Health Technology Assessment approach, we simulated the potential of the widespread application of PA pressure monitoring on the German healthcare system for the period 2009-2021. RESULTS: This healthcare economic simulation formulated input assumptions based on results from the CHAMPION Trial, a multicenter, prospective, randomized controlled U.S. trial that demonstrated a 37% reduction of hospitalizations in persistently symptomatic previous HF patients. Based on these results, an estimated 114,800 hospitalizations would expected to be avoided. This effect would potentially save an estimated €522 million, an equivalent of $575 million, during the entire simulation period. CONCLUSION: This healthcare economic modeling of the PA pressure monitoring system's impact demonstrates substantial clinical and economic benefits in the German healthcare system.

Activation of nuclear factor-κB in acinar cells increases the severity of pancreatitis in mice.

BACKGROUND & AIMS: Nuclear factor-κB (NF-κB) is activated during early stages of pancreatitis. This transcription factor regulates genes that control many cell activities, including inflammation and survival. There is evidence that activation of NF-κB protects against pancreatitis, and, in other cases, that it promotes this disease. We compared the effects of NF-κB in different mouse models of pancreatitis to understand these complications. METHODS: To model constitutive activation of NF-κB, we expressed a transgene that encodes its p65 subunit or the inhibitor of κB kinase (IKK)2 in pancreatic acinar cells of mice. We analyzed effects on pancreatic tissues and levels of NF-κB target genes in these mice and compared them with mice that did not express transgenic p65 or IKK2 (controls). RESULTS: Transgenic expression of p65 led to compensatory expression of the inhibitory subunit IKB-α and, therefore, no clear phenotype. However, p65 transgenic mice given injections of cerulein, to induce acute pancreatitis, had higher levels of NF-κB activity in acinar cells, greater levels of inflammation, and more severe outcomes than control mice. In contrast, constitutive expression of IKK2 directly increased the activity of NF-κB in acinar cells and induced pancreatitis. Prolonged activity of IKK2 (3 months) resulted in activation of stellate cells, loss of acinar cells, and fibrosis, which are characteristics of chronic pancreatitis. Co-expression of IKK2 and p65 greatly increased the expression of inflammatory mediators and the severity of pancreatitis, compared with control mice. CONCLUSIONS: The level of NF-κB activation correlates with the severity of acute pancreatitis in mice. Longer periods of activation (3 months) lead to chronic pancreatitis. These findings indicate that strategies to inactivate NF-κB might be used to treat patients with acute or chronic pancreatitis.

Epidemiology of primary hip and knee arthroplasties in Germany: 2004 to 2008.

The objective of this study was to describe the trend in utilization of primary joint arthroplasties in Germany. Between 2004 and 2008, the number of total knee arthroplasties (TKAs) increased faster than that of total hip arthroplasties (THAs). In 2008, 159000 primary THAs and 146000 primary TKAs were performed. This represented a 15% increase in THAs and a 33% increase in TKAs compared to 2004. The annual increase in number of surgeries was 4500 for THAs and 9,000 for TKAs. Although older adults remained the main recipients of joint arthroplasties, incidence rate increased faster in non-elderly(18-64 years) compared with elderly (≥65 years) in both THAs and TKAs. Obesity, more strongly associated with TKAs than with THAs, could be a contributor to the recent steeper growth in TKAs in Germany.

Intracellular activation of trypsinogen in transgenic mice induces acute but not chronic pancreatitis.

BACKGROUND AND AIMS: Premature intra-acinar activation of trypsinogen is widely considered key for both the initiation of acute pancreatitis and the development of chronic pancreatitis. However, the biological consequences of intracellular trypsinogen activation have not been directly examined. To do so, a new mouse model was developed. METHODS: Mice were engineered to conditionally express an endogenously activated trypsinogen within pancreatic acinar cells (PACE-tryp(on)). Hallmarks of pancreatitis were determined and findings were correlated to the level (zygosity) and extent (temporal and spatial) of conditional PACE-tryp(on) expression. Furthermore, the impact of acinar cell death in PACE-tryp(on) mice was assessed and compared with a model of selective diphtheria toxin (DT)-mediated induction of acinar apoptosis. RESULTS: Initiation of acute pancreatitis was observed with high (homozygous), but not low (heterozygous) levels of PACE-tryp(on) expression. Subtotal (maximal-rapid induction) but not limited (gradual-repetitive induction) conditional PACE-tryp(on) expression was associated with systemic complications and mortality. Rapid caspase-3 activation and apoptosis with delayed necrosis was observed, and loss of acinar cells led to replacement with fatty tissue. Chronic inflammation or fibrosis did not develop. Selective depletion of pancreatic acinar cells by apoptosis using DT evoked similar consequences. CONCLUSIONS: Intra-acinar activation of trypsinogen is sufficient to initiate acute pancreatitis. However, the primary response to intracellular trypsin activity is rapid induction of acinar cell death via apoptosis which facilitates resolution of the acute inflammation rather than causing chronic pancreatitis. This novel model provides a powerful tool to improve our understanding of basic mechanisms occurring during pancreatitis.

Contractile cell forces exerted on rigid substrates.

Adhesive cells including fibroblasts produce contractile forces to the underlying substrate for their locomotion. Such forces are not only high enough to deform compliant membranes such as silicone but also bend rigid micro-cantilevers. The cell-induced bending of silicon micro-cantilevers was determined using laser deflection during trypsin treatment. The observed cantilever relaxation corresponds to a contractile cell force of (16±7) µN per rat-2 fibroblast. The cantilever bending approach represents a unique method for the determination of contractile cell forces on any kind of rigid substrate in desired (physiological) environment. Hence, the fundamental technique allows building cell-based biosensors or should provide a quantitative parameter for characterising the cyto-compatibility of load bearing implant surfaces.

Ras activity levels control the development of pancreatic diseases.

BACKGROUND & AIMS: Differentiated pancreatic acinar cells expressing endogenous levels of mutant K-Ras do not spontaneously develop pancreatic ductal adenocarcinoma (PDAC). However, we hypothesized that acinar cells would develop PDAC in the presence of Ras activity levels mimicking those of human tumor cells. METHODS: We measured Ras activity in PDAC cells from mice and humans using a Raf pull-down assay. We compared the effects of acinar cell expression of mutant K-Ras at endogenous and elevated levels on Ras activity and on the development of PDAC. RESULTS: Ras activity was greatly elevated in PDAC cells compared with nontransformed cells expressing endogenous levels of mutant K-Ras. Expression of endogenous levels of mutant K-Ras in differentiated acinar cells resulted in moderately elevated Ras activity and in sparse murine pancreatic intraepithelial neoplasias (mPanINs) that did not spontaneously advance to PDAC unless the tumor suppressor p53 was simultaneously deleted. In contrast, expression of mutant K-Ras at higher levels generated Ras activity equal to that in PDAC. High Ras activity mimicking levels in PDAC led to acinar cell senescence and generated inflammation and fibrosis resembling the histologic features of chronic pancreatitis. With higher Ras activity in acinar cells, abundant mPanINs formed and spontaneously progressed to both cystic papillary carcinoma and metastatic PDAC. CONCLUSIONS: There is an important relationship between Ras activity levels and the progression of PDAC. Sufficient Ras activity in pancreatic acinar induces several important pancreatic disease manifestations not previously reported and supports a potential direct linkage between chronic pancreatitis, cystic papillary carcinoma, and PDAC.

Robust acinar cell transgene expression of CreErT via BAC recombineering.

Pancreatic acinar cells are critical in gastrointestinal physiology and pancreatitis and may be involved in pancreatic cancer. Previously, a short rat pancreatic elastase promoter has been widely utilized to control acinar cell transgene expression. However, this partial sequence does not confer robust and stable expression. In this study, we tested the hypothesis that a transgene employing bacterial-artificial-chromosome (BAC) technology to express a tamoxifen-regulated Cre recombinase from a full-length mouse elastase gene (BAC-Ela-CreErT) would be more robust and stable. When founders were crossed with Rosa26 reporter mice nearly 100% of acini expressed beta-galactosidase after tamoxifen treatment. The expression was specific for pancreatic acinar cells and these characteristics have remained stable for 2 years. However, because of high levels of expression in differentiated acinar cells, this construct is tamoxifen independent in approximately 50% of adult acinar cells. This model of pancreatic acinar specific Cre expression is a powerful tool for future transgenic and knockout studies.

Cardiac implant registries 2006-2016: a systematic review and summary of global experiences.

OBJECTIVES: The importance of Cardiac Implant Registry (CIR) for ensuring a long-term follow-up in postmarket surveillance has been recognised and approved, but there is lack of consensus standards on how to establish a CIR. The aim of this study is to investigate the structure and key elements of CIRs in the past decade (2006-2016) and to provide recommendations on 'best practice' approaches. SETTINGS AND PARTICIPANTS: A systematic search on CIR was employed in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. The following databases were searched: the PubMed (Medline), ScienceDirect and the Scopus database, EMBASE. After identifying the existing CIRs, an aggregative approach will be used to explore key elements emerging in the identified registries. RESULTS: The following 82 registries were identified: 18 implantable cardioverterdefibrillator (ICD) registries, 7 cardiac resynchronisation therapy (CRT) registries, 5 pacemaker registries and 6 cardiovascular implantable electronic device registries which combined ICD, pacemaker and CRT implantation data; as well as 22 coronary stent registries and 24 transcatheteraortic heart valve implantation registries. While 71 national or local registries are from a single country, 44 are from European countries and 9 are located in USA. The following criteria have been summarised from the identified registries, including: registry working group, ethic issues, transparency, research objective, inclusion criteria, compulsory participation, endpoint, sample size, data collection basement, data collection methods, data entry, data validation and statistical analysis. CONCLUSIONS: Registries provide a 'real-world' picture for patients, physicians, manufacturers, payers, decision-makers and other stakeholders. CIRs are important for regulatory decisions concerning the safety and therefore approval issues of the medical device; for payers CIRs provide evidence on the medical device benefit and drive the decision whether the product should be reimbursed or not; for hospitals CIRs' data are important for sound procurement decisions, and CIRs also help patients and their physicians to joint decision-making which of the products is the most appropriate.

Characterisation of a transgenic mouse expressing R122H human cationic trypsinogen.

BACKGROUND: The R122H mutation of the cationic trypsinogen was found in patients with hereditary pancreatitis. A transgenic animal carrying this mutation could be useful as a genetic model system of pancreatitis. METHODS: Mice transgenic for the human R122H cationic trypsinogen were generated using the -205 fragment of the rat elastase promoter. The presence of the transgene was assayed in the DNA, in pancreatic mRNA and in zymogen granule lysates. Serum levels of amylase, lipase and cytokines (MCP-1, IL-6) were monitored and the histological appearance of the tissue was investigated. Pancreatitis was induced by 7 hourly injections of 50 mug/kg cerulein. The procedure was repeated twice weekly for 10 consecutive weeks. The animals were sacrificed 24 (n = 8) and 48 hours (n = 8) after the first injection and at the end of the whole treatment (n = 7). RESULTS: The transgene was detected at the genomic level and in pancreatic mRNA. The corresponding protein was found in low amounts in zymogen granule lysates. R122H mice showed elevated pancreatic lipase, but there was no spontaneous development of pancreatitis within 18 months. After induction of pancreatitis, levels of lipase (after 24 hours) and amylase (after 48 hours) were higher in R122H mice compared to controls. Repeated treatment with cerulein resulted in a slightly more severe pancreatitis in R122H animals. Amylase, lipase, and the cytokine levels were similar to controls. CONCLUSION: The R122H transgenic mouse failed to develop a spontaneous pancreatitis but a repeatedly provoked cerulein-induced pancreatitis led to a slightly more severe pancreatitis. The rather small difference in comparison to controls could be due to the low expression of the transgene in the mouse pancreas.

Understanding nano-anatomy of healthy and carious human teeth: a prerequisite for nanodentistry.

The anatomy of human teeth reflects its usage. Spatially resolved X-ray scattering permits quantitative studies of the characteristic arrangement of the anisotropic calcium phosphate crystallites and the collagen fibers within the hard tissues of the crown. The present study summarizes the distinctive nanometer-sized anatomical features of the tooth hard tissues including their interface taking advantage of spatially resolved synchrotron radiation-based small-angle X-ray scattering. The comparison of slices from eight teeth indicates a long-range organization of tooth nanostructures.

An NF-κB pathway-mediated positive feedback loop amplifies Ras activity to pathological levels in mice.

Genetic mutations that give rise to active mutant forms of Ras are oncogenic and found in several types of tumor. However, such mutations are not clear biomarkers for disease, since they are frequently detected in healthy individuals. Instead, it has become clear that elevated levels of Ras activity are critical for Ras-induced tumorigenesis. However, the mechanisms underlying the production of pathological levels of Ras activity are unclear. Here, we show that in the presence of oncogenic Ras, inflammatory stimuli initiate a positive feedback loop involving NF-κB that further amplifies Ras activity to pathological levels. Stimulation of Ras signaling by typical inflammatory stimuli was transient and had no long-term sequelae in wild-type mice. In contrast, these stimuli generated prolonged Ras signaling and led to chronic inflammation and precancerous pancreatic lesions (PanINs) in mice expressing physiological levels of oncogenic K-Ras. These effects of inflammatory stimuli were disrupted by deletion of inhibitor of NF-κB kinase 2 (IKK2) or inhibition of Cox-2. Likewise, expression of active IKK2 or Cox-2 or treatment with LPS generated chronic inflammation and PanINs only in mice expressing oncogenic K-Ras. The data support the hypothesis that in the presence of oncogenic Ras, inflammatory stimuli trigger an NF-κB-mediated positive feedback mechanism involving Cox-2 that amplifies Ras activity to pathological levels. Because a large proportion of the adult human population possesses Ras mutations in tissues including colon, pancreas, and lung, disruption of this positive feedback loop may be an important strategy for cancer prevention.

Intracellular trypsin induces pancreatic acinar cell death but not NF-kappaB activation.

Premature intracellular activation of the digestive enzyme trypsinogen is considered to be the initiating event in pancreatitis. However, the direct consequences of intracellular trypsin activity have not previously been examined. In the current study, a mutant trypsinogen (paired basic amino acid cleaving enzyme (PACE)-trypsinogen), which is activated intracellularly by the endogenous protease PACE, was developed. This new construct allowed for the first time direct examination of the effects of intracellular trypsin on pancreatic acinar cells. We found that PACE-trypsinogen was expressed in the secretory pathway and was activated within acinar cells. Expression of PACE-trypsinogen induced apoptosis of HEK293 cells and pancreatic acinar cells, as indicated by histology, DNA laddering, PARP cleavage, and caspase-3 activation. Cell death was blocked by the trypsin inhibitor Pefabloc but not by the pancaspase inhibitor benzyloxycarbonyl-VAD, indicating that caspase-independent pathways were also involved. However, intracellular trypsin had no significant effect on the activity of the proinflammatory transcription factor NF-kappaB. In contrast, extracellular trypsin caused cell damage and dramatically increased NF-kappaB activity. These data indicate that localization of active trypsin determines its effects on pancreatic acinar cells. This new model will greatly improve our understanding of the role of active trypsin in pancreatitis and its associated inflammatory response.

The cardioprotective effect of mesenchymal stem cells is mediated by IGF-I and VEGF.

Emerging evidence suggests that adipose tissue-derived stem cells (ASCs) can be used for the treatment of ischemic heart diseases. However, the mechanisms underlying their therapeutic effects have not been clearly defined. In this study cytokines released by ASCs were detected by ELISA and pro-angiogenic effects were assessed by tube formation assay. To define the anti-apoptotic effect of ASCs, neonatal rat cardiomyocytes were subjected to hypoxia condition in a co-culture system. Our data show that ASCs secrete significant amounts of VEGF (810.65+/-56.92 pg/microg DNA) and IGF-I (328.33+/-22.7 pg/microg DNA). Cardiomyocytes apoptosis was significantly prevented by ASCs and 62.5% of the anti-apoptotic effect was mediated by IGF-I and 34.2% by VEGF. ASCs promoted endothelial cell tube formation by secreting VEGF. In conclusion we demonstrated that ASCs have a marked impact on anti-apoptosis and angiogenesis and helps to explain data of stem cells benefit without transdifferentiation.

Clusterin is protective in pancreatitis through anti-apoptotic and anti-inflammatory properties.

Clusterin is overexpressed in pancreas during the acute phase of pancreatitis. We intended to clarify the role of clusterin expression in stressed exocrine pancreas. We performed in vitro experiments in transfected AR4-2J cells with modified expression levels of clusterin and in vivo studies in clusterin-deficient mice. AR4-2J cells were exposed to agents mimicking cell-stress during pancreatitis (cerulein, hydrogen peroxide, staurosporine or lysophosphatidylcholine). Clusterin-overexpressing AR4-2J cells showed higher viability after cell stress and accordingly reduced rates of apoptosis and lessened caspase-3 activation. Blockage of endogenous clusterin expression reduced viability and enhanced apoptosis. Presence of clusterin reduced NF-kappaB activation and expression of the NF-kappaB target genes TNF-alpha and MOB-1 under cell stress. Clusterin-deficient mice showed a more severe course of acute experimental pancreatitis with enhanced rates of apoptosis and inflammatory cell infiltration. We concluded that clusterin was protective during inflammation of exocrine pancreas because of its anti-apoptotic and anti-inflammatory functions.

Expression of mutated cationic trypsinogen reduces cellular viability in AR4-2J cells.

Mutations in the human cationic trypsinogen are associated with hereditary pancreatitis. The cDNA coding for human cationic trypsinogen was subcloned into the expression vector pcDNA3. The mutations R122H, N29I, A16V, D22G, and K23R were introduced by site directed mutagenesis. We constructed an expression vector coding for active trypsin by subcloning the cDNA of trypsin lacking the coding region for the trypsin activating peptide behind an appropriate signal peptide. Expression of protein was verified by Western blot and measurement of enzymatic activity. AR4-2J cells were transiently transfected with the different expression vectors and cell viability and intracellular caspase-3 activity were quantified. In contrast to wild-type trypsinogen, expression of active trypsin and mutated trypsinogens reduced cell viability of AR4-2J cells. Expression of trypsin and R122H trypsinogen induced caspase-3 activity. Acinar cells might react to intracellular trypsin activity by triggering apoptosis.

The stress response of the exocrine pancreas.

Most attacks of acute pancreatitis display a self-limiting course. This suggests that pancreatic acinar cells may be able to protect themselves against cellular injury thus preventing further progression of the disease. In this review we describe several genes overexpressed in acute experimental pancreatitis which take part in the pancreatic stress response. We discuss the possible function of the pancreatitis-associated protein 1, the small nuclear protein p8, the glycoprotein clusterin, different heat shock proteins, the p53-dependent stress proteins TP53INP1alpha and TP53INP1beta, the vacuole membrane protein-1, as well as the interferon-inducible protein-15, the antiproliferative p53-dependent protein PC3/TIS21/BTG2, and the pancreatitis-induced protein-49. The implications of these proteins in pathophysiological processes like apoptosis regulation, regeneration, cell cycle and growth control, regulation of inflammation, and vacuole formation are discussed. Study of the function of stress proteins expressed in response to pancreatitis could widen our understanding of the pathophysiology of the disease and enable us to develop new rational therapeutic strategies.