dithizone and Diabetes-Mellitus--Type-1

Type 1 diabetes mellitus is an autoimmune disorder leading to loss of beta cells. There is a dire need to inhibit apoptosis and induce regeneration of new beta cells. There are plants in the Indian medicine system having the potential for rejuvenation. In the present study, we have attempted to evaluate the capacity of aqueous extract of Tinospora cordifolia to regenerate beta cells from PANC-1 ductal cells. After differentiation, the characterization of β-cell phenotype was carried out using dithizone and Gomori's staining and further confirmed by mRNA expression study of insulin, Pdx-1, and carbonic anhydrase-9. Insulin production was estimated with ELISA. Aqueous extract of Tinospora cordifolia at 15 μg/ml concentration can effectively induce differentiation of PANC-1 cells into beta cells. The morphological observations showed brownish-colored dithizone and purple-colored Gomori's staining. The β-cells demonstrated significant mRNA expression of insulin and Pdx-1 and downregulation of carbonic anhydrase-9. The functionality of beta cells was demonstrated by 1.5-fold increase in insulin secretion in response to high glucose. Tinospora cordifolia has potential to differentiate PANC-1 ductal cells into functional beta cells and can be a lead towards non-invasive treatment of type 1 diabetes mellitus.

Topics: Animals; Carbonic Anhydrases; Diabetes Mellitus, Type 1; Dithizone; Humans; Insulins; Pancreatic Ducts; Phenotype; Plant Extracts; RNA, Messenger; Tinospora

Transplantation of pancreatic islets is considered a potential curative treatment of type 1 diabetes. Electron microscopy plays a major role in the evaluation of pancreatic islets. The aim of this study was to study the reversal of diabetes by xenotransplantation of monkey pancreatic islets into diabetic recipient by functional and structural findings.. Islets of Langerhans were isolated from monkeys by collagenase digestion method. Two days after the induction of diabetes in rats with streptozotocin, diabetes was confirmed. Freshly isolated islets were transplanted under the renal capsule of the diabetic rats. The recipients received cyclosporin A (30 mg/kg) every day. Fasting plasma glucose was estimated on days 3, 7, and 14 after transplantation. The presence of glucose and ketone in urine was checked. After 14 days, the grafts were removed and processed for light and electron microscopic study.. After the induction of diabetes, the mean fasting plasma glucose was 347.20 mg/dL. On day 3 after transplantation, the mean fasting plasma glucose value was 100 mg/dL. The mean fasting plasma glucose was 94.6 mg/dL on day 7 and 94.8 mg/dL on day 14. Histology of the monkey islet grafts after 14 days showed the survival of pancreatic islets. Ultrastructure of the same grafts showed the presence of alpha, beta, and delta granules similar to those of native pancreatic islets with the other cellular organelles.. The diabetic state of rats can be reversed by xenotransplantation of isolated monkey islets. Ultrastructural study confirms the normal synthesis and release of islet hormones. The released insulin from transplanted monkey islets had lowered the plasma glucose level of recipient rats.

Topics: Animals; Blood Glucose; Chelating Agents; Diabetes Mellitus, Type 1; Dithizone; Female; Glucagon-Secreting Cells; Graft Survival; Insulin-Secreting Cells; Islets of Langerhans Transplantation; Macaca radiata; Male; Microscopy, Electron; Rats; Rats, Wistar; Staining and Labeling; Transplantation, Heterologous

The determination of islet mass is important for the normalization of islet experiments in the laboratory and for the precise dosing of islets for transplantation. The common microscopical analysis is based on individual islet sizing, calculation of the frequency distribution, and conversion into islet equivalents (IEQ), which is the volume of a spherical islet with a diameter of 150 microm. However, islets are of irregular form, which makes this determination user dependent, and the analysis is irreproducible once the original sample is discarded. This routine technique of islet quantification was compared with the analysis of areal density measurements. It was assumed that the entire area occupied by islets can be expressed in IEQ without sizing and counting individual islets. Porcine islets were isolated by continuous digestion/filtration and purified by gradient centrifugation. Purified islets were stained with dithizone and were repeatedly pictured under the microscope with random area selection. A total of 51 pictures was taken from 11 different purifications and stained islets were detected by digital image analysis. The correlation coefficient (r) between bothanalyses was 0.977 with an underestimation of islet yield by areal density detection (slope: 0.75 +/- 0.03). Areal density analysis per picture took about 1 min, which is about 10 times faster than the traditional method without increasing the method error (CV 2.1% vs. 2.7%). In summary, areal density measurements allow a rapid and reproducible estimation of IEQ without counting individual islets. It can be performed in a single step analysis without computer programming and is valuable for online determinations of islet yield preceding transplantation.

Topics: Animals; Cell Count; Cell Culture Techniques; Cell Division; Cell Size; Cells, Cultured; Diabetes Mellitus, Type 1; Dithizone; Female; Image Processing, Computer-Assisted; Insulin; Insulin Secretion; Islets of Langerhans; Islets of Langerhans Transplantation; Male; Pancreas, Artificial; Reproducibility of Results; Sus scrofa

The aim of this study was to develop techniques to obtain monodispersed, human islet cells in large quantities, since these constitute a potentially transplantable beta cell mass with which to treat established type 1 diabetes, as well as provide the most appropriate substrate for studying the immune pathogenesis of the disease. Human islets were isolated from the pancreas of beating-heart organ donors by collagenase digestion. Enzymatic (collagenase types II, IV, V, and XI, trypsin, DNAse, and hyaluronidase) and chemical (EDTA and EGTA) conditions were then used to find the optimum requirements for digestion of intact human islets into their constituent cells. The combination of trypsin with EDTA provided the highest yield of monodispersed islet cells (963 cells/islet) and highest viability (88%). DNAse with EGTA gave high yields (710 cells/islet) but viability was low (55%). Lower yields and viability were obtained using collagenase types II, IV, V, and XI (47-243 cells/islet; viability 45-62%), hyaluronidase (410 cells/islet; 75% viability), and EDTA alone (253 cells/islet; viability 43%). Human islet cells monodispersed using trypsin 0.125 mg/ml in 0.1 mM EDTA retained an insulin secretory response to glucose, and had intact surface class I MHC molecules when analyzed immediately after digestion by flow cytofluorimetry. Our results indicate that functionally intact, single, human islet cells may be obtained in abundance, and provide a potential substrate for islet cell transplantation in the treatment of patients with type 1 diabetes.

Topics: Cell Separation; Cell Survival; Collagenases; Diabetes Mellitus, Type 1; Dithizone; Flow Cytometry; Fluorescent Antibody Technique; Glucose; Humans; Insulin; Insulin Secretion; Islets of Langerhans; Islets of Langerhans Transplantation; Pancreas; Staining and Labeling