Ryosuke Misawa

Altered Islet Composition and Disproportionate Loss of Large Islets in Patients with Type 2 Diabetes

Human islets exhibit distinct islet architecture with intermingled alpha- and beta-cells particularly in large islets. In this study, we quantitatively examined pathological changes of the pancreas in patients with type 2 diabetes (T2D). Specifically, we tested a hypothesis that changes in endocrine cell mass and composition are islet-size dependent. A large-scale analysis of cadaveric pancreatic sections from T2D patients (n = 12) and non-diabetic subjects (n = 14) was carried out combined with semi-automated analysis to quantify changes in islet architecture. The method provided the representative islet distribution in the whole pancreas section that allowed us to examine details of endocrine cell composition in individual islets. We observed a preferential loss of large islets (>60 µm in diameter) in T2D patients compared to non-diabetic subjects. Analysis of islet cell composition revealed that the beta-cell fraction in large islets was decreased in T2D patients. This change was accompanied by a reciprocal increase in alpha-cell fraction, however total alpha-cell area was decreased along with beta-cells in T2D. Delta-cell fraction and area remained unchanged. The computer-assisted quantification of morphological changes in islet structure minimizes sampling bias. Significant beta-cell loss was observed in large islets in T2D, in which alpha-cell ratio reciprocally increased. However, there was no alpha-cell expansion and the total alpha-cell area was also decreased. Changes in islet architecture were marked in large islets. Our method is widely applicable to various specimens using standard immunohistochemical analysis that may be particularly useful to study large animals including humans where large organ size precludes manual quantitation of organ morphology.

Regional Differences in Islet Distribution in the Human Pancreas - Preferential Beta-Cell Loss in the Head Region in Patients with Type 2 Diabetes

While regional heterogeneity in islet distribution has been well studied in rodents, less is known about human pancreatic histology. To fill gaps in our understanding, regional differences in the adult human pancreas were quantitatively analyzed including the pathogenesis of type 2 diabetes (T2D). Cadaveric pancreas specimens were collected from the head, body and tail regions of each donor, including subjects with no history of diabetes or pancreatic diseases (n = 23) as well as patients with T2D (n = 12). The study further included individuals from whom islets were isolated (n = 7) to study islet yield and function in a clinical setting of islet transplantation. The whole pancreatic sections were examined using an innovative large-scale image capture and unbiased detailed quantitative analyses of the characteristics of islets from each individual (architecture, size, shape and distribution). Islet distribution/density is similar between the head and body regions, but is >2-fold higher in the tail region. In contrast to rodents, islet cellular composition and architecture were similar throughout the pancreas and there was no difference in glucose-stimulated insulin secretion in islets isolated from different regions of the pancreas. Further studies revealed preferential loss of large islets in the head region in patients with T2D. The present study has demonstrated distinct characteristics of the human pancreas, which should provide a baseline for the future studies integrating existing research in the field and helping to advance bi-directional research between humans and preclinical models.

Coating Human Pancreatic Islets With Cd4+cd25highcd127− Regulatory T Cells as a Novel Approach for the Local Immunoprotection

Objectives:To develop a novel approach for local immunoprotection using CD4+CD25highCD127– T regulatory cells (Tregs) attached to the surface of the islets before transplantation. Background:Tregs expanded ex vivo can control allo and autoreactivity, therefore, Treg-based therapy may offer more effective protection for transplanted islets from immunologic attack than currently used immunosuppression. Local application of Tregs can make such therapy more clinically feasible and efficient. Methods:Human islets were isolated and coated with allogeneic ex vivo expanded Tregs using biotin-poly(ethylene glycol)-N-hydroxysuccinimide ester (biotin-PEG-NHS) and streptavidin as binding molecules. Results:Coating pancreatic islets with Tregs did not affect islet viability (>90% fluorescein diacetate/propidium iodide) or the insulin secretion profile in dynamic islet perifusion assays. After in vitro incubation with allogeneic T effector cells, Treg-coated islets revealed preserved function with higher insulin secretion compared with controls-native islets, coated islets with T effector cells or when Tregs were added to the culture, but not attached to islets (P < 0.05). In addition, the Enzyme-linked immunosorbent spot (ELISPOT) assay revealed suppression of interferon (IFN)-&ggr; secretion, when T effector cells were challenged with Treg-coated islets comparing to controls (99 ± 7 vs 151 ± 8 dots, respectively; P < 0.01). Conclusions:We demonstrated, for the first time, the ability to bind immune regulatory cells to target cells with preservation of their viability and function and protective activity against immune attack. If successfully tested in an animal model, local delivery of immunoprotective Tregs on the surface of transplanted pancreatic islets may be an alternative or improvement to the currently used immunosuppression.

Enhancing Pancreatic Beta-Cell Regeneration In Vivo with Pioglitazone and Alogliptin

Aims/Hypothesis Pancreatic beta-cells retain limited ability to regenerate and proliferate after various physiologic triggers. Identifying therapies that are able to enhance beta-cell regeneration may therefore be useful for the treatment of both type 1 and type 2 diabetes. Methods In this study we investigated endogenous and transplanted beta-cell regeneration by serially quantifying changes in bioluminescence from beta-cells from transgenic mice expressing firefly luciferase under the control of the mouse insulin I promoter. We tested the ability of pioglitazone and alogliptin, two drugs developed for the treatment of type 2 diabetes, to enhance beta-cell regeneration, and also defined the effect of the immunosuppression with rapamycin and tacrolimus on transplanted islet beta mass. Results Pioglitazone is a stimulator of nuclear receptor peroxisome proliferator-activated receptor gamma while alogliptin is a selective dipeptidyl peptidase IV inhibitor. Pioglitazone alone, or in combination with alogliptin, enhanced endogenous beta-cell regeneration in streptozotocin-treated mice, while alogliptin alone had modest effects. In a model of syngeneic islet transplantation, immunosuppression with rapamycin and tacrolimus induced an early loss of beta-cell mass, while treatment with insulin implants to maintain normoglycemia and pioglitazone plus alogliptin was able to partially promote beta-cell mass recovery. Conclusions/Interpretation These data highlight the utility of bioluminescence for serially quantifying functional beta-cell mass in living mice. They also demonstrate the ability of pioglitazone, used either alone or in combination with alogliptin, to enhance regeneration of endogenous islet beta-cells as well as transplanted islets into recipients treated with rapamycin and tacrolimus.

Distinct function of the head region of human pancreas in the pathogenesis of diabetes

The large size of the human pancreas challenges unbiased quantitative analyses that require a practical stereological approach. While many histological studies of the pancreas in the past lacked regional information, we have shown marked heterogeneity within an individual, where islet distribution/density is relatively low in the head and gradually increases through the body toward the tail region by > 2-fold.1, 2 Studies focusing on the tail region may be prone to overestimation of β-cell/islet mass when normalizing measured values per person by using pancreas weight or volume. In this article, beyond technical issues, we discuss the pathophysiological importance of studying the head region of the human pancreas regarding its unique characteristics in early development, and the anatomical disposition that may lead to a preferential loss of β-cells in patients with type 2 diabetes and the development of pancreatic cancer.

Application of Digital Image Analysis to Determine Pancreatic Islet Mass and Purity in Clinical Islet Isolation and Transplantation.

Pancreatic islet mass, represented by islet equivalent (IEQ), is the most important parameter in decision making for clinical islet transplantation. To obtain IEQ, the sample of islets is routinely counted under a microscope and discarded thereafter. Islet purity, another parameter in islet processing, is routinely assessed by estimation only. In this study, we validated our digital image analysis (DIA) system by using the software of Image Pro Plus and a custom-designed Excel template to assess islet mass and purity to better comply with current good manufacturing practice (cGMP) standards. Human islet samples (60 collected from a single isolation and 24 collected from 12 isolations) were captured as calibrated digital images for the permanent record. Seven trained technicians participated in determination of IEQ and purity by the manual counting method (manual image counting, Manual I) and DIA. IEQ count showed statistically significant correlations between the Manual I and DIA in all sample comparisons (r > 0.819 and p < 0.0001). A statistically significant difference in IEQ between Manual I and DIA was not found in all sample groups (p > 0.05). In terms of purity determination, statistically significant differences between assessment and DIA measurement were found in high-purity 100-μl samples (p < 0.005) and low-purity 100-μl samples (p < 0.001) of the single isolation. In addition, islet particle number (IPN) and the IEQ/IPN ratio did not differ statistically between Manual I and DIA. In conclusion, the DIA used in this study is a reliable technique to determine IEQ and purity. Islet sample preserved as a digital image and results produced by DIA can be permanently stored for verification, technical training, and information exchange among islet centers. Therefore, DIA complies better with cGMP requirements than the manual counting method. We propose DIA as a quality control tool to supplement the established standard manual method for islet counting and purity estimation.

Impact of culture medium on CD4+ CD25highCD127lo/neg Treg expansion for the purpose of clinical application

A recently discovered population of lymphocytes, called T regulatory cells (Tregs), is characterized by expression of transcription factor Forkhead box P3 (FoxP3). These cells have been successfully used as therapeutic treatments and prophylaxis for graft-versus-host disease (GVHD) and diabetes and might become an attractive alternative to traditional immunotherapy. Here we evaluated how the type of culture medium and the type of serum can influence yield and quality of Tregs after in vitro expansion. We compared Treg fold of expansion and their phenotypical characteristics including expression of FoxP3, CD25, CD127, CD62L and CD45RA in three commercially available culture media (RPMI 1640 (Cellgro; Manassas VA, USA), SCGM (Cellgenix; Freiburg, Germany), and X-VIVO 20 (Lonza; Walkersville, MD, USA)) with addition of human serum (HS, 10%) or fetal bovine serum (FBS, 10%). Among the tested media, X-VIVO 20 supplemented with HS produced the highest yield after 17days of in vitro expansion (a median of 86-fold expansion, range 30-1365) and highest level of FoxP3 expression (a median of 66.8% of positive cells, range 56-84.8%) in CD4(+) CD25(hi)CD127(lo/neg) FACS sorted polyclonal Tregs. There was no difference in Tregs yield whether HS or FBS serum was used. In conclusion, the yield of the ex vivo expanded Tregs is related to the type of media applied. Supplementation of the culture with FBS or human serum is equally beneficial.