Inflammation and ventricular-vascular coupling in hypertensive patients with metabolic syndrome.

BACKGROUND AND AIMS: Metabolic syndrome (MetS) is currently considered to raise the risk for type 2 diabetes and cardiovascular events. It has been suggested that part of this risk excess may be due to a cluster of additional factors associated with MetS. We aimed to investigate the role of inflammation on the ventricular-vascular coupling in patients with MetS. METHODS AND RESULTS: We enrolled a total of 227 hypertensive patients (106 with MetS and 121 without MetS) matched for age and gender. Aortic pulse wave velocity (aPWV), intima-media thickness (IMT) and high sensitivity C-reactive protein (CRP) increased according to the number of MetS components. Patients with MetS showed increased aPWV (11.5 ± 3.7 vs. 10.3 ± 2.5 m/s, P = 0.03) compared with controls. In a model adjusted for age, sex, heart rate and mean blood pressure, aPWV resulted increased in patients with CKD (beta 1.29 m/s, 95%CI 0.61-1.96 m/s, P < 0.001) and MetS (beta 0.89 m/s, 95%CI 0.28-1.51 m/s, P = 0.005). After additional adjustment for CRP and IMT, the slope of aPWV was respectively reduced by 16% and 62%, suggesting that inflammation and intima-media thickening could contribute to aortic stiffening in patients with MetS. In these patients, aPWV was also associated with left-ventricular mass index (beta 0.79 g/m2.7, 95%CI 0.05-1.52 g/m2.7, P = 0.05). CONCLUSION: MetS is characterized by an inflammation-dependent acceleration in cardiovascular ageing. This pattern of pathophysiological abnormalities may contribute to amplify the burden of cardiovascular risk in patients with MetS.

Childhood infectious diseases and risk of multiple myeloma: an analysis of the Italian multicentre case-control study.

Common childhood infectious diseases have been associated with a reduced risk of following haematopoietic malignancies, but investigations on multiple myeloma (MM) are scarce. Information about 213 MM cases and 1128 healthy controls were obtained from a multicentre population-based Italian case-control study. The association between chickenpox, measles, mumps, pertussis and rubella and the MM risk was estimated by unconditional logistic regression, adjusting for age, gender and residence area. No association was found between MM risk and any considered infectious disease. The number of infections was slightly inversely associated with the risk of MM, but statistical significance was not reached (OR 0.87, 95% CI 0.55-1.4 for 1-2 diseases vs. none and OR 0.68, 95% CI 0.41-1.1 for 3-5 diseases, respectively, P = 0.131). We did not find a clear evidence that common infections during childhood are associated with the subsequent risk of developing MM.

Detecting familial hypercholesterolemia by serum lipid profile screening in a hospital setting: Clinical, genetic and atherosclerotic burden profile.

BACKGROUND AND AIMS: Familial hypercholesterolemia (FH) is underdiagnosed and public cholesterol screening may be useful to find new subjects. In this study, we aim to investigate the prevalence of FH patients in a hospital screening program and evaluate their atherosclerotic burden using intima-media thickness (IMT). METHODS AND RESULTS: We screened 1575 lipid profiles and included for genetic analysis adults with a low-density lipoprotein (LDL) cholesterol >190 mg/dL and triglycerides <200 mg/dL and first-degree child relatives with LDL cholesterol >160 mg/dL and triglycerides <200 mg/dL. The diagnosis of FH was presumed by Dutch Lipid Clinic Network (DLCN) criteria and confirmed by the presence of the genetic variant. Mean common carotid intima-media thickness (IMT) was assessed using consensus criteria. After confirming LDL cholesterol value and excluding secondary hypercholesterolemia, 56 subjects with a DLCN ≥4 performed genetic analysis. Of these, 26 had an FH genetic variant. The proportion of patients with a mutation having a DLCN score of 6-8 was 75%; in individuals with a DLCN score >8 it was 100%. Mean IMT was higher in FH patients compared to non FH (0.73 [0.61-0.83] vs 0.71 [0.60-0.75] mm, p < 0.01). Moreover, we detected two mutations not previously described. Finally, simple regression analysis showed a correlation of IMT with LDL cholesterol >190 mg/dL and corneal arcus (p < 0.01 and p < 0.001, respectively). CONCLUSIONS: A hospital screening was useful to detect FH subjects with increased atherosclerosis. Also, next-generation sequencing was able to detect new FH mutations.

High intake of dietary advanced glycation end-products is associated with increased arterial stiffness and inflammation in subjects with type 2 diabetes.

BACKGROUND AND AIMS: Modern diets are high in advanced glycation end-products (dAGEs), derived from processing methods, exerting a pivotal role in promoting atherosclerotic risk. In this cross-sectional study we investigate the relationship between dAGE intake, arterial stiffness, inflammatory profile and macronutrient composition, in subjects with type 2 diabetes without overt cardiovascular disease. METHODS AND RESULTS: Arterial stiffness, carboxy-methyl-lysine, endogenous secretory receptor for AGEs (esRAGE), high sensitivity C reactive protein (hs-CRP), S100A12 and macronutrient intake were evaluated in 85 subjects with type 2 diabetes. The subjects were stratified into two groups according to dAGE consumption: high and low dAGE intake (≥ or <15.000 kU/day, respectively). Subjects with high dAGE intake (n = 45) showed a higher augmentation, augmentation index and pulse wave velocity (PWV) compared with those subjects with low dAGE intake (18 ± 5.4 vs 12.2 ± 6.3 mmHg, P < 0.05; 38.3 ± 5.4 vs 29.3 ± 10%; 9.2 ± 1.4 m/sec vs 7.9 ± 1.7, P < 0.05, respectively). hs-CRP were higher in subjects with high dAGE intake [0.42 (0.18-0.54) vs 0.21 (0.14-0.52) mg/dL, P < 0.05] whereas esRAGE plasma levels were lower [0.16 (0.23-0.81) vs 0.2 (0.14-0.54) ng/dL, P < 0.05]. Simple regression analysis showed a correlation between dAGEs and fat intake. Multivariate analysis showed an independent association between augmentation, systolic blood pressure (BP) and dAGE consumption; BMI and esRAGE were the major determinants of PWV. CONCLUSIONS: Our data suggests that a chronic high dAGE diet could lead to a vascular dysfunction and inflammatory activation, contributing to the development of vascular complications in subjects with type 2 diabetes. Testing this hypothesis may represent a direction of future research.

Intracellular and extracellular miRNome deregulation in cellular models of NAFLD or NASH: Clinical implications.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) represents the most common chronic liver disease in industrialized countries. NAFLD has the potential to progress through the inflammatory phase of nonalcoholic steatohepatitis (NASH) to fibrosis, cirrhosis, and hepatocellular carcinoma. Identifying patients at risk for this transition is a relevant clinical challenge. The complexity of these phenotypes in vivo made necessary the development of in vitro models in order to dissect the molecular signalling affected in NAFLD and NASH, but also to identify potential circulating biomarkers. METHODS AND RESULTS: We profiled the expression of 754 cellular and medium-secreted human miRNAs in HepG2 cells after lipotoxic (Palmitate, model of NASH) or not-lipotoxic stimuli (Oleate-Palmitate, model of NAFLD). Results were validated through Single TaqMan assays. We performed computational analysis of miRNA targets and pathways. Oleate-palmitate treatment induced a variation of 2.8% and 10% of total miRNAs in cells and medium, respectively; palmitate treatment caused 10% and 19% intracellular and extracellular miRNA deregulation, respectively. We validated miR-126, miR-150, miR-223, miR-483-3p, miR-1226*, and miR-1290 deregulation. Through computational analysis, we observed that targets of both intracellular and extracellular DE miRNAs were involved in processes associated with the onset and progression of NAFLD and NASH, such as fatty acid metabolism, apoptosis and inflammation. CONCLUSIONS: These data would be useful to elucidate the role of miRNAs in the pathogenesis and progression of the NAFLD spectrum, but they also allow the identification of novel potential biomarkers for differential diagnosis to be tested in vivo.

Lipid and liver abnormalities in haemoglobin A1c-defined prediabetes and type 2 diabetes.

BACKGROUND AND AIMS: We aimed to investigate lipid abnormalities and liver steatosis in patients with HbA1c-defined prediabetes and type 2 diabetes compared to individuals with HbA1c-defined normoglycaemia. METHODS AND RESULTS: Ninety-one subjects with prediabetes according to HbA1c, i.e. from 5.7 to 6.4% (39-46 mmol/mol), 50 newly diagnosed patients with HbA1c-defined type 2 diabetes (HbA1c ≥6.5% [≥48 mmol/mol]), and 67 controls with HbA1c lower than 5.7% (<39 mmol/mol), were studied. Fasting blood samples for lipid profiles, fatty liver index (FLI), bioimpedance analysis, ultrasound scan of the liver, and BARD (body mass index, aspartate aminotransferase/alanine aminotransferase ratio, diabetes) score for evaluation of liver fibrosis, were performed in all subjects. In comparison to controls, subjects with prediabetes were characterised by: lower apolipoprotein AI and HDL cholesterol levels, higher blood pressure, triglycerides levels and apolipoprotein B/apolipoprotein AI ratio, higher FLI, increased prevalence of and more severe hepatic steatosis, similar BARD score, and higher total body fat mass. In comparison to subjects with diabetes, subjects with prediabetes exhibited: similar blood pressure and apolipoprotein B/apolipoprotein AI ratio, similar FLI, reduced prevalence of and less severe hepatic steatosis, lower BARD score, increased percent fat and lower total body muscle mass. In comparison to controls, subjects with diabetes showed: lower apolipoprotein AI and HDL cholesterol levels, higher blood pressure and triglycerides levels, higher FLI, increased prevalence of and more severe hepatic steatosis, higher BARD score, and higher total body muscle mass. Moreover, HbA1c was correlated with BMI, HOMA-IR, triglycerides, HDL cholesterol, AST, and ALT. CONCLUSIONS: Subjects with HbA1c-defined prediabetes and type 2 diabetes, respectively, are characterised by abnormalities in lipid profile and liver steatosis, thus exhibiting a severe risk profile for cardiovascular and liver diseases.

A local glucagon-like peptide 1 (GLP-1) system in human pancreatic islets.

AIMS/HYPOTHESIS: Glucagon-like peptide 1 (GLP-1) is a major incretin, mainly produced by the intestinal L cells, with beneficial actions on pancreatic beta cells. However, while in vivo only very small amounts of GLP-1 reach the pancreas in bioactive form, some observations indicate that GLP-1 may also be produced in the islets. We performed comprehensive morphological, functional and molecular studies to evaluate the presence and various features of a local GLP-1 system in human pancreatic islet cells, including those from type 2 diabetic patients. METHODS: The presence of insulin, glucagon, GLP-1, proconvertase (PC) 1/3 and PC2 was determined in human pancreas by immunohistochemistry with confocal microscopy. Islets were isolated from non-diabetic and type 2 diabetic donors. GLP-1 protein abundance was evaluated by immunoblotting and matrix-assisted laser desorption-ionisation-time of flight (MALDI-TOF) mass spectrometry. Single alpha and beta cell suspensions were obtained by enzymatic dissociation and FACS sorting. Glucagon and GLP-1 release were measured in response to nutrients. RESULTS: Confocal microscopy showed the presence of GLP-1-like and PC1/3 immunoreactivity in subsets of alpha cells, whereas GLP-1 was not observed in beta cells. The presence of GLP-1 in isolated islets was confirmed by immunoblotting, followed by mass spectrometry. Isolated islets and alpha (but not beta) cell fractions released GLP-1, which was regulated by glucose and arginine. PC1/3 (also known as PCSK1) gene expression was shown in alpha cells. GLP-1 release was significantly higher from type 2 diabetic than from non-diabetic isolated islets. CONCLUSIONS/INTERPRETATION: We have shown the presence of a functionally competent GLP-1 system in human pancreatic islets, which resides in alpha cells and might be modulated by type 2 diabetes.

Separate impact of metabolic syndrome and altered glucose tolerance on early markers of vascular injuries.

OBJECTIVE: We investigated the separate impact of metabolic syndrome (MS) and altered glucose tolerance on early markers of vascular injuries. METHODS: Intima-media thickness (IMT) and pulse wave analysis (PWA), were evaluated in 132 overweight or obese subjects, with (MS(+)) or without (MS(-)) MS; subjects were further classified as normotolerant (NT) or with altered glucose tolerance (AGT) according to a 2 h oral glucose tolerance test (OGTT). RESULTS: In MS(+) patients, IMT was higher than in the MS(-) group, and PWA revealed higher augmentation pressure (Aug, the contribution that wave reflection makes to systolic arterial pressure) and lower subendocardial viability ratio (SEVR, an estimate of myocardial perfusion). When analyzed according to glucose tolerance, IMT was higher in MS(+)NT subjects and AGT patients with and without MS, vs. MS(-)NT subjects. Logistic regression modeling showed that both AGT and MS were independently associated with increased IMT. However, only MS remained associated with IMT after adjustment for age. SEVR was reduced only in MS(+) patients, independently of glucose tolerance. In both groups, Aug and AugI were higher in the AGT group, but the correlation with 2 h-plasma glucose disappeared when corrected for age. CONCLUSION: Both MS and AGT altered IMT, but the effect of AGT disappears when age is added to the multiple regression model. In contrast, arterial stiffness was affected differently in the two categories: in subjects with MS, the subendocardial viability ratio (an estimate of myocardial perfusion) was impaired, while in subjects with AGT, both Aug and AugI were increased. These data suggest that applying the definition of MS might help to better characterize cardiovascular risk in subjects with altered glucose tolerance or obesity.

Effects of metformin on oxidative stress, adenine nucleotides balance, and glucose-induced insulin release impaired by chronic free fatty acids exposure in rat pancreatic islets.

BACKGROUND: In rat pancreatic islets, chronic exposure to high free fatty acid (FFA) levels impairs insulin secretion and ß cell mass. The mechanisms underlying this defect are not completely understood. Since islets have intrinsically low anti-oxidant enzyme defense, oxidative stress might be responsible for ß cell damage. AIM: In this study, we investigated if FFA could induce oxidative stress in rat pancreatic islets and if metformin might reverse adverse effects. MATERIAL AND METHODS: We cultured rat pancreatic islets in the presence or absence of FFA (oleate/palmitate 2:1, 2 mM) for 72 h. In some experiments, we used metformin (2.5 µg/ml) during the last 24 h. RESULTS: In our model, glucosestimu lated insulin release was markedly reduced (p<0.005) after chronic FFA exposure, and the ATP/ADP ratio was altered (p<0.05). We observed a significant increase of reactive oxygen species (ROS) (p<0.001), malondialdehyde a lipid peroxidation product (p<0.01) and nitric oxide (NO) levels in the culture media (p<0.001). Inducible NO synthase (iNOS) and heat shock protein-70 (HSP-70) protein expression were also increased (p<0.001 and p<0.01, respectively). When metformin was present during the last 24 h of culture, insulin secretion was restored, and the ATP/ADP ratio was normalized. ROS production, NO production, lipid peroxidation, iNOS and HSP-70 protein expression levels had decreased. CONCLUSIONS: These data indicate that, in rat pancreatic islets, chronic exposure to high FFA induces oxidative stress and that metformin, by reducing this effect, may have a direct beneficial effect on insulin secretion impaired by lipotoxicity.

Early phase insulin secretion is increased in subjects with normal fasting glucose and metabolic syndrome: a premature feature of beta-cell dysfunction.

BACKGROUND AND AIMS: Metabolic syndrome (MS) has been mainly related to insulin resistance, but the role of changes in insulin secretion has not been thoroughly investigated. METHODS AND RESULTS: Using an oral glucose tolerance test (OGTT) we studied beta-cell function and insulin sensitivity in subjects with normal fasting glucose with and without MS, and their relationship to fatty liver which was evaluated by abdominal-ultrasonography. In MS early phase insulin secretion, as measured by insulinogenic index (IG(30)), was increased (p<0.05) independently from insulin sensitivity. Furthermore IG(30) was progressively higher as the number of factors needed for the diagnosis of MS increased (p<0.01). Insulin and C-peptide AUC were also increased (p<0.01 and p<0.05, respectively) but, in contrast to IG(30), these differences disappeared when ISI was used as a covariate. After OGTT, 51% of the subjects with MS had altered post-load glucose tolerance compared to 24.9% without MS (p<0.01). In both groups, the altered glucose tolerance was associated with a similar IG(30) reduction. In normo-tolerant subjects with MS the IG(30) was higher (+54.1%, p<0.01), and this elevation occurred irrespective of ISI; however, the beta-cell compensatory capacity for insulin resistance (disposition index) was impaired (p<0.001). Fatty liver was more frequent (p<0.001) and more severe (p<0.01) in MS, and it was significantly related to total AUC-insulin (p<0.001), independently from ISI. CONCLUSION: These findings indicate that the prevalence of altered tolerance is more frequent in subjects with normal fasting glucose and MS. The hyperinsulinemia might not only be an adaptive response to insulin resistance, but a primary defect of beta-cell function contributing to glucose intolerance.

Therapeutic options for elderly diabetic subjects: open label, randomized clinical trial of insulin glargine added to oral antidiabetic drugs versus increased dosage of oral antidiabetic drugs.

Glycemic control in elderly persons with type 2 diabetes mellitus (T2DM) is challenging because they are more likely to have other age-associated medical conditions and to experience hypoglycemia during intensive therapy. A best therapeutic strategy for these patients has not yet been defined. We investigated the efficacy and safety of adding once-daily insulin glargine to patients' current oral antidiabetic drugs (OAD) regimen, compared to increasing the OAD doses. The study enrolled patients aged 65 years or more, with poor glycemic control. Patients were randomized to two groups and entered a 3-week titration period in which their actual therapy was adjusted to meet the study's glycemic goals, by either adding insulin glargine to current therapy (group A, 27 patients) or increasing current OAD dosages (group B, 28 patients). Thereafter, therapies were continued unchanged for a 24-week observation period. The mean therapeutic dosage of insulin glargine in group A was 14.9 IU/day (SD = 5.0 IU/day). During the observation period, mean levels of glycosylated hemoglobin (HbA1c) reduced by 1.5% in group A and 0.6% in group B (P = 0.381). An HbA1c level <7.0% was achieved by five patients in each group. Mean fasting blood glucose levels reduced by 29 and 15% in groups A and B, respectively (P = 0.029). Group A had fewer total hypoglycemic events (23 vs. 79, P = 0.030) and fewer patients experiencing any such event (9 vs. 17, P = 0.045). Neither a serious hypoglycemic event nor other adverse event occurred. These results suggest that, compared to increasing OAD dosage, the addition of insulin glargine to current OAD therapy is as effective but safer in terms of the risk for hypoglycemia in elderly patients with T2DM.

Molecular determinants of insulin resistance, cell apoptosis and lipid accumulation in non-alcoholic steatohepatitis.

BACKGROUNDS AND AIMS: Non-alcoholic-steatohepatitis (NASH) is closely related to insulin resistance, but it is unknown whether insulin resistance may be localized in hepatocytes. This study investigates insulin signalling in liver tissue from NASH, and the molecular mechanisms by which insulin-resistance could lead to liver damage (apoptosis). Moreover, to investigate the mechanisms of lipid overload we studied key enzymes in hepatocytes lipid metabolism. METHODS AND RESULTS: In liver specimens from 11 patients with NASH and 7 histological normal livers, we measured total and phosphorylated Akt (active form), Bax and Bcl-2 by Western-blot analysis. In addition, we studied AMP-activated protein Kinase and Carnitine-Palmitoyl-Transferase-1 gene expression, key regulators of non-esterified fatty acid synthesis and oxidation, by reverse transcription polymerase chain reaction. In NASH, phosphorylated Akt was impaired (104.3+/-10.6 vs 152.6+/-22.4 AU, p<0.002) and correlated with necroinflammatory score (r=-0.62; p<0.05). Bax/Bcl-2 ratio was increased in NASH. Moreover, we observed a decrease of AMP-activated protein Kinase (10.74+/-6 vs 144.7+/-41.6 AU, p<0.0001) and Carnitine-Palmitoyl-Transferase-1 gene expression (38.7+/-14.6 vs 192.1+/-26.2 AU, p<0.0001), and both were correlated with steatosis score (r=-0.56, p<0.05, r=-0.87, p<0.05 respectively). CONCLUSIONS: Akt, a key molecule of insulin signalling and cell apoptosis is impaired in NASH, suggesting an important role of hepatic insulin resistance in liver failure. Moreover, decreased non-esterified fatty acid oxidation may cause hepatic lipid overload.

Effects of n-3 polyunsaturated fatty acids in subjects with nonalcoholic fatty liver disease.

BACKGROUND: Long-chain polyunsaturated fatty acid omega-3 levels are decreased in the hepatic tissue of patients with nonalcoholic fatty liver disease. Polyunsaturated fatty acids are negative regulators of hepatic lipogenesis and attenuate the inflammatory response in mice. AIM: To investigate whether polyunsaturated fatty acid may be effective in the treatment of nonalcoholic fatty liver disease. METHODS: Forty patients with nonalcoholic fatty liver disease were randomized into two groups for treatment of 6 months duration. Group DP (n=20) received an AHA recommended diet and polyunsaturated fatty acid 2g/day; Group D (n=20) received only the AHA regular diet. Outcome measurements were fatty liver assessed by abdominal ultrasound, liver aminotransferase and tumour necrosis factor-alpha serum levels, and insulin resistance assessed by HOMA(IR). RESULTS: After 6 months of treatment, the DP group displayed a decrease in alanine aminotransferase levels (p<0.01), as well as in triglyceride levels (p<0.01), serum tumour necrosis factor-alpha levels (p<0.05) and in HOMA(IR) (p<0.05). In the D group, no significant modification was observed. In the DP group, complete fatty liver regression was observed in 33.4% of the patients, and an overall reduction in 50%. In contrast, no patient achieved complete regression in the D group, whereas some amount of reduction occurred in 27.7% of the patients; the remaining 72.2% did not change. CONCLUSION: Our results indicate that alanine aminotransferase, triglyceride and serum tumour necrosis factor-alpha levels, as well as fatty liver improved after polyunsaturated fatty acid administration.

Cellular and molecular effects of protons: apoptosis induction and potential implications for cancer therapy.

Due to their ballistic precision, apoptosis induction by protons could be a strategy to specifically eliminate neoplastic cells. To characterize the cellular and molecular effects of these hadrons, we performed dose-response and time-course experiments by exposing different cell lines (PC3, Ca301D, MCF7) to increasing doses of protons and examining them with FACS, RT-PCR, and electron spin resonance (ESR). Irradiation with a dose of 10 Gy of a 26,7 Mev proton beam altered cell structures such as membranes, caused DNA double strand breaks, and significantly increased intracellular levels of hydroxyl ions, are active oxygen species (ROS). This modified the transcriptome of irradiated cells, activated the mitochondrial (intrinsic) pathway of apoptosis, and resulted in cycle arrest at the G2/M boundary. The number of necrotic cells within the irradiated cell population did not significantly increase with respect to the controls. The effects of irradiation with 20 Gy were qualitatively as well as quantitatively similar, but exposure to 40 Gy caused massive necrosis. Similar experiments with photons demonstrated that they induce apoptosis in a significantly lower number of cells and in a temporally delayed manner. These data advance our knowledge on the cellular and molecular effects of proton irradiation and could be useful for improving current hadrontherapy protocols.

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

AIMS/HYPOTHESIS: Little 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. METHODS: Islets 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. RESULTS: Compared 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/INTERPRETATION: In 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.

Th2 cytokines have a partial, direct protective effect on the function and survival of isolated human islets exposed to combined proinflammatory and Th1 cytokines.

Studies in rodents have suggested that Th2 and Th3 cytokines can be effective in reducing proinflammatory and Th1 cytokine-induced islet damage. Whether this is the case with human islets and might be due to a direct action of Th2 and Th3 cytokines is not known. In the present study, we evaluated whether Th2 (500 U/ml IL-4 plus 100 U/ml IL-10) or Th3 (5 ng/ml TGF-1beta) cytokines may prevent the derangements induced on isolated human islets by prolonged (12 or 72 h) exposure to combined proinflammatory (50 U/ml IL-1beta, 1000 U/ml TNF alpha) and Th1 (1000 U/ml interferon gamma) cytokines. Compared with control islets, cells preincubated for 12 or 72 h with proinflammatory and Th1 cytokines showed a significant decrease of glucose-stimulated insulin secretion and a significant increase of nitrites production. The addition of IL-4 plus IL-10 or TGF-1beta in the medium prevented these cytostatic effects in the 12-h incubation experiments, but not after the 72-h exposure period. IL-1beta, interferon gamma, and TNF alpha caused no major change in either islet cell survival or Bcl-2 and Bax mRNA expression after a 12-h incubation; however, a marked increase in the amount of dead cells, with a major decrease of Bcl-2 mRNA expression, was observed after 72 h. The presence of Th2, but not of Th3, cytokines significantly reduced beta-cell death, without any major effect on Bcl-2 and Bax mRNA expression. These results suggest that Th2 and (at lower extent) Th3 cytokines may have a partial, direct protective effect on isolated human islets exposed to the cytostatic and cytotoxic action of proinflammatory and Th1 cytokines.

Chronic exposure to high leucine impairs glucose-induced insulin release by lowering the ATP-to-ADP ratio.

Exposure of rat pancreatic islets to 20 mM leucine for 24 h reduced insulin release in response to glucose (16.7 and 22.2 mM). Insulin release was normal when the same islets were stimulated with leucine (40 mM) or glyburide (1 microM). To investigate the mechanisms responsible for the different effect of these secretagogues, we studied several steps of glucose-induced insulin secretion. Glucose utilization and oxidation rates in leucine-precultured islets were similar to those of control islets. Also, the ATP-sensitive K(+) channel-independent pathway of glucose-stimulated insulin release, studied in the presence of 30 mM K(+) and 250 microM diazoxide, was normal. In contrast, the ATP-to-ADP ratio after stimulation with 22.2 mM glucose was reduced in leucine-exposed islets with respect to control islets. The decrease of the ATP-to-ADP ratio was due to an increase of ADP levels. In conclusion, prolonged exposure of pancreatic islets to high leucine levels selectively impairs glucose-induced insulin release. This secretory abnormality is associated with (and might be due to) a reduced ATP-to-ADP ratio. The abnormal plasma amino acid levels often present in obesity and diabetes may, therefore, affect pancreatic islet insulin secretion in these patients.

Melanosynthesis, differentiation, and apoptosis in Kupffer cells from Rana esculenta.

It is well known that amphibian Kupffer cells (KCs) contain eumelanins. In this paper, we demonstrate through a molecular analysis that Rana esculenta KCs synthesize high levels of mRNA for tyrosinase and through cytochemistry that they possess dopa oxidase activity: both these data prove that frog KCs are capable of autonomously synthesizing eumelanins. On the other hand, by using a highly sensitive reverse transcription-polymerase chain reaction assay we clearly show that in mammalian KCs the tyrosinase gene is not expressed. Quite unexpectedly, we have detected tyrosinase mRNA in Rana esculenta spleen, lung, and heart; to explain this finding, we suggest that it could be due to the presence of pigmented macrophages within the spleen, that probably behave as KCs, and of melanophores in lung and heart. It also may be hypothesized that the Rana esculenta tyrosinase gene, as opposed to its mammalian counterpart, is expressed in many cell types because its promoter contains sequences that are recognized by widely synthesized transcription factors. Our experiments also demonstrate that there is an inverse correlation between the amount of tyrosinase mRNA and melanin content, and that populations of terminally differentiated KCs are characterized by a high degree of apoptosis. Based on these data, we propose that differentiating KCs start accumulating eumelanins, as a result of previous expression of high levels of tyrosinase and of dopa oxidase activity, acquire the full KC phenotype (characterized by both phagocytic and melanosynthetic ability), and then undergo apoptosis. Accordingly, we propose that these cells could represent an interesting model to study, at the molecular level, the relationship between differentiation, specific gene expression, and programmed cell death in higher eukaryotes.

Hospital-acquired malaria transmitted by contaminated gloves.

We describe two cases of malaria occurring in a malaria-free zone in two in-patients, two weeks after a case of Plasmodium falciparum malaria, acquired in Burkina Faso, had been admitted to the same ward. After reviewing the techniques used by nursing staff, we conclude that transmission probably occurred via gloves contaminated following manipulation of venous cannulae and drip lines of the patient with Burkina Faso-acquired malaria and which had not been discarded before manipulating the intravenous lines of the other two patients. Nosocomial transmission of unusual and potentially life-threatening infections should be taken into consideration in those settings where compliance with universal precautions is not rigorous.

Bovine islets are less susceptible than human islets to damage by human cytokines.

BACKGROUND: The potential benefits of islet xenografting in type 1 diabetes include the intriguing, but still unanswered, possibility that the grafted xenoislets may be less subjected to human autoimmune attack. Cytokines may play a major role in the pathogenesis of autoimmune diabetes by causing impairment of insulin release and pancreatic islet cell toxicity. METHODS: We compared insulin secretion, islet cell death and survival, inducible nitric oxide synthase (iNOS) mRNA expression, nitrite production, and Bcl-2 and Bax mRNA expression in isolated human and large mammal (bovine) islets exposed to 50 U/ml recombinant human interleukin-1, 1,000 U/ml recombinant human tumor necrosis factor-alpha and 1,000 U/ml recombinant human interferon-gamma. RESULTS: After 24-hr exposure, a marked decrease of glucose-stimulated insulin secretion was observed with human, but not with bovine islets. After 48-hr exposure, human, but not bovine, pancreatic islets showed a significantly higher percentage of apoptotic cells compared to controls. Treatment of human islets with human cytokines induced up-regulation of iNOS mRNA, increased levels of nitrites, and down-regulation of Bcl-2 mRNA, with unchanged levels of Bax mRNA. These parameters were not affected by cytokines in bovine islets. CONCLUSIONS: Bovine islets are less susceptible than human islets to the effects of human cytokines, which may be a potential advantage of xenotransplantation.