Ugo Boggi

Coxsackie B4 virus infection of β cells and natural killer cell insulitis in recent-onset type 1 diabetic patients

Type 1 diabetes is characterized by T cell-mediated autoimmune destruction of pancreatic β cells. Several studies have suggested an association between Coxsackie enterovirus seroconversion and onset of disease. However, a direct link between β cell viral infection and islet inflammation has not been established. We analyzed pancreatic tissue from six type 1 diabetic and 26 control organ donors. Immunohistochemical, electron microscopy, whole-genome ex vivo nucleotide sequencing, cell culture, and immunological studies demonstrated Coxsackie B4 enterovirus in specimens from three of the six diabetic patients. Infection was specific of β cells, which showed nondestructive islet inflammation mediated mainly by natural killer cells. Islets from enterovirus-positive samples displayed reduced insulin secretion in response to glucose and other secretagogues. In addition, virus extracted from positive islets was able to infect β cells from human islets of nondiabetic donors, causing viral inclusions and signs of pyknosis. None of the control organ donors showed signs of viral infection. These studies provide direct evidence that enterovirus can infect β cells in patients with type 1 diabetes and that infection is associated with inflammation and functional impairment.

MicroRNA-21 in Pancreatic Cancer: Correlation with Clinical Outcome and Pharmacologic Aspects Underlying Its Role in the Modulation of Gemcitabine Activity

MicroRNA-21 (miR-21) was reported to be overexpressed and contributes to invasion and gemcitabine resistance in pancreatic ductal adenocarcinoma (PDAC). The aim of this study was to evaluate whether miR-21 expression was associated with the overall survival (OS) of PDAC patients treated with gemcitabine and to provide mechanistic insights for new therapeutic targets. miR-21 expression was evaluated in cells (including 7 PDAC cell lines, 7 primary cultures, fibroblasts, and a normal pancreatic ductal cell line) and tissues (neoplastic specimens from 81 PDAC patients and normal ductal samples) isolated by laser microdissection. The role of miR-21 on the pharmacologic effects of gemcitabine was studied with a specific miR-21 precursor (pre-miR-21). Patients with high miR-21 expression had a significantly shorter OS both in the metastatic and in the adjuvant setting. Multivariate analysis confirmed the prognostic significance of miR-21. miR-21 expression in primary cultures correlated with expression in their respective tissues and with gemcitabine resistance. Pre-miR-21 transfection significantly decreased antiproliferative effects and apoptosis induction by gemcitabine, whereas matrix metalloproteinase (MMP)-2/MMP-9 and vascular endothelial growth factor expression were upregulated. Addition of inhibitors of phosphoinositide 3-kinase and mammalian target of rapamycin resulted in decrease of phospho-Akt and prevented pre-miR-21-induced resistance to the proapoptotic effects of gemcitabine. miR-21 expression correlated with outcome in PDAC patients treated with gemcitabine. Modulation of apoptosis, Akt phosphorylation, and expression of genes involved in invasive behavior may contribute to the role of miR-21 in gemcitabine chemoresistance and to the rational development of new targeted combinations.

Functional and morphological alterations of mitochondria in pancreatic beta cells from type 2 diabetic patients

Aims/hypothesisLittle information is available on the insulin release properties of pancreatic islets isolated from type 2 diabetic subjects. Since mitochondria represent the site where important metabolites that regulate insulin secretion are generated, we studied insulin release as well as mitochondrial function and morphology directly in pancreatic islets isolated from type 2 diabetic patients.MethodsIslets were prepared by collagenase digestion and density gradient purification, and insulin secretion in response to glucose and arginine was assessed by the batch incubation method. Adenine nucleotides, mitochondrial membrane potential, the expression of UCP-2, complex I and complex V of the respiratory chain, and nitrotyrosine levels were evaluated and correlated with insulin secretion.ResultsCompared to control islets, diabetic islets showed reduced insulin secretion in response to glucose, and this defect was associated with lower ATP levels, a lower ATP/ADP ratio and impaired hyperpolarization of the mitochondrial membrane. Increased protein expression of UCP-2, complex I and complex V of the respiratory chain, and a higher level of nitrotyrosine were also found in type 2 diabetic islets. Morphology studies showed that control and diabetic beta cells had a similar number of mitochondria; however, mitochondrial density volume was significantly higher in type 2 diabetic beta cells.Conclusions/interpretationIn pancreatic beta cells from type 2 diabetic subjects, the impaired secretory response to glucose is associated with a marked alteration of mitochondrial function and morphology. In particular, UCP-2 expression is increased (probably due to a condition of fuel overload), which leads to lower ATP, decreased ATP/ADP ratio, with consequent reduction of insulin release.

Inflammatory cells contribute to the generation of an angiogenic phenotype in pancreatic ductal adenocarcinoma

Background: Inflammatory cells contribute to the growth and spread of human malignancies by producing molecules that enhance tumour invasiveness. Aims: To characterise the inflammatory infiltrate in pancreatic ductal adenocarcinoma and to analyse its contribution to angiogenesis and its prognostic relevance. Methods: Immunohistochemistry was used to identify inflammatory cells and evaluate the expression of proangiogenic and prolymphangiogenic molecules (vascular endothelial growth factor A (VEGF-A), VEGF-C, and basic fibroblast growth factor (bFGF)) by inflammatory and cancer cells in 137 pancreatic cancers. Intratumorous microvessel density (IMD) was assessed using CD34 as an endothelial cell marker. Results: There were significantly more mast cells and macrophages in pancreatic cancers than in normal pancreas and the number of mast cells directly correlated with the presence of lymph node metastases. However, there was no relation between numbers of infiltrating inflammatory cells and the presence of chronic pancreatitis (CP)-like changes in the parenchyma surrounding the tumour. Double immunostaining revealed that both pancreatic mast cells and macrophages express VEGF-A, VEGF-C, and bFGF. These factors were also expressed in the tumour cells in many cases. The numbers of VEGF-A expressing tumour cells and bFGF expressing tumour and inflammatory cells significantly correlated with IMD. Moreover, tumours with higher IMD had higher numbers of infiltrating mast cells and macrophages. Conclusions: Mononuclear inflammatory cells of the non-specific immune response are recruited to pancreatic cancer tissues independent of the presence of CP-like changes, may influence the metastatic capacity of the cancer cells, and may contribute to the development of tumours with high angiogenic activity.

Autophagy in human type 2 diabetes pancreatic beta cells

Aims/hypothesisBeta cell loss contributes to type 2 diabetes, with increased apoptosis representing an underlying mechanism. Autophagy, i.e. the physiological degradation of damaged organelles and proteins, may, if altered, be associated with a distinct form of cell death. We studied several features of autophagy in beta cells from type 2 diabetic patients and assessed the role of metabolic perturbation and pharmacological intervention.MethodsPancreatic samples were obtained from organ donors and isolated islets prepared both by collagenase digestion and density gradient centrifugation. Beta cell morphology and morphometry were studied by electron microscopy. Gene expression studies were performed by quantitative RT-PCR.ResultsUsing electron microscopy, we observed more dead beta cells in diabetic (2.24 ± 0.53%) than control (0.66 ± 0.52%) samples (p < 0.01). Massive vacuole overload (suggesting altered autophagy) was associated with 1.18 ± 0.54% dead beta cells in type 2 diabetic samples and with 0.36 ± 0.26% in control samples (p < 0.05). Density volume of autophagic vacuoles and autophagosomes was significantly higher in diabetic beta cells. Unchanged gene expression of beclin-1 and ATG1 (also known as ULK1), and reduced transcription of LAMP2 and cathepsin B and D was observed in type 2 diabetic islets. Exposure of non-diabetic islets to increased NEFA concentration led to a marked increase of vacuole accumulation, together with enhanced beta cell death, which was associated with decreased LAMP2 expression. Metformin ameliorated autophagy alterations in diabetic beta cells and beta cells exposed to NEFA, a process associated with normalisation of LAMP2 expression.Conclusions/interpretationBeta cells in human type 2 diabetes have signs of altered autophagy, which may contribute to loss of beta cell mass. To preserve beta cell mass in diabetic patients, it may be necessary to target multiple cell-death pathways.

Islet inflammation and CXCL10 in recent-onset type 1 diabetes

Type 1 diabetes results from a T cell‐mediated destruction of insulin‐producing pancreatic β cells. Little is known on local factors contributing to migration of T cells to pancreatic tissue. We recently demonstrated evidence of viral infection in β cells in several recent‐onset type 1 diabetes patients. Islet inflammation was analysed in a series of new‐ or recent‐onset type 1 diabetic patients and non‐diabetic control subjects. Autoimmune T cell reactivity was studied in lymphocytes derived from pancreas‐draining lymph nodes of one recent‐onset type 1 diabetes patient in partial clinical remission. Insulitic lesions were characterized by presence of β cells, elevated levels of the chemokine CXCL10 and infiltration of lymphocytes expressing the corresponding chemokine receptor CXCR3 in all pancreatic lesions of type 1 diabetes patients, regardless of enterovirus infection of β cells. CXCR3 and CXCL10 were undetectable in pancreata of non‐diabetic control subjects. T cells isolated from draining lymph nodes of a recent‐onset patient with virally infected β cells and in clinical remission reacted with multiple islet autoantigens and displayed a mixed interferon (IFN)‐γ/interleukin (IL)‐10 cytokine pattern. Our data point to CXCL10 as an important cytokine in distressed islets that may contribute to inflammation leading to insulitis and β cell destruction, regardless of local viral infection. We demonstrate further pro‐ and anti‐inflammatory islet autoreactivity, indicating that different adaptive and innate immune responses may contribute to insulitis and β cell destruction.

Gliclazide protects human islet beta-cells from apoptosis induced by intermittent high glucose.

Decreased beta‐cell mass, mainly due to apoptosis, is crucial for the development and progression of type 2 diabetes. Chronic exposure to high glucose levels is a probable underlying mechanism, whereas the role of oral anti‐diabetic agents (sulphonylureas in particular) is still unsettled.

Kidney preservation with university of Wisconsin and Celsior solution: a prospective multicenter randomized study.

Background. Although the University of Wisconsin (U.W.) solution continues to be the most commonly used for intra-abdominal organs, a new solution, Celsior, already used for heart and lungs, has been proposed for kidney and liver preservation. The aim of this research was to assess the effect of Celsior as compared with U.W. on immediate graft function and a 2-year follow-up of kidney transplants. Methods. A prospective multicenter randomized study was designed to evaluate the efficacy of the Celsior solution in the clinical preservation of the kidney. In this report, we present the data collected as of September 2000. One hundred donors were included in the trial resulting in 187 renal transplants. Ninety-nine kidneys were stored in Celsior solution and 88 in U.W. solution. The groups were comparable with regard to donor and recipient characteristics. Results. Delayed graft function occurred in 31.3% of the Celsior group and in 33.9% of the U.W. group (P =n.s.). Mean serum creatinine levels and mean daily urinary output were also comparable. Two year graft survival in kidneys preserved with Celsior was 84% as compared with 75% for U.W.-preserved kidneys without any significant statistical difference. Conclusions. Our data show that the preservation of kidneys in Celsior solution in a clinical setting is equivalent to that of U.W. solution. When using Celsior during multiple-organ donor harvesting it would be possible to perform an in situ flush of all intra-abdominal and intrathoracic organs with a single cold storage solution.

Effects of pancreas-kidney transplantation on diabetic retinopathy

The effects of pancreas transplantation (PTx) on diabetic retinopathy (DR) are still debated. We studied the course of DR in 48 patients (age: 40 ± 7 years; males/females 26/22, body mass index (BMI): 23.0 ± 2.4 kg/m2, duration of diabetes: 24 ± 8 years) bearing a successful PTx (combined with a kidney). Follow‐up ranged 6–60 months (median: 17 months). Before transplantation, according to the Eurodiab Study classification, 12 patients (25%) had nonproliferative retinopathy (NPDR; mild, moderate or severe), and 36 patients (75%) had laser‐treated and/or proliferative retinopathy (LT/PDR). During the follow‐up, in the NPDR group improvement/deterioration was defined as regression/progression to a lower/higher retinopathy grade; in the LT/PTD group, stabilization was defined as no new neo‐vessel formation or development of new lesions requiring laser‐treatment. In the NPDR group, five (41.7%) patients improved of one or more lesion grading, three (25%) patients showed no change, and four (33.3%) patients progressed of one grade. In the LT/PDR group, the post‐transplant data were: stabilization in 35 (97%) patients, and worsening in one (3%) patient. The number of improved/stabilized patients was significantly higher in the transplanted than in a control group of nontransplanted type 1 diabetic patients. In conclusion, despite a relatively short follow‐up period, successful PTx in our cohort of patients was associated with improvement and/or stabilization of DR in the majority of recipients.

Pancreas preservation with University of Wisconsin and Celsior solutions: a single center, prospective, randomized pilot study

Background. Celsior is an extracellular-type, low-viscosity, preservation solution already used for heart, lung, liver, and kidney transplantation. We report the results of a single-center, prospective, randomized pilot study specifically designed to compare the safety profile of Celsior solution with University of Wisconsin (UW) solution in clinical pancreas transplantation. Methods. A total of 105 consecutive procurements were randomized to graft preservation with UW (n=53) solution or Celsior (n=52) solution. The groups were comparable with regard to all donor and recipient characteristics. Results. Five grafts were discarded and 100 grafts (50 UW vs. 50 Celsior) were transplanted. Mean cold and warm ischemia times were 11.0±2.1 hr and 37.2±6.0 min for UW compared with 10.8±1.8 hr and 38.1±5.9 min for Celsior (P =not significant). Delayed endocrine pancreas function was recorded in one graft preserved with UW solution. Eleven recipients (UW 12% vs. Celsior 10%, P =not significant) required a relaparotomy. The mean serum levels of glucose, amylase, and lipase remained comparable between the study arms at equivalent intervals after transplantation. One recipient died with functioning grafts in each study arm; two further grafts were lost to arterial thrombosis (Celsior) and chronic rejection (UW), respectively. Actuarial 1-year patient and graft survival rates overlapped in the two study arms (98% and 96%, respectively). Conclusions. Within the range of cold ischemia time reported in this study, UW and Celsior solutions have similar safety profiles for pancreas preservation.