Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted.

Thesis (Ph. D.)--University of Louisville, 2003. / Department of Pharmacology and Toxicology. Vita. "December 2003." Includes bibliographical references (leaves 136-154).

Islet transplantation has proved to be a useful treatment for Type 1 diabetes mellitus, but inadequate supplies of transplantable donor tissue have intensified the need to find a renewable source of β-cells. Human embryonic stem cells (HESC) are pluripotent and may offer a viable alternative to donor islets, but their targeted differentiation to a more specific β-cell phenotype is proving challenging. One strategy to restore β-cell mass is through activation of progenitor cells present in the pancreas. The aim of this study was to isolate and characterise progenitor cells from pancreatic tissue obtained from patients with Congenital Hyperinsulinism of Infancy (CHI). An enzymatic digestion was used to isolate islets from four patients with CHI and CHI-derived cell lines (NES139, NES140, NES140 and NES144) were subsequently derived that had the potential to proliferate in vitro. The previously-described cell line NES2Y was utlised as a control cell line for comparison. Using RT-PCR, exon array, qPCR, immunocytochemistry and Ca2+ microfluorimetry techniques this thesis examines both the molecular and physiological characteristics of these four CHI-derived cell lines to establish their potential as populations of pancreatic progenitor cells. Genotyping revealed that all of the patients carried mutations in the SUR1 gene, ABCC8. Pancreatic endocrine progenitor markers (e.g. PDX1, SOX9 and HLBX9) as well as islet precursor markers (e.g. NKX2.2, NKX6.1, NEUROD1, PAX6 and FOXA2) were identified and their expression was stable over continuous cell culture. However, each of the cell lines failed to express other markers, specifically NGN3, PAX4, and ISLET1. Cell lines developed from each patient then underwent a fibroblast to epithelial-like morphological transition. High-throughput exon array analysis revealed a significant down regulation of ACTA2, VIM and upregulation of CDH1 (q value < 0.05), a gene expression pattern associated with a mesenchymal-to-epithelial transition. Analysis at the mRNA level identified that CHI-derived cell lines expressed those channels and transporters associated with the β-cell function of glucose-stimulated insulin secretion (GSIS). Yet, when expression of all five endocrine hormones was investigated, mRNA expression was undetectable in three CHI-derived cell lines, except for the expression of insulin in NES143. Protein level assessment, however, failed to detect any expression of insulin. Functional studies examining whole cellular calcium dynamics and those underlying GSIS revealed that, whilst ATP (0.1 mM) and histamine (0.1 mM) readily raised intracellular Ca2+, each of the cell lines failed consistently to respond to tolbutamide (0.1 mM), glucose (20 mM), diazoxide (0.1 mM) and KCl (40 mM), except for NES140 which responded to applications of acetylcholine (0.1 mM). Given the display of cellular plasticity, molecular and physiological characteristics, the data show CHI-derived cell lines mimic pancreatic progenitor cell populations. More importantly, they represent islet precursor cells of the secondary transition phase of pancreatic development. Future studies should concentrate on the inductive potential of these cells to produce mature insulin-secreting β-cells.

Background: Current targeted therapies in pancreatic cancer have been ineffective. The tumor stroma, including intra- and peri-tumoral inflammation and fibrosis, is increasingly implicated in pancreatic cancer. Pancreatic cancer is characterized by a highly fibrotic tumor environment resulting in stromal resistance to chemotherapy. Peroxisome proliferator-activated receptor-alpha (PPARα), a ligand-activated nuclear receptor/transcription factor, is a negative regulator of inflammation. In PPARα deficient mice, stromal processes inhibit tumor growth, resulting in dormant tumors. The presence of PPARα in the tumor cells as well as in the host is necessary for unabated tumor growth. Objective: We hypothesized that blocking the PPARα pathway with a small molecule PPARα antagonist (NXT) may prevent pancreatic cancer progression by targeting tumor cells as well as non-neoplastic cells in the tumor microenvironment. Methods: Growth inhibitory activity of the PPARα antagonist was assessed in murine as well as human pancreatic tumor cell lines (Panc0H7 and BxPC3) and in a murine macrophage cell line (RAW 264.7). Cell viability was determined by trypan blue exclusion assay. AKT, P-AKT, PCNA, BAX, and p27 levels were analyzed by western blot analysis. Cell cycle changes were detected by flow cytometry. Cellular senescence was determined by senescence-associated β-gal (SA-β-gal) staining. Results: The PPARα antagonist inhibited cell growth in macrophages and in pancreatic tumor cells as confirmed by reduced protein level expression of PCNA and activated AKT. Treatment of the PPARα antagonist was non-cytotoxic to tumor cells. Inhibition of PPARα induced cell cycle arrest at G0/G1 in tumor cells and macrophages. The induction of cellular senescence was observed in pancreatic cancer cells. Interestingly, we observed a reduction in protein level expression of BAX, a marker for apoptosis, and p27, an inhibitor of the cell cycle. Conclusion: We now demonstrate that a PPARα antagonist exerts its anti-growth activity by inducing G0/G1 cell cycle arrest, thereby inducing cellular senescence without cell death. These findings provide a mechanism for the anti-tumorigenic activity of PPARα inhibition, and the rationale to use PPARα antagonists as a novel therapeutic approach to pancreatic cancer. / 2016-11-03T00:00:00Z

No description available.

Context: Pseudocysts are a common complication of acute and chronic pancreatitis. These are usually located within the pancreas but they can occur at other sites as well, including the mediastinum, neck, pelvis and rarely in the liver as in our case. The diagnosis of intrahepatic pancreatic pseudocyst relies on the demonstration of a high amylase level in the sampled cystic fluid in the absence of infection or neoplasm. Case report: A 60-year-old man with a history of chronic pancreatitis presents with a clinical and laboratory picture suggestive of acute exacerbation of his pancreatitis. A computed tomogram (CT) scan of the abdomen revealed a pancreatic pseudocyst and a cystic lesion involving both lobes of the liver. CT diagnostic aspiration of the intrahepatic cyst revealed high amylase level (greater than 20,000 U/L). The cyst was treated with percutaneous drainage with complete resolution of the cyst. Conclusion: In the setting of pancreatitis, intrahepatic pancreatic pseudocyst should be considered in the differential diagnosis of cystic lesion of the liver.

In this project we study the role of RAGE in the melanoma and pancreatic cancer progression. Based on published studies, we hypothesized that RAGE localization in melanoma varies with different cellular architectures. To test this hypothesis, we utilized an in vitro spheroid model and a lung colonization mice model to compare the RAGE localization in 3D architecture vs 2D monolayer culture. RAGE was found at the cell surface in WM115 and B16F10 spheroids, whereas RAGE is mostly distributed intracellularly in WM266. We also observed that RAGE is present at the surface of B16F10 melanoma cells within tumor nodules in the lungs of mice colonized with B16F10 cells. Previously, our group has demonstrated that RAGE promotes pancreatic tumor cell survival under normoxic conditions, upon gemcitabine administration. Hypoxia is also associated with increased tumor aggressiveness. Based on published reports, we hypothesized that RAGE upregulation under hypoxic conditions contributes to autophagy and migration in pancreatic cancer cells. We observed that autophagy decreases after RAGE inhibition by FPSZM1. Moreover, we observed decreased cell migration after RAGE blockage, indicating that RAGE also mediates migration under hypoxia. We also investigated Advanced Glycation Endproducts (AGEs) on proliferation and migration of pancreatic cancer cells. Based on published reports, we hypothesized that RAGE activation by AGEs contributes to the proliferation and migration in pancreatic cancer cells. We employed ribose modified BSA to activate RAGE in the murine KPC 5517 pancreatic cancer cell line. We observed that AGE-treated samples showed significant increase in migration but no change in proliferation. As RAGE is involved in the progression of melanoma and pancreatic cancer, our results will help researchers to better understand the biology of RAGE. Our research can help to design RAGE-specific antibodies and inhibitors that could target RAGE more effectively. Moreover, our findings on AGE-RAGE interactions, and on the role of RAGE in pancreatic cancer progression under hypoxia, may contribute to reduce the progression of pancreatic cancer. Our results showing that a RAGE inhibitor can reduce autophagy and migration of pancreatic tumor cells, suggest that FPS-ZM1 could be utilized as a potential therapeutic aid for the treatment of pancreatic cancer.

Background: Endoscopic retrograde pancreatography (ERP) has various applications in the diagnosis and management of pancreatic trauma. The utility of ERP in pancreatic trauma presenting to a level 1 equivalent trauma centre was analysed. Methods: Patients who sustained pancreatic trauma and underwent ERP were identified. Patient demographics, mechanism of injury, time to presentation, diagnostic modalities, associated injuries, clinical management, endoscopic interventions and their timing, surgical treatment and patient outcomes were recorded. Results: Forty-eight patients with pancreatic trauma were referred for ERP after blunt (26), gunshot (15), or stab (7) injury. The average time from injury to ERP was 38 days (range 2 – 365). An ERP visualized the duct in 47 patients. Twenty-four patients had a pancreatic fistula, 12 patients had a main pancreatic duct stricture or cut-off and 10 patients had a pseudocyst. Endoscopic interventions were pancreatic duct sphincterotomy (15), pancreatic duct stent (7) or pseudocyst drainage (6). Ten patients demonstrated minor injuries and no interventions were performed. One patient had a normal pancreatogram. Ten patients required pancreatic surgery following ERP (distal pancreatectomy n=6, pancreaticojejenostomy n=3 and cystjejenostomy n=1). One patient unable to tolerate ERP had a distal pancreatectomy. Conclusion: The majority of ERPs were performed post surgery or after a delayed presentation. Diagnostic success was high and in conjunction with therapeutic interventions 77% of patients avoided surgery for their pancreatic complications. ERP is an effective tool in the delayed management of the local complications of pancreatic trauma.

Indiana University-Purdue University Indianapolis (IUPUI) / Type 2 diabetes mellitus is a chronic disorder characterized by hyperglycemia, insulin resistance, and insufficient insulin secretion from the pancreatic β cells. To maintain adequate levels of insulin secretion, β cells rely on highly coordinated control of luminal ER Ca2+. Stromal Interaction Molecule 1 (STIM1) is an ER Ca2+ sensor that serves to replenish ER Ca2+ stores in response to depletion by gating plasmalemmal Orai1 channels in a process known as store-operated calcium entry (SOCE). We developed a method for the direct measurement of SOCE in pancreatic β cells and found that deletion of STIM1 in INS-1 cells (STIM1KO) is sufficient to block Ca2+ influx in response to store-depletion. To determine the physiological importance of β cell STIM1, we created mice with pancreatic β cell specific deletion of STIM1 (STIM1Δβ) and placed them on a high fat diet (HFD) with 60% of kilocalories derived from fat. After 8 weeks of HFD, female, but not male, STIM1Δβ mice exhibited increased body weight and fat mass as well as significant glucose intolerance and impaired insulin secretion without observable differences in insulin tolerance. Immunohistochemical analysis revealed a reduction of β cell mass and an increase of α cell mass; ELISA of islet lysates revealed a similar significant reduction in insulin content and increased glucagon content. RNA-sequencing performed on STIM1Δβ islets revealed differentially expressed genes for functions related to apoptosis, lipid metabolism, and epithelial cell differentiation, as well as loss of β cell identity. Proteomics analysis of STIM1KO cells phenocopied the metabolic findings, revealing significantly increased glucagon expression. Analysis of islet RNA-sequencing results showed modulation of pathways related to 17-β estradiol (E2) signaling, with notable downregulation of G-protein coupled estrogen receptor 1 (GPER1) expression. Consistently, treatment of female wild-type islets with pharmacological SOCE inhibitors led to reduced expression GPER1, while STIM1KO cells showed lower mobilization of intracellular cAMP levels in response to GPER agonist treatment. Taken together, these findings identify a novel interaction between SOCE and E2 signaling in the female islet and suggest that loss of STIM1 and impairments in SOCE may contribute to diabetes pathophysiology through loss of β cell identity. / 2022-12-28