Effect of partial pancreatectomy on diabetic status in BALB/c mice.

Pancreatic regeneration after pancreatectomy has been well documented in animal models. However, the phenomenon of pancreatic regeneration in diabetes has not been exploited as yet. We report here the restoration of euglycaemic status in streptozotocin (STZ)-induced diabetic BALB/c mice, after 50% pancreatectomy. We observed that, after pancreatectomy, STZ-diabetic mice showed a rapid improvement in glycaemic status, starting from the 8th postoperative day, and remained normoglycaemic throughout a 90-day follow-up study. STZ-induced diabetic and control non-diabetic BALB/c mice underwent pancreatectomy and were monitored regularly for changes in body weight, plasma glucose and serum insulin concentrations and histological status of the pancreas. All the pancreatectomised animals showed euglycaemic status from about 20 days after operation, whereas a majority (around 70%) of the diabetic, sham-operated animals died of sustained hyperglycaemia by 20-30 days after operation. Examination of the regenerating pancreas indicated nesidioblastotic activity and supported the theory of a ductal origin of islet stem cells. Islets isolated from the regenerating pancreas showed a progressive increase in islet area (1227.9+/-173.2 micrometer(2) on day 5 compared with 2473.8+/-242.0 micrometer(2) by day 20). The increment in insulin concentrations and subsequent decrement in glycaemia of the diabetic pancreatectomised animals indicate islet neogenesis occurring after the operative insult, leading to a normoglycaemic status, probably recapitulating ontogeny. We have shown that induction of a regenerative stimulus (pancreatectomy) in conditions of STZ-induced diabetes may trigger pancreatic regenerative processes, thereby restoring a functional pancreas, in STZ-diabetic mice

Manipulating cellular microRNAs and analyzing high-dimensional gene expression data using machine learning workflows.

MicroRNAs (miRNAs) are elements of the gene regulatory network and manipulating their abundance is essential toward elucidating their role in patho-physiological conditions. We present a detailed workflow that identifies important miRNAs using a machine learning algorithm. We then provide optimized techniques to validate the identified miRNAs through over-expression/loss-of-function studies. Overall, these protocols apply to any field in biology where high-dimensional data are produced. For complete details on the use and execution of this protocol, please refer to Wong et al. (2021a)

Increased myofibroblasts in the small airways, and relationship to remodelling and functional changes in smokers and COPD patients: potential role of epithelial-mesenchymal transition

Introduction: Previous reports have shown epithelial–mesenchymal transition (EMT) as an active processthat contributes to small airway fibrotic pathology. Myofibroblasts are highly active pro-fibrotic cells thatsecrete excessive and altered extracellular matrix (ECM). Here we relate small airway myofibroblastpresence with airway remodelling, physiology and EMT activity in smokers and COPD patients.Methods: Lung resections from nonsmoker controls, normal lung function smokers and COPD currentand ex-smokers were stained with anti-human α-smooth muscle actin (SMA), collagen 1 and fibronectin.αSMA+ cells were computed in reticular basement membrane (Rbm), lamina propria and adventitia andpresented per mm of Rbm and mm2 of lamina propria. Collagen-1 and fibronectin are presented as apercentage change from normal. All analyses including airway thickness were measured using Image-proplus 7.0.Results: We found an increase in subepithelial lamina propria (especially) and adventitia thickness in allpathological groups compared to nonsmoker controls. Increases in αSMA+ myofibroblasts were observedin subepithelial Rbm, lamina propria and adventitia in both the smoker and COPD groups compared tononsmoker controls. Furthermore, the increase in the myofibroblast population in the lamina propria wasstrongly associated with decrease in lung function, lamina propria thickening, increase in ECM proteindeposition, and finally EMT activity in epithelial cells.Conclusions: This is the first systematic characterisation of small airway myofibroblasts in COPD based ontheir localisation, with statistically significant correlations between them and other pan-airway structural,lung function and ECM protein changes. Finally, we suggest that EMT may be involved in such changes

Human Blood Vessel–Derived Endothelial Progenitors for Endothelialization of Small Diameter Vascular Prosthesis

BACKGROUND:Coronary bypass graft failure as a result of acute thrombosis and intimal hyperplasia has been the major challenge in surgical procedures involving small-diameter vascular prosthesis. Coating synthetic grafts with patients' own endothelial cells has been suggested to improve the patency rate and overall success of bypass surgeries. METHODOLOGY/PRINCIPAL FINDINGS:We isolated endothelial progenitor cells (EPCs) from leftover pieces of human saphenous vein/mammary artery. We demonstrate that EPCs can be expanded to generate millions of cells under low-density culture conditions. Exposure to high-density conditions induces differentiation to endothelial cell phenotype. EPC-derived endothelial cells show expression of CD144high, CD31, and vWF. We then assessed the ability of differentiated endothelial cells to adhere and grow on small diameter expanded polytetrafluoroethylene (ePTFE) tubings. Since ePTFE tubings are highly hydrophobic, we optimized protocols to introduce hydrophilic groups on luminal surface of ePTFE tubings. We demonstrate here a stepwise protocol that involves introduction of hydrophilic moieties and coating with defined ECM components that support adhesion of endothelial cells, but not of blood platelets. CONCLUSION/SIGNIFICANCE:Our data confirms that endothelial progenitors obtained from adult human blood vessels can be expanded in vitro under xenoprotein-free conditions, for potential use in endothelialization of small diameter ePTFE grafts. These endothelialized grafts may represent a promising treatment strategy for improving the clinical outcome of small-caliber vascular grafts in cardiac bypass surgeries

Islet-Like Cell Aggregates Generated from Human Adipose Tissue Derived Stem Cells Ameliorate Experimental Diabetes in Mice

BACKGROUND: Type 1 Diabetes Mellitus is caused by auto immune destruction of insulin producing beta cells in the pancreas. Currently available treatments include transplantation of isolated islets from donor pancreas to the patient. However, this method is limited by inadequate means of immuno-suppression to prevent islet rejection and importantly, limited supply of islets for transplantation. Autologous adult stem cells are now considered for cell replacement therapy in diabetes as it has the potential to generate neo-islets which are genetically part of the treated individual. Adopting methods of islet encapsulation in immuno-isolatory devices would eliminate the need for immuno-suppressants. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we explore the potential of human adipose tissue derived adult stem cells (h-ASCs) to differentiate into functional islet like cell aggregates (ICAs). Our stage specific differentiation protocol permit the conversion of mesodermic h-ASCs to definitive endoderm (Hnf3β, TCF2 and Sox17) and to PDX1, Ngn3, NeuroD, Pax4 positive pancreatic endoderm which further matures in vitro to secrete insulin. These ICAs are shown to produce human C-peptide in a glucose dependent manner exhibiting in-vitro functionality. Transplantation of mature ICAs, packed in immuno-isolatory biocompatible capsules to STZ induced diabetic mice restored near normoglycemia within 3-4 weeks. The detection of human C-peptide, 1155±165 pM in blood serum of experimental mice demonstrate the efficacy of our differentiation approach. CONCLUSIONS: h-ASC is an ideal population of personal stem cells for cell replacement therapy, given that they are abundant, easily available and autologous in origin. Our findings present evidence that h-ASCs could be induced to differentiate into physiologically competent functional islet like cell aggregates, which may provide as a source of alternative islets for cell replacement therapy in type 1 diabetes

Pig-to-Nonhuman Primates Pancreatic Islet Xenotransplantation: An Overview

The therapy of type 1 diabetes is an open challenging problem. The restoration of normoglycemia and insulin independence in immunosuppressed type 1 diabetic recipients of islet allotransplantation has shown the potential of a cell-based diabetes therapy. Even if successful, this approach poses a problem of scarce tissue supply. Xenotransplantation can be the answer to this limited donor availability and, among possible candidate tissues for xenotransplantation, porcine islets are the closest to a future clinical application. Xenotransplantation, with pigs as donors, offers the possibility of using healthy, living, and genetically modified islets from pathogen-free animals available in unlimited number of islets. Several studies in the pig-to-nonhuman primate model demonstrated the feasibility of successful preclinical islet xenotransplantation and have provided insights into the critical events and possible mechanisms of immune recognition and rejection of xenogeneic islet grafts. Particularly promising results in the achievement of prolonged insulin independence were obtained with newly developed, genetically modified pigs islets able to produce immunoregulatory products, using different implantation sites, and new immunotherapeutic strategies. Nonetheless, further efforts are needed to generate additional safety and efficacy data in nonhuman primate models to safely translate these findings into the clinic

Blood Glucose Levels Regulate Pancreatic β-Cell Proliferation during Experimentally-Induced and Spontaneous Autoimmune Diabetes in Mice

Type 1 diabetes mellitus is caused by immune-mediated destruction of pancreatic beta-cells leading to insulin deficiency, impaired intermediary metabolism, and elevated blood glucose concentrations. While at autoimmune diabetes onset a limited number of beta-cells persist, the cells' regenerative potential and its regulation have remained largely unexplored. Using two mouse autoimmune diabetes models, this study examined the proliferation of pancreatic islet ss-cells and other endocrine and non-endocrine subsets, and the factors regulating that proliferation.We adapted multi-parameter flow cytometry techniques (including DNA-content measurements and 5'-bromo-2'-deoxyuridine [BrdU] incorporation) to study pancreatic islet single cell suspensions. These studies demonstrate that beta-cell proliferation rapidly increases at diabetes onset, and that this proliferation is closely correlated with the diabetic animals' elevated blood glucose levels. For instance, we show that when normoglycemia is restored by exogenous insulin or islet transplantation, the beta-cell proliferation rate returns towards low levels found in control animals, yet surges when hyperglycemia recurs. In contrast, other-than-ss endocrine islet cells did not exhibit the same glucose-dependent proliferative responses. Rather, disease-associated alterations of BrdU-incorporation rates of delta-cells (minor decrease), and non-endocrine islet cells (slight increase) were not affected by blood glucose levels, or were inversely related to glycemia control after diabetes onset (alpha-cells).We conclude that murine beta-cells' ability to proliferate in response to metabolic need (i.e. rising blood glucose concentrations) is remarkably well preserved during severe, chronic beta-cell autoimmunity. These data suggest that timely control of the destructive immune response after disease manifestation could allow spontaneous regeneration of sufficient beta-cell mass to restore normal glucose homeostasis

Chronic Obstructive Pulmonary Disease and Lung Cancer: Underlying Pathophysiology and New Therapeutic Modalities

Chronic obstructive pulmonary disease (COPD) and lung cancer are major lung diseases affecting millions worldwide. Both diseases have links to cigarette smoking and exert a considerable societal burden. People suffering from COPD are at higher risk of developing lung cancer than those without, and are more susceptible to poor outcomes after diagnosis and treatment. Lung cancer and COPD are closely associated, possibly sharing common traits such as an underlying genetic predisposition, epithelial and endothelial cell plasticity, dysfunctional inflammatory mechanisms including the deposition of excessive extracellular matrix, angiogenesis, susceptibility to DNA damage and cellular mutagenesis. In fact, COPD could be the driving factor for lung cancer, providing a conducive environment that propagates its evolution. In the early stages of smoking, body defences provide a combative immune/oxidative response and DNA repair mechanisms are likely to subdue these changes to a certain extent; however, in patients with COPD with lung cancer the consequences could be devastating, potentially contributing to slower postoperative recovery after lung resection and increased resistance to radiotherapy and chemotherapy. Vital to the development of new-targeted therapies is an in-depth understanding of various molecular mechanisms that are associated with both pathologies. In this comprehensive review, we provide a detailed overview of possible underlying factors that link COPD and lung cancer, and current therapeutic advances from both human and preclinical animal models that can effectively mitigate this unholy relationship