Hideki Asakawa

Examination of islets in the pancreas biopsy specimens from newly diagnosed Type 1 (insulin-dependent) diabetic patients

SummaryWe attempted to examine the immunopathological change of the pancreatic islets of newly diagnosed Type 1 (insulin-dependent) diabetic patients and thereby to obtain useful information for the therapy of the patients. For this purpose, pancreas biopsy under laparoscopy was performed 2–4 months after the onset of Type 1 diabetes in seven newly diagnosed patients. All biopsies were performed safely without any complications. Immunohistochemical examination of the biopsy specimens revealed a marked decrease of insulin-containing cells, preservation of glucagon-containing cells, and various degrees of expression of MHC class I and class II antigens in islet cells and in endothelial cells within and around the islets. Signs of active autoimmune phenomena, e. g. lymphocytic infiltration or immunoglobulin deposition in islets, were not detected in any of these patients by light microscopical evaluation. We conclude that pancreas biopsy under laparoscopy has shown various immunological changes in the islets of newly diagnosed Type 1 diabetic patients. Pancreas biopsy, however, may not be suitable under the present protocol for the selection of patients for immunotherapy because of problems including sampling errors.

Elevation of fibrinogen and thrombin–antithrombin III complex levels of type 2 diabetes mellitus patients with retinopathy and nephropathy

Diabetes is associated with a hypercoagulable state. Blood hypercoagulability may accelerate atherosclerosis and the diabetic microvascular complications. Thrombin-antithrombin III complex (TAT) and fibrinogen levels are parameters of coagulation and fibrinolysis. In the present study, we examined the risk factors for the diabetic microangiopathy including TAT and fibrinogen levels. To investigate the relationship between the clinical parameters and microangiopathy in type 2 diabetic patients, the clinical parameters of subjects with microangiopathy were compared with those of subjects without microangiopathy. The clinical parameters were as follows: age at examination, duration of diabetes, fasting plasma glucose (FPG) level, HbA(1C) level, insulin level, TAT level, fibrinogen level, lipoprotein (a) (Lp(a)) level, total cholesterol level, triglyceride level, HDL cholesterol level and existence of hypertension. The plasma TAT and fibrinogen levels were significantly higher in patients with retinopathy or nephropathy than in patients without these complications. Moreover, fibrinogen levels of patients with microalbuminuria or background retinopathy were significantly higher than those of patients with normoalbuminuria or no retinopathy. The duration of diabetes was significantly longer in patients with any microangiopathy than in patients without it. Multiple regression analyses showed that duration and fibrinogen level were independent factors associated with the existence of retinopathy or nephropathy. Our data show that the disorder of coagulation and fibrinolysis is significantly associated with diabetic retinopathy and nephropathy and exists at the early stage of microangiopathy.

Effect of mosapride on glycemic control and gastric emptying in type 2 diabetes mellitus patients with gastropathy.

Delay of gastric emptying is one of the factors responsible for unfavorable glycemic control. We investigated the possible effects of mosapride, a digestive tract prokinetic agent, on glycemic control in diabetic patients complicated with gastropathy. Enrolled were 36 type II diabetic patients presenting with mild digestive tract symptoms. They were given mosapride 15 mg per day for 6 months. Seventeen cases were subjected to gastric emptying test according to marker method (administration of a capsule containing 20 pieces of radiopaque marker during breakfast, followed by abdominal X-ray imaging 3 and 5 h later). In 18 cases, HbA(1C) was improved by more than 0.3% for 6 months, whereby these 18 cases were defined as the improvement group. The remaining 18 cases were defined as the non-improvement group. In the gastric emptying study, basal number of the residual markers before administration of mosapride was determined 3 and 5 h later to show 18.3+/-1.8 and 7.6+/-5.1, respectively, in the improvement group while after administration, they were reduced down to 11.2+/-5.1 and 1.4+/-2.5, respectively. In sharp contrast, the basal counterparts in the non-improvement group were 19.1+/-1.5, and 16.4+/-3.4, respectively, whereas administration failed to reduce the number of the residual markers and they remained to be as high as 19.0+/-1.4 and 11.1+/-6.4, respectively. Gastric motility in the improvement group was much more improved by mosapride administration relative to those in the non-improvement group. Mosapride might elicit improvement in the glycemic control in the patients with diabetic gastropathy.

High glucose and hyperosmolarity increase secretion of interleukin-1 β in cultured human aortic endothelial cells

Interleukin-1 (IL-1) is secreted by endothelial cells (ECs) and smooth-muscle cells (SMCs), which are two major component cells of vessels and detected in atherosclerotic lesions. To evaluate the effect of hyperglycemia on the secretion of IL-1 beta in endothelial cells in diabetic patients, we investigated the effects of high glucose and hyperosmolar conditions on the secretion of IL-1 beta from cultured human aortic endothelial cells (HAECs). HAECs were treated with high concentration of glucose or hyperosmolar condition for 3 days. IL-1 beta in the supernatant was measured by high sensitive enzyme-linked immunosorbent assay (ELISA). Under high concentration of glucose (16.6 mmol/L) and hyperosmolar condition (glucose 5.5 mmol/L + mannitol 11.1 mmol/L), the secretion of IL-1 beta was significantly increased (41.0 +/- 2.8 and 26.3 +/- 5.9% increase, respectively, compared with that of 5.5 mmol/L glucose). In conclusion, high glucose and hyperosmolar condition increase the secretion of IL-1 beta in HAECs. The results suggest that diabetic macroangiopathies might be accelerated partly through the increase of IL-1 beta secretion in HAECs.

Induction of Insulitis by Adoptive Transfer With L3T4+ Lyt2− T-Lymphocytes in T-Lymphocyte–Depleted NOD Mice

To clarify the pathogenesis of insulitis in the nonobese diabetic (NOD) mouse, an animal model for human insulin-dependent diabetes mellitus, T-lymphocyte-depleted NOD mice (B mice) were adoptively transferred with spleen and lymph node cells from cyclophosphamide-treated NOD mice after separating the cells with monoclonal antibodies against various T-lymphocyte surface antigens plus complement. Light-microscopic and immunohistochemical studies were also performed to investigate the lymphocytic infiltrations. The incidence of insulitis detected in B mice was much lower when compared with that of the lesion naturally occurring in the NOD mouse. However, higher incidence of insulitis was inducible in B mice by transferring unfractionated lymphoid cells from NOD mice. When the Thy1+ cell-depleted fraction was transferred into the B mice, no increase in the incidence of insulitis was observed. The Lyt1+ or L3T4+ cell-eliminated fraction was also unable to transfer insulitis. Conversely, donor cells depleted of Lyt2+ components successfully induced insulitis in the recipient B mice. These data were consistent with the immunohistochemical study, which showed that the main phenotype of the cells infiltrating the islets was L3T4+. These results suggest the importance of L3T4+Lyt2− T-lymphocytes in the pathogenesis of insulitis in NOD mice.

Relationship of leptin level with metabolic disorders and hypertension in Japanese type 2 diabetes mellitus patients

Leptin is considered to play an important role in the regulation of body weight and metabolism in obese individuals. However, the relationship of leptin with metabolic disorders or vascular complications in type 2 diabetic patients has yet to be elucidated. In this study, we investigated the association of leptin levels with clinical parameters (glycemic control, lipid levels, abdominal fat distribution) and investigated the leptin levels of diabetic patients with and without vascular complications in Japanese diabetic patients. In male and female patients, leptin levels were significantly associated with body mass index (BMI), percent body fat, insulin level, triglyceride (TG) level, total abdominal fat area (TFA), visceral fat area (VFS), and subcutaneous fat area (SFA). Only in male patients, leptin levels were inversely correlated with HDL-cholesterol, fasting plasma glucose (FPG), and HbA(1C). Leptin levels in male and female patients with hypertension were higher than in those without hypertension. Leptin levels of both males and females with angiopathy were not statistically different from those without angiopathy. In conclusion, leptin is involved in various metabolic disorders and hypertension, and we speculate that it may not be strongly associated with vascular complications in Japanese diabetic individuals.

Comparison of risk factors of macrovascular complications: Peripheral vascular disease, cerebral vascular disease, and coronary heart disease in Japanese Type 2 diabetes mellitus patients

Although macroangiopathies such as peripheral vascular disease (PVD), cerebral vascular disease (CVD), and coronary heart disease (CHD) can often be observed in patients with diabetes mellitus, they are not specific for diabetes mellitus. Moreover, it is unclear whether their progressive mechanism is different. In the present study, we compared the risk factors among the diabetic macrovascular complications. Univariate analyses showed that in all patients, age at examination, duration of diabetes, thrombin-antithrombin III complex (TAT) level, fibrinogen level, lipoprotein (a) (Lp(a)) level, total cholesterol (T-Chol) level, and existence of microagiopathy were risk factors for PVD. Age, duration of diabetes, insulin level, TAT level, fibrinogen level, HDL cholesterol (HDL-Chol) level, hypertension, and nephropathy were risk factors for CVD. Only fibrinogen level was a risk factor for CHD. Moreover, Lp(a) level was a risk factor for PVD and CVD in male patients, but not in females. On the other hand, insulin level was a risk factor for CVD in female patients, but not in males. Multivariate analyses showed that TAT level, T-Chol level, and neuropathy were independent variables for PVD and that age, TAT level, and HDL-Chol level were independent variables for CVD. On the other hand, only fibrinogen level was the independent variable for CHD in males. Our results suggest that the progressive mechanism of PVD and CVD might be different from that of CHD and might differ according to gender in Japanese diabetic patients.

Plasma brain natriuretic peptide levels in normotensive Type 2 diabetic patients without cardiac disease and macroalbuminuria

To clarify the relationship of the plasma brain natriuretic peptide (BNP) levels to diabetic complications, we studied plasma BNP levels in 100 normotensive diabetic patients without clinical cardiac disease and macroalbuminuria. The values of plasma BNP levels were not significantly different between patients with microalbuminuria and those with normoalbuminuria (12.2 +/- 2.0 vs. 12.3 +/- 1.3 pg/ml, means +/- S.E.M.), and neither were the BNP levels of patients with and without retinopathy significantly different (15.7 +/- 3.4 vs. 11.4 +/- 1.0 pg/ml). BNP levels of the subjects with cerebral vascular disease (CVD) were not statistically different from those of subjects without CVD (17.5 +/- 5.5 vs. 11.7 +/- 1.0 pg/ml), although mean BNP value of subjects with CVD was higher than that of subjects without it. With regard to peripheral vascular disease (PVD), BNP levels of the subjects with PVD were not statistically different from those of subjects without PVD (13.5 +/- 2.3 vs. 12.1 +/- 1.2 pg/ml). We also studied radial arterial oxygen tension of 45 patients and compared these levels between those with and without diabetic complications. However, we could not find statistical differences between them. In conclusion, our study suggests that BNP and arterial oxygen tension levels will not be affected by retinopathy, microalbuminuria, CVD, and PVD in normotensive diabetic patients without clinical cardiac disease and macroalbuminuria. Therefore, when normotensive diabetic patients without macroalbuminuria show increased plasma level of BNP, we should examine their cardiac function in detail, considering subclinical cardiac disease.

Multistep carcinogenesis in anaplastic thyroid carcinoma: a case report

Summary We previously established an anaplastic thyroid carcinoma cell line (KOA2) that had double mutations: an N‐ras mutation and a p53 gene mutation. To clarify multistep carcinogenesis, we analysed surgical material from the patient from whom KOA2 was derived for abnormalities in the N‐ras and p53 genes. The resected material had two histologically different lesions: a follicular neoplasm and an anaplastic carcinoma. The N‐ras mutation was observed in both lesions, but the p53 gene mutation only in the anaplastic lesion. These facts indicate that an N‐ras mutation may induce follicular neoplasm and a subsequent p53 mutation may have caused the follicular neoplasm to transform to anaplastic carcinoma in this patient. This report suggests direct evidence for multistep carcinogenesis in anaplastic thyroid carcinoma.Abbreviations: APC, adenomatous polyposis coli; DCC, deleted in colorectal cancer; PCR‐SSCP, polymerase chain reaction‐single strand conformation polymorphism.

ESTABLISHMENT OF A HUMAN ANAPLASTIC THYROID CANCER CELL LINE SECRETING GRANULOCYTE COLONY-STIMULATING FACTOR IN RESPONSE TO CYTOKINES

SummaryA human anaplastic thyroid cancer cell line K-119, derived from a 77-yr-old woman who had developed marked neutrophilia and underwent surgery for anaplastic thyroid cancer, has been established. The spindlelike and polygonal cells in shape are stably proliferating since the beginning of its culture 2 yr ago. The cells grow rapidly and the population doubling time is 26 h. The chromosomes show many abnormalities and many marker chromosomes have been observed. Heterotransplantation of the cells into nude mice has resulted in the formation of tumors that are histologically interpreted as anaplastic cancer. The most noteworthy characteristics of the cell line are the many Ki-67-positive cells (86.3%) and that the cell line spontaneously secretes granulocyte colony-stimulating factor (G-CSF) and releases increased amounts of G-CSF in response to the stimulation of tumor necrosis factor, interleukin 1α, and interleukin 1β. The conditioned medium obtained from K-119 cells contains an autocrine factor stimulating the proliferation of themselves.