Unlocking Clinical Insights: Lymphocyte-Specific Protein Tyrosine Kinase Candidates as Promising Therapeutic Targets for Pancreatic Cancer Risk Stratification.

Background: Uncover the pivotal link between lymphocyte-specific protein tyrosine kinase (Lck)-related genes and clinical risk stratification in pancreatic cancer. Methods: This study identifies shared genes between differentially expressed genes (DEGs) and Lck-related genes in pancreatic cancer using a methodological framework rooted in The Cancer Genome Atlas database. Feature gene selection is accomplished and a signature model is constructed. Statistical significant clinical endpoints such as overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) were defined. Results: After performing random survival forest, Lasso regression, and multivariate Cox regression model, 7 trait genes out of 272 Lck-associated DEGs are selected to create a signature model that is independent of other clinical factors and can predict OS and DSS. It appears that high-risk patients have activated the TP53 signaling pathway and the cell cycle signaling pathway. LAMA3 turned out to be the hub gene of the signature with high expression in pancreatic cancer. Patients with increased expression of LAMA3 had a short OS, DSS, and PFI in comparison. The candidate competing endogenous RNA network of LAMA3 turned out to be OPI5-AS1/hsa-miR-186-5p/LAMA3 axis. Conclusions: A characteristic signature of seven Lck-related genes, especially LAMA3, has been shown to be a key factor in clinical risk stratification for pancreatic cancer.

Integrating a microRNA signature as a liquid biopsy-based tool for the early diagnosis and prediction of potential therapeutic targets in pancreatic cancer.

INTRODUCTION: Pancreatic cancer is a highly aggressive cancer, and early diagnosis significantly improves patient prognosis due to the early implementation of curative-intent surgery. Our study aimed to implement machine-learning algorithms to aid in early pancreatic cancer diagnosis based on minimally invasive liquid biopsies. MATERIALS AND METHODS: The analysis data were derived from nine public pancreatic cancer miRNA datasets and two sequencing datasets from 26 pancreatic cancer patients treated in our medical center, featuring small RNAseq data for patient-matched tumor and non-tumor samples and serum. Upon batch-effect removal, systematic analyses for differences between paired tissue and serum samples were performed. The robust rank aggregation (RRA) algorithm was used to reveal feature markers that were co-expressed by both sample types. The repeatability and real-world significance of the enriched markers were then determined by validating their expression in our patients' serum. The top candidate markers were used to assess the accuracy of predicting pancreatic cancer through four machine learning methods. Notably, these markers were also applied for the identification of pancreatic cancer and pancreatitis. Finally, we explored the clinical prognostic value, candidate targets and predict possible regulatory cell biology mechanisms involved. RESULTS: Our multicenter analysis identified hsa-miR-1246, hsa-miR-205-5p, and hsa-miR-191-5p as promising candidate serum biomarkers to identify pancreatic cancer. In the test dataset, the accuracy values of the prediction model applied via four methods were 94.4%, 84.9%, 82.3%, and 83.3%, respectively. In the real-world study, the accuracy values of this miRNA signatures were 82.3%, 83.5%, 79.0%, and 82.2. Moreover, elevated levels of these miRNAs were significant indicators of advanced disease stage and allowed the discrimination of pancreatitis from pancreatic cancer with an accuracy rate of 91.5%. Elevated expression of hsa-miR-205-5p, a previously undescribed blood marker for pancreatic cancer, is associated with negative clinical outcomes in patients. CONCLUSION: A panel of three miRNAs was developed with satisfactory statistical and computational performance in real-world data. Circulating hsa-miRNA 205-5p serum levels serve as a minimally invasive, early detection tool for pancreatic cancer diagnosis and disease staging and might help monitor therapy success.

Establishment and Thorough Characterization of Xenograft (PDX) Models Derived from Patients with Pancreatic Cancer for Molecular Analyses and Chemosensitivity Testing.

Patient-derived xenograft (PDX) tumor models are essential for identifying new biomarkers, signaling pathways and novel targets, to better define key factors of therapy response and resistance mechanisms. Therefore, this study aimed at establishing pancreas carcinoma (PC) PDX models with thorough molecular characterization, and the identification of signatures defining responsiveness toward drug treatment. In total, 45 PC-PDXs were generated from 120 patient tumor specimens and the identity of PDX and corresponding patient tumors was validated. The majority of engrafted PDX models represent ductal adenocarcinomas (PDAC). The PDX growth characteristics were assessed, with great variations in doubling times (4 to 32 days). The mutational analyses revealed an individual mutational profile of the PDXs, predominantly showing alterations in the genes encoding KRAS, TP53, FAT1, KMT2D, MUC4, RNF213, ATR, MUC16, GNAS, RANBP2 and CDKN2A. Sensitivity of PDX toward standard of care (SoC) drugs gemcitabine, 5-fluorouracil, oxaliplatin and abraxane, and combinations thereof, revealed PDX models with sensitivity and resistance toward these treatments. We performed correlation analyses of drug sensitivity of these PDX models and their molecular profile to identify signatures for response and resistance. This study strongly supports the importance and value of PDX models for improvement in therapies of PC.

Combination of the biomarkers for aging and cancer? - Challenges and current status.

The proportion of patients diagnosed with cancer has been shown to rise with the increasing aging global population. Advanced age is a major risk factor for morbidity and mortality in older adults. As individuals experience varying health statuses, particularly with age, it poses a challenge for medical professionals in the cancer field to obtain standardized treatment outcomes. Hence, relying solely on chronological age and disease-related parameters is inadequate for clinical decision-making for elderly patients. With functional, multimorbidity-related, and psychosocial changes that occur with aging, oncologic diseases may develop and be treated differently from younger patients, leading to unique challenges in treatment efficacy and tolerance. To overcome this challenge, personalized therapy using biomarkers has emerged as a promising solution. Various categories of biomarkers, including inflammatory, hematological, metabolic, endocrine, and DNA modification-related indicators, may display features related to both cancer and aging, aiding in the development of innovative therapeutic approaches for patients with cancer in old age. Furthermore, physical functional measurements as non-molecular phenotypic biomarkers are being investigated for their potential complementary role in structured multidomain strategies to combat age-related diseases such as cancer. This review provides insight into the current developments, recent discoveries, and significant challenges in cancer and aging biomarkers, with a specific focus on their application in advanced age.

Concept of a fully-implantable system to monitor tumor recurrence.

Current treatment for glioblastoma includes tumor resection followed by radiation, chemotherapy, and periodic post-operative examinations. Despite combination therapies, patients face a poor prognosis and eventual recurrence, which often occurs at the resection site. With standard MRI imaging surveillance, histologic changes may be overlooked or misinterpreted, leading to erroneous conclusions about the course of adjuvant therapy and subsequent interventions. To address these challenges, we propose an implantable system for accurate continuous recurrence monitoring that employs optical sensing of fluorescently labeled cancer cells and is implanted in the resection cavity during the final stage of tumor resection. We demonstrate the feasibility of the sensing principle using miniaturized system components, optical tissue phantoms, and porcine brain tissue in a series of experimental trials. Subsequently, the system electronics are extended to include circuitry for wireless energy transfer and power management and verified through electromagnetic field, circuit simulations and test of an evaluation board. Finally, a holistic conceptual system design is presented and visualized. This novel approach to monitor glioblastoma patients is intended to early detect recurrent cancerous tissue and enable personalization and optimization of therapy thus potentially improving overall prognosis.

Transient receptor potential channels as predictive marker and potential indicator of chemoresistance in colon cancer.

Transient receptor potential (TRP) channels are strongly associated with colon cancer development and progression. This study leveraged a multivariate Cox regression model on publicly available datasets to construct a TRP channels-associated gene signature, with further validation of signature in real world samples from our hospital treated patient samples. Kaplan-Meier (K-M) survival analysis and receiver operating characteristic (ROC) curves were employed to evaluate this gene signature's predictive accuracy and robustness in both training and testing cohorts, respectively. Additionally, the study utilized the CIBERSORT algorithm and single-sample gene set enrichment analysis to explore the signature's immune infiltration landscape and underlying functional implications. The support vector machine algorithm was applied to evaluate the signature's potential in predicting chemotherapy outcomes. The findings unveiled a novel three TRP channels-related gene signature (MCOLN1, TRPM5, and TRPV4) in colon adenocarcinoma (COAD). The ROC and K-M survival curves in the training dataset (AUC = 0.761; p = 1.58e-05) and testing dataset (AUC = 0.699; p = 0.004) showed the signature's robust predictive capability for the overall survival of COAD patients. Analysis of the immune infiltration landscape associated with the signature revealed higher immune infiltration, especially an increased presence of M2 macrophages, in high-risk group patients compared to their low-risk counterparts. High-risk score patients also exhibited potential responsiveness to immune checkpoint inhibitor therapy, evident through increased CD86 and PD-1 expression profiles. Moreover, the TRPM5 gene within the signature was highly expressed in the chemoresistance group (p = 0.00095) and associated with poor prognosis (p = 0.036) in COAD patients, highlighting its role as a hub gene of chemoresistance. Ultimately, this signature emerged as an independent prognosis factor for COAD patients (p = 6.48e-06) and expression of model gene are validated by public data and real-world patients. Overall, this bioinformatics study provides valuable insights into the prognostic implications and potential chemotherapy resistance mechanisms associated with TRPs-related genes in colon cancer.

COL10A1 allows stratification of invasiveness of colon cancer and associates to extracellular matrix and immune cell enrichment in the tumor parenchyma.

Background: Treatment options for metastatic colorectal cancer (CRC) are mostly ineffective. We present new evidence that tumor tissue collagen type X alpha 1 (COL10A1) is a relevant candidate biomarker to improve this dilemma. Methods: Several public databases had been screened to observe COL10A1 expression in transcriptome levels with cell lines and tissues. Protein interactions and alignment to changes in clinical parameters and immune cell invasion were performed, too. We also used algorithms to build a novel COL10A1-related immunomodulator signature. Various wet-lab experiments were conducted to quantify COL10A1 protein and transcript expression levels in disease and control cell models. Results: COL10A1 mRNA levels in tumor material is clinical and molecular prognostic, featuring upregulation compared to non-cancer tissue, increase with histomorphological malignancy grading of the tumor, elevation in tumors that invade perineural areas, or lymph node invasion. Transcriptomic alignment noted a strong positive correlation of COL10A1 with transcriptomic signature of cancer-associated fibroblasts (CAFs) and populations of the immune compartment, namely, B cells and macrophages. We verified those findings in functional assays showing that COL10A1 are decreased in CRC cells compared to fibroblasts, with strongest signal in the cell supernatant of the cells. Conclusion: COL10A1 abundance in CRC tissue predicts metastatic and immunogenic properties of the disease. COL10A1 transcription may mediate tumor cell interaction with its stromal microenvironment.

Overcoming Barriers in Neurosurgical Education: A Novel Approach to Practical Ventriculostomy Simulation.

BACKGROUND: In the high-risk, high-stakes specialty of neurosurgery, traditional teaching methods often fail to provide young residents with the proficiency needed to perform complex procedures in stressful situations, with direct effects on patient outcomes. Physical simulators provide the freedom of focused, hands-on training in a more controlled environment. However, the adoption of simulators in neurosurgical training remains a challenge because of high acquisition costs, complex production processes, and lack of realism. OBJECTIVE: To introduce an easily reproducible, cost-effective simulator for external ventricular drain placements through various ventriculostomy approaches with life-like tactile brain characteristics based on real patients' data. METHODS: Whole brain and skull reconstruction from patient's computed tomography and MRI data were achieved using freeware and a desktop 3-dimensional printer. Subsequently, a negative brain silicone mold was created. Based on neurosurgical expertise and rheological measurements of brain tissue, gelatin in various concentrations was tested to cast tactilely realistic brain simulants. A sample group of 16 neurosurgeons and medical students tested and evaluated the simulator in respect to realism, haptics, and general usage, scored on a 5-point Likert scale. RESULTS: We saw a rapid and significant improvement of accuracy among novice medical students. All participants deemed the simulator as highly realistic, effective, and superior to conventional training methods. CONCLUSION: We were able to demonstrate that building and implementing a high-fidelity simulator for one of the most important neurosurgical procedures as an effective educational and training tool is achievable in a timely manner and without extensive investments.

Sensory Ion Channel Candidates Inform on the Clinical Course of Pancreatic Cancer and Present Potential Targets for Repurposing of FDA-Approved Agents.

Background: Transient receptor potential channels (TRPs) have been demonstrated to take on functions in pancreatic adenocarcinoma (PAAD) biology. However, little data are available that validate the potential of TRP in a clinical translational setting. Methods: A TRPs-related gene signature was constructed based on the Cox regression using a TCGA-PAAD cohort and receiver operating characteristic (ROC) was used to evaluate the predictive ability of this model. Core genes of the signature were screened by a protein-to-protein interaction (PPI) network, and expression validated by two independent datasets. The mutation analysis and gene set enrichment analysis (GSEA) were conducted. Virtual interventions screening was performed to discover substance candidates for the identified target genes. Results: A four TRPs-related gene signature, which contained MCOLN1, PKD1, TRPC3, and TRPC7, was developed and the area under the curve (AUC) was 0.758. Kaplan−Meier analysis revealed that patients with elevated signature score classify as a high-risk group featuring significantly shorter recurrence free survival (RFS) time, compared to the low-risk patients (p < 0.001). The gene prediction model also had a good predictive capability for predicting shortened overall survival (OS) and disease-specific survival (DSS) (AUC = 0.680 and AUC = 0.739, respectively). GSEA enrichment revealed the core genes of the signature, TRPC3 and TRPC7, were involved in several cancer-related pathways. TRPC3 mRNA is elevated in cancer tissue compared to control tissue and augmented in tumors with lymph node invasion compared to tumors without signs of lymph node invasion. Virtual substance screening of FDA approved compounds indicates that four small molecular compounds might be potentially selective not only for TRPC3 protein but also as a potential binding partner to TRPC7 protein. Conclusions: Our computational pipeline constructed a four TRP-related gene signature that enables us to predict clinical prognostic value of hitherto unrecognized biomarkers for PAAD. Sensory ion channels TRPC3 and TRPC7 could be the potential therapeutic targets in pancreatic cancer and TRPC3 might be involved in dysregulating mitochondrial functions during PAAD genesis.

The IL-17A/IL-17RA Axis Is Not Related to Overall Survival and Cancer Stem Cell Modulation in Pancreatic Cancer.

(1) Background: IL-17A accelerates pancreatic intraepithelial neoplasia (PanIN) progression. In this study, we examined whether IL-17A/IL-17RA promotes pancreatic ductal adenocarcinoma (PDAC) aggressiveness in terms of survival and cancer stem cell modulation. (2) Methods: In vitro, the wound-healing assay, the sphere formation assay, and flow cytometry were applied to assess cancer stem cell features. In vivo, pancreatic tumors were induced in C57BL/6 mice using electroporation with oncogenic plasmids (P53-/- R172H; KrasG12V). Anti-IL-17 antibodies were administered as immunotherapy. We analyzed IL-17A/IL-17RA related survival using publicly available transcriptomic data (n = 903). (3) Results: IL-17A/IL-17RA expression was not related to survival in PDAC patients. IL-17A neither induces stem cell markers nor increases sphere formation and cell motility in vitro. Blocking the IL-17A/IL-17RA axis in a murine pancreatic cancer model did not improve the survival of mice, but reduced the tumor burden slightly. (4) Conclusions: IL-17A does not promote stem cell expansion in PDAC cell lines. Blocking IL-17A/IL-17RA signaling does not interfere with pancreatic cancer development and progression and may not be considered as a promising monotherapy for PDAC.

Early trypsin activation develops independently of autophagy in caerulein-induced pancreatitis in mice.

Premature intrapancreatic trypsinogen activation is widely regarded as an initiating event for acute pancreatitis. Previous studies have alternatively implicated secretory vesicles, endosomes, lysosomes, or autophagosomes/autophagolysosomes as the primary site of trypsinogen activation, from which a cell-damaging proteolytic cascade originates. To identify the subcellular compartment of initial trypsinogen activation we performed a time-resolution analysis of the first 12 h of caerulein-induced pancreatitis in transgenic light chain 3 (LC3)-GFP autophagy reporter mice. Intrapancreatic trypsin activity increased within 60 min and serum amylase within 2 h, but fluorescent autophagosome formation only by 4 h of pancreatitis in parallel with a shift from cytosolic LC3-I to membranous LC3-II on Western blots. At 60 min, activated trypsin in heavier subcellular fractions was co-distributed with cathepsin B, but not with the autophagy markers LC3 or autophagy protein 16 (ATG16). Supramaximal caerulein stimulation of primary pancreatic acini derived from LC3-GFP mice revealed that trypsinogen activation is independent of autophagolysosome formation already during the first 15 min of exposure to caerulein. Co-localization studies (with GFP-LC3 autophagosomes versus Ile-Pro-Arg-AMC trypsin activity and immunogold-labelling of lysosomal-associated membrane protein 2 [LAMP-2] versus trypsinogen activation peptide [TAP]) indicated active trypsin in autophagolysosomes only at the later timepoints. In conclusion, during the initiating phase of caerulein-induced pancreatitis, premature protease activation develops independently of autophagolysosome formation and in vesicles arising from the secretory pathway. However, autophagy is likely to regulate overall intracellular trypsin activity during the later stages of this disease.

Levels of the Autophagy-Related 5 Protein Affect Progression and Metastasis of Pancreatic Tumors in Mice.

BACKGROUND AND AIMS: Cells in pancreatic ductal adenocarcinoma (PDAC) undergo autophagy, but its effects vary with tumor stage and genetic factors. We investigated the consequences of varying levels of the autophagy related 5 (Atg5) protein on pancreatic tumor formation and progression. METHODS: We generated mice that express oncogenic Kras in primary pancreatic cancer cells and have homozygous disruption of Atg5 (A5;Kras) or heterozygous disruption of Atg5 (A5+/-;Kras), and compared them with mice with only oncogenic Kras (controls). Pancreata were analyzed by histology and immunohistochemistry. Primary tumor cells were isolated and used to perform transcriptome, metabolome, intracellular calcium, extracellular cathepsin activity, and cell migration and invasion analyses. The cells were injected into wild-type littermates, and orthotopic tumor growth and metastasis were monitored. Atg5 was knocked down in pancreatic cancer cell lines using small hairpin RNAs; cell migration and invasion were measured, and cells were injected into wild-type littermates. PDAC samples were obtained from independent cohorts of patients and protein levels were measured on immunoblot and immunohistochemistry; we tested the correlation of protein levels with metastasis and patient survival times. RESULTS: A5+/-;Kras mice, with reduced Atg5 levels, developed more tumors and metastases, than control mice, whereas A5;Kras mice did not develop any tumors. Cultured A5+/-;Kras primary tumor cells were resistant to induction and inhibition of autophagy, had altered mitochondrial morphology, compromised mitochondrial function, changes in intracellular Ca2+ oscillations, and increased activity of extracellular cathepsin L and D. The tumors that formed in A5+/-;Kras mice contained greater numbers of type 2 macrophages than control mice, and primary A5+/-;Kras tumor cells had up-regulated expression of cytokines that regulate macrophage chemoattraction and differentiation into M2 macrophage. Knockdown of Atg5 in pancreatic cancer cell lines increased their migratory and invasive capabilities, and formation of metastases following injection into mice. In human PDAC samples, lower levels of ATG5 associated with tumor metastasis and shorter survival time. CONCLUSIONS: In mice that express oncogenic Kras in pancreatic cells, heterozygous disruption of Atg5 and reduced protein levels promotes tumor development, whereas homozygous disruption of Atg5 blocks tumorigenesis. Therapeutic strategies to alter autophagy in PDAC should consider the effects of ATG5 levels to avoid the expansion of resistant and highly aggressive cells.

Targeting cancer stem cells and their niche: perspectives for future therapeutic targets and strategies.

A small subpopulation of cells within the bulk of tumors share features with somatic stem cells, in that, they are capable of self-renewal, they differentiate, and are highly resistant to conventional therapy. These cells have been referred to as cancer stem cells (CSCs). Recent reports support the central importance of a cancer stem cell-like niche that appears to help foster the generation and maintenance of CSCs. In response to signals provided by this microenvironment, CSCs express the tumorigenic characteristics that can drive tumor metastasis by the induction of epithelial-mesenchymal-transition (EMT) that in turn fosters the migration and recolonization of the cells as secondary tumors within metastatic niches. We summarize here recent advances in cancer stem cell research including the characterization of their genetic and epigenetic features, metabolic specialities, and crosstalk with aging-associated processes. Potential strategies for targeting CSCs, and their niche, by regulating CSCs plasticity, or therapeutic sensitivity is discussed. Finally, it is hoped that new strategies and related therapeutic approaches as outlined here may help prevent the formation of the metastatic niche, as well as counter tumor progression and metastatic growth.

Legumain is activated in macrophages during pancreatitis.

Pancreatitis is an inflammatory disease of the pancreas characterized by dysregulated activity of digestive enzymes, necrosis, immune infiltration, and pain. Repeated incidence of pancreatitis is an important risk factor for pancreatic cancer. Legumain, a lysosomal cysteine protease, has been linked to inflammatory diseases such as atherosclerosis, stroke, and cancer. Until now, legumain activation has not been studied during pancreatitis. We used a fluorescently quenched activity-based probe to assess legumain activation during caerulein-induced pancreatitis in mice. We detected activated legumain by ex vivo imaging, confocal microscopy, and gel electrophoresis. Compared with healthy controls, legumain activity in the pancreas of caerulein-treated mice was increased in a time-dependent manner. Legumain was localized to CD68(+) macrophages and was not active in pancreatic acinar cells. Using a small-molecule inhibitor of legumain, we found that this protease is not essential for the initiation of pancreatitis. However, it may serve as a biomarker of disease, since patients with chronic pancreatitis show strongly increased legumain expression in macrophages. Moreover, the occurrence of legumain-expressing macrophages in regions of acinar-to-ductal metaplasia suggests that this protease may influence reprogramming events that lead to inflammation-induced pancreatic cancer.

Cathepsin B Activity Initiates Apoptosis via Digestive Protease Activation in Pancreatic Acinar Cells and Experimental Pancreatitis.

Pancreatitis is associated with premature activation of digestive proteases in the pancreas. The lysosomal hydrolase cathepsin B (CTSB) is a known activator of trypsinogen, and its deletion reduces disease severity in experimental pancreatitis. Here we studied the activation mechanism and subcellular compartment in which CTSB regulates protease activation and cellular injury. Cholecystokinin (CCK) increased the activity of CTSB, cathepsin L, trypsin, chymotrypsin, and caspase 3 in vivo and in vitro and induced redistribution of CTSB to a secretory vesicle-enriched fraction. Neither CTSB protein nor activity redistributed to the cytosol, where the CTSB inhibitors cystatin-B/C were abundantly present. Deletion of CTSB reduced and deletion of cathepsin L increased intracellular trypsin activation. CTSB deletion also abolished CCK-induced caspase 3 activation, apoptosis-inducing factor, as well as X-linked inhibitor of apoptosis protein degradation, but these depended on trypsinogen activation via CTSB. Raising the vesicular pH, but not trypsin inhibition, reduced CTSB activity. Trypsin inhibition did not affect apoptosis in hepatocytes. Deletion of CTSB affected apoptotic but not necrotic acinar cell death. In summary, CTSB in pancreatitis undergoes activation in a secretory, vesicular, and acidic compartment where it activates trypsinogen. Its deletion or inhibition regulates acinar cell apoptosis but not necrosis in two models of pancreatitis. Caspase 3-mediated apoptosis depends on intravesicular trypsinogen activation induced by CTSB, not CTSB activity directly, and this mechanism is pancreas-specific.

Complement Component 5 Mediates Development of Fibrosis, via Activation of Stellate Cells, in 2 Mouse Models of Chronic Pancreatitis.

BACKGROUND & AIMS: Little is known about the pathogenic mechanisms of chronic pancreatitis. We investigated the roles of complement component 5 (C5) in pancreatic fibrogenesis in mice and patients. METHODS: Chronic pancreatitis was induced by ligation of the midpancreatic duct, followed by a single supramaximal intraperitoneal injection of cerulein, in C57Bl6 (control) and C5-deficient mice. Some mice were given injections of 2 different antagonists of the receptor for C5a over 21 days. In a separate model, mice were given injections of cerulein for 10 weeks to induce chronic pancreatitis. Direct effects of C5 were studied in cultured primary cells. We performed genotype analysis for the single-nucleotide polymorphisms rs 17611 and rs 2300929 in C5 in patients with pancreatitis and healthy individuals (controls). Blood cells from 976 subjects were analyzed by transcriptional profiling. RESULTS: During the initial phase of pancreatitis, levels of pancreatic damage were similar between C5-deficient and control mice. During later stages of pancreatitis, C5-deficient mice and mice given injections of C5a-receptor antagonists developed significantly less pancreatic fibrosis than control mice. Primary pancreatic stellate cells were activated in vitro by C5a. There were no differences in the rs 2300929 SNP between subjects with or without pancreatitis, but the minor allele rs17611 was associated with a significant increase in levels of C5 in whole blood. CONCLUSIONS: In mice, loss of C5 or injection of a C5a-receptor antagonist significantly reduced the level of fibrosis of chronic pancreatitis, but this was not a consequence of milder disease in early stages of pancreatitis. C5 might be a therapeutic target for chronic pancreatitis.

Lipidomic analysis of molecular cardiolipin species in livers exposed to ischemia/reperfusion.

Transient hepatic ischemia can cause significant liver injury. A central and early event in ischemia/reperfusion (I/R) injury is the impairment of mitochondria. The phospholipid cardiolipin (CL) is required for efficient mitochondrial function. The aim of this study was to analyze composition, content, and oxidation of CL in dependence of I/R stress. Therefore, we exposed rat livers to 20 min ischemia by interrupting the perfusion with Krebs-Ringer solution in situ. Tissue histology as well as increased activities of LDH, GLDH, and ASAT analysed in the efflux after 50 min reperfusion indicated impairment of the liver. For the analysis of local CL distribution the liver homogenate was separated according to density into 11 fractions. The fractions displayed different contents of CL and citrate synthase peaking at density of about 1.07 g/cm(3). Among the fractions, the distribution of molecular CL species significantly differed. I/R caused loss of about 30 % CL and 17 % citrate synthase activity. Further, I/R shifted the CL and citrate synthase activity profile toward lower densities. Oxidized CL was exclusively found in fractions with high CL and citrate synthase content after I/R stress. I/R treatment caused significant changes in the distribution of molecular CL species. Our data demonstrate that I/R causes significant decrease in CL content and increase of oxidized CL that may be of impact for impairment of mitochondrial function by I/R. These results lead to the suggestion that strategies supporting anti-oxidative defence and CL synthesis may be beneficial to reduce I/R injury of the liver.

Impaired autophagy induces chronic atrophic pancreatitis in mice via sex- and nutrition-dependent processes.

BACKGROUND & AIMS: Little is known about the mechanisms of the progressive tissue destruction, inflammation, and fibrosis that occur during development of chronic pancreatitis. Autophagy is involved in multiple degenerative and inflammatory diseases, including pancreatitis, and requires the protein autophagy related 5 (ATG5). We created mice with defects in autophagy to determine its role in pancreatitis. METHODS: We created mice with pancreas-specific disruption of Atg5 (Ptf1aCreex1;Atg5F/F mice) and compared them to control mice. Pancreata were collected and histology, immunohistochemistry, transcriptome, and metabolome analyses were performed. ATG5-deficient mice were placed on diets containing 25% palm oil and compared with those on a standard diet. Another set of mice received the antioxidant N-acetylcysteine. Pancreatic tissues were collected from 8 patients with chronic pancreatitis (CP) and compared with pancreata from ATG5-deficient mice. RESULTS: Mice with pancreas-specific disruption of Atg5 developed atrophic CP, independent of ß-cell function; a greater proportion of male mice developed CP than female mice. Pancreata from ATG5-deficient mice had signs of inflammation, necrosis, acinar-to-ductal metaplasia, and acinar-cell hypertrophy; this led to tissue atrophy and degeneration. Based on transcriptome and metabolome analyses, ATG5-deficient mice produced higher levels of reactive oxygen species than control mice, and had insufficient activation of glutamate-dependent metabolism. Pancreata from these mice had reduced autophagy, increased levels of p62, and increases in endoplasmic reticulum stress and mitochondrial damage, compared with tissues from control mice; p62 signaling to Nqo1 and p53 was also activated. Dietary antioxidants, especially in combination with palm oil-derived fatty acids, blocked progression to CP and pancreatic acinar atrophy. Tissues from patients with CP had many histologic similarities to those from ATG5-deficient mice. CONCLUSIONS: Mice with pancreas-specific disruption of Atg5 develop a form of CP similar to that of humans. CP development appears to involve defects in autophagy, glutamate-dependent metabolism, and increased production of reactive oxygen species. These mice might be used to identify therapeutic targets for CP.

Autoimmune pancreatitis in MRL/Mp mice is a T cell-mediated disease responsive to cyclosporine A and rapamycin treatment.

BACKGROUND: Autoimmune pancreatitis (AIP) in humans invariably responds to steroid treatment, but little is known about the underlying pathogenesis and the benefits of alternative treatments. OBJECTIVE: To study the pathogenesis, and the efficacy of alternative immunosuppressant agents in the MRL/Mp mouse model of AIP. DESIGN: MRL/Mp mice were pretreated for 4 weeks with polyinosinic:polycytidylic acid to induce AIP. Pancreatic sections of mice genetically deleted for CTLA-4 were analysed. Blockage of CTLA-4 was achieved by intraperitoneal antibody treatment with 2 µg/g anti-mouse-CD152. Subsequent therapeutic studies were performed for a period of 4 weeks using cyclosporine A (40 µg/g), rapamycin (1 µg/g) or azathioprine (15 µg/g). RESULTS: Blockage of CTLA-4 in MRL/Mp mice suppressed regulatory T cell (Treg) function and raised the effector T cell (Teff) response with subsequent histomorphological organ destruction, indicating that AIP is a T cell-driven disease. Using an established histopathological score, we found that dexamethasone, cyclosporine A and rapamycin, but less so azathioprine, reduced pancreatic damage. However, the beneficial effects of cyclosporine A and rapamycin were achieved via different mechanisms: cyclosporine A inhibited Teff activation and proliferation whereas rapamycin led to selective expansion of Tregs which subsequently suppressed the Teff response. CONCLUSIONS: The calcineurin inhibitor cyclosporine A and the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, improve the course of AIP in MRL/Mp mice via different mechanisms. These findings further support the concept of autoreactive T cells as key players in the pathogenesis of AIP and suggest that cyclosporine A and rapamycin should be considered for treatment of AIP in humans.