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The role of temozolomide concurrent with and adjuvant to radiotherapy (RT/TMZ) in elderly patients with glioblastoma (GBM) remains unclear. We evaluated the outcome of patients >70 years in the context of the Project of Emilia-Romagna Region in Neuro-Oncology (PERNO), the first Italian prospective observational population-based study in neuro-oncology. For this analysis the criteria for selecting patients enrolled in the PERNO study were: age >70 years; PS 0–3; histologically confirmed GBM; postoperative radiotherapy (RT) after surgery with or without concomitant temozolomide (TMZ) or postsurgical TMZ alone. Between January 2009 and December 2010, 76 GBM elderly patients were identified in the prospective PERNO study. Twenty-three patients did not receive any treatment after surgery, and 53 patients received postsurgical treatments (25 patients received RT alone and 28 patients RT/TMZ). Median survival was 11.1 months (95 % CI 8.8–13.5), adding temozolomide concomitant and adjuvant to radiotherapy it was 11.6 months (95 % CI 8.6–14.6), and 9.3 months (95 % CI 8.1–10.6) in patients treated with RT alone (P = 0.164). However, patients with MGMT methylated treated with RT/TMZ obtained a better survival (17.2 months, 95 % CI 11.5–22.9) (P = 0.042). No difference in terms of survival were observed if patients with MGMT unmethylated tumor received RT alone, or RT/TMZ or, in MGMT methylated tumor, if patients received radiotherapy alone. In elderly patients RT/TMZ represent a widely used approach but it is effective with methylated MGMT tumors only.

Unfortunately, some of the participants of the PERNO Study Group are missing in the original publication of the article. The correct details are given below: Participants: Agati R., Ambrosetto G., Bacci A., Baldin E., Baldrati A., Barbieri E., Bartolini S., Bellavista E., Bisulli F., Bonora E., Bunkheila F., Carelli V., Cerasoli S., Crisci M., Dall’Occa P., de Biase D., Ferro S., Franceschi C., Frezza G., GrassoV., Leonardi M., Marucci G., Morandi L., Mostacci B., Palandri G., Pasini E., Pastore Trossello M., Pession A., Poggi R., Riguzzi P., Rinaldi R., Rizzi S., Romeo G., Spagnolli F., Tinuper P., Trocino C., Visani M. (Bologna), Dall’Agata M., Faedi M., Frattarelli M., Gentili G., Giovannini A., Iorio P., Pasquini U., Galletti G., Guidi C., Neri W., Patuelli A.., Strumia S. (ForlÍ-Cesena), Casmiro M., Gamboni A., Rasi F. (Faenza, RA), Cruciani G. (Lugo, RA), Cenni P., Dazzi C., Guidi A.R., Zumaglini F. (Ravenna), Amadori A., Pasini G., Pasquinelli M., Pasquini E., Polselli A., Ravasio A., Viti B. (Rimini), Sintini M. (Cattolica, RN), Ariatti A., Bertolini F., Bigliardi G., Carpeggiani P., Cavalleri F., Meletti S., Nichelli P., Pettorelli E., Pinna G., Zunarelli E. (Modena), Artioli F., Bernardini I., Costa M., Greco G., Guerzoni R., Stucchi C. (Carpi, MO), Iaccarino C., Ragazzi M., Rizzi R., Zuccoli G. (Reggio Emilia), Api P., Cartei F., Fallica E., Granieri E., Latini F., Lelli G., Monetti C., Saletti A., Schivalocchi R., Seraceni S., Tola M.R., Urbini B. (Ferrara), Giorgi C., Montanari E. (Fidenza, PR), Cerasti D., Crafa P., Dascola I., Florindo I., Giombelli E., Mazza S., Ramponi V., Servadei F., Silini EM., Torelli P. (Parma), Immovilli P., Morelli N., Vanzo C. (Piacenza), Nobile C. (Padova).

Background: Clinical and molecular factors are essential to define the prognosis in patients with glioblastoma (GBM). O6-methylguanine-DNA methyltransferase (MGMT) methylation status, age, Karnofsky Performance Status (KPS), and extent of surgical resection are the most relevant prognostic factors. Our investigation of the role of gender in predicting prognosis shows a slight survival advantage for female patients. Methods: We performed a prospective evaluation of the Project of Emilia Romagna on Neuro-Oncology (PERNO) registry to identify prognostic factors in patients with GBM who received standard treatment. Results: A total of 169 patients (99 males [58.6%] and 70 females [41.4%]) were evaluated prospectively. MGMT methylation was evaluable in 140 patients. Among the male patients, 36 were MGMT methylated (25.7%) and 47 were unmethylated (33.6%); among the female patients, 32 were methylated (22.9%) and 25 were unmethylated (17.9%). Survival was longer in the methylated females compared with the methylated males (P = 0.028) but was not significantly different between the unmethylated females and the unmethylated males (P = 0.395). In multivariate analysis, gender and MGMT methylation status considered together (methylated females vs. methylated males; hazard ratio [HR], 0.459; 95% confidence interval [CI], 0.242-0.827; P = 0.017), age (HR, 1.025; 95% CI, 1.002-1.049; P = 0.032), and KPS (HR, 0.965; 95% CI, 0.948-0.982; P < 0.001) were significantly correlated with survival. Conclusions: Survival was consistently longer among MGMT methylated females compared with males. Gender can be considered as a further prognostic factor.

International audience; no abstract

Several biomarkers have been proposed as useful parameters to better specify the prognosis or to delineate new target therapy strategies for glioblastoma patients. MicroRNAs could represent putative target molecules, considering their role in tumorigenesis, cancer progression and their specific tissue expression. Although several studies have tried to identify microRNA signature for glioblastoma, a microRNA profile is still far from being well-defined. In this work the expression of 19 microRNAs (miR-7, miR-9, miR-9∗, miR-10a, miR-10b, miR-17, miR-20a, miR-21, miR-26a, miR-27a, miR-31, miR-34a, miR-101, miR-137, miR-182, miR-221, miR-222, miR-330, miR-519d) was evaluated in sixty formalin-fixed and paraffin-embedded glioblastoma samples using a locked nucleic acid real-time PCR. Moreover, a comparison of miRNA expressions was performed between primary brain neoplasias of different grades (grades IV-I). The analysis of 14 validated miRNA expression in the 60 glioblastomas, using three different non-neoplastic references as controls, revealed a putative miRNA signature: mir-10b and miR-21 were up-regulated, while miR-7, miR-31, miR-101, miR-137, miR-222 and miR-330 were down-regulated in glioblastomas. Comparing miRNA expression between glioblastoma group and gliomas of grades I-III, 3 miRNAs (miR-10b, mir-34a and miR-101) showed different regulation statuses between high-grade and low-grade tumors. miR-10b was up-regulated in high grade and significantly down-regulated in low-grade gliomas, suggesting that could be a candidate for a GBM target therapy. This study provides further data for the identification of a miRNA profile for glioblastoma and suggests that different-grade neoplasia could be characterized by different expression of specific miRNAs.

Baruzzi A. (Chair), Albani F., Calbucci F., D’Alessandro R., Michelucci R. (IRCCS Institute of Neurological Sciences, Bologna, Italy), Brandes A. (Department of Medical Oncology, Bellaria-Maggiore Hospitals, Bologna, Italy), Eusebi V. (Department of Hematology and Oncological Sciences ‘‘L. & A. Seragnoli’’, Section of Anatomic Pathology at Bellaria Hospital, Bologna, Italy), Ceruti S., Fainardi E., Tamarozzi R. (Neuroradiology Unit, Department of Neurosciences and Rehabilitation, S. Anna Hospital, Ferrara, Italy), Emiliani E. (Istituto Oncologico Romagnolo, Department of Medical Oncology, Santa Maria delle Croci Hospital, Ravenna, Italy), Cavallo M. (Division of Neurosurgery, Department of Neurosciences and Rehabilitation, S. Anna Hospital, Ferrara, Italy).

Glioblastoma is the most aggressive brain tumor that may occur in adults. Regardless of the huge improvements in surgery and molecular therapy, the outcome of neoplasia remains poor. MicroRNAs are small molecules involved in several cellular processes, and their expression is altered in the vast majority of tumors. Several studies reported the expression of different miRNAs in glioblastoma, but one of the most critical point in understanding glioblastoma miRNAs profile is the comparison of these studies. In this paper, we focused our attention on the non-neoplastic references used for determining miRNAs expression. The aim of this study was to investigate if using three different non-neoplastic brain references (normal adjacent the tumor, commercial total RNA, and epileptic specimens) could provide discrepant results. The analysis of 19 miRNAs was performed using Real-Time PCR, starting from the set of samples described above and the expression values compared. Moreover, the three different normal RNAs were used to determine the miRNAs profile in 30 glioblastomas. The data showed that different non-neoplastic controls could lead to different results and emphasize the importance of comparing miRNAs profiles obtained using the same experimental condition.

BackgroundAlbeit several studies pointed out the pivotal role that CD4+T cells have in Multiple Sclerosis, the CD8+ T cells involvement in the pathology is still in its early phases of investigation. Proteasome degradation is the key step in the production of MHC class I-restricted epitopes and therefore its activity could be an important element in the activation and regulation of autoreactive CD8+ T cells in Multiple Sclerosis.Methodology/principal findingsImmunoproteasomes and PA28-alphabeta regulator are present in MS affected brain area and accumulated in plaques. They are expressed in cell types supposed to be involved in MS development such as neurons, endothelial cells, oligodendrocytes, macrophages/macroglia and lymphocytes. Furthermore, in a genetic study on 1262 Italian MS cases and 845 controls we observed that HLA-A*02+ female subjects carrying the immunoproteasome LMP2 codon 60HH variant have a reduced risk to develop MS. Accordingly, immunoproteasomes carrying the LMP2 60H allele produce in vitro a lower amount of the HLA-A*0201 restricted immunodominant epitope MBP(111-119).Conclusion/significanceThe immunoproteasome LMP2 60HH variant reduces the risk to develop MS amongst Italian HLA-A*02+ females. We propose that such an effect is mediated by the altered proteasome-dependent production of a specific MBP epitope presented on the MHC class I. Our observations thereby support the hypothesis of an involvement of immunoproteasome in the MS pathogenesis.

Cell death is as important as cell proliferation for cell turn-over, and susceptibility to cell death is affected by a number of parameters that change with time. A time-dependent derangement of such a crucial process, or even the simple cell loss mediated by cell death impinges upon aging and longevity. In this review we will discuss how cell death phenomena are modulated during aging and what is their possible role in the aging process. We will focus on apoptosis and autophagy, which affect mostly proliferating and post-mitotic cells, respectively, and on mitochondrial degradation in long living cells. Since the "decisional process" that leads the cell to death is very complex, we will also discuss the possibility to address this topic with a systems biology approach.

Many epidemiological data indicate the presence of a strong familial component of longevity that is largely determined by genetics, and a number of possible associations between longevity and allelic variants of genes have been described. A breakthrough strategy to get insight into the genetics of longevity is the study of centenarians, the best example of successful ageing. We review the main results regarding nuclear genes as well as the mitochondrial genome, focusing on the investigations performed on Italian centenarians, compared to those from other countries. These studies produced interesting results on many putative "longevity genes". Nevertheless, many discrepancies are reported, likely due to the population-specific interactions between gene pools and environment. New approaches, including large-scale studies using high-throughput techniques, are urgently needed to overcome the limits of traditional association studies performed on a limited number of polymorphisms in order to make substantial progress to disentangle the genetics of a trait as complex as human longevity.

The Sarcolab pilot study of 2 crewmembers, investigated before and after a 6-mo International Space Station mission, has demonstrated the substantial muscle wasting and weakness, along with disruption of muscle's oxidative metabolism. The present work aimed at evaluating the pro/anti-inflammatory status in the same 2 crewmembers (A, B). Blood circulating (c-)microRNAs (miRs), c-proteasome, c-mitochondrial DNA, and cytokines were assessed by real-time quantitative PCR or ELISA tests. Time series analysis was performed ( i.e., before flight and after landing) at 1 and 15 d of recovery (R+1 and R+15, respectively). C-biomarkers were compared with an age-matched control population and with two-dimensional proteomic analysis of the 2 crewmembers' muscle biopsies. Striking differences were observed between the 2 crewmembers at R+1, in terms of inflamma-miRs (c-miRs-21-5p, -126-3p, and -146a-5p), muscle specific (myo)-miR-206, c-proteasome, and IL-6/leptin, thus making the 2 astronauts dissimilar to each other. Final recovery levels of c-proteasome, c-inflamma-miRs, and c-myo-miR-206 were not reverted to the baseline values in crewmember A. In both crewmembers, myo-miR-206 changed significantly after recovery. Muscle biopsy of astronaut A showed an impressive 80% increase of ?-1-antitrypsin, a target of miR-126-3p. These results point to a strong stress response induced by spaceflight involving muscle tissue and the proinflammatory setting, where inflamma-miRs and myo-miR-206 mediate the systemic recovery phase after landing.-Capri, M., Morsiani, C., Santoro, A., Moriggi, M., Conte, M., Martucci, M., Bellavista, E., Fabbri, C., Giampieri, E., Albracht, K., Flück, M., Ruoss, S., Brocca, L., Canepari, M., Longa, E., Di Giulio, I., Bottinelli, R., Cerretelli, P., Salvioli, S., Gelfi, C., Franceschi, C., Narici, M., Rittweger, J. Recovery from six-month spaceflight at the International Space Station: muscle-related stress into a proinflammatory setting.

Inflamm-aging, that is the age-associated inflammatory status, is considered one of the most striking consequences of immunosenescence, as it is believed to be linked to the majority of age-associated diseases sharing an inflammatory basis. Nevertheless, evidence is emerging that inflamm-aging is at least in part independent from immunological stimuli. Moreover, centenarians who avoided or delayed major inflammatory diseases display markers of inflammation. In this paper we proposed a reappraisal of the concept of inflamm- aging, suggesting that its pathological effects can be independent from the total amount of pro-inflammatory mediators, but they would be rather associated with the anatomical district and type of cells where they are produced and where they primarily act.

Proteasomes (including constitutive proteasome, immunoproteasome, and their regulatory complexes) are multicatalytic complexes crucial for cell and body homeostasis and survival, being responsible for a consistent part of protein degradation. In the central nervous system (CNS), the activity of proteasomes affects a variety of crucial brain activities. While proteasome alteration (content and activity) during aging has been studied in several tissues and cellular models, few data are available regarding human CNS, and the identification of an appropriate and reliable model for the role of proteasome in human brain aging is still lacking. In this review, the available data on proteasome and brain aging in rodents, as well as the few data on non human primates, are critically revised. On the whole, the data regarding changes of proteasome activity and content with age are far from being clear, not only due to the heterogeneity of the models (differences between species, among strains of the same species) but also due to the brain areas considered. We paid particular attention to recent data obtained in human brain of non-demented donors and subjects affected by Alzheimer Disease (AD) demented subjects, as well as to new data on non human primate brain. The study of the possible role of proteasomes in brain aging, and the identification of reliable animal models, is pivotal for the development of possible ad hoc therapeutic interventions capable of retarding/counteracting brain aging and age-related brain pathologies. The therapeutic capability and limits of Vitamin E, the possible set up of proteasome modifiers (activators) as well as the effects on proteasomes of other drugs used for AD therapy are discussed within a scenario which deserves more attention and further investigations. © 2007 Bentham Science Publishers Ltd.

Aging is considered the major risk factor for cancer, one of the most important mortality causes in the western world. Inflammaging, a state of chronic, low-level systemic inflammation, is a pervasive feature of human aging. Chronic inflammation increases cancer risk and affects all cancer stages, triggering the initial genetic mutation or epigenetic mechanism, promoting cancer initiation, progression and metastatic diffusion. Thus, inflammaging is a strong candidate to connect age and cancer. A corollary of this hypothesis is that interventions aiming to decrease inflammaging should protect against cancer, as well as most/all age-related diseases. Epidemiological data are concordant in suggesting that the Mediterranean Diet (MD) decreases the risk of a variety of cancers but the underpinning mechanism(s) is (are) still unclear. Here we review data indicating that the MD (as a whole diet or single bioactive nutrients typical of the MD) modulates multiple interconnected processes involved in carcinogenesis and inflammatory response such as free radical production, NF-κB activation and expression of inflammatory mediators, and the eicosanoids pathway. Particular attention is devoted to the capability of MD to affect the balance between pro- and anti-inflammaging as well as to emerging topics such as maintenance of gut microbiota (GM) homeostasis and epigenetic modulation of oncogenesis through specific microRNAs.

Sarcopenia, the progressive loss of muscle mass and strength, is a phenomenon characterizing human aging whose etiology is still not clear. While there is increasing evidence for the influence of inter-muscular adipose tissue infiltration in the development of sarcopenia, much less is known about a possible role for intra-muscular triglycerides (IMTG). IMTG accumulate in form of lipid droplets decorated by proteins such as Perilipins (Plins). In skeletal muscle the most abundant are Plin2 and Plin5. In this study we compared the expression of these two Plins in Vastus lateralis muscle samples of subjects of different age, both healthy donors (HD) and patients with limited lower limb mobility (LLMI). These latter are characterized by a condition of chronic physical inactivity. Plin2 expression resulted higher in old age for both HD and LLMI patients, while Plin5 slightly decreased only in LLMI patients. Moreover, in these patients, only Plin2 was associated with the decrease of muscle strength and the expression of factors related to muscle atrophy (MuRF1, Atrogin and p53). An increase in Plin2 and a concomitant decrease of Plin5 was also observed when we considered animal model of disuse-induced muscle atrophy. As a whole, these data indicate that Plin2 and Plin5 have a different expression pattern during muscle aging and inactivity, and only Plin2 appears to be associated with functional alterations of the muscle.

Background As yet, no population-based prospective studies have been conducted to investigate the incidence and clinical outcome of glioblastoma (GBM) or the diffusion and impact of the current standard therapeutic approach in newly diagnosed patients younger than aged 70 years. Methods Data on all new cases of primary brain tumors observed from January 1, 2009, to December 31, 2010, in adults residing within the Emilia-Romagna region were recorded in a prospective registry in the Project of Emilia Romagna on Neuro-Oncology (PERNO). Based on the data from this registry, a prospective evaluation was made of the treatment efficacy and outcome in GBM patients. Results Two hundred sixty-seven GBM patients (median age, 64 y; range, 29–84 y) were enrolled. The median overall survival (OS) was 10.7 months (95% CI, 9.2–12.4). The 139 patients ≤aged 70 years who were given standard temozolomide treatment concomitant with and adjuvant to radiotherapy had a median OS of 16.4 months (95% CI, 14.0–18.5). With multivariate analysis, OS correlated significantly with KPS (HR = 0.458; 95% CI, 0.248–0.847; P = .0127), MGMT methylation status (HR = 0.612; 95% CI, 0.388–0.966; P = .0350), and treatment received in a high versus low-volume center (HR = 0.56; 95% CI, 0.328–0.986; P = .0446). Conclusions The median OS following standard temozolomide treatment concurrent with and adjuvant to radiotherapy given to (72.8% of) patients aged ≤70 years is consistent with findings reported from randomized phase III trials. The volume and expertise of the treatment center should be further investigated as a prognostic factor.

The ubiquitin-proteasome system is the major intracellular molecular machinery for protein degradation and maintenance of protein homeostasis in most human cells. As ubiquitin-proteasome system plays a critical role in the regulation of the immune system, it might also influence the development and progression of multiple sclerosis (MS). Bothex vivoanalyses and animal models suggest that activity and composition of ubiquitin-proteasome system are altered in MS. Proteasome isoforms endowed of immunosubunits may affect the functionality of different cell types such as CD8+and CD4+T cells and B cells as well as neurons during MS development. Furthermore, the study of proteasome-related biomarkers, such as proteasome antibodies and circulating proteasomes, may represent a field of interest in MS. Proteasome inhibitors are already used as treatment for cancer and the recent development of inhibitors selective for immunoproteasome subunits may soon represent novel therapeutic approaches to the different forms of MS. In this review we describe the current knowledge on the potential role of proteasomes in MS and discuss thepro et contraof possible therapies for MS targeting proteasome isoforms.

International audience; Human aging is associated with a progressive loss of muscle mass and strength and a concomitant fat accumulation in form of inter-muscular adipose tissue, causing skeletal muscle function decline and immobilization. Fat accumulation can also occur as intra-muscular triglycerides (IMTG) deposition in lipid droplets, which are associated with perilipin proteins, such as Perilipin2 (Plin2). It is not known whether Plin2 expression changes with age and if this has consequences on muscle mass and strength. We studied the expression of Plin2 in the vastus lateralis (VL) muscle of both healthy subjects and patients affected by lower limb mobility limitation of different age. We found that Plin2 expression increases with age, this phenomenon being particularly evident in patients. Moreover, Plin2 expression is inversely correlated with quadriceps strength and VL thickness. To investigate the molecular mechanisms underpinning this phenomenon, we focused on IGF-1/p53 network/signalling pathway, involved in muscle physiology. We found that Plin2 expression strongly correlates with increased p53 activation and reduced IGF-1 expression. To confirm these observations made on humans, we studied mice overexpressing muscle-specific IGF-1, which are protected from sarcopenia. These mice resulted almost negative for the expression of Plin2 and p53 at two years of age. We conclude that fat deposition within skeletal muscle in form of Plin2-coated lipid droplets increases with age and is associated with decreased muscle strength and thickness, likely through an IGF-1- and p53-dependent mechanism. The data also suggest that excessive intramuscular fat accumulation could be the initial trigger for p53 activation and consequent loss of muscle mass and strength.

Inflammation is part of a complex physiological response to harmful stimuli and pathogenic stress. The five components of the Nuclear Factor κB (NF-κB) family are prominent mediators of inflammation, acting as key transcriptional regulators of hundreds of genes. Several signaling pathways activated by diverse stimuli converge on NF-κB activation, resulting in a regulatory system characterized by high complexity. It is increasingly recognized that the number of components that impinges upon phenotypic outcomes of signal transduction pathways may be higher than those taken into consideration from canonical pathway representations. Scope of the present analysis is to provide a wider, systemic picture of the NF-κB signaling system. Data from different sources such as literature, functional enrichment web resources, protein-protein interaction and pathway databases have been gathered, curated, integrated and analyzed in order to reconstruct a single, comprehensive picture of the proteins that interact with, and participate to the NF-κB activation system. Such a reconstruction shows that the NF-κB interactome is substantially different in quantity and quality of components with respect to canonical representations. The analysis highlights that several neglected but topologically central proteins may play a role in the activation of NF-κB mediated responses. Moreover the interactome structure fits with the characteristics of a bow tie architecture. This interactome is intended as an open network resource available for further development, refinement and analysis.

Immunoproteasome has been associated to neurodegenerative and autoimmune diseases as a marker and regulator of inflammatory mechanisms. Its expression in the brain may occur upon neuroinflammation in different cell types and affect a variety of homeostatic and inflammatory pathways including the oxidized protein clearance and the self-antigen presentation. In the present study we investigated the immunoproteasome expression in hippocampi and cortex of patients affected by different histopathological forms of pharmaco-resistent mesial temporal lobe epilepsy. We identified a pathology-specific pattern of immunoproteasome expression, which could provide insight into the complex neuroinflammatory pathogenic components of this disease.

Acetylcholinesterase inhibitors (AChEIs) have been used to improve cognitive status and disability in patients with mild to moderate Alzheimer's disease (AD). However, while the efficacy of AChEIs (i.e. how they act in randomized controlled trials) in this setting is widely accepted, their effectiveness (i.e. how they behave in the real world) remains controversial. To compare the effects of three AChEIs, donepezil (Aricept), galantamine (Reminyl) and rivastigmine (Exelon), in an Italian national, prospective, observational study representative of the 'real world' clinical practice of AChEI treatment for AD. 938 patients with mild to moderate AD collected within the framework of the Italian National Cronos Project (CP), involving several UVAs (AD Evaluation Units) spread over the entire national territory, who were receiving donepezil, galantamine or rivastigmine were followed for 36 weeks by measuring: (i) function, as determined by the Activities of Daily Living (ADL) and Instrumental Activities of Daily Living (IADL) scales; (ii) cognition, as measured by the Mini-Mental State Examination (MMSE) and the Alzheimer's Disease Assessment Scale-cognitive subscale (ADAS-cog) [primary outcome measures]; and (iii) behaviour, as measured on the Neuropsychiatric Inventory (NPI) and Clinical Dementia Rating (CDR) scale. Moreover, all patients were genotyped for apolipoprotein E (apoE) genetic variants. No statistically significant improvement in the primary outcome measures (MMSE and ADAS-Cog) was observed with drug therapy at 36 weeks, at which point all groups had lost, on average, 1 point on the MMSE and gained 2-3 points on the ADAS-Cog scale compared with baseline. On the secondary outcome measures at week 36, all treatment groups showed a significant worsening on the ADL and IADL scales compared with baseline, while on the NPI scale there were no significant differences from baseline except for the galantamine-treated group which worsened significantly. Moreover, patients receiving galantamine worsened significantly compared with the donepezil-treated group on the IADL scale. ApoE epsilon4 allele did not influence the effect of drug therapy. Over a 36-week follow-up period, no significant difference in the effects of donepezil, galantamine and rivastigmine on a variety of functional and cognitive parameters was observed in a large number of apoE-genotyped patients with mild to moderate AD recruited within the framework of a national project representative of the scenario usually encountered in actual clinical practice in Italy. The limitations (possibility of administration of lower drug doses than are used in clinical trials, relatively short follow-up period and the lack of randomization) and strengths (large number of patients, concomitant observation of the three drugs and the number of parameters assessed, including apoE genotype) of the present study are acknowledged. Our type of naturalistic study should complement clinical trials because 'real world' practice operates in the face of the numerous variables (e.g. health status and co-morbidities) associated with a complex disease such as AD in elderly people.

BackgroundAlzheimer's Disease (AD) is the most common neurodegenerative disease and the leading cause of dementia among senile subjects. It has been proposed that AD can be caused by defects in mitochondrial oxidative phosphorylation. Given the fundamental contribution of the mitochondrial genome (mtDNA) for the respiratory chain, there have been a number of studies investigating the association between mtDNA inherited variants and multifactorial diseases, however no general consensus has been reached yet on the correlation between mtDNA haplogroups and AD.Methodology/principal findingsWe applied for the first time a high resolution analysis (sequencing of displacement loop and restriction analysis of specific markers in the coding region of mtDNA) to investigate the possible association between mtDNA-inherited sequence variation and AD in 936 AD patients and 776 cognitively assessed normal controls from central and northern Italy. Among over 40 mtDNA sub-haplogroups analysed, we found that sub-haplogroup H5 is a risk factor for AD (OR=1.85, 95% CI:1.04-3.23) in particular for females (OR=2.19, 95% CI:1.06-4.51) and independently from the APOE genotype. Multivariate logistic regression revealed an interaction between H5 and age. When the whole sample is considered, the H5a subgroup of molecules, harboring the 4336 transition in the tRNAGln gene, already associated to AD in early studies, was about threefold more represented in AD patients than in controls (2.0% vs 0.8%; p=0.031), and it might account for the increased frequency of H5 in AD patients (4.2% vs 2.3%). The complete re-sequencing of the 56 mtDNAs belonging to H5 revealed that AD patients showed a trend towards a higher number (p=0.052) of sporadic mutations in tRNA and rRNA genes when compared with controls.ConclusionsOur results indicate that high resolution analysis of inherited mtDNA sequence variation can help in identifying both ancient polymorphisms defining sub-haplogroups and the accumulation of sporadic mutations associated with complex traits such as AD.

Human aging and longevity are complex and multi-factorial traits that result from a combination of environmental, genetic, epigenetic and stochastic factors, each contributing to the overall phenotype. The multi-factorial process of aging acts at different levels of complexity, from molecule to cell, from organ to organ systems and finally to organism, giving rise to the dynamic "aging mosaic". At present, an increasing amount of experimental data on genetics, genomics, proteomics and other -omics are available thanks to new high-throughput technologies but a comprehensive model for the study of human aging and longevity is still lacking. Systems biology represents a strategy to integrate and quantify the existing knowledge from different sources into predictive models, to be later tested and then implemented with new experimental data for validation and refinement in a recursive process. The ultimate goal is to compact the new acquired knowledge into a single picture, ideally able to characterize the phenotype at systemic/organism level. In this review we will briefly discuss the aging phenotype in a systems biology perspective, showing four specific examples at different levels of complexity, from a systemic process (inflammation) to a cascade-process pathways (coagulation) and from cellular organelle (proteasome) to single gene-network (PON-1), which could also represent targets for anti-aging strategies.

Despite the central role of proteasomes in relevant physiological pathways and pathological processes, this topic is unexpectedly largely unexplored in human liver. Here we present data on the presence of proteasome and immunoproteasome in human livers from normal adults, fetuses and patients affected by major hepatic diseases such as cirrhosis and chronic active hepatitis. Immunohistochemistry for constitutive (alpha4 and beta1) and inducible (LMP2 and LMP7) proteasome subunits, and for the PA28alphabeta regulator, was performed in liver samples from 38 normal subjects, 6 fetuses, 2 pediatric cases, and 19 pathological cases (10 chronic active hepatitis and 9 cirrhosis). The immunohistochemical data have been validated and quantified by Western blotting analysis. The most striking result we found was the concomitant presence in hepatocyte cytoplasm of all healthy subjects, including the pediatric cases, of constitutive proteasome and immunoproteasome subunits, as well as PA28alphabeta. At variance, immunoproteasome was not present in hepatocytes from fetuses, while a strong cytoplasmic and nuclear positivity for LMP2 and LMP7 was found in pathological samples, directly correlated to the histopathological grade of inflammation. At variance from other organs such as the brain, immunoproteasome is present in livers from normal adult and pediatric cases, in apparent absence of pathological processes, suggesting the presence of a peculiar regulation of the proteasome/immunoproteasome system, likely related to the physiological stimuli derived from the gut microbiota after birth. Other inflammatory stimuli contribute in inducing high levels of immunoproteasome in pathological conditions, where its role deserve further attention.

Mobile Visual Search is a new technology that aims at linking physical objects with digital information by taking pictures of them using a mobile device with on-board camera. In this paper we present an easily extendible and adaptable framework for Mobile Visual Search applications that takes advantage of the Smart-M3 interoperability platform. After introducing Smart-M3, we describe the proposed architecture and illustrate its use in two novel visual search scenarios.

How many animal models are adequate to study human aging? Aging is an adaptive process performed by an integrated panel of evolutionarily selected mechanisms aimed at maintaining soma integrity. The possibility of extrapolating results from animal models to human beings has to be addressed in an ecological context. Model systems fit basic requirements of scientific research, and experimental animals show a series of advantages for the study of aging and longevity in humans. However, animal models have intrinsic constraints because they are artificial: Humans are not inbred and live in different conditions from both an environmental and a socio-anthropological-cultural point of view. Even if research on aging and longevity has been performed primarily in model systems such as yeast, worms, and flies, results obtained in humans are not only of basic importance but are also largely unexpected, probably because of the peculiar characteristics of humans (protected environment, culture, economic conditions, stochasticity). In some cases, studies in animal models or humans have led to analogous results, largely because basic mechanisms involved in aging have been conserved throughout evolution. In other cases, results are different or even opposite, as is described in this chapter. Animal models are often not sufficiently adequate for the study of human longevity, but their usefulness in achieving knowledge at different levels (molecular, cellular, physiological, behavioral) is unquestionable. Thus, it seems that the concomitant and integrated use of ad hoc models, also comparing different species, together with new in silico and high throughput strategies, will be the general framework within which studies on human aging and longevity should be performed to accelerate the identification of new determinants of healthy aging and longevity.

Proteasomes are fundamental for the degradation of intracellular proteins, having a key role in several important metabolic and signaling pathways, in the cell cycle and in antigen presentation. In vitro proteasomal digestion assays are widely used in molecular biology and immunology. We developed a model, ProteaMAlg (proteasome modeling algorithm) that describes the kinetics of specific protein fragments generated by 20S proteasomes in different conditions, once the substrate cleavage strengths are provided. ProteaMAlg was tested on a variety of data available in the literature as well as on new degradation experiments performed with polypeptides of different sequences and lengths. The comparison between in vitro and in silico experiments was used to quantify the effect on degradation of the sequence and the length of target polypeptides, of the presence of regulatory molecules such as PA28-alphabeta, and of the type of 20S proteasome (constitutive- or immunoproteasome). The model showed that the effect of the PA28 regulatory subunit results in a modification of the gating functions of the proteasome core particle. Immunoproteasome digestion experiments suggested that this form of proteasome, which is involved in generating MHC-class I epitopes, presents modified cleavage and gating activities. Our analysis improves the current understanding of the kinetics of proteasome functioning, and provides a tool to quantify and predict the effect of key parameters during in vitro digestion. ProteaMAlg is publicly available on the web (http://www.proteamalg.com).

In this study, we investigated proteasome composition and activity in the brain of Macaca fascicularis, in order to test whether this nonhuman primate species might be a suitable animal model for anti-aging therapies in the central nervous system, addressed to the ubiquitin-proteasome system. We detected the catalytic beta subunits of constitutive proteasome, as well as the PA28 regulator and a subunit of immunoproteasome (i.e., beta1i [LMP2]), in seven adult, six old, and one young nonhuman primate brains. Subunit expression and proteasome activity were not influenced by the age of the animal in any of the brain regions (temporal and frontal cortex and cerebellum) we studied. However, an area-specific susceptibility to aged-related oxidative stress emerged. On the whole, the results suggest that, compared to humans, Macaca fascicularis primates may have a different age-dependent regulation of the ubiquitin-proteasome system and, possibly, of neuroinflammation in the brain. An in silico model of the 20S immunoproteasome containing the Macaca fascicularis alpha and beta subunits, present in database or identified by our group (i.e., LMP2), has been developed. Additional information was obtained by de novo sequencing of the beta1 (delta) subunit of Macaca fascicularis. A comparison with humans suggests that in multiprotein complexes some functional subunits, such as alpha subunits, appear to be preferentially conserved during evolution.

The immunoproteasome subunit low molecular weight protein 2 (LMP2) codon 60 polymorphism has been associated with autoimmune diseases. It has also been demonstrated to influence susceptibility to TNF-α-induced apoptosis in blood cells and proteasome activity in aged human brain. In the present study, anin silicomodel of immunoproteasome was used to examine the effect of the R60H polymorphism in the LMP2 subunit. The investigation of immunoproteasome expression, activity and intracellular localisation in anin vitrocellular model, namely lymphoblastoid cell lines, showed no major variations in functionality and amount, while a significant difference in antibody affinity was apparent. These data were integrated with previous results obtained in different tissues and combined with a structural model of the LMP2 polymorphism. Accordingly, we identified three prospective mechanisms that could explain the biological data for the polymorphism, such as modulation of the binding affinity of a putative non-catalytic modifier site on the external surface of the immunoproteasome core, or the modification of any channel between α and β rings.

Aging is a complex process which is accompanied with the decline and the reshaping of different functions of the body. In particular the immune system is characterized, during ageing (immunosenescence) by a remodeling of innate immunity (well preserved, up-regulated) and clonotypical immunity (severely altered) and by the occurrence of a chronic inflammatory process (inflammaging) which are, at least in part, genetically controlled. In this scenario, it can be anticipated that a crucial role is played by age-related structural and functional alterations and modifications of proteasomes and immunoproteasomes, the last being a key component of antigen processing and MHC class I antigen presentation. A variety of experimental data are available, suggesting that proteasomes are affected by age, and that in centenarians they are relatively preserved. On the contrary, few data are available on immunoproteasomes, likely as a consequence of the poverty of suitable cellular models. Lymphoblastoid cell lines from EBV immortalized B cells from old donors is envisaged as a possible model for the study of immunoproteasomes in humans and their changes with age. Thus, basic questions such as those related to possible consequences, for immune responses in infectious diseases and cancer, of age-related alterations of antigen processing and presenting, change with age of self-antigen repertoire, and the genetic basis of immunoprotesome activity and its change with age, remain largely unanswered. © 2003 Elsevier Ireland Ltd. All rights reserved.

In this study, we investigated the presence and role of immunoproteasome and its LMP2 subunit polymorphism at codon 60 in Alzheimer's disease (AD). Immunoproteasome was present in brain areas such as hippocampus and cerebellum and localized in neurons, astrocytes and endothelial cells. A higher expression of immunoproteasome was found in brain of AD patients than in brain of non-demented elderly, being its expression in young brain negligible or absent. Furthermore, AD affected regions showed a partial decrease in proteasome trypsin-like activity. The study of LMP2 polymorphism (R/H) showed that it does not influence LMP2 expression (neither the mRNA nor mature protein) in brain tissue. However, control brain areas of AD patients carrying the RR genotype showed an increased proteasome activity in comparison with RH carriers. To test whether this effect of the genotype might be related to AD onset we performed a genetic study, which allowed us to exclude an association of LMP2 codon 60 polymorphism with AD onset, despite its influence on the proteasome activity in human brain.

Immunoproteasome is an emerging biological target that constitutes a key element not only in antigen presentation but also in T cell and cytokine regulation as well as cellular homeostasis. Respect to standard proteasome, the inducible expression and different sensitivity towards activity modulators of immunoproteasome render it a potential therapeutic target for tumours and central nervous system diseases. In this review we report the cutting edge studies for understanding when immunoproteasome expression is induced and how it regulates pivotal pathways involved in tumours and neuropathologies, including apoptosis and inflammation. We emphasize its role as a new pharmacological target by describing the recent medicinal chemistry efforts aimed at design selective small-molecule modulators of both standard- and immuno-proteasome forms. Finally, we also present an in silico model of the human immunoproteasome structure by the major molecular differences with the 20S standard proteasome and discuss the perspective for the design of novel specific smallmolecule modulators for the different proteasome isoforms.