Quantification of islet size and architecture

Human islets exhibit distinct islet architecture particularly in large islets that comprise of a relatively abundant fraction of α-cells intermingled with β-cells, whereas mouse islets show largely similar architecture of a β-cell core with α-cells in the periphery. In humans, islet architecture is islet-size dependent. Changes in endocrine cell mass preferentially occurred in large islets as demonstrated in our recent study on pathological changes of the pancreas in patients with type 2 diabetes.¹ Kilimnik G, Zhao B, Jo J, Periwal V, Witkowski P, Misawa R, et al. Altered islet composition and disproportionate loss of large islets in patients with type 2 diabetes. PLoS One 2011; 6:e27445; http://dx.doi.org/10.1371/journal.pone.0027445; PMID: 22102895 [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] The size dependency of human islets in morphological changes prompted us to develop a method to capture the representative islet distribution in the whole pancreas section combined with a semi-automated analysis to quantify changes in islet architecture. The computer-assisted quantification allows detailed examination of endocrine cell composition in individual islets and minimizes sampling bias. The standard immunohistochemistry based method is widely applicable to various specimens, which is particularly useful for large animal studies but is also applied to a large-scale analysis of the whole organ section from mice. In this article, we describe the method of image capture, parameters measured, data analysis and interpretation of the data.     

Quantification of islet size and architecture

Human islets exhibit distinct islet architecture particularly in large islets that comprise of a relatively abundant fraction of α-cells intermingled with β-cells, whereas mouse islets show largely similar architecture of a β-cell core with α-cells in the periphery. In humans, islet architecture is islet-size dependent. Changes in endocrine cell mass preferentially occurred in large islets as demonstrated in our recent study on pathological changes of the pancreas in patients with type 2 diabetes. ( 1) The size dependency of human islets in morphological changes prompted us to develop a method to capture the representative islet distribution in the whole pancreas section combined with a semi-automated analysis to quantify changes in islet architecture. The computer-assisted quantification allows detailed examination of endocrine cell composition in individual islets and minimizes sampling bias. The standard immunohistochemistry based method is widely applicable to various specimens, which is particularly useful for large animal studies but is also applied to a large-scale analysis of the whole organ section from mice. In this article, we describe the method of image capture, parameters measured, data analysis and interpretation of the data.     

Risks and benefits of transplantation in the cure of type 1 diabetes: whole pancreas versus islet transplantation. A single center study

BACKGROUND: Pancreas and islet transplantation are the only available options to replace beta-cell function in patients with type 1 diabetes. Great variability in terms of rate of success for both approaches is reported in the literature and it is difficult to compare the respective risks and benefits. OBJECTIVES: The aim of this study was to analyze risks and benefits of pancreas transplantation alone (PTA) and islet transplantation alone (ITA) by making use of the long-term experience of a single center where both transplantations are performed. We focused on the risks and benefits of both procedures, with the objective of better defining indications and providing evidence to support the decision-making process. The outcomes of 33 PTA and 33 ITA were analyzed, and pancreas and islet function (i.e., insulin independence), perioperative events, and long-term adverse events were recorded. RESULTS: We observed a higher rate of insulin independence in PTA (75%) versus ITA (59%), with the longer insulin independence among PTA patients receiving tacrolimus. The occurrence of adverse events was higher for PTA patients in terms of hospitalization length and frequency, re-intervention for surgical and immunological acute complications, CMV reactivation, and other infections. CONCLUSIONS: In conclusion, these results support the practice of listing patients for PTA when the metabolic control and the progression of chronic complications require a rapid normalization of glucose levels, with the exception of patients with cardiovascular disease, because of the high surgical risks. ITA is indicated when replacement of beta-cell mass is needed in patients with a high surgical risk.     

Current indications for pancreas or islet transplant

Pancreas or islet transplantation can provide good glycaemic control and insulin independence. Pancreas transplantation has been associated with improvement in diabetic retinopathy, nephropathy, neuropathy and vasculopathy, but has the associated morbidity of major surgery. Both forms of therapy require long-term immunosuppression and its attendant risks and both achieve insulin independence rates of about 80% at 1 year. Pancreas transplantation at the same time as a renal transplant is a worthwhile option to employ, especially if the diabetes has been difficult to control. Diabetes associated with frequent severe hypoglycaemia or extreme lability, despite optimization of diabetes management, may benefit from either pancreas or islet transplant alone with the latter being the lower-risk procedure. More quantitative measures of hypoglycaemia and lability are now available to facilitate the assessment of the severity of these problems with glucose control. Diabetic patients with renal involvement (macroproteinuria, but no major elevation of creatinine) and unstable diabetes may be helped with an islet or pancreas transplant, but this approach should still be considered experimental and such a transplant may hasten the need for renal replacement therapy. In the setting of well-controlled diabetes and intact renal function, it is difficult to justify pancreas or islet transplant alone given the risks of immunosuppression.     

From immunohistological to anatomical alterations of human pancreas in type 1 diabetes: New concepts on the stage

The histological analysis of human pancreatic samples in type 1 diabetes (T1D) has been proven essential to move forward in the evaluation of in situ events characterizing T1D. Increasing availability of pancreatic tissues collected from diabetic multiorgan donors by centralized biorepositories, which have shared tissues among researchers in the field, has allowed a deeper understanding of T1D pathophysiology, using novel immunohistological and high‐throughput methods. In this review, we provide a comprehensive update of the main recent advancements in the characterization of cellular and molecular events involving endocrine and exocrine pancreas as well as the immune system in the onset and progression of T1D. Additionally, we underline novel elements, which provide evidence that T1D pathological changes affect not only islet β‐cells but also the entire pancreas.     

The IKEM pancreas and islet transplant program as part of healthcare for type 1 diabetes patients: retrospective analysis of outcome from 1983 to 2010

Currently, 25-30 pancreas transplantations per year are carried out in type 1 diabetes (T1D) recipients residing in Czech Republic. Most of the recipients are transplanted together with kidney allografts, but pancreas is also transplanted alone in selected patients with brittle diabetes. Since 2005, the Institute for Clinical and Experimental Medicine (IKEM) islet transplant program was initiated as complementary therapeutic modality. The aim of this paper was to analyze the transplant program at our clinical center, and to examine the survival of recipients, and their pancreas, kidney, and islet grafts. Patient and graft survival rates were evaluated in the following three categories using Kaplan-Meier test: simultaneous pancreas and kidney transplantation (SPKTx), pancreas transplantation alone (PTA), and islet transplantation (ITx). Three hundred and ninety SPKTx, 34 PTA and 44 ITx were carried out between 1983 and 2010. One- and 5-year patient survival rates were 92 % and 81% in SPKTx, respectively. In SPKTx, the 1-year survival rate of pancreas grafts was 78%, and the 5-year rate was 66%. Kidney graft survival rates were 89% and 79%, respectively, after the same follow-up periods. In the PTA category, recipient survivals were 100% after 1 year, and 92% after 3 years. 70% and 65% of pancreatic grafts were working properly at 1 and 3-year follow-ups, respectively. To date, we have carried out 44 islet transplantations in 31 recipients. Islet function (C-peptide ≥ 0.2 ng/ml) was documented in 60% of recipients after 12 months. So far, only 3 patients remained free of exogenous insulin. While SPKTx is a well established treatment for uremic T1D patients, ITx represents an emerging complementary treatment modality. The latter is especially suitable for high-risk recipients, but routine clinical application is still hampered by the limited availability of usable organ transplants and viability of transplanted islets.     

Malignant glucagonoma of the pancreas diagnosed through anemia and diabetes mellitus

Glucagonoma of the pancreas is a rare tumor with distinct clinical manifestations, such as necrolytic migratory erythema, weight loss, anemia, diabetes mellitus, and hypoamino-acidemia. We report the case of a 68-year-old Japanese man who underwent curative resection for malignant glucagonoma of the pancreas diagnosed through anemia and diabetes mellitus. The patient had had diabetes mellitus for 20 years. Anemia was diagnosed in 1998. On admission, the hemoglobin level was 8.3?g/dl, but the levels of serum iron, vitamin B12, and erythropoietin and, the number of reticulocytes were within normal limits. The levels of carcinoembryonic antigen (CEA), carbohydrate antigen (CA)19-9, and DUPAN-2 were also within normal limits, and exocrine function of the pancreas (PFD, 75%) was normal. Ultrasonography (US) revealed a hypoechoic tumor in the distal pancreas. Computed tomography (CT) demonstrated a high-density area 4?cm in diameter with calcification. The serum glucagon level was very high (2360?pg/ml), but the levels of other hormones such as somatostatin or gastrin were within normal limits, while insulin was low. Glucagonoma of the pancreas was diagnosed, and distal pancreatectomy with splenectomy was performed. Histological examination revealed a malignant endocrine tumor, which was immunohistochemically positive for chromogranin A and glucagon. Two months after the operation, the serum glucagon level had decreased to within normal limits and the hemoglobin level had increased to 10.4?g/dl. The case of glucagonoma reported here was found through diagnostic examinations of anemia and treated by surgical resection, by which the patient's anemia was largely alleviated. Therefore, we recommend checking patients who have diabetes mellitus and anemia in order to diagnose and treat glucagonoma in its early statge.     

Islet cell surface antibodies in Type 1 (insulin-dependent) diabetes mellitus: Use of human fetal pancreas cultures as substrate

Summary Sixteen pancreases from 11–24 week old human fetuses were cultured for up to 11 days to investigate islet cell surface antibodies. Hormonal content and presence of cytoplasmic autoantigen were assessed by immunofluorescence with specific antihormone sera and high titre cytoplasmic islet cell antibody positive sera. Viable islet cells cultured on coverslips were tested with 21 islet cell antibody positive sera from Type 1 (insulin-dependent) diabetics, one islet cell antibody positive serum from a non-diabetic and four normal control sera. Surface binding immunoglobulins were detected by indirect immunofluorescence in nine out of 11 newly diagnosed Type 1 diabetics and in two out of ten longstanding diabetics with another coexistent autoimmune endocrinopathy. The four-layer double immunofluorescence technique showed that the surface antibody stained insulin secreting cells, but owing to rarity of A and D cells in the fetal cultures it has not yet been possible to exclude the reactivity of islet cell surface antibodies with glucagon or somatostatin cells.     

Structural similarities and differences between the human and the mouse pancreas

Mice remain the most studied animal model in pancreas research. Since the findings of this research are typically extrapolated to humans, it is important to understand both similarities and differences between the 2 species. Beside the apparent difference in size and macroscopic organization of the organ in the 2 species, there are a number of less evident and only recently described differences in organization of the acinar and ductal exocrine tissue, as well as in the distribution, composition, and architecture of the endocrine islets of Langerhans. Furthermore, the differences in arterial, venous, and lymphatic vessels, as well as innervation are potentially important. In this article, the structure of the human and the mouse pancreas, together with the similarities and differences between them are reviewed in detail in the light of conceivable repercussions for basic research and clinical application.     

Structural similarities and differences between the human and the mouse pancreas

Mice remain the most studied animal model in pancreas research. Since the findings of this research are typically extrapolated to humans, it is important to understand both similarities and differences between the 2 species. Beside the apparent difference in size and macroscopic organization of the organ in the 2 species, there are a number of less evident and only recently described differences in organization of the acinar and ductal exocrine tissue, as well as in the distribution, composition, and architecture of the endocrine islets of Langerhans. Furthermore, the differences in arterial, venous, and lymphatic vessels, as well as innervation are potentially important. In this article, the structure of the human and the mouse pancreas, together with the similarities and differences between them are reviewed in detail in the light of conceivable repercussions for basic research and clinical application.     

Establishment of a prolonged pancreas preservation model for islet isolation research in mice

Establishing a prolonged pancreas preservation model in a small animal is important for islet isolation research. Use of a rat pancreas model has been reported, but no published reports have used a mouse pancreas for prolonged cold preservation prior to islet isolation. For the model, a mouse is preferred over a rat because of its small size, well-known immune characterization and variety of gene-modulated models. In the present study, we established a prolonged pancreas preservation model in a mouse for islet isolation research. The collagenase solution was injected successfully after 24 and 48 h cold preservations in University of Wisconsin solution, and islets could be isolated from both groups of preserved pancreata. The islet yields from the control, 24 h preserved, and 48 h preserved pancreata were 183.9 ± 13.9, 128.5 ± 15.5, and 24.6 ± 12.9 per pancreas, respectively. The propidium iodide–positive area assay was significantly increased in both preserved groups, and insulin secretion levels in response to 20.0 mM glucose and stimulation indices were significantly decreased in the 48 h preserved group. Inflammation-related genes mRNA levels were significantly upregulated in the 24 h preserved group, as previously shown in the human model. Thus, this model might be useful for prehuman islet isolation screening research, reserving research using human pancreata for the most promising approaches.     

Establishment of a prolonged pancreas preservation model for islet isolation research in mice

Establishing a prolonged pancreas preservation model in a small animal is important for islet isolation research. Use of a rat pancreas model has been reported, but no published reports have used a mouse pancreas for prolonged cold preservation prior to islet isolation. For the model, a mouse is preferred over a rat because of its small size, well-known immune characterization, and variety of gene-modulated models. In the present study, we established a prolonged pancreas preservation model in a mouse for islet isolation research. The collagenase solution was injected successfully after 24 and 48 h cold preservations in University of Wisconsin solution, and islets could be isolated from both groups of preserved pancreata. The islet yields from the control, 24 h preserved, and 48 h preserved pancreata were 183.9 ± 13.9, 128.5 ± 15.5, and 24.6 ± 12.9 per pancreas, respectively. The propidium iodide-positive area assay was significantly increased in both preserved groups, and insulin secretion levels in response to 20.0 mM glucose and stimulation indices were significantly decreased in the 48 h preserved group. Inflammation-related genes mRNA levels were significantly upregulated in the 24 h preserved group, as previously shown in the human model. Thus, this model might be useful for prehuman islet isolation screening research, reserving research using human pancreata for the most promising approaches.     

ERCP diagnosis of tumors in the region of the head of the pancreas

During the period 1974–1983, 320 patients with pancreas carcinoma, papilla of Vater carcinoma, bile duct bifurcation carcinoma, or duodenal carcinoma were examined by ERCP. Using 30 ERCP criteria, a radiological diagnosis was made. A valid pathological diagnosis was available in 200 patients (62.5%). In 183 of the 200 patients (91.5%), the ERCP diagnosis and the pathological diagnosis were identical. We then performed an analysis using 52 ERCP criteria. In 192 of the 200 patients (96.0%), the ERCP diagnosis based on this reanalysis and the pathological diagnosis were identical. By discriminant analysis, 13 ERCP criteria with a maximal discriminatory value were selected in patients in whom all diagnostic structures (bile ducts, pancreatic duct, and duodenum) were visible. Using these 13 criteria selected by discriminant analysis, the diagnostic score was 98.9%. A computer program based on these 13 ERCP criteria was designed for use in practice. The diagnostic accuracy of this computer program was 98.4%. Finally we tested this computer program on 171 new patients who were seen in the period 1983– 1986. In 143 of the 171 patients, a valid pathological diagnosis was available (83.6%). Comparing the ERCP diagnosis in all patients (even if not all structures were visible) with a valid pathological diagnosis, the prospective score of the computer program was 91.6%. Using this program it was possible to evaluate examinations in which not all structures were visible. When the ERCP diagnosis was uncertain, the doubt could be quantified.     

Increased O-GlcNAc transferase in pancreas of rats with streptozotocin-induced diabetes

Aims/hypothesis. Streptozotocin (STZ), a chemically reactive analogue of N-acetylglucosamine, induces necrosis of the beta cells, resulting in diabetes mellitus. Glucose-induced insulin resistance is mediated by increased activity of the hexosamine pathway. We aimed to examine the regulation of O-GlcNAc transferase expression and activity in the normal and streptozotocin diabetic pancreas.¶Methods. Rats were made diabetic by an injection of streptozotocin (65 mg/kg). The expression of O-GlcNAc transferase protein was examined by immunoblot analysis. Activity of O-GlcNAc transferase was assayed by the incorporation of [³H]GlcNAc into the synthetic peptide. Localization of O-GlcNAc transferase was done by immunohistochemistry. The change of O-GlcNAc modification of proteins was examined by immunoblot analysis.¶Results. In the STZ-induced diabetic pancreas, a severe loss of beta cells was observed, whereas alpha cells had increased in number. The diabetic pancreas showed an increase in the expression of O-GlcNAc transferase at the protein level and the O-GlcNAc transferase activity in it was increased significantly (p < 0.05). An increase in the immunostaining intensity in the cytoplasm of islet beta cells was also observed in the diabetic pancreas, whereas exocrine cells and islet cells other than beta cells showed little change in immunostaining intensity. The pancreas of STZ-diabetic rats showed a 3.1-fold increase in total cellular O-GlcNAc-modified proteins.¶Conclusion/interpretation. These findings indicate that O-GlcNAc transferase plays an important part in the modulation of O-GlcNAc concentrations in the pancreas and suggest that the increase in O-GlcNAc modification of the proteins correlates closely with diabetes. [Diabetologia (2000) 43: 1239–1247]     

Spontaneous hypoglycaemia after pancreas transplantation in Type 1 diabetes mellitus

Hypoglycaemia is an important complication of insulin treatment in Type 1 diabetes mellitus (DM). Pancreas transplantation couples glucose sensing and insulin secretion, attaining a distinctive advantage over insulin treatment. We tested whether successful transplantation can avoid hypoglycaemia in Type 1 DM. Combined kidney and pancreas transplanted Type 1 DM who complied with good function criteria (KP-Tx, n = 55), and isolated kidney or liver transplanted non-diabetic subjects on the same immunosuppressive regimen (CON-Tx, n = 14), underwent 1-day metabolic profiles in the first 3 years after transplantation, sampling plasma glucose (PG) and pancreatic hormones every 2 hours. KP-Tx had lower PG than CON-Tx in the night and in the morning and higher insulin concentrations throughout the day. KP-Tx had lower PG nadirs than CON-Tx (4.40 ± 0.05 vs 4.96 ± 0.16 mmol l⁻¹, ANOVA p = 0.001). Nine per cent of KP-Tx had hypoglycaemic values (PG ≤3.0 mmol l⁻¹) in the profiles, both postprandial and postabsorptive, whereas none of CON-Tx did (p < 0.02). In conclusion, after pancreas transplantation, mild hypoglycaemia is frequent, although its clinical impact is limited. Compared to insulin treatment in Type 1 DM, pancreas transplantation improves but cannot eliminate hypoglycaemia. Copyright © 1998 John Wiley & Sons, Ltd.     

Transgenic and other experimental models of pancreas and islet damage

To unravel the cellular and molecular mechanisms involved in β-cell renewal and expansion throughout life, several different experimental models were devised in the past. A number of experimental approaches and transgenic models have been engineered to trigger specifically pancreatic injury and thus explore regeneration. Globally, three main strategies are followed to induce pancreas damage: surgical, chemical and genetic. Some of the most relevant studies regarding these three approaches are briefly summarized in this short overview. Although significant progress has been achieved in recent years, there is much room for improving our understanding of many fundamental processes regulating β-cell mass maintenance.     

Islet separation and islet cell culture in vitro from human embryo pancreas

INTRODUCTlONDiabetesmellitusisthemostc0mmondiseaseanditsdeathrateranksthe8thintheworld.Upt0n0w,itsincidencehasatendencytoincrease[11.Sincedisorderofsugarmetabolismmightresultinmicrovasculardegenerati0nandinjuryofimportanthumanorgans,itwillendangerhumanhealthseri0usly-Inl969,Younszaifirstreportedthemethodthatcoulddecreasediabetessymptomsbyislettissuetransplantation.In1981,ourcountrybegantotreatdiabetestype1bytransplantingculturedislettissues.Inourstudy,wedigestedthepiecesofpancreaswithcol…     

Islet vs. pancreas transplantation in Brazil: Defining criteria for pancreas allocation decision

Background: Many studies have evaluated whether there are characteristics related to pancreas donors and the islet isolation process that can influence in pancreatic islet yield. However, this analysis has not yet been performed in Brazil, one of the world leaders in whole pancreas organ transplantation (WOPT), where pancreas allocation for pancreatic islet transplantation (PIT) has no officially defined criteria. Definition of parameters that would predict the outcome of islet isolation from local pancreas donors would be useful for defining allocation priority in Brazil. Objective: To analyze the relationship between multiple donor-related and islet isolation variables with the total number of isolated pancreatic islet equivalents (IEQ) in a Brazilian sample of pancreas donors. Methods: Several variables were analyzed in 74 pancreata relative to the outcome of total IEQs obtained at the end of the process. Results: In univariate analysis, body mass index (BMI) (p = 0.003), the presence of fatty infiltrates in the pancreas as observed during harvesting (p = 0.042) and pancreas digestion time (p = 0.046) were identified as variables related to a greater IEQ yield. In a multivariate analysis a statistically significant contribution to the variability of islet yield was found only for the BMI (p = 0.017). A ROC curve defined a BMI = 30 as a cut-off point, with pancreata from donors with BMI > 30 yielding more islets than donors with BMI < 30 (p < 0.001). Conclusion: These data reinforces the importance of the donor BMI as a defining parameter for successful islet isolation and establishes this variable as a potential pancreas allocation criterion in Brazil, where there is an unequal competition for good quality organs between WOPT and PIT.     

Normal response to pregnancy in rats cured of streptozotocin diabetes by transplantation of one fetal pancreas

Summary We have investigated glucose homeostasis and insulin response to glucose in seven rats before, during and after pregnancy, who were previously successfully transplanted with a single fetal pancreas. Increased need for insulin during pregnancy provides an opportunity to test the reserve capacity of the transplanted organ. Plasma glucose in seven rats was normal before pregnancy (7.3±0.7 mmol/l), during pregnancy (6.6 ±1 mmol/l) and after parturition (6.7 ±0.3 mmol/l). Fasting plasma glucose was lower after parturition (5.1±1 mmol/l) than before pregnancy (6.1±0.7 mmol/l). The disappearance rate of injected glucose was the same before (2.3±0.2%/min) as after pregnancy (2.6±0.2%/min). Basal plasma insulin before pregnancy was elevated and there was no rise from glucose; after parturition the basal and pattern of response was normal. The total insulin content of the transplants (859±154 mU) was only 21% of that of normal rats; we conclude that this provides a reserve adequate for the needs of pregnancy.