Publications by authors named "Gopalakrishnan Loganathan"

Background: Alcoholic chronic pancreatitis (ACP) is a serious inflammatory disorder of the exocrine pancreatic gland. A previous study from this laboratory showed that ethanol (EtOH) causes cytotoxicity, dysregulates AMPKα and ER/oxidative stress signaling, and induces inflammatory responses in primary human pancreatic acinar cells (hPACs). Here we examined the differential cytotoxicity of EtOH and its oxidative (acetaldehyde) and nonoxidative (fatty acid ethyl esters; FAEEs) metabolites in hPACs was examined to understand the metabolic basis and mechanism of ACP.

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Primary toxicity targets of alcohol and its metabolites in the pancreas are cellular energetics and endoplasmic reticulum (ER). Therefore, the role of AMP-Activated Protein Kinase (AMPKα) in amelioration of ethanol (EtOH)-induced pancreatic acinar cell injury including ER/oxidative stress, inflammatory responses, the formation of fatty acid ethyl esters (FAEEs) and mitochondrial bioenergetics were determined in human pancreatic acinar cells (hPACs) and AR42J cells incubated with/without AMPKα activator [5-aminoimidazole-4-carboxamide ribonucleotide (AICAR)]. EtOH treated hPACs showed concentration and time-dependent increases for FAEEs and inactivation of AMPKα, along with the upregulation of ACC1 and FAS (key lipogenic proteins) and downregulation of CPT1A (involved β-oxidation of fatty acids).

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Purpose: Human islet isolation requires a defined collagenase-protease enzyme combination for obtaining a successful islet yield. While different islet laboratories use different enzyme combinations, a systematic methodology to identify optimal enzyme combinations and their concentrations within a single donor pancreas has not been tested. In this study, we designed a trisected pancreas model to test efficacy of three clinical grade enzyme blends (VitaCyte, Roche, SERVA) within a single pancreas.

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Background And Aims: Successful clinical human allo or auto-islet transplantation requires the recovery of a sufficient number of functional islets from either brain-dead or chronic pancreatitis pancreases respectively.

Methods: In the last two decades (2000-2019), significant progress has been made in improving the human islet isolation procedures and in standardizing the use of different tissue dissociation enzyme (TDE; a mixture of collagenase and protease enzymes) blends to recover higher islet yields.

Results And Conclusions: This review presents information focusing on properties and role of TDE blends during the islet isolation process, particularly emphasizing on the current developments, associated challenges and future perspectives within the field.

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Islet cell auto-transplantation is a novel strategy for maintaining blood glucose levels and improving the quality of life in patients with chronic pancreatitis (CP). Despite the many recent advances associated with this therapy, obtaining a good yield of islet infusate still remains a pressing challenge. Reprogramming technology, by making use of the pancreatic exocrine compartment, can open the possibility of generating novel insulin-producing cells.

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Human islet isolation from young donor pancreases (YDP) utilizing the current purified standard dose of collagenase-protease enzyme mixtures often results in the release of a high percentage of mantled islets. Mantled islets are those surrounded by exocrine tissue and are difficult to purify by density gradient centrifugation, leading to poor islet recovery. Based on difference in extracellular matrix, and total collagen content between YDP and old donor pancreas (ODP, > 35 Y) led us to compare results from islet isolation using increased collagenase combination (ICC) or increased protease combination (IPC), to the standard enzyme combination (SEC) in a "trisected" pancreas model to overcome the donor-to-donor variability.

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The intra-islet endothelial cells (ECs), the building blocks of islet microvasculature, govern a number of cellular and pathophysiological processes associated with the pancreatic tissue. These cells are key to the angiogenic process and essential for islet revascularization after transplantation. Understanding fundamental mechanisms by which ECs regulate the angiogenic process is important as these cells maintain and regulate the intra-islet environment facilitated by a complex signaling crosstalk with the surrounding endocrine cells.

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A high number of human islets can be isolated by using modern purified tissue dissociation enzymes; however, this requires the use of >20 Wunsch units (WU)/g of pancreas for digestion. Attempts to reduce this dose have resulted in pancreas underdigestion and poor islet recovery but improved islet function. In this study, we achieved a high number of functional islets using a low dose of recombinant collagenase enzyme mixture (RCEM-1200 WU rC2 and 10 million collagen-degrading activity [CDA] U of rC1 containing about 209 mg of collagenase to digest a 100-g pancreas).

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Purpose Of Review: The current article reviews the rationale, sources and preparation of pig islets for xenotransplantation, and presents current progress in solving the problems associated with establishing pig islet transplant as a clinical treatment for type 1 diabetes.

Summary: Islet transplantation is an effective treatment option for type 1 diabetes, but the available supply of human pancreases is insufficient to meet the need and demand for obtaining islets. Pig islets provide a readily available source for islet transplantation, with trials in non-human primates demonstrating their potential to reverse diabetes.

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The intra-islet microvasculature is a critical interface between the blood and islet endocrine cells governing a number of cellular and pathophysiological processes associated with the pancreatic tissue. A growing body of evidence indicates a strong functional and physical interdependency of β-cells with endothelial cells (ECs), the building blocks of islet microvasculature. Intra-islet ECs, actively regulate vascular permeability and appear to play a role in fine-tuning blood glucose sensing and regulation.

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Unlabelled: Isolation following a good manufacturing practice-compliant, human islet product requires development of a robust islet isolation procedure where effective limits of key reagents are known. The enzymes used for islet isolation are critical but little is known about the doses of class I and class II collagenase required for successful islet isolation.

Methods: We used a factorial approach to evaluate the effect of high and low target activities of recombinant class I (rC1) and class II (rC2) collagenase on human islet yield.

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Selection of an optimal donor pancreas is the first key task for successful islet isolation. We conducted a retrospective multicenter study in 11 centers in North America to develop an islet donor scoring system using donor variables. The data set consisting of 1,056 deceased donors was used for development of a scoring system to predict islet isolation success (defined as postpurification islet yield >400,000 islet equivalents).

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Thiamin (vitamin B1), a member of the water-soluble family of vitamins, is essential for normal cellular functions; its deficiency results in oxidative stress and mitochondrial dysfunction. Pancreatic acinar cells (PAC) obtain thiamin from the circulation using a specific carrier-mediated process mediated by both thiamin transporters -1 and -2 (THTR-1 and THTR-2; encoded by the SLC19A2 and SLC19A3 genes, respectively). The aim of the current study was to examine the effect of chronic exposure of mouse PAC in vivo and human PAC in vitro to nicotine (a major component of cigarette smoke that has been implicated in pancreatic diseases) on thiamin uptake and to delineate the mechanism involved.

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Background: Reliable in vitro islet quality assessment assays that can be performed routinely, prospectively, and are able to predict clinical transplant outcomes are needed. In this paper we present data on the utility of an assay based on cellular oxygen consumption rate (OCR) in predicting clinical islet autotransplant (IAT) insulin independence (II). IAT is an attractive model for evaluating characterization assays regarding their utility in predicting II due to an absence of confounding factors such as immune rejection and immunosuppressant toxicity.

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Background: In the absence of a reliable islet potency assay, nude mice (NM) transplantation is the criterion standard to assess islet quality for clinical transplantation. There are factors other than islet quality that affect the transplant outcome.

Methods: Here, we analyzed the transplant outcomes in 335 NM receiving islets from human (n=103), porcine (n=205), and nonhuman primate (NHP; n=27) donors.

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Background: The optimal enzyme blend that maximizes human islet yield for transplantation remains to be determined. In this study, we evaluated eight different enzyme combinations (ECs) in an attempt to improve islet yield. The ECs consisted of purified, intact or truncated class 1 (C1) and class 2 (C2) collagenases from Clostridium histolyticum (Ch), and neutral protease (NP) from Bacillus thermoproteolyticus rokko (thermolysin) or Ch (ChNP).

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Islets isolated from cadaveric donor pancreas are functionally viable and can be transplanted in diabetic patients to reduce insulin requirements. This therapeutic approach is less efficient because a significant portion of functional islets is lost due to oxidative stress, inflammation, and hypoxia. Exendin-4, a glucagon-like peptide-1 receptor agonist, is known to improve islet survival through activation of the transcription factor, cAMP response element binding protein (CREB).

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Background: Pancreatic acinar cells are commonly cotransplanted along with islets during auto- and allotransplantations. The aims of this study were to identify how acinar cell proteases cause human islet cell loss before and after transplantation of impure islet preparations and to prevent islet loss and improve function with supplementation of α-1 antitrypsin (A1AT).

Methods: Acinar cell protease activity, insulin levels, and percent islet loss were measured after culture of pure and impure clinical islet preparations.

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Background: Using standard density gradient (SDG) ranges for human islet purification frequently results in islet loss and transplantation of lower islet mass. Measuring the densities of islet and acinar tissue beforehand to customize the gradient range for the actual COBE 2991 cell processor (COBE) purification is likely to maximize the recovery of islets. We developed an analytical test gradient system (ATGS) for predicting pancreatic tissue densities before COBE purification to minimize islet loss during purification.

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Background: Purified tissue dissociation enzymes (TDEs) are critical to successful human islet isolation required for clinical transplantation, but little is known about the characteristics of the key enzymes-class I (C1) and class II (C2) collagenase from Clostridium histolyticum-used in these procedures. Here, we show the differences between the C1 collagenase found in purified collagenase products manufactured by three suppliers and the impact of differences in C1 between two suppliers on human islet yield.

Methods: Collagenase from Roche, Serva/Nordmark (Uetersen, Germany), and VitaCyte (Indianapolis, IN) were analyzed by analytical high-performance liquid chromatography and collagen degradation activity (CDA), an assay that preferentially detects intact C1 collagenase.

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