Beta cell nuclear musculoaponeurotic fibrosarcoma oncogene family A (MafA) is deficient in type 2 diabetes.

AIMS/HYPOTHESIS: The beta cell transcriptional factor musculoaponeurotic fibrosarcoma oncogene family A (MafA) regulates genes important for beta cell function. Loss of nuclear MafA has been implicated in beta cell dysfunction in animal models of type 2 diabetes. We sought to establish if nuclear MafA is less abundant in beta cell nuclei in humans with type 2 diabetes. METHODS: Pancreas obtained at surgery from five non-diabetic individuals and six individuals with type 2 diabetes was immunostained for insulin, glucagon and MafA. RESULTS: Beta cell nuclear MafA was markedly decreased in type 2 diabetes (1.6 ± 1.2% vs 46.3 ± 8.3%, p < 0.001). CONCLUSIONS/INTERPRETATION: Beta cell nuclear MafA is markedly decreased in humans with type 2 diabetes, which may contribute to impaired beta cell dysfunction.

A low frequency of pancreatic islet insulin-expressing cells derived from cord blood stem cell allografts in humans.

AIMS/HYPOTHESIS: We sought to establish if stem cells contained in cord blood cell allografts have the capacity to differentiate into insulin-expressing beta cells in humans. METHODS: We studied pancreases obtained at autopsy from individuals (n = 11) who had prior opposite-sex cord blood transplants to reconstitute haematopoiesis. Pancreatic tissue sections were stained first by XY-fluorescence in situ hybridisation and then insulin immunohistochemistry. Pancreases obtained at autopsy from participants without cord blood cell infusions served as controls (n = 11). RESULTS: In the men with prior transplant of female cord blood, there were 3.4 ± 0.3% XX-positive insulin-expressing islet cells compared with 0.32 ± 0.05% (p < 0.01) in male controls. In women with prior transplant of male cord blood cells we detected 1.03 ± 0.20% XY insulin-expressing islet cells compared with 0.03 ± 0.03 in female controls (p < 0. 001). CONCLUSIONS/INTERPRETATION: Cord blood stem cells have the capacity to differentiate into insulin-expressing cells in non-diabetic humans. It remains to be established whether these cells have the properties of beta cells.

Pancreatic duct replication is increased with obesity and type 2 diabetes in humans.

AIMS/HYPOTHESIS: In a high-fat-fed rat model of type 2 diabetes we noted increased exocrine duct replication. This is a predisposing factor for pancreatitis and pancreatic cancer, both of which are more common in type 2 diabetes. The aim of the study reported here was to establish if obesity and/or type 2 diabetes are associated with increased pancreatic ductal replication in humans. METHODS: We obtained pancreas at autopsy from 45 humans, divided into four groups: lean (BMI <25 kg/m(2)); obese (BMI >27 kg/m(2)); non-diabetic; and with type 2 diabetes. Pancreases were evaluated after immunostaining for the duct cell marker cytokeratin and Ki67 for replication. RESULTS: We show for the first time that both obesity and type 2 diabetes in humans are associated with increased pancreatic ductal replication. Specifically, we report that (1) replication of pancreatic duct cells is increased tenfold by obesity, and (2) lean subjects with type 2 diabetes demonstrate a fourfold increase in replication of pancreatic duct cells compared with their lean non-diabetic controls. CONCLUSIONS/INTERPRETATION: Pancreatic duct cell replication is increased in humans in response to both obesity and type 2 diabetes, potentially providing a mechanism for the increased risk of pancreatitis and pancreatic cancer in those with obesity and/or type 2 diabetes.

Adaptive changes in pancreatic beta cell fractional area and beta cell turnover in human pregnancy.

AIMS/HYPOTHESIS: We sought to establish the extent and basis for adaptive changes in beta cell numbers in human pregnancy. METHODS: Pancreas was obtained at autopsy from women who had died while pregnant (n = 18), post-partum (n = 6) or were not pregnant at or shortly before death (controls; n = 20). Pancreases were evaluated for fractional pancreatic beta cell area, islet size and islet fraction of beta cells, beta cell replication (Ki67) and apoptosis (TUNEL), and indirect markers of beta cell neogenesis (insulin-positive cells in ducts and scattered beta cells in pancreas). RESULTS: The pancreatic fractional beta cell area was increased by approximately 1.4-fold in human pregnancy, with no change in mean beta cell size. In pregnancy there were more small islets rather than an increase in islet size or beta cells per islet. No increase in beta cell replication or change in beta cell apoptosis was detected, but duct cells positive for insulin and scattered beta cells were increased with pregnancy. CONCLUSIONS/INTERPRETATION: The adaptive increase in beta cell numbers in human pregnancy is not as great as in most reports in rodents. This increase in humans is achieved by increased numbers of beta cells in apparently new small islets, rather than duplication of beta cells in existing islets, which is characteristic of pregnancy in rodents.

Relationship between fractional pancreatic beta cell area and fasting plasma glucose concentration in monkeys.

AIMS/HYPOTHESIS: We sought to establish the relationship between fasting plasma glucose concentrations and pancreatic fractional beta cell area in adult cynomolgus monkeys (Macaca fascicularis). METHODS: Fasting plasma glucose and pancreatic fractional beta cell area were measured in 18 control and 17 streptozotocin-treated adult primates (17.0 +/- 1.2 vs 15.4 +/- 1.2 years old). RESULTS: Fasting plasma glucose was increased (12.0 +/- 2.0 vs 3.4 +/- 0.1 mmol/l, p < 0.01) and fractional beta cell area was decreased (0.62 +/- 0.13% vs 2.49 +/- 0.35%, p < 0.01) in streptozotocin-treated monkeys. The relationship between fasting plasma glucose and pancreatic fractional beta cell area was described by a wide range of beta cell areas in controls. In streptozotocin-treated monkeys there was an inflection of fasting blood glucose at approximately 50% of the mean beta cell area in controls with a steep increase in blood glucose for each further decrement in beta cell area. CONCLUSIONS/INTERPRETATION: In adult non-human primates a decrement in fractional beta cell area of approximately 50% or more leads to loss of glycaemic control.

CHOP Contributes to, But Is Not the Only Mediator of, IAPP Induced ß-Cell Apoptosis.

The islet in type 2 diabetes is characterized by ß-cell loss, increased ß-cell apoptosis, and islet amyloid derived from islet amyloid polypeptide (IAPP). When protein misfolding protective mechanisms are overcome, human IAPP (h-IAPP) forms membrane permeant toxic oligomers that induce ß-cell dysfunction and apoptosis. In humans with type 2 diabetes (T2D) and mice transgenic for h-IAPP, endoplasmic reticulum (ER) stress has been inferred from nuclear translocation of CCAAT/enhancer-binding protein homologous protein (CHOP), an established mediator of ER stress. To establish whether h-IAPP toxicity is mediated by ER stress, we evaluated diabetes onset and ß-cell mass in h-IAPP transgenic (h-TG) mice with and without deletion of CHOP in comparison with wild-type controls. Diabetes was delayed in h-TG CHOP(-/-) mice, with relatively preserved ß-cell mass and decreased ß-cell apoptosis. Deletion of CHOP attenuates dysfunction of the autophagy/lysosomal pathway in ß-cells of h-TG mice, uncovering a role for CHOP in mediating h-IAPP-induced dysfunction of autophagy. As deletion of CHOP delayed but did not prevent h-IAPP-induced ß-cell loss and diabetes, we examined CHOP-independent stress pathways. JNK, a target of the IRE-1pTRAF2 complex, and the Bcl-2 family proapoptotic mediator BIM, a target of ATF4, were comparably activated by h-IAPP expression in the presence and absence of CHOP. Therefore, although these studies affirm that CHOP is a mediator of h-IAPP-induced ER stress, it is not the only one. Therefore, suppression of CHOP alone is unlikely to be a durable therapeutic strategy to protect against h-IAPP toxicity because multiple stress pathways are activated.

Islet amyloid polypeptide in human insulinomas. Evidence for intracellular amyloidogenesis.

Amyloid deposits that characteristically form in the pancreatic islets of patients with non-insulin-dependent diabetes mellitus (NIDDM) and in insulinomas are both derived from islet amyloid polypeptide (IAPP). Evidence from previous studies has suggested that deposition of IAPP-derived amyloid is related to inherent amyloidogenic sequences present within normal human IAPP, together with an increased production and local concentration of IAPP. However, whether the aggregation of IAPP to form amyloid fibrils is primarily an intra- or extracellular event is not clear. To address this question, we studied 20 human insulinomas by light and electron microscopy. By light microscopy, amyloid deposits were demonstrated in 13 of 20 (65%) human insulinomas. Furthermore, evaluation of Congo red-stained tumor sections showed small, globular or irregular, congophilic amyloid deposits within the cytoplasm of many tumor cells in 10 of 13 (77%) amyloid-containing insulinomas. Dense, punctate areas of IAPP immunoreactivity within tumor cells corresponded with the congophilic intracellular deposits. Ubiquitin immunoreactivity also was observed as punctate intracellular labeling and within large extracellular amyloid deposits. Among the 10 insulinomas available for electron microscopic evaluation, pathological IAPP-immunoreactive (immunogold) deposits were found in 3 of 5 insulinomas in which amyloid was demonstrated by light microscopy and in none of 5 tumors found negative for amyloid by light microscopy. Morphology of IAPP-immunoreactive deposits varied from those with the classical distinct 7- to 10-nm diameter nonbranching fibrils to those with distinct but faint fibrillarity to those without discernable fibrils.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a 15;17 translocation in every patient with acute promyelocytic leukemia.

Cytogenetic specimens were obtained from blood or bone marrow in 27 patients with acute promyelocytic leukemia, including four with the microgranular variant. A 15;17 translocation was identified in 21 to 100 percent of metaphase cells from all 27 patients. The structural rearrangement was identical in every case, and the breakpoints were assigned to 15q22 and 17q21.1. Twelve patients had complete remission, and two (both with the microgranular variant) have had unmaintained continuous remission longer than four years. These data indicate that the 15;17 translocation may be found in every patient with acute promyelocytic leukemia if optimal chromosome analysis is performed.

Lymphoepithelioma-like carcinoma of the lung.

Originally described in the nasopharynx, lymphoepitheliomas are undifferentiated carcinomas with prominent lymphoid infiltration. Recently this tumor has been described in a number of other sites. An association with prior infection by the Epstein-Barr virus has been recognized in cases from the nasopharynx. We report four cases of lymphoepithelioma-like carcinoma arising in the lung. Epstein-Barr virus serology indicated prior infection in two cases, one of which was also found by in situ hybridization to have the virus genome. Lymphoepithelioma-like carcinoma must be considered in the differential diagnosis of primary lung tumors, particularly when an extensive infiltrate by lymphoid tissue is present and may even suggest lymphoma.

Analysis of lymphoepithelioma and lymphoepithelioma-like carcinomas for Epstein-Barr viral genomes by in situ hybridization.

Lymphoepithelioma of the nasopharynx has a strong association with Epstein-Barr virus (EBV). To test the hypothesis that lymphoepithelioma-like carcinomas occurring at other sites are also associated with EBV virus, we used in situ hybridization to analyze 20 cases of lymphoepithelioma and histologically similar lesions and five basaloid squamous cell carcinomas for evidence of EBV genomes. EBV genomes were demonstrated in six of six lymphoepitheliomas of the nasopharynx but in none of five basaloid squamous cell carcinoma. Only one of 14 lymphoepithelioma-like carcinomas was found to contain EBV genomes. The single positive lymphoepithelioma-like carcinoma occurred in the lung of an Asian patient, suggesting that ethnic or geographic influences may be important in determining whether EBV is associated with these nonnasopharyngeal neoplasms. Despite their histologic similarity, most lymphoepithelioma-like carcinomas probably have a different pathogenesis from nasopharyngeal lymphoepithelioma.

Lymphocytic mastopathy associated with infiltrating lobular breast carcinoma.

A case of lymphocytic mastopathy (LM) and concomitant infiltrating lobular breast carcinoma is described. The inflammatory infiltrate cuffed areas of carcinoma. The LM changes also affected lobules that had not been infiltrated by the carcinoma, although adjacent areas contained both in situ and infiltrating tumour. A minor portion of the inflammation was distributed throughout the ducts. The striking lobulocentricity of the inflammatory infiltrate corresponds to previous reports of LM, and distinguishes this case from the inflammatory response commonly associated with breast carcinomas. It is highly likely that the breast carcinoma induced the LM changes in lobules not yet harbouring tumour.

Modestly increased beta cell apoptosis but no increased beta cell replication in recent-onset type 1 diabetic patients who died of diabetic ketoacidosis.

AIMS/HYPOTHESIS: Type 1 diabetes is characterised by a deficit in beta cell mass thought to be due to immune-mediated increased beta cell apoptosis. Beta cell turnover has not been examined in the context of new-onset type 1 diabetes with diabetic ketoacidosis. METHODS: Samples of pancreas were obtained at autopsy from nine patients, aged 12 to 38 years (mean 24.3+/-3.4 years), who had had type 1 diabetes for less than 3 years before death due to diabetic ketoacidosis. Samples of pancreas obtained at autopsy from nine non-diabetic cases aged 11.5 to 38 years (mean 24.2+/-3.4 years) were used as control. Fractional beta cell area (insulin staining), beta cell replication (insulin and Ki67 staining) and beta cell apoptosis (insulin and TUNEL staining) were measured. RESULTS: In pancreas obtained at autopsy from recent-onset type 1 diabetes patients who had died of diabetic ketoacidosis, the beta cell deficit varied from 70 to 99% (mean 90%). The pattern of beta cell loss was lobular, with almost all beta cells absent in most pancreatic lobules; islets in lobules not devoid of beta cells had reduced or a near-normal complement of beta cells. Beta cell apoptosis was increased in recent-onset type 1 diabetes, but to a surprisingly modest degree given the marked hyperglycaemia (30 mmol/l), acidosis and presumably high NEFA. Beta cell replication, scattered pancreatic beta cells and beta cells in exocrine ducts were not increased in recent-onset type 1 diabetes. CONCLUSIONS/INTERPRETATION: These findings do not support the notion of active beta cell regeneration by replication in new-onset type 1 diabetes under conditions of diabetic ketoacidosis. The gluco-lipotoxicity reported in isolated human islets may be less evident in vivo.

Pancreas volumes in humans from birth to age one hundred taking into account sex, obesity, and presence of type-2 diabetes.

Our aims were (1) by computed tomography (CT) to establish a population database for pancreas volume (parenchyma and fat) from birth to age 100 years, (2) in adults, to establish the impact of gender, obesity, and the presence or absence of type-2 diabetes on pancreatic volume (parenchyma and fat), and (3) to confirm the latter histologically from pancreatic tissue obtained at autopsy with a particular emphasis on whether pancreatic fat is increased in type-2 diabetes. We measured pancreas volume in 135 children and 1,886 adults (1,721 nondiabetic and 165 with type-2 diabetes) with no history of pancreas disease who had undergone abdominal CT scan between 2003 and 2006. Pancreas volume was computed from the contour of the pancreas on each CT image. In addition to total pancreas volume, parenchymal volume, fat volume, and fat/parenchyma ratio (F/P ratio) were determined by CT density. We also quantified pancreatic fat in autopsy tissue of 47 adults (24 nondiabetic and 23 with type-2 diabetes). During childhood and adolescence, the volumes of total pancreas, pancreatic parenchyma, and fat increase linearly with age. From age 20-60 years, pancreas volume reaches a plateau (72.4 +/- 25.8 cm(3) total; 44.5 +/- 16.5 cm(3) parenchyma) and then declines thereafter. In adults, total ( approximately 32%), parenchymal ( approximately 13%), and fat ( approximately 68%) volumes increase with obesity. Pancreatic fat content also increases with aging but is not further increased in type-2 diabetes. We provide lifelong population data for total pancreatic, parenchymal, and fat volumes in humans. Although pancreatic fat increases with aging and obesity, it is not increased in type-2 diabetes.

Increased islet beta cell replication adjacent to intrapancreatic gastrinomas in humans.

AIMS/HYPOTHESIS: Type 1 and type 2 diabetes are characterised by a beta cell deficit. Islet hyperplasia has been described in patients with Zollinger-Ellison syndrome secondary to gastrin-producing tumours (gastrinomas), and gastrin therapy has increased beta cell mass in rodents and human islets in vitro. In the present studies we addressed the following questions: (1) In pancreas specimens from gastrinoma cases, is the fractional beta cell area increased? (2) If so, is this restricted to tumour-adjacent islets or also present in tumour-distant islets? (3) Is new beta cell formation (beta cell replication and islet neogenesis) increased and beta cell apoptosis decreased in pancreas specimens from gastrinoma cases? METHODS: Pancreas was obtained at surgery from four patients with Zollinger-Ellison syndrome caused by pancreatic gastrinomas and 15 control subjects at autopsy. RESULTS: Islet fractional beta cell area (p<0.001), islet size (p<0.001) and beta cell replication (Ki67 staining) (p<0.05) were increased in islets adjacent to the tumours, but not in tumour-distant pancreas, compared with control subjects. We did not observe any differences in beta cell apoptosis or in the number of insulin-positive cells in ducts either adjacent to or distant from the tumour. CONCLUSIONS/INTERPRETATION: One or more factors released by human gastrinomas increase beta cell replication in islets immediately adjacent to the tumour, but not in tumour-distant islets. While these findings demonstrate that adult human beta cells can be driven into the cell cycle, they caution against the therapeutic usefulness of gastrin, since islets located >1 cm away from the gastrinomas did not exhibit changes in beta cell turnover, despite markedly elevated systemic gastrin levels sufficient to cause severe gastrointestinal symptoms.

Direct evidence of attempted beta cell regeneration in an 89-year-old patient with recent-onset type 1 diabetes.

AIMS/HYPOTHESIS: We investigated whether there was evidence of attempted beta cell regeneration in the pancreas obtained from a patient with recent-onset type 1 diabetes, and if so by what mechanism this occurred. SUBJECTS, MATERIALS AND METHODS: We examined pancreas tissue from a lean 89-year-old patient (BMI 18.0 kg/m(2)) with recent-onset type 1 diabetes who had had a distal pancreatectomy to remove a low-grade pancreatic intraepithelial neoplasia. RESULTS: In the tumour-free tissue, the fractional beta cell area was 0.54+/-0.2% of pancreas area (about one-third of that in non-diabetic humans). CD3-positive T lymphocytes and macrophages had infiltrated the majority of the islets. Subclassification of the T cell population revealed a predominance of CD8-positive cells over CD4-positive cells. Beta cell apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling [TUNEL] staining) was greatly increased, consistent with ongoing immune-mediated beta cell destruction. There was also a marked increase (more than approximately 100-fold) in the frequency of beta cell replication (0.69+/-0.15% Ki67-positive beta cells) in all blocks examined. CONCLUSIONS/INTERPRETATION: The present report provides direct evidence of attempted beta cell regeneration through the mechanism of beta cell replication in a case of newly diagnosed type 1 diabetes, and affirms that beta cell apoptosis is an important mechanism for beta cell loss in type 1 diabetes.

Sustained beta cell apoptosis in patients with long-standing type 1 diabetes: indirect evidence for islet regeneration?

AIMS/HYPOTHESIS: Type 1 diabetes is widely held to result from an irreversible loss of insulin-secreting beta cells. However, insulin secretion is detectable in some people with long-standing type 1 diabetes, indicating either a small population of surviving beta cells or continued renewal of beta cells subject to ongoing autoimmune destruction. The aim of the present study was to evaluate these possibilities. MATERIALS AND METHODS: Pancreatic sections from 42 individuals with type 1 diabetes and 14 non-diabetic individuals were evaluated for the presence of beta cells, beta cell apoptosis and replication, T lymphocytes and macrophages. The presence and extent of periductal fibrosis was also quantified. RESULTS: Beta cells were identified in 88% of individuals with type 1 diabetes. The number of beta cells was unrelated to duration of disease (range 4-67 years) or age at death (range 14-77 years), but was higher (p<0.05) in individuals with lower mean blood glucose. Beta cell apoptosis was twice as frequent in type 1 diabetes as in control subjects (p<0.001), but beta cell replication was rare in both groups. The increased beta cell apoptosis in type 1 diabetes was accompanied by both increased macrophages and T lymphocytes and a marked increase in periductal fibrosis (p<0.001), implying chronic inflammation over many years, consistent with an ongoing supply of beta cells. CONCLUSIONS/INTERPRETATION: Most people with long-standing type 1 diabetes have beta cells that continue to be destroyed. The mechanisms underlying increased beta cell death may involve both ongoing autoimmunity and glucose toxicity. The presence of beta cells despite ongoing apoptosis implies, by definition, that concomitant new beta cell formation must be occurring, even after long-standing type 1 diabetes. We conclude that type 1 diabetes may be reversed by targeted inhibition of beta cell destruction.

Ultrastructural characterization of de novo and secondary leukemias.

Cells from 95 patients with acute leukemia were studied by cytochemistry, light microscopy, and transmission electron microscopy (TEM), and were classified according to the French-American-British (FAB) guidelines. This group included 63 patients with acute nonlymphocytic leukemia (ANLL) de novo, 18 with acute lymphocytic leukemia (ALL), and 14 with ANLL as a second malignancy. In addition, 13 cases of chronic myelocytic leukemia in blast crisis were studied. Ultrastructural examination resulted in reclassification of 6 cases of ANLL de novo; two of these were reclassified from M2 (myeloblastic leukemia with maturation) to M3 variant (microgranular variant of hypergranular promyelocytic leukemia). The classification of the cases of CML in blast crisis was identical by light microscopy and TEM. IN 1 case of myeloblastic crisis, however, basophilic granules were demonstrated by TEM but were not appreciated by light microscopy. Classification of the cases of secondary leukemia was possible by light microscopy and cytochemistry in all 14 cases, but was often difficult since the cytochemical reactions were usually less intense than in de novo ANLL. This was particularly true in those cases classified as M1, and in such cases, TEM was required to confirm the diagnosis.