Pancreatic islet transplantation is a physiologically advantageous and minimally invasive procedure for the treatment of type 1 diabetes mellitus. Here, we describe the first reported case of successful allogeneic islet transplantation alone, using single-donor, marginal-dose islets in a Korean patient. A 59-yr-old patient with type 1 diabetes mellitus, who suffered from recurrent severe hypoglycemia, received 4,163 islet equivalents/kg from a single brain-death donor. Isolated islets were infused intraportally without any complications. The immunosuppressive regimen was based on the Edmonton protocol, but the maintenance dosage was reduced because of mucositis and leukopenia. Although insulin independence was not achieved, the patient showed stabilized blood glucose concentration, reduced insulin dosage and reversal of hypoglycemic unawareness, even with marginal dose of islets and reduced immunosuppressant. Islet transplantation may successfully improve endogenous insulin production and glycemic stability in subjects with type 1 diabetes mellitus.
Blood Glucose/analysis ; Diabetes Mellitus, Type 1/*surgery ; Female ; Humans ; Hypoglycemia/*surgery ; Immunosuppression/methods ; Immunosuppressive Agents/therapeutic use ; Islets of Langerhans/physiology/*surgery ; Islets of Langerhans Transplantation/*methods ; Middle Aged ; Republic of Korea ; Tissue Donors

Type 1 diabetes is an autoimmune disease caused by permanent destruction of insulin-producing pancreatic beta cells and requires lifelong exogenous insulin therapy. Recently, islet transplantation has been developed, and although there have been significant advances, this approach is not widely used clinically due to the poor survival rate of the engrafted islets. We hypothesized that improving survival of engrafted islets through ex vivo genetic engineering could be a novel strategy for successful islet transplantation. We transduced islets with adenoviruses expressing betacellulin, an epidermal growth factor receptor ligand, which promotes beta-cell growth and differentiation, and transplanted these islets under the renal capsule of streptozotocin-induced diabetic mice. Transplantation with betacellulin-transduced islets resulted in prolonged normoglycemia and improved glucose tolerance compared with those of control virus-transduced islets. In addition, increased microvascular density was evident in the implanted islets, concomitant with increased endothelial von Willebrand factor immunoreactivity. Finally, cultured islets transduced with betacellulin displayed increased proliferation, reduced apoptosis and enhanced glucose-stimulated insulin secretion in the presence of cytokines. These experiments suggest that transplantation with betacellulin-transduced islets extends islet survival and preserves functional islet mass, leading to a therapeutic benefit in type 1 diabetes.
Animals ; Apoptosis ; Betacellulin ; Cell Proliferation ; Diabetes Mellitus, Experimental/*surgery ; Glucose Intolerance/*surgery ; Humans ; Insulin-Secreting Cells/*metabolism/physiology ; Intercellular Signaling Peptides and Proteins/genetics/*metabolism ; *Islets of Langerhans Transplantation ; Mice ; Mice, Inbred C57BL ; Rats

OBJECTIVETo study the effect of pretreatment with apoptotic donor spleen cells on spleen lymphocyte function of recipient rats undergoing islet transplantation to explore new approaches to prolong islet graft survival.

METHODSApoptotic spleen cells from donor rats were obtained by exposure to γ-ray irradiation from (60)Co. Diabetic SD rat models were randomly divided into 4 groups to receive tail vein injections with saline (group A), normal cells (group B), apoptotic donor cells (group C), or necrotic donor cells (group D). One week later, orthotopic transplantation of islets under the renal capsule was performed. Before and at 1 and 2 weeks after islet transplantation, the recipient rats were examined for proliferative activity of spleen lymphocytes with CFSE cell staining and for IL-2 and IL-10 expressions in the cells using ELISA.

RESULTSPretreatment with donor apoptotic cells significantly suppressed the proliferative activity of recipient spleen lymphocytes before and at 1 and 2 weeks after islet transplantation as compared with the other three groups (P<0.05). The level of IL-2 was significantly decreased while IL-10 increased in apoptotic donor cell pretreatment group compared with those in the other 3 groups at each time point of observation.

CONCLUSIONThe effect of pretreatment with apoptotic donor cells on recipient spleen lymphocytes suggest an important role of apoptotic donor spleen cells in immune tolerance of grafts.

Animals ; Apoptosis ; immunology ; Cell Proliferation ; Cobalt Radioisotopes ; Diabetes Mellitus, Experimental ; metabolism ; pathology ; surgery ; Gamma Rays ; Graft Survival ; Immune Tolerance ; Interleukin-10 ; metabolism ; Interleukin-2 ; metabolism ; Islets of Langerhans Transplantation ; Lymphocyte Transfusion ; Lymphocytes ; metabolism ; pathology ; radiation effects ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Spleen ; cytology ; metabolism ; radiation effects

Recent evidence has suggested that human skin fibroblasts may represent a novel source of therapeutic stem cells. In this study, we report a 3-stage method to induce the differentiation of skin fibroblasts into insulin-producing cells (IPCs). In stage 1, we establish the isolation, expansion and characterization of mesenchymal stem cells from human labia minora dermis-derived fibroblasts (hLMDFs) (stage 1: MSC expansion). hLMDFs express the typical mesenchymal stem cell marker proteins and can differentiate into adipocytes, osteoblasts, chondrocytes or muscle cells. In stage 2, DMEM/F12 serum-free medium with ITS mix (insulin, transferrin, and selenite) is used to induce differentiation of hLMDFs into endoderm-like cells, as determined by the expression of the endoderm markers Sox17, Foxa2, and PDX1 (stage 2: mesenchymal-endoderm transition). In stage 3, cells in the mesenchymal-endoderm transition stage are treated with nicotinamide in order to further differentiate into self-assembled, 3-dimensional islet cell-like clusters that express multiple genes related to pancreatic beta-cell development and function (stage 3: IPC). We also found that the transplantation of IPCs can normalize blood glucose levels and rescue glucose homeostasis in streptozotocin-induced diabetic mice. These results indicate that hLMDFs have the capacity to differentiate into functionally competent IPCs and represent a potential cell-based treatment for diabetes mellitus.
Animals ; Biological Markers/metabolism ; *Cell Culture Techniques ; *Cell Differentiation ; Cell Proliferation/drug effects ; Cell Separation ; Cells, Cultured ; Dermis/*cytology/drug effects ; Diabetes Mellitus, Experimental/*surgery ; Female ; Fibroblasts/*cytology/drug effects ; Genitalia, Female/*cytology ; Glucose/metabolism ; Hepatocyte Nuclear Factor 3-beta/metabolism ; Homeodomain Proteins/metabolism ; Humans ; Insulin/pharmacology/secretion ; Insulin-Secreting Cells/*cytology/metabolism ; *Islets of Langerhans Transplantation ; Mesenchymal Stem Cells/*cytology/drug effects/metabolism ; Mice ; Mice, Nude ; Niacinamide/pharmacology ; Recovery of Function ; SOXF Transcription Factors/metabolism ; Sodium Selenite/pharmacology ; Trans-Activators/metabolism ; Transferrin/pharmacology

Up till 2000 when Edmonton group introduced islet transplant procedure in conjunction with a novel glucocorticoid-free immunosuppressive regimen rendering 100% (n=7) of patients with type 1 diabetes insulin-independent for at least 1 year, islet transplant was taken into the clinic. Although significant progress in clinical islet transplant has occurred during recent years, challenges remain, including shortage of available donor organs, technical aspects of islet preparation and transplantation, immunological rejection post-transplant, unclear long-term outcomes of islet transplantation. Special attention is given to current limitation in islet transplantation together with new possible strategies that raise expectations for the widespread use of islet transplantation in the future.
Diabetes Mellitus ; surgery ; Humans ; Immunosuppressive Agents ; therapeutic use ; Islets of Langerhans Transplantation ; immunology ; methods ; trends

OBJECTIVETo investigate the effects of Gastric bypass surgery on the apoptosis of islet β-cells in type 2 nonobese diabetic (NOD) rats and its mechanisms.

METHODSSeventy-two 8-week-old GK rats were randomly divided into four groups:operation group (group O, n = 18), sham operation group (group S, n = 18), diet control group (group F, n = 18) and control group (group C, n = 18). The levels of fasting, postprandial blood glucose, insulin and glucagon-like peptide-1 (GLP-1) were measured and compared among the 4 groups before the operation and at 1, 2, 4 and 8 weeks following the operation. The blood samples were collected at 2, 4 and 8 weeks after the operation for the measurement of postprandial blood glucose, and then the rats in batches (6 rats in each group) were decapitated to retrieve the pancreas. The apoptosis of the islet β-cells was detected by using TUNEL assay, and the expression of apoptosis-related proteins Bcl-2, Bax was measured with immunohistochemistry.

RESULTSAs for group O, the fasting blood glucose level decreased from (16.2 ± 0.8) mmol/L before the operation to respectively (9.2 ± 0.6) mmol/L and (9.7 ± 0.7) mmol/L at 4 and 8 weeks after the operation; postprandial blood glucose decreased from (31.1 ± 1.1) mmol/L before the operation to respectively (13.1 ± 0.7) mmol/L and (12.3 ± 0.7) mmol/L at 4 and 8 weeks after the operation. Fasting insulin level increased from (28.0 ± 1.2) mU/L before the operation to respectively (62.8 ± 1.9) mU/L and (61.7 ± 1.4) mU/L at 4 and 8 weeks after the operation; and at 4 and 8 weeks after the operation postprandial insulin level was (77.4 ± 1.1) mU/L and (77.1 ± 1.0) mU/L. At 2 weeks from the operation, the fasting GLP-1 in group O increased from (10.7 ± 1.0) pmol/L to (13.5 ± 0.8) pmol/L, and respectively to (26.1 ± 0.9) pmol/L and (25.3 ± 1.2) pmol/L at 4 and 8 weeks after the operation. The differences in the above-mentioned items before and after the operation were all significant in group O (P < 0.05), and the differences in the items among group O and the other three groups (P < 0.05) were all significant as well. In group O, the apoptosis rate of pancreatic islet cell decreased to (5.9 ± 0.7)% at 4 weeks from the operation, and (6.3 ± 1.1)% at 8 weeks from the operation (P < 0.05). The expression of Bcl-2 protein in group O was 31.3 ± 1.5, 35.7 ± 1.0 and 35.8 ± 0.8 at 2, 4 and 8 weeks post operation, which was significantly higher in statistics than those of the same time point in the other three groups (P < 0.05). The expression of Bax protein in group O was 13.3 ± 0.9, 10.8 ± 0.9 and 10.9 ± 1.1 at 2, 4 and 8 weeks from the operation, which was significantly lower in statistics than those of the same time point in the other three groups (P < 0.05).

CONCLUSIONSGastric bypass surgery can significantly reduce the blood glucose level and promote the secretion of GLP-1, and therefore inhibit the apoptosis of the islet β cells in diabetic rats through the Bcl-2 pathway.

Animals ; Apoptosis ; Blood Glucose ; Diabetes Mellitus, Type 2 ; pathology ; surgery ; Disease Models, Animal ; Gastric Bypass ; Glucagon-Like Peptide 1 ; blood ; Insulin ; blood ; Islets of Langerhans ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo study the protective effect of islet xenograft and its possible mechanism of high expression of heme oxygenase-1 (HO-1) in donor pancreas islet induced by cobalt protoporphyrin (CoPP).

METHODSMale SD rats and C57BL/6 mouse were used as donors and recipients respectively. Donors were divided into 3 groups according to different pretreatment 24 hours before donation: control group (injected intraperitoneally with NaCl), induce group [injected intraperitoneally with cobalt-protoporphyrin (CoPP)], block group (injected intraperitoneally with CoPP and zinc protoporphyrin simultaneously). A modified approach was used for islet isolation.Recipients were rendered diabetic by intraperitoneal injection of streptozotocin. Islets were transplanted into mouse subrenal capsule. Postoperative mouse glycemia were monitored daily and normoglycemia time was compared among each group. The receptor mouse serum IL-10 was detected by ELISA approach, and real-time PCR was used to check the expression of IL-10 mRNA in islet graft tissues. The graft tissues were observed for the lymphocyte infiltration after HE staining.

RESULTSDiabetes mice accepted islets untreated, induced or blocked maintained the euglycemia for (9.3 +/- 1.4), (16.3 +/- 1.5) and (9.7 +/- 1.0) d respectively. The xeno-islets presented HO-1 over-expression survived much longer than that absent (P < 0.05), it was no significance between control group and block group (P > 0.05). The mouse islet serum IL-10 content after induction was (73.0 +/- 9.7) pg/ml, significantly higher than (30.6 +/- 3.9) pg/ml of the untreated group and (32.1 +/- 5.9) pg/ml of the blocked group (P < 0.05), there was no difference between control group and block group (P > 0.05). Moreover, the IL-10 mRNA expression up-regulated statistic significantly in HO-1 induced islet xeno-graft. Pathological examination showed that the graft lymphocyte infiltration of the induced group was obviously less serious than the other two groups.

CONCLUSIONSThe higher expression of HO-1 induced by CoPP in vivo would significantly prolong graft survival time and its mechanism could be related to immune modulation of IL-10.

Animals ; Diabetes Mellitus, Experimental ; metabolism ; pathology ; surgery ; Graft Survival ; Heme Oxygenase-1 ; drug effects ; metabolism ; Interleukin-10 ; metabolism ; Islets of Langerhans ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Pancreas Transplantation ; Protoporphyrins ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Subrenal Capsule Assay ; Transplantation, Heterologous

In the conventional treatments of type I diabetes, there are various problems. As a new adequate treatment of diabetes, cell replacement therapy of diabetes has been applied and given research priority. We have investigated the applications of cell transplantation in the treatment of diabetes and have retrieved the relevant articles on cells transplantation for the treatment of diabetes. In this paper, we review the history, development, merits and demerits of cell transplantation and the recent advances in pancreatic islet transplantation research. The latest progress in the induction of stem cell to differentiate into the insulin-producing cells was also introduced.
Animals ; Diabetes Mellitus, Type 1 ; surgery ; therapy ; Humans ; Insulin-Secreting Cells ; cytology ; Islets of Langerhans Transplantation ; methods ; Stem Cell Transplantation

OBJECTIVETo study the effects of gene transfer cytotoxic T lymphocyte associated antigen 4 immunoglobulin (CTLA4-Ig) and anti-cluster of differentiation 154 (CD154) mAb on the rejection of rat islet xenografts.

METHODSHuman islets were infected with the recombinant adenoviruses containing CTLA4-Ig gene. Transduced islets were transplanted under the left kidney capsule of diabetic rats. And then the animal model were treated with anti-CD154 monoclonal antibody. The changes of blood sugar were measured and the survival rates of grafts and transplantation rats were observed after transplantation. The morphological changes of grafts were observed. Expression of CTLA4-Ig and insulin were detected by immunohistochemical staining and cytokines were quantified by ELISA.

RESULTS(1) The blood glucose of transplantation rats decreased to normal level on 2nd day post-transplantation. The average level blood glucose of control group A, anti-CD154mAb treatment group B, transfected group C and associated treatment group D increased on day 8, 18, 25, 36, post-transplantation respectively. (2) The grafts of group A, B, C and D survived for (10.0 +/- 2.1) d, (22.0 +/- 8.2) d, (28.0 +/- 6.5) d and (37.0 +/- 9.3) d respectively. The survival of grafts in group D was significant longer than that in group A, B and C, respectively; The survival of group B and C were significantly prolonged compared with group A and the survival of group B was significantly different with group C (P < 0.05). The survival of transplantation rats were (21.0 +/- 5.7) d, (35.0 +/- 6.5) d, (48.0 +/- 8.5) d and (65.0 +/- 12.5) d in group A, B, C and D, respectively. The survival of transplantation rats compared each other among four groups were same as the survival of grafts (P < 0.05). (3) In control animals (group A), serum IL-2 and TNF-alpha concentration were elevated to a high level within seven days post-transplantation and significantly increased compared with that before transplantation (P < 0.01). (4) Hematoxylin-eosin staining of grafts showed a lot of islets under the kidney capsule of transplantation rats, no inflammatory cell infiltrate and immunohistochemical staining of grafts demonstrated expression of insulin protein at islets in group B, C and D. These grafts positively stained for CTLA4-Ig in group C and D.

CONCLUSIONSGene transfer CTLA4-Ig and anti-CD154mAb treatment can inhibit the rejection of rat islet xenografts and treatment Ad-CTLA4-Ig and anti-CD154 mAb could induce immune tolerance of islet xenografts.

Adenoviridae ; genetics ; Animals ; Antibodies, Monoclonal ; therapeutic use ; CD40 Ligand ; immunology ; Diabetes Mellitus, Experimental ; surgery ; Genetic Vectors ; Graft Rejection ; immunology ; prevention & control ; Humans ; Immunoconjugates ; genetics ; Islets of Langerhans Transplantation ; Rats ; Rats, Wistar ; Transfection ; Transplantation, Heterologous

The goals of this research are to improve the functionality (insulin secretion rate and pattern) and to expand the life-span of immunoprotected pancreatic islets. The low functionality (less than 15% of the insulin release rate of native islets in pancreas) required a large number of islets within the implant, which causes complications in surgery and discomfort for patients. The limited life-span of the islets in a biohybrid artificial pancreas (BAP) may require frequent cell reseeding and cause further supply problems in islet transplantation. Improved islet functionality and prolonged life-span will minimize the volume of the BAP by reducing the number of islets needed for diabetic patients to achieve normoglycaemia and reduce problems associated with islet supply. It is hypothesized in this research that 1) by mimicking facilitated oxygen transport in avascular tissues, the immunoprotected islets release a higher amount of insulin, recover their intrinsic biphasic release pattern, and prolong their life-span, and 2) insulinotropic agents further promote insulin secretion from islets. Based on these hypotheses, a new BAP system will be designed which contains the water-soluble polymeric conjugates of oxygen carriers (or oxygen binding vehicles) and islet stimulants of sulfonylurea compounds and glucagon-like insulinotropic peptide-1 with entrapped islets in the BAP. The research examines their effects on islet viability, the amount of insulin secretion, the insulin release profile, and the life-span of immunoprotected pancreatic islets. Especially, the combined synergy effects of both hypotheses will be emphasized. The successful results in improving functionality and life- span of islets entrapped in an immunoprotected membrane can be applied in the delivery of microencapsulated therapeutic cells and to the miniaturization of a BAP. In addition, the approaches proposed in this research will provide a potential solution to the shortage problem of human cell or tissue sources.
Diabetes Mellitus, Type 1/*surgery ; Diffusion Chambers, Culture ; Humans ; Islets of Langerhans ; *Pancreas, Artificial