Increased soluble APPalpha, Abeta 1-42, and anti-Abeta 1-42 antibodies in plasma from down syndrome patients.

Down syndrome (DS) is the most common genetic disorder characterized by an extra copy of chromosome 21. DS subjects show signs of progressive cognitive decline, and most of them develop Alzheimer's type dementia at the age of 50 to 55 years. The aim of this study was to evaluate amyloid precursor protein (APP) metabolites and anti-Abeta 1-42 antibodies plasma levels in DS as possible biomarkers of Abeta accumulation potentially leading to neurodegeneration. We investigated plasma levels of sAPPalpha, Abeta 1-42, and anti-Abeta 1-42 antibodies by enzyme-linked immunosorbent assay in 24 DS subjects, 10 non-DS mentally retarded subjects and 18 age-matched controls. We found that sAPPalpha levels were about 1.5-fold higher and Abeta 1-42 levels were about 6-fold higher in DS respect to mentally retarded patients and to controls. DS patients showed Abeta 1-42 antibodies levels 4-fold higher than non-DS mentally retarded group and 2-fold higher than controls. Moreover, anti-Abeta 1-42 antibodies levels were inversely correlated with age in DS subjects. Our results suggested sAPPalpha as a possible peripheral marker for the alteration in APP metabolism in DS and highlighted an alteration in anti-abeta antibodies, for the first time evaluated in plasma from DS subjects.

Enhanced folate binding of cultured fibroblasts from Alzheimer's disease patients.

We compared the levels of serum folate from Alzheimer's disease (AD) patients and from age-matched healthy subjects and used primary cultures of fibroblasts, obtained from the two groups, to assess possible differences in their ability to bind folate. The results show that the levels of circulating folate are significantly (p<0.01; n=30) lower in AD patients than in controls (4.91+/-2.44 and 7.56+/-2.5 ng/mL, respectively). Moreover, the folate binding of AD fibroblasts is significantly (p<0.01; n=8) higher (2-4-fold) with respect to controls. RT-PCR experiments suggest that the higher folate binding could be due to an enhanced expression in AD fibroblasts of folate receptor alpha.

Increased plasma glutamate in stroke patients might be linked to altered platelet release and uptake.

Experimental studies have shown the role of excitotoxicity in the pathogenesis of ischemic brain lesions, and glutamate levels have been found to be elevated in CSF and plasma from patients, early after stroke. In this study, we investigated whether platelets could be involved in the mechanism of altered plasma glutamate levels after stroke. Forty four patients, from 6 hours to 9 months after ischemic stroke, 15 age-related healthy controls and 15 controls with stroke risk factors or previous transient ischemic attack were enrolled. Glutamate plasma levels, platelet glutamate release after aggregation and platelet glutamate uptake were assessed. Plasma glutamate levels were increased up to 15 days after the ischemic event in stroke patients, and the levels at day 3 were inversely correlated with the neurologic improvement between day 3 and 15. Ex vivo platelet glutamate release was decreased by 70% in stroke patients, suggesting previous in vivo platelet activation. Moreover, platelet glutamate uptake in these patients was decreased by 75% up to 15 days and was still reduced 90 days after stroke. Our data show a prolonged increase of glutamate in plasma after stroke, which might presumably be linked to altered platelet functions, such as excessive release of the amino acid or impaired uptake.

Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury In Vitro.

Several biomedical applications, such as xenotransplantation, require multiple genes simultaneously expressed in eukaryotic cells. Advances in genetic engineering technologies have led to the development of efficient polycistronic vectors based on the use of the 2A self-processing oligopeptide. The aim of this work was to evaluate the protective effects of the simultaneous expression of a novel combination of anti-inflammatory human genes, ENTPD1, E5NT and HO-1, in eukaryotic cells. We produced an F2A system-based multicistronic construct to express three human proteins in NIH3T3 cells exposed to an inflammatory stimulus represented by tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine which plays an important role during inflammation, cell proliferation, differentiation and apoptosis and in the inflammatory response during ischemia/reperfusion injury in several organ transplantation settings. The protective effects against TNF-α-induced cytotoxicity and cell death, mediated by HO-1, ENTPD1 and E5NT genes were better observed in cells expressing the combination of genes as compared to cells expressing each single gene and the effect was further improved by administrating enzymatic substrates of the human genes to the cells. Moreover, a gene expression analyses demonstrated that the expression of the three genes has a role in modulating key regulators of TNF-α signalling pathway, namely Nemo and Tnfaip3, that promoted pro-survival phenotype in TNF-α injured cells. These results could provide new insights in the research of protective mechanisms in transplantation settings.

Glutamate transporters in platelets: EAAT1 decrease in aging and in Alzheimer's disease.

Platelets release glutamate upon activation and are an important clearance system of the amino acid from blood, through high-affinity glutamate uptake, similar to that described in brain synaptosomes. Since platelet glutamate uptake is decreased in neurodegenerative disorders, we performed a morphological and molecular characterization of platelet glutamate transporters. The three major brain glutamate transporters EAAT1, EAAT2 and EAAT3 are expressed in platelets, with similar molecular weight, although at lower density than brain. A Na(+)-dependent-high-affinity glutamate uptake was competitively inhibited by known inhibitors but not by dihydrokainic acid, suggesting platelet EAAT2 does not play a major role in glutamate uptake at physiological conditions. We observed decreased glutamate uptake V(max), without modification of transporter affinity, in aging, which could be linked to the selective decrease of EAAT1 expression and mRNA. Moreover, in AD patients we found a further EAAT1 reduction compared to age-matched controls, which could explain the decrease of platelet uptake previously described. Platelet glutamate transporters may be used as peripheral markers to investigate the role of glutamate in patients with neuropsychiatric disorders.

Peripheral cytokine release in Alzheimer patients: correlation with disease severity.

Various studies suggested that inflammation is involved in the pathogenesis of Alzheimer's disease (AD). We investigated cytokine release from LPS-stimulated blood cells of 32 AD patients, with different disease severity, compared to 16 age-related controls. A significant decrease of IL-1beta and IL-6 secretion was observed in severely demented patients; TNF-alpha release was also decreased, but not significantly. By contrast, mild and moderate patients showed a cytokine release similar to controls. IL-1beta, IL-6 and TNF-alpha secretion was negatively correlated with the severity of dementia, quantified by the MMSE. Our data suggest that alterations of the immune profile are associated with AD progression.

Increased susceptibility to plasma lipid peroxidation in Alzheimer disease patients.

Oxidative stress, linked to Abeta-lipid interactions, plays a pathogenetic role in Alzheimer's disease. We investigated modifications of lipid peroxidation products in plasma of 52 AD patients, 42 healthy controls and 16 patients with amyotrophic lateral sclerosis, a neurodegenerative disease where oxidative stress also plays a pathogenetic role. Final lipid peroxidation products were measured in plasma by thiobarbituric acid reactive substances (TBARS) assay before and after ex vivo oxidative stress catalysed by copper. There were no significant changes at basal conditions, but after copper-induced oxidation TBARS levels were higher in AD patients (19.0 microM +/- 2.2) versus both controls (5.2 microM +/- 0.8, p<0.001) and ALS patients (7.6 microM +/- 2.1, p<0.01). Stimulated TBARS levels were significantly higher in mild and moderate AD (p<0.0001) with respect to controls, but not in severe AD patients, with a significant inverse correlation between disease severity and lipid peroxidation (p<0.005, r2=0.21). Treatment of a subgroup (13) of mild and moderate AD patients with vitamin C and E for three months decreased plasma lipoperoxidation susceptibility by 60%. Thus, oxidative stress, expressed as ex vivo susceptibility to lipid peroxidation, appears to be an early phenomenon, probably related to AD pathogenetic mechanisms.

Increased tissue factor pathway inhibitor and homocysteine in Alzheimer's disease.

We investigated the possible involvement of vascular damage in the pathogenesis of Alzheimer's disease (AD), by assessment of plasma levels of tissue factor pathway inhibitor (TFPI), a serine protease inhibitor induced by endothelial injury, and homocysteine (Hcy), a known risk factor for cerebrovascular disorders, folate levels were also measured. 110 probable AD, 38 mild cognitive impairment, 31 patients affected by idiopathic Parkinson's disease (without dementia) and 100 healthy controls, who displayed no vascular disorders were enrolled. TFPI and Hcy were significantly higher in AD patients with respect to other groups. The levels of TFPI and Hcy were positively correlated in hyperhomocysteinemic AD and mild cognitive impairment subjects, and were negatively correlated with folate levels. Our findings suggest that an impairment of endothelial function associated with high Hcy levels may occur in AD patients, despite the absence of manifest cerebrovascular lesions. Therefore, TFPI may represent a candidate marker of endothelial damage in AD and might be used for the identification and monitoring of patients that would benefit from folate supplementation treatment.

Valproate induces epigenetic modifications in lymphomonocytes from epileptic patients.

Valproate (VPA) is an anti-epileptic and mood-stabilizing drug with a broad range of action and which mechanism of action still remains in part elusive. Recently the discovery that VPA modifies the epigenome increasing the transcriptional rate of target genes raises the issue of understanding the exact role of this mechanism. In this work we tested the possibility that VPA could modify the epigenome of lymphomonocytes (PBMC) obtained from epileptic patients chronically treated in monotherapy with VPA and phenobarbital. Acetyl-histone H3 expression was assessed by western blotting and global DNA methylation by incorporation of [³H]dCTP. A significant increase in histone acetylation and a correlated decrease of global DNA methylation were shown at VPA therapeutically relevant plasma concentrations. This effect was drug-related, since it was not demonstrated in PBMC obtained from phenobarbital-treated patients. Moreover, a VPA dose-response curve was performed on PBMC obtained from healthy controls, demonstrating an increase of acetyl-histone H3 content. We suggest that the epigenetic properties of VPA expressed on PBMC at these concentrations might be operative in different tissues, with possible implications for the field of neuropsychiatric disorders.

Mutual information optimization for mass spectra data alignment.

"Signal" alignments play critical roles in many clinical setting. This is the case of mass spectrometry data, an important component of many types of proteomic analysis. A central problem occurs when one needs to integrate (mass spectrometry) data produced by different sources, e.g., different equipment and/or laboratories. In these cases some form of "data integration'" or "data fusion'" may be necessary in order to discard some source specific aspects and improve the ability to perform a classification task such as inferring the "disease classes'" of patients. The need for new high performance data alignments methods is therefore particularly important in these contexts. In this paper we propose an approach based both on an information theory perspective, generally used in a feature construction problem, and on the application of a mathematical programming task (i.e. the weighted bipartite matching problem). We present the results of a competitive analysis of our method against other approaches. The analysis was conducted on data from plasma/ethylenediaminetetraacetic acid (EDTA) of "control" and Alzheimer patients collected from three different hospitals. The results point to a significant performance advantage of our method with respect to the competing ones tested.

Cholinesterase inhibitor use is associated with increased plasma levels of anti-Aß 1-42 antibodies in Alzheimer's disease patients.

Acetyl-cholinesterase inhibitors (AChEI) are drugs frequently prescribed for the treatment of Alzheimer's disease (AD), exerting an effect on cognition, as well as on behavioural and psychological symptoms of dementia and activities of daily living. The efficacy of AChEI may be ascribed not only to the activation of cholinergic transmission, but also to other mechanisms, among which a putative regulation of the immune response has already been hypothesized. In the present study, we evaluated, in a cross-sectional sample of 66AD patients and 48 healthy controls, the putative influence of AChEI on anti-Abeta 1-42 antibody plasma levels by ELISA assay. AD patients receiving AChEI therapy showed increased plasma levels of anti-Abeta 1-42 antibodies respect to untreated AD patients and antibodies levels similar to those of healthy controls, both before and after normalization by total IgG values. Our results support a potential role of AChEI in the modulation of the immune response against Abeta. We suggest that a strategy aimed at increasing the endogenous response against this peptide might represent an interesting therapeutic target to be further investigated.

Post-methionine load test: A more sensitive tool to reveal hyperhomocysteinemia in Alzheimer patients?

OBJECTIVE: To identify the real number of hyperhomocysteinemic Alzheimer's patients who may benefit from homocysteine-lowering therapy. METHODS: Basal and post-methionine load homocysteine levels were assessed by rp-HPLC system. RESULTS: PML test revealed twice as many hyperhomocysteinemic AD subjects with respect to the fasting analysis. CONCLUSION: PML test resulted useful in detecting higher number of hyperhomocysteinemic AD patients who may have the chance of an early folate treatment.

Is M129V of PRNP gene associated with Alzheimer's disease? A case-control study and a meta-analysis.

The methionine/valine (M/V) polymorphism at codon 129 within the prion protein gene (PRNP) represents a known risk factor for Creutzfeldt-Jakob disease (CJD). Few authors reported also the effects of this polymorphism on the risk of Alzheimer's disease (AD), although with controversial results. To better clarify this issue, we performed a novel case-control study and a meta-analysis of published association studies between PRNP and AD. Our findings argue against PRNP as a susceptibility gene for developing AD in the Italian population but support the hypothesis that the V allele influences cognitive performances. The meta-analysis, revealed that Caucasian subjects homozygous at codon 129 had a 1.3-fold increased risk [95% CI: 1.0-1.6, p = 0.05] of developing AD compared to heterozygous individuals. We also observed that MM genotype and M allele represent a risk factor for AD, independently from the ethnic background, providing a significant but modest association between this polymorphism and AD.

Vascular endothelial growth factor gene variability is associated with increased risk for AD.

Converging evidence points to a pivotal role of vascular endothelial growth factor (VEGF) in neuronal protection and a lack of its activity in neurodegenerative disorders. To investigate this possible association, we screened the VEGF gene promoter for various well-known single-nucleotide polymorphisms in a series of 249 consecutively recruited Italian patients with sporadic Alzheimer's disease (AD). Genetic analysis indicated different distributions of two single-nucleotide polymorphisms in the AD population compared with healthy control subjects. In particular, the frequencies of -2578A/A and -1198C/T genotypes were significantly greater in AD patients than in control subjects (23.7 vs 14.7% and 2.8 vs 0%, respectively). The -2578A/A genotype was associated with an increased risk for disease, independently of apolipoprotein E genotype. The risk was significantly increased with respect to various VEGF genotype combinations. In contrast, no difference in serum VEGF levels was detected comparing 96 patients and 49 control subjects. These findings suggest that polymorphisms within the promoter region of the VEGF gene confer greater risk for AD, probably by reducing its neuroprotective effect, and confirm the biological role of VEGF in neurodegenerative processes.