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Demyelination is a key pathogenic feature ofmultiple sclerosis (MS). Here, we evaluated the astrocyte contribution to myelin loss and focused on the neurotrophin receptor TrkB, whose up-regulation on the astrocyte finely demarcated chronic demyelinated areas in MS and was paralleled by neurotrophin loss. Mice lacking astrocyte TrkB were resistant to demyelination induced by autoimmune or toxic insults, demonstrating that TrkB signaling in astrocytes fostered oligodendrocyte damage. In vitro and ex vivo approaches highlighted that astrocyte TrkB supported scar formation and glia proliferation even in the absence of neurotrophin binding, indicating TrkB transactivation in response to inflammatory or toxic mediators. Notably, our neuropathological studies demonstrated copper dysregulation in MS and model lesions and TrkB-dependent expression of copper transporter (CTR1) on glia cells during neuroinflammation. In vitro experiments evidenced that TrkB was critical for the generation of glial intracellular calcium flux and CTR1 up-regulation induced by stimuli distinct from neurotrophins. These events led to copper uptake and release by the astrocyte, and in turn resulted in oligodendrocyte loss. Collectively, these data demonstrate a pathogenic demyelination mechanism via the astrocyte release of copper and open up the possibility of restoring copper homeostasis in the white matter as a therapeutic target in MS.

Effects of Kidney-Pancreas Transplantation on Atherosclerotic Risk Factors and Endothelial Function in Patients With Uremia and Type 1 Diabetes Paolo Fiorina 1 , Ennio La Rocca 1 , Massimo Venturini 2 , Fabio Minicucci 3 , Isabella Fermo 4 , Rita Paroni 4 , Armando D’Angelo 4 , Marisa Sblendido 3 , Valerio Di Carlo 5 , Marco Cristallo 5 , Alessandro Del Maschio 2 , Guido Pozza 1 and Antonio Secchi 1 1 Internal Medicine 2 Radiology 3 Neurophysiology 4 Laboratory Medicine, and 5 General Surgery, San Raffaele Scientific Institute, Universitâ Vita e Salute, Milan, Italy Abstract Cardiovascular disease and the development of coronary artery disease play a pivotal role in increasing mortality in patients with type 1 diabetes. The aim of our study was to evaluate the effects of pancreas transplantation on atherosclerotic risk factors, endothelial-dependent dilation (EDD), and progression of intima media thickness (IMT) in patients with uremia and type 1 diabetes after kidney-alone (KA) or kidney-pancreas (KP) transplantation. A cross-sectional study comparing two groups of patients with type 1 diabetes was performed. Sixty patients underwent KP transplantation and 30 patients underwent KA transplantation. Age and cardiovascular risk profile were comparable in patients before transplantation. In all patients, atherosclerotic risks factors (lipid profile, fasting and postmethionine load plasma homocysteine, von Willebrand factor levels, d -dimer fragments, and fibrinogen) were assessed and Doppler echographic evaluation of IMT and endothelial function with flow-mediated and nitrate dilation of the brachial artery was performed. Twenty healthy subjects were chosen as controls (C) for EDD. Compared with patients undergoing KA transplantation, patients undergoing KP transplantation showed lower values for HbA 1c (KP = 6.2 ± 0.1% vs. KA = 8.4 ± 0.5%; P < 0.01), fasting homocysteine (KP = 14.0 ± 0.7 μmol/l vs. KA = 19.0 ± 2.0 μmol/l; P = 0.02), von Willebrand factor levels (KP = 157.9 ± 8.6% vs. KA = 212.5 ± 16.2%; P < 0.01), d -dimer fragments (KP = 0.29 ± 0.02 μg/ml vs. KA = 0.73 ± 0.11 μg/ml; P < 0.01), fibrinogen (KP = 363.0 ± 11.1 mg/dl vs. KA = 397.6 ± 19.4 mg/dl; NS), triglycerides (KP = 122.7 ± 8.6 mg/dl vs. KA = 187.0 ± 30.1 mg/dl; P = 0.01), and urinary albumin excretion rate (KP = 13.5 ± 1.9 mg/24 h vs. KA = 57.3 ± 26.3 mg/24 h; P < 0.01). Patients undergoing KP transplantation showed a normal EDD (KP = 6.21 ± 2.42%, KA = 0.65 ± 2.74%, C = 8.1 ± 2.1%; P < 0.01), whereas no differences were observed in nitrate-dependent dilation. Moreover, IMT was lower in patients undergoing KP transplantation than in patients undergoing KA transplantation (KP = 0.74 ± 0.03 mm vs. KA = 0.86 ± 0.09 mm; P = 0.04). Our study showed that patients with type 1 diabetes have a lower atherosclerotic risk profile after KP transplantation than after KA transplantation. These differences are tightly correlated with metabolic control, fasting homocysteine levels, lower d -dimer fragments, and lower von Willebrand factor levels. Normal endothelial function and reduction of IMT was observed only in patients undergoing KP transplantation. C, control DDF, d-dimer fragments EDD, endothelial-dependent dilation IMT, intima media thickness KA, kidney alone KP, kidney-pancreas tHcy, homocysteine vWF, von Willebrand factor Footnotes Address correspondence and reprint requests to Antonio Secchi, MD, Medicina I, San Raffaele Scientific Institute, Via Olgettina 60, 20132, Milano, Italy. E-mail: antonio.secchi{at}hsr.it . Received for publication 24 July 2000 and accepted in revised form 31 October 2000.

The plasma concentration of asymmetrical dimethylarginine (ADMA), an inhibitor of nitric-oxide synthase, which has been linked to endothelial dysfunction and atherosclerosis in the general population, is raised in patients with end-stage renal disease and could contribute to the high cardiovascular risk in patients with chronic renal failure. We investigated the relation between cardiovascular risk factors and plasma ADMA concentration in a cohort of haemodialysis patients (n=225), and tested the predictive power of ADMA for mortality and cardiovascular outcomes. Patients had standard dialysis three times a week. We accurately recorded cardiovascular events over a mean follow-up of 33·4 months (SD 14·6); these events were reviewed by a panel of physicians. We identified correlates of plasma ADMA by univariate and multivariate analyses. On univariate analysis, ADMA concentration in plasma was directly related to concentrations of fibrinogen and L-arginine in plasma, duration of dialysis treatment, and serum cholesterol concentration, and was inversely related to serum albumin concentration. On multivariate analysis, only plasma fibrinogen (p=0·0001) and serum albumin (p=0·04) concentrations were independently related to plasma ADMA concentration (multiple r=0·44, p=0·0001). 83 patients died, 53 (64%) by cardiovascular causes. In a Cox's proportional-hazards model, plasma ADMA ranked as the second factor predicting overall mortality (hazard ratio 1·26, 95% Cl 1·11–1·41, p=0·0001) and cardiovascular events (1·17, 1·04–1·33, p=0·008). In haemodialysis patients, plasma ADMA is a strong and independent predictor of overall mortality and cardiovascular outcome. These findings lend support to the hypothesis that accumulation of ADMA is an important risk factor for cardiovascular disease in chronic renal failure.

Hyperhomocysteinemia predicts cardiovascular outcomes in hemodialysis patients. We prospectively tested the prediction power of homocysteinemia for all-cause and cardiovascular outcomes in a cohort of 175 hemodialysis patients followed for 29 ± 12 months. Survival analysis was performed by the Cox's proportional hazard model and data were expressed as hazard ratio and 95% confidence interval (CI). During the follow-up period 51 patients died, 31 of them (61%) of cardiovascular causes and 16 patients developed non-fatal atherothrombotic complications. Plasma total homocysteine was an independent predictor of cardiovascular mortality (P = 0.01). Combined analysis of fatal and non-fatal atherothrombotic events showed that homocysteine was a strong and independent predictor of these outcomes because the risk of these events was 8.2 times higher (95% CI 1.9 to 32.2) in patients in the third homocysteine tertile than in those in the first tertile (P = 0.005). There is a clear association between hyperhomocysteinemia and incident cardiovascular mortality and atherothrombotic events in hemodialysis patients. Intervention studies are needed to determine whether the accumulation of this substance has a causal role in the pathogenesis of cardiovascular damage in patients undergoing hemodialysis.

Objective We investigated whether preventing hyperglycemia in septic patients affected the plasma concentration of asymmetric-dimethylarginine and if this was associated with clinical benefit. Design A prospective, multicenter, randomized, controlled, clinical study. Setting Intensive care units (ICU) in three university hospitals. Patients A total of 72 patients admitted for severe sepsis or septic shock, who stayed at least 3 days in the ICU. At admission the patients were assigned to receive either tight or conventional glycemic control. Interventions Determination of circulating levels of asymmetric-dimethylarginine, arginine, interleukin-6, C-reactive-protein and tumor-necrosis-factor-α. Measurements and results Blood was sampled at admission (no differences between groups), and on the 3rd, 6th, 9th, and 12th (T12) days. Sequential organ failure assessment was scored at each sampling time. All the data were analyzed on an intention-to-treat basis. The control and treatment groups received the same energy intake, glycemia (110.4 ± 17.3 vs. 163.0 ± 28.9 mg/dL, P  < 0.001) and insulin ( P  = 0.02) supply differed. No differences were found in high plasma levels of asymmetric-dimethylarginine ( P  = 0.812) at any time during the ICU stay. The clinical course, as indicated by markers of inflammation, average and maximum organ failure score, ICU stay and ICU and 90-day mortality, was the same. Conclusions Intensive insulin treatment, while achieving glucose control, did not reduce asymmetric-dimethylarginine in high-risk septic patients fed with no more than 25 kcal/kg per day to limit ventilatory demand and to simplify glucose control. Descriptor 45 (SIRS/sepsis: clinical studies).

Background: Mutations in the retina-specific ABC transporter (ABCA4) gene have been associated with several forms of macular degenerations. Because the high complexity of the molecular genotype makes scanning of the ABCA4 gene cumbersome, we describe here the first use of denaturing HPLC (DHPLC) to screen for ABCA4 mutations. Methods: Temperature conditions were designed for all 50 exons based on effective separation of 83 samples carrying 86 sequence variations and 19 mutagenized controls. For validation, samples from 23 previously characterized Stargardt patients were subjected to DHPLC profiling. Subsequently, samples from a cohort of 30 patients affected by various forms of macular degeneration were subjected to DHPLC scanning under the same conditions. Results: DHPLC profiling not only identified all 132 sequence alterations previously detected by double-gradient denaturing gradient gel electrophoresis but also identified 5 sequence alterations that this approach had missed. Moreover, DHPLC scanning of an additional panel of 30 previously untested patients led to the identification of 26 different mutations and 29 polymorphisms, accounting for 203 sequence variations on 29 of the 30 patients screened. In total, the DHPLC approach allowed us to identify 16 mutations that had never been reported before. Conclusions: These results provide strong support for the use of DHPLC for molecular characterization of the ABCA4 gene.

Denaturing HPLC (DHPLC) is a useful technique for the fast screening of known and unknown heterozygous gene mutations. Most DNA mutations causing genetic disorders consist of nucleotide substitutions, but insertions and deletions occur, albeit less frequently. The heteroduplexes with insertions/deletions have gaps that may affect molecular stability differently from the mismatches caused by substitutions. Therefore, gaps and mismatches may be distinguished by DHPLC analysis, which is based on the differential thermal stability of amplicons with different characteristics. To verify this hypothesis, we examined 12 DNA samples containing insertions and deletions of different sizes (one to 29 residues) from four different genes (ABCA4, CFTR, FTL, and SLC11A3). We found that all of them were detected by DHPLC runs at 50°C, which is considered a non‐denaturing temperature, as well as by runs at the temperature optimized for mismatch recognition. The finding confirms that gaps reduce heteroduplex stability more than mismatches, and indicates that DHPLC analysis at low temperature may be applied to distinguish DNA deletions/insertions from substitutions. Hum Mutat 22:98–102, 2003. © 2003 Wiley‐Liss, Inc.

To the Editor: Mandel et al. (March 21 issue) 1 suggest that contributing factors, such as resistance to activated protein C, may be needed for thrombosis to occur in patients with moderate increases in plasma levels of homocysteine. However, isolated hyperhomocysteinemia after fasting or a methionine load has been identified as an independent risk factor for venous thrombosis in two studies of patients with early-onset venous or arterial thromboembolic disease (or both), 2 , 3 and isolated hyperhomocysteinemia after fasting is a proven risk factor for deep-vein thrombosis in the general population. 4 Because of the high prevalence of both resistance to activated protein . . .