Tatsuo Tomita

Apoptosis in pancreatic β-islet cells in Type 2 diabetes

Apoptosis plays important roles in the pathophysiology of Type 2 diabetes mellitus (T2DM). The etiology of T2DM is multifactorial, including obesity-associated insulin resistance, defective insulin secretion, and loss of β-cell mass through β-cell apoptosis. β-cell apoptosis is mediated through a milliard of caspase family cascade machinery in T2DM. The glucose-induced insulin secretion is the principle pathophysiology of diabetes and insufficient insulin secretion results in chronic hyperglycemia, diabetes. Recently, hyperglycemia-induced β-cell apoptosis has been extensively studied on the balance of pro-apoptotic Bcl-2 proteins (Bad, Bid, Bik, and Bax) and anti-apoptotic Bcl family (Bcl-2 and Bcl-xL) toward apoptosis in vitro isolated islets and insulinoma cell culture. Apoptosis can only occur when the concentration of pro-apoptotic Bcl-2 exceeds that of anti-apoptotic proteins at the mitochondrial membrane of the intrinsic pathway. A bulk of recent research on hyperglycemia-induced apoptosis on β-cells unveiled complex details on glucose toxicity on β-cells in molecular levels coupled with cell membrane potential by adenosine triphosphate generation through K+ channel closure, opening Ca2+ channel and plasma membrane depolarization. Furthermore, animal models using knockout mice will shed light on the basic understanding of the pathophysiology of diabetes as a glucose metabolic disease complex, on the balance of anti-apoptotic Bcl family and pro-apoptotic genes. The cumulative knowledge will provide a better understanding of glucose metabolism at a molecular level and will lead to eventual prevention and therapeutic application for T2DM with improving medications.Apoptosis plays important roles in the pathophysiology of Type 2 diabetes mellitus (T2DM). The etiology of T2DM is multifactorial, including obesity-associated insulin resistance, defective insulin secretion, and loss of β-cell mass through β-cell apoptosis. β-cell apoptosis is mediated through a milliard of caspase family cascade machinery in T2DM. The glucose-induced insulin secretion is the principle pathophysiology of diabetes and insufficient insulin secretion results in chronic hyperglycemia, diabetes. Recently, hyperglycemia-induced β-cell apoptosis has been extensively studied on the balance of pro-apoptotic Bcl-2 proteins (Bad, Bid, Bik, and Bax) and anti-apoptotic Bcl family (Bcl-2 and Bcl-xL) toward apoptosis in vitro isolated islets and insulinoma cell culture. Apoptosis can only occur when the concentration of pro-apoptotic Bcl-2 exceeds that of anti-apoptotic proteins at the mitochondrial membrane of the intrinsic pathway. A bulk of recent research on hyperglycemia-induced apoptosis on β-cells unveiled complex details on glucose toxicity on β-cells in molecular levels coupled with cell membrane potential by adenosine triphosphate generation through K+ channel closure, opening Ca2+ channel and plasma membrane depolarization. Furthermore, animal models using knockout mice will shed light on the basic understanding of the pathophysiology of diabetes as a glucose metabolic disease complex, on the balance of anti-apoptotic Bcl family and pro-apoptotic genes. The cumulative knowledge will provide a better understanding of glucose metabolism at a molecular level and will lead to eventual prevention and therapeutic application for T2DM with improving medications.

Immunocytochemical localisation of caspase-3 in pancreatic islets from type 2 diabetic subjects

Aims: Caspase‐3 has been recognised as a main effector caspase of the apoptotic cascade. Involvement of caspase‐3 has been implicated in a β‐cell cloned cell line from type 1 diabetic subjects and in isolated islets from type 2 diabetic subjects. This study aimed to immunocytochemically identify cleaved caspase‐3 positive islet cells in type 2 diabetic subjects compared with control subjects. Methods: Using commercially available rabbit anti‐cleaved caspase‐3 antibody, immunocytochemical staining was performed on 16 cases of pancreatic tissues from type 2 diabetic subjects compared with age‐matched controls. Results: Control islets revealed cleaved caspase‐3 positive cells in about 4.7% in total islet cells with large and small islets positive at 4.1% and 7.0%, respectively. Islets from type 2 diabetic subjects showed higher immunostaining percentage at 8.7% in total islets with large and small islets positive for cleaved caspase‐3 at 7.7% and 12%, respectively, at about twice that of the control values. Islets from type 2 diabetics were generally insulin cell‐less and glucagon cell‐rich, but insulin cells still remained. Type 2 diabetic islets showed various stromal amyloid deposits, displacing the residual islet cells. Cleaved caspase‐3 positive cells were more in the less amyloid deposited islets than in the islet cell deficient islets containing more amyloid deposits; the latter correspond to the end‐stage of type 2 diabetic islets. Conclusions: The more cleaved caspase‐3 immunostained islets from type 2 diabetics may implicate an accelerated apoptotic cascade in the islets, accompanied by increasing amyloid deposits, before proceeding to ultimate cell death.

Localization of Nerve Fibers in Colonic Polyps, Adenomas, and Adenocarcinomas by Immunocytochemical Staining for PGP 9.5

BackgroundPGP 9.5 is a cytoplasmic protein and is a specific marker for neurites and neurons.AimsUsing anti-PGP 9.5, this study aimed to localize nerve fibers in normal colons, polyps, adenomas and adenocarcinomas.Methods Colonic polyps, adenomas and T1 to T3 adenocarcinomas with adjacent normal colon were immunostained for PGP 9.5 using rabbit anti-PGP 9.5.ResultsIn normal colon, numerous nerve fibers were localized in inner and outer muscles, from which submucosa and lamina propria were innervated. In hyperplastic polyps and tubular adenomas, the stalk revealed Meissner’s plexus and large-diameter nerve fibers, and fine nerve fibers innervated abundantly in lamina propria of hyperplastic polyps and small tubular adenomas. In villous adenomas, large-diameter nerve fibers and Meissner’s plexus were localized in the stalk whereas a few or no fine nerve fibers were localized in fine stroma. In adenocarcinomas, more fine fibers were localized in submucosal stroma adjacent to the invading carcinoma in T1 carcinomas but there were no nerve fibers in the midst of tumors in T2 and T3 carcinomas. There were focally and sporadically increased nerve fibers adjacent to invading cancer nests in 5 of 8 T2 cases. In T3 carcinomas, fragmented Auerbach’s plexus were noted in cancer-invaded colonic muscles and there were no increased fine nerve fibers in the cancer-invaded subserosa in the majority of cases. PGP 9.5 immunostaining revealed tumor-associated neurogenesis in submucosa but no obviously increased nerve fibers within cancer-invaded muscles.Conclusions This lack of tumor-associated neurogenesis supports insidious and often silent clinical presentation of colonic carcinomas until invading through the colonic wall to adjacent organs.

Immunocytochemical Localization of Lymphatic and Venous Vessels in Colonic Polyps and Adenomas

Histopathological localization of lymphatic vessels has been hindered because of a lack of suitable immunocytochemical markers for lymphatic vessels. Using lymphatic vessels endothelial hyaluronan receptor-1 (LYVE-1) immunocytochemical staining, hyperplastic polyps, tubular adenomas to villous adenomas, were investigated for lymphatic vessels compared with immunostained blood vessels using factor-8. Four cases each of hyperplastic polyps, tubular adenomas to villous adenomas, were routinely fixed in formalin and embedded in paraffin and were immunostained using goat anti-LYVE-1 for lymphatic vessels and rabbit anti-factor-8 for blood vessels. In normal colon and hyperplastic polyps, slender lymphatic vessels were noted in muscularis mucosa, which spread into the base of colonic crypt, whereas round venous vessels, they extend into lamina propria. In tubular adenomas, small lymphatic and venous vessels were noted in broad fibrous stalks. In villous adenomas, smaller lymphatic and venous vessels were noted in fine intervillous stroma. In normal colon and hyperplastic polyps, slender, irregularly shaped lymphatic vessels were present in muscularis mucosa, spreading into the base of the colonic crypt. In tubular adenomas, small lymphatic and venous vessels were noted in fibrous stalks. In villous adenomas, smaller lymphatic and venous vessels were noted in intervillous stroma. There are no increased lymphatic and venous vessels in intermucosal stroma and stalks of adenomas compared with normal colon.

LYVE-1 immunocytochemical staining for gastrointestinal carcinoids

Aims: By immunocytochemical staining for lymphatic vessels using anti‐lymphatic vessel hyaluronan receptor (LYVE‐1) antibody, pancreatic islets and some pancreatic endocrine tumours (PETs) were positively stained for the cytoplasm in addition to lymphatic vessels. The current study was extended to investigate possible immunostaining of gastrointestinal carcinoids using goat antihuman LYVE‐1 antibody. Methods: Lymphatic vessels were immunostained by LYVE‐1 and blood vessels were immunostained by factor 8 (F‐8). Results: Among 27 gastrointestinal carcinoids, six cases (22%) including five primary and one metastatic carcinoids were positive in the cytoplasm for LYVE‐1 in addition to lymphatic vessels and there were abundant lymphatic and blood vessels at the margin of the carcinoids. These six LYVE‐1 positive cases consisted of three of 10 small intestinal, one of five appendiceal and two of seven large intestinal carcinoids. There was no LYVE‐1 positive staining in the adjacent normal gastro‐intestinal mucosa. Conclusions: The presence of LYVE‐1 immunostaining in some gastrointestinal carcinoids may support a structure‐function relationship of lymphatic vessels/hyaluronan receptor for modulating synthesis and secretion of hormones and amines by carcinoid tumour cells.

Apoptosis of pancreatic β-cells in Type 1 diabetes

Type 1 diabetes mellitus (T1DM) results from autoimmune destruction of pancreatic β-cells after an asymptomatic period over years. Insulitis activates antigen presenting cells, which trigger activating CD4+ helper-T cells, releasing chemokines/cytokines. Cytokines activate CD8+ cytotoxic-T cells, which lead to β-cell destruction. Apoptosis pathway consists of extrinsic (receptor-mediated) and intrinsic (mitochondria-driven) pathway. Extrinsic pathway includes Fas pathway to CD4+-CD8+ interaction, whereas intrinsic pathway includes mitochondria-driven pathway at a balance between anti-apoptotic B-cell lymphoma (Bcl)-2 and Bcl-xL and pro-apoptotic Bad, Bid, and Bik proteins. Activated cleaved caspse-3 is the converging point between extrinsic and intrinsic pathway. Apoptosis takes place only when pro-apoptotic proteins exceed anti-apoptotic proteins. Since the concordance rate of T1DM in identical twins is about 50%, environmental factors are involved in the development of T1DM, opening a door to find means to detect and prevent further development of autoimmune β-cell destruction for a therapeutic application.

Lymphatic Vessels in the Human Endometrium: Are they Present or Absent?

The presence and structure-function relationship of lymphatic vessels in the human endometrium are still sketchy and unsettled: some authors claimed that there were no lymphatic vessels [1,2] but more reports agreed that lymphatic vessels are present in the human endometrium, especially in the basalis but may not be in the functionalis [3-6]. Recently more reliable Immunocytochemical markers for lymphatic vessels became available and have been widely utilized using D2-40 [7] and lymphatic epithelial hyaluronan receptor-1 (LYVE-1) [8]. D2-40 recognizes the collecting lymphatic vessels and LYVE-1 recognizes mainly lymphatic capillaries [9]. With routinely formalin-fixed and paraffin-embedded sections, not all lymphatic sections are immunostained but frozen sections provide consistently much more lymphatic and venous vessels. There are numerous lymphatic vessels in the myometrium and basalis in all menstrual phases. In the Day 3 endometrium from the menstrual period, there were numerous small lymphatic vessels in the remaining basalis by both D2-40 and LYVE-1. There are cyclic changes of lymphatic vessels in the functionalis: The early proliferative phase endometrium, Day 5 showed very few, small lymphatic vessels in the lower functionalis (Figure 1) and Day 9 endometrium revealed few lymphatic vessels in the upper endometrium.