Claudia Placidi

Immunomodulatory Function of Bone Marrow-Derived Mesenchymal Stem Cells in Experimental Autoimmune Type 1 Diabetes

Human clinical trials in type 1 diabetes (T1D) patients using mesenchymal stem cells (MSC) are presently underway without prior validation in a mouse model for the disease. In response to this void, we characterized bone marrow-derived murine MSC for their ability to modulate immune responses in the context of T1D, as represented in NOD mice. In comparison to NOD mice, BALB/c-MSC mice were found to express higher levels of the negative costimulatory molecule PD-L1 and to promote a shift toward Th2-like responses in treated NOD mice. In addition, transfer of MSC from resistant strains (i.e., nonobese resistant mice or BALB/c), but not from NOD mice, delayed the onset of diabetes when administered to prediabetic NOD mice. The number of BALB/c-MSC trafficking to the pancreatic lymph nodes of NOD mice was higher than in NOD mice provided autologous NOD-MSC. Administration of BALB/c-MSC temporarily resulted in reversal of hyperglycemia in 90% of NOD mice (p = 0.002). Transfer of autologous NOD-MSC imparted no such therapeutic benefit. We also noted soft tissue and visceral tumors in NOD-MSC-treated mice, which were uniquely observed in this setting (i.e., no tumors were present with BALB/c- or nonobese resistant mice-MSC transfer). The importance of this observation remains to be explored in humans, as inbred mice such as NOD may be more susceptible to tumor formation. These data provide important preclinical data supporting the basis for further development of allogeneic MSC-based therapies for T1D and, potentially, for other autoimmune disorders.

Pancreatic islet amyloidosis, β-cell apoptosis, and α-cell proliferation are determinants of islet remodeling in type-2 diabetic baboons

β-Cell dysfunction is an important factor in the development of hyperglycemia of type-2 diabetes mellitus, and pancreatic islet amyloidosis (IA) has been postulated to be one of the main contributors to impaired insulin secretion. The aim of this study was to evaluate the correlation of IA with metabolic parameters and its effect on islets of Langerhans remodeling and relative endocrine-cell volume in baboons. We sequenced the amylin peptide, determined the fibrillogenic propensities, and evaluated pancreatic histology, clinical and biochemical characteristics, and endocrine cell proliferation and apoptosis in 150 baboons with different metabolic status. Amylin sequence in the baboon was 92% similar to humans and showed superimposable fibrillogenic propensities. IA severity correlated with fasting plasma glucose (FPG) (r = 0.662, P < 0.001) and HbA1c (r = 0.726, P < 0.001), as well as with free fatty acid, glucagon values, decreased homeostasis model assessment (HOMA) insulin resistance, and HOMA-B. IA severity was associated with a decreased relative β-cell volume, and increased relative α-cell volume and hyperglucagonemia. These results strongly support the concept that IA and β-cell apoptosis in concert with α-cell proliferation and hypertrophy are key determinants of islets of Langerhans “dysfunctional remodeling” and hyperglycemia in the baboon, a nonhuman primate model of type-2 diabetes mellitus. The most important determinants of IA were age and FPG (R2 = 0.519, P < 0.0001), and different FPG levels were sensitive and specific to predict IA severity. Finally, a predictive model for islet amyloid severity was generated with age and FPG as required variables.

Altered Insulin Receptor Signalling and β-Cell Cycle Dynamics in Type 2 Diabetes Mellitus

Insulin resistance, reduced β-cell mass, and hyperglucagonemia are consistent features in type 2 diabetes mellitus (T2DM). We used pancreas and islets from humans with T2DM to examine the regulation of insulin signaling and cell-cycle control of islet cells. We observed reduced β-cell mass and increased α-cell mass in the Type 2 diabetic pancreas. Confocal microscopy, real-time PCR and western blotting analyses revealed increased expression of PCNA and down-regulation of p27-Kip1 and altered expression of insulin receptors, insulin receptor substrate-2 and phosphorylated BAD. To investigate the mechanisms underlying these findings, we examined a mouse model of insulin resistance in β-cells – which also exhibits reduced β-cell mass, the β-cell-specific insulin receptor knockout (βIRKO). Freshly isolated islets and β-cell lines derived from βIRKO mice exhibited poor cell-cycle progression, nuclear restriction of FoxO1 and reduced expression of cell-cycle proteins favoring growth arrest. Re-expression of insulin receptors in βIRKO β-cells reversed the defects and promoted cell cycle progression and proliferation implying a role for insulin-signaling in β-cell growth. These data provide evidence that human β- and α-cells can enter the cell-cycle, but proliferation of β-cells in T2DM fails due to G1-to-S phase arrest secondary to defective insulin signaling. Activation of insulin signaling, FoxO1 and proteins in β-cell-cycle progression are attractive therapeutic targets to enhance β-cell regeneration in the treatment of T2DM.

Chronic hyperglycemia impairs insulin secretion by affecting insulin receptor expression, splicing, and signaling in RIN beta cell line and human islets of Langerhans.

Recent evidence suggests that insulin signaling through the insulin receptor A type (Ex11−), regulates insulin gene transcription. Because chronic hyperglycemia negatively affects insulin receptor function and regulates alternative splicing of the insulin receptor, we inquired whether chronic exposure of pancreatic β‐cells to high glucose results in alterations in insulin signaling due to changes in insulin receptor expression and relative abundance of its spliced isoforms. Our results demonstrate that the insulin receptor is localized in insulin secretory vescicles in human pancreatic β‐cells. Furthermore, we find that alterations in insulin expression and secretion caused by chronic exposure to high glucose are paralleled by decreased insulin receptor expression and increased relative abundance of the Ex11+ isoform in both human islets and RIN β‐cells. PDX‐1 and HMGI(Y) transcription factors are down‐regulated by high glucose. These changes are associated with defects in insulin signaling involving insulin receptor‐associated PI 3‐kinase/Akt/PHAS‐I pathway in RIN β‐cells. Re‐expression in RIN β‐cells chronically exposed to high glucose of the Ex11−, but not the Ex11+, isoform restored insulin mRNA expression. These data suggest that changes in early steps of insulin receptor signaling may play a role in determining β‐cell dysfunction caused by chronic hyperglycemia.

Proteomics Reveals Novel Oxidative and Glycolytic Mechanisms in Type 1 Diabetic Patients' Skin Which Are Normalized by Kidney-Pancreas Transplantation

Background In type 1 diabetes (T1D) vascular complications such as accelerated atherosclerosis and diffused macro-/microangiopathy are linked to chronic hyperglycemia with a mechanism that is not yet well understood. End-stage renal disease (ESRD) worsens most diabetic complications, particularly, the risk of morbidity and mortality from cardiovascular disease is increased several fold. Methods and Findings We evaluated protein regulation and expression in skin biopsies obtained from T1D patients with and without ESRD, to identify pathways of persistent cellular changes linked to diabetic vascular disease. We therefore examined pathways that may be normalized by restoration of normoglycemia with kidney-pancreas (KP) transplantation. Using proteomic and ultrastructural approaches, multiple alterations in the expression of proteins involved in oxidative stress (catalase, superoxide dismutase 1, Hsp27, Hsp60, ATP synthase δ chain, and flavin reductase), aerobic and anaerobic glycolysis (ACBP, pyruvate kinase muscle isozyme, and phosphoglycerate kinase 1), and intracellular signaling (stratifin-14-3-3, S100-calcyclin, cathepsin, and PPI rotamase) as well as endothelial vascular abnormalities were identified in T1D and T1D+ESRD patients. These abnormalities were reversed after KP transplant. Increased plasma levels of malondialdehyde were observed in T1D and T1D+ESRD patients, confirming increased oxidative stress which was normalized after KP transplant. Conclusions Our data suggests persistent cellular changes of anti-oxidative machinery and of aerobic/anaerobic glycolysis are present in T1D and T1D+ESRD patients, and these abnormalities may play a key role in the pathogenesis of hyperglycemia-related vascular complications. Restoration of normoglycemia and removal of uremia with KP transplant can correct these abnormalities. Some of these identified pathways may become potential therapeutic targets for a new generation of drugs.

TTF1 expression in normal lung neuroendocrine cells and related tumors: immunohistochemical study comparing two different monoclonal antibodies

Thyroid transcription factor-1 (TTF1) regulates lung morphogenesis and differentiation, and its immunohistochemical expression is used for identifying lung neoplasms. The 8G7G3/1 antibody has been used in previous studies, but a different and more sensitive anti-TTF1 antibody, named SPT24, has become commercially available. Since the immunohistochemical expression of TTF1 in normal lung neuroendocrine (NE) cells has not been previously investigated and its utility in the diagnosis of lung NE tumors is a controversial issue, we studied the TTF1 expression in normal adult and fetal lungs, in 83 pulmonary NE neoplasms, in 131 non-lung NE tumors and in 36 metastases from these neoplasms using these two antibodies. A TTF1 immunoreactivity was demonstrated in normal fetal and adult NE cells when using the SPT24 clone. Conversely, using the 8G7G3/1 antibody, only rare fetal neuroendocrine cells were TTF1 positive while adult NE cells were negative. The SPT24 clone identified TTF1 expression in more carcinoids, most of them peripherally located, and poorly differentiated NE carcinomas than the 8G7G3/1 clone. Non-pulmonary well-differentiated NE tumors were negative for both antibodies. Among the 45 non-pulmonary poorly differentiated NE carcinomas 11% were positive for 8G7G3/1 and 18% for SPT24. TTF1 expression in metastases perfectly reflected that detected in the related primary tumors. Our results indicate that the SPT24 antibody is more sensitive than the 8G7G3/1 clone for labeling lung carcinoids and it appears particularly useful in detecting peripheral neoplasms. In addition, the expression of TTF1 in normal NE cells suggests a possible role for the transcription factor in their development and differentiation.

The Glial Glutamate Transporter 1 (GLT1) Is Expressed by Pancreatic β-Cells and Prevents Glutamate-induced β-Cell Death

Glutamate is the major excitatory neurotransmitter of the central nervous system (CNS) and may induce cytotoxicity through persistent activation of glutamate receptors and oxidative stress. Its extracellular concentration is maintained at physiological concentrations by high affinity glutamate transporters of the solute carrier 1 family (SLC1). Glutamate is also present in islet of Langerhans where it is secreted by the α-cells and acts as a signaling molecule to modulate hormone secretion. Whether glutamate plays a role in islet cell viability is presently unknown. We demonstrate that chronic exposure to glutamate exerts a cytotoxic effect in clonal β-cell lines and human islet β-cells but not in α-cells. In human islets, glutamate-induced β-cell cytotoxicity was associated with increased oxidative stress and led to apoptosis and autophagy. We also provide evidence that the key regulator of extracellular islet glutamate concentration is the glial glutamate transporter 1 (GLT1). GLT1 localizes to the plasma membrane of β-cells, modulates hormone secretion, and prevents glutamate-induced cytotoxicity as shown by the fact that its down-regulation induced β-cell death, whereas GLT1 up-regulation promoted β-cell survival. In conclusion, the present study identifies GLT1 as a new player in glutamate homeostasis and signaling in the islet of Langerhans and demonstrates that β-cells critically depend on its activity to control extracellular glutamate levels and cellular integrity.

Serotonin-producing enterochromaffin cell tumors of the pancreas: clinicopathologic study of 15 cases and comparison with intestinal enterochromaffin cell tumors

Objectives: Serotonin-producing tumors of the pancreas are rare endocrine neoplasms composed of enterochromaffin (EC) cells that have been mainly described in the literature as case reports. This study analyzes the clinicopathologic features of a series of pancreatic EC cell neoplasms and their similarities to and differences from intestinal EC cell tumors. Methods: The morphological, immunohistochemical, ultrastructural, and fluorescent in situ hybridization features of 15 pancreatic and 20 intestinal serotonin-producing neoplasms were compared. In addition, we reviewed the literature on pancreatic serotonin-producing tumors to better understand the clinicopathologic features of this rare tumor type. Results: The lack of substance P and acidic fibroblast growth factor immunoreactivity; the low immunohistochemical expression of CDX2, vesicular monoamine transporter 1, connective tissue growth factor, and prostatic acid phosphatase; the lack of S100-positive sustentacular cells; the strong expression of vesicular monoamine transporter 2; and peculiar ultrastructural features characterize pancreatic EC cell tumors and differentiate them from intestinal ones, although both categories show similar chromosome 18 cytogenetic alterations. The review of the literature indicates that pancreatic functioning tumors associated with the carcinoid syndrome arise in younger patients and are larger, more frequently malignant, and more aggressive neoplasms than pancreatic nonfunctioning ones. Conclusions: Pancreatic EC cell tumors show several different morphological features compared with related intestinal tumors despite similar cytogenetic alterations on chromosome 18.

Morphological and ultrastructural features of human islet grafts performed in diabetic nude mice

Islet transplantation is a new therapeutic approach to type 1 diabetes mellitus. However, in several patients insulin levels are not restored and the glycemic control is inadequate. To clarify the cause of graft failure, the authors investigated with light and electron microscopy some human islet grafts before and after transplantation under the kidney capsule of streptozotocin-induced diabetic nude mice. In isolated islets, both pre- and post-transplantation, the endocrine component was scarcely represented, the β/α cell ratio was reduced, and β cells showed degenerative aspects such as apoptosis, immature secretory granules, and amylin fibrils deposition. The authors conclude that islet graft failure may be due to an insufficent β cell mass related to their distress probably caused by anoxia and/or overstimulation.

Disproportionate Hyperproinsulinemia, β-Cell Restricted Prohormone Convertase 2 Deficiency, and Cell Cycle Inhibitors Expression by Human Islets Transplanted into Athymic Nude Mice: Insights into Nonimmune-Mediated Mechanisms of Delayed Islet Graft Failure:

To learn more about nonimmune-mediated islet graft failure, we transplanted different preparations (preps) of isolated human islets under the kidney capsule of streptozotocin (STZ)-diabetic nude mice. One month after the implantation of 1,000 or 2,000 islets, grafts were harvested for morphological, immunohistochemical, and ultrastructural analysis. Only a single islet prep cured the diabetes out of all the recipients, while the remaining preps showed only partial function after the implantation of 2,000 islets. Transplanted mice showed high circulating proinsulin levels but, with the exclusion of those bearing curative grafts, relatively low mature insulin levels. Engrafted β-cells showed positive carboxypeptidase E (CPE) and prohormone convertase 1 (PC1) staining, while prohormone convertase 2 (PC2) was undetectable. In contrast, PC2 was abundantly expressed by engrafted α-cells. Moreover, engrafted β-cells did not show evidence of replication, and preapoptotic β-cells, with intra- and extracellular amyloid deposition, were detected with electron microscopy. Cell cycle inhibitors p16INK4, p21WAF1, and p27Kip1 were abundantly expressed in the islet grafts and showed a predominant nuclear localization. In conclusion, diabetic nude mice transplanted with human islets showed disproportionate hyperproinsulinemia and graft evidence of β-cell restricted PC2 depletion, amyloid deposition and β-cell death, and lack of β-cell replication with nuclear translocation of p27Kip1 and p21WAF1 that together may contribute to delayed graft failure.