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collaboration au projet H2020 European Cooperation in Science and Technology (COST) program European Network on Microvesicles and Exosomes in Health and Disease (ME-HAD); International audience; Recent research has demonstrated that all body fluids assessed contain substantial amounts of vesicles that range in size from 30 to 1000 nm and that are surrounded by phospholipid membranes containing different membrane microdomains such as lipid rafts and caveolae. The most prominent representatives of these so-called extracellular vesicles (EVs) are nanosized exosomes (70-150 nm), which are derivatives of the endosomal system, and microvesicles (100-1000 nm), which are produced by outward budding of the plasma membrane. Nanosized EVs are released by almost all cell types and mediate targeted intercellular communication under physiological and pathophysiological conditions. Containing cell-type specific signatures, EVs have been proposed as biomarkers in a variety of diseases. Furthermore, according to their physical functions, EVs of selected cell types have been used as therapeutic agents in immune therapy, vaccination trials, regenerative medicine, and drug delivery. Undoubtedly, the rapidly emerging field of basic and applied EV research will significantly influence the biomedicinal landscape in the future. In this Perspective, we, a network of European scientists from clinical, academic, and industry settings collaborating through the H2020 European Cooperation in Science and Technology (COST) program European Network on Microvesicles and.Exosomes in Health and Disease (ME-HAD), demonstrate the high potential of nanosized EVs for both diagnostic and therapeutic (i.e., theranostic) areas of nanomedicine.

In the mature central nervous system (CNS), oligodendrocytes provide support and insulation to axons thanks to the production of a myelin sheath. During their maturation to myelinating cells, oligodendroglial precursors (OPCs) follow a very precise differentiation program, which is finely orchestrated by transcription factors, epigenetic factors and microRNAs (miRNAs), a class of small non-coding RNAs involved in post-transcriptional regulation. Any alterations in this program can potentially contribute to dysregulated myelination, impaired remyelination and neurodegenerative conditions, as it happens in multiple sclerosis (MS). Here, we identify miR-125a-3p, a developmentally regulated miRNA, as a new actor of oligodendroglial maturation, that, in the mammalian CNS regulates the expression of myelin genes by simultaneously acting on several of its already validated targets. In cultured OPCs, over-expression of miR-125a-3p by mimic treatment impairs while its inhibition with an antago-miR stimulates oligodendroglial maturation. Moreover, we show that miR-125a-3p levels are abnormally high in the cerebrospinal fluid of MS patients bearing active demyelinating lesions, suggesting that its pathological upregulation may contribute to MS development, at least in part by blockade of OPC differentiation leading to impaired repair of demyelinated lesions.

Multiple sclerosis (MS) is a neurodegenerative disease in which axonal loss is the major cause of irreversible neurological disability. Neurological deficits in MS patients are related to inflammatory demyelination and axonal degeneration. Axonal transection occurs at sites of inflammation and begins at disease onset but is clinically silent because the central nervous system (CNS) compensates for neuronal loss. Neuronal injury in the course of chronic neuroinflammation is a key factor in determining long-term disability in patients. Viewing MS as an autoimmune, inflammatory, and neurodegenerative disease has major implications for therapy, and CNS protection and repair is needed in addition to controlling inflammation. The availability of novel tools in molecular neurogenetics and increasingly sophisticated neuroimaging technologies, together with the revitalization of MS neuropathology research, has created a new paradigm for the multidisciplinary study of this disease. This chapter summarizes epidemiological, clinical, diagnostic tools, and current therapies for MS and other important neurodegenerative diseases such as neuromyelitis optica.

We have previously demonstrated that multiple sclerosis (MS) patients with KIR2DL2 expression on Natural killer (NK) cells are more susceptible to herpes simplex virus 1 (HSV-1) infection. We explored cytokine expression by NK cells during HSV-1 infection in association with KIR2DL2 expression. MS KIR2DL2(+) NK cells failed to control HSV-1 infection and secreted high levels of Th17 cytokines, while MS KIR2DL2(-) NK cells released Th1 cytokines, mainly IFN-gamma. Our data showed, for the first time, a peculiar Th17 cytokine secretion by MS KIR2DL2(+) NK cells in the presence of HSV-1 infection, that could be implicated in MS pathogenesis.

Interleukin-25 (IL-25) is the only anti-inflammatory cytokine of the IL-17 family, and it has been shown to be efficacious in inhibiting neuroinflammation. Known for its effects on cells of the adaptive immune system, it has been more recently described to be effective also on cells of the innate immune system, namely macrophages. We used a lentiviral-mediated gene therapy approach to deliver IL-25 to the central nervous system (CNS) in two mouse models of neuroinflammation, entorhinal cortex lesion and experimental autoimmune encephalomyelitis. In both, we found that IL-25 gene therapy was able to modulate CNS myeloid cells, either infiltrating macrophages or resident microglia, towards an anti-inflammatory, tissue-protective phenotype, as testified by the increase in markers such as Arginase-1 (Arg1), Mannose receptor 1 (CD206) and Chitinase 3-like 3 (Ym1). As a consequence, neuroinflammation was partly inhibited and the CNS protected from immune-mediated damage. To our knowledge, this is the first example of M2 shift (alternative activation) induced in vivo on CNS-resident myeloid cells by gene therapy, and may constitute a promising strategy to investigate the potential role of protective microglia in neurological disorders.

We tried to identify the target/s of autoantibodies to basal ganglia neurons found in a patient with hyperkinetic movement disorders (HMD) characterized by rapid, rhythmic involuntary movements or spasms in both face and neck. Patient and control sera were used in Western blot to probe mouse brain homogenates. Two-dimensional gel electrophoresis (2-DE) SDS-PAGE protein spots recognized by the patient's antibodies were excised and sequenced by mass spectrometry analysis, and the glycolytic enzyme aldolase A was identified as the antigen recognized by the patient's autoantibodies. To assess relevance and specificity of these antibodies to the identified targets as biomarkers of autoimmunity in movement disorders, autoantibody responses to the identified target were then measured by ELISA in various diseases of the central nervous system. Anti-aldolase A autoantibodies were associated mainly with HMD (7/17, 41%) and Parkinson's disease (4/30, 13%) patients, and undetectable in subjects with other inflammatory and non-inflammatory central nervous system diseases. We, thus, identified aldolase A as an autoantigen in a sub-group of patients with HMD, a clinically ill-defined syndrome. Anti-aldolase A antibodies may represent a useful biomarker of autoimmunity in HMD patients.

BACKGROUND AND PURPOSE Alterations of glutamate-mediated synaptic transmission occur early during neuroinflammatory insults, and lead to degenerative neuronal damage in multiple sclerosis (MS) and also in experimental autoimmune encephalomyelitis (EAE), which is a murine model of MS. Fingolimod is an effective orally active agent for the treatment of MS, affecting lymphocyte invasion of the brain. However, it is still unclear if fingolimod can be neuroprotective in this disorder. EXPERIMENTAL APPROACH Using neurophysiological recordings and morphological evaluation of dendritic integrity, we evaluated the effects of oral fingolimod on the clinical score of EAE mice in order to determine whether the compound was associated with preservation of synaptic transmission. KEY RESULTS Oral fingolimod prevented and reversed the pre- and postsynaptic alterations of glutamate transmission in EAE mice. These effects were associated with a clear amelioration of the clinical deterioration seen in EAE mice, and with a significant inhibition of neuronal dendritic pathology. Fingolimod did not alter the spontaneous excitatory postsynaptic currents in control animals, suggesting that only the pathological processes behind the inflammation-induced defects in glutamate transmission were modulated by this compound. CONCLUSIONS AND IMPLICATIONS The beneficial effects of fingolimod on the clinical, synaptic and dendritic abnormalities of murine EAE might correlate with the neuroprotective actions of this agent, as observed in MS patients. LINKED ARTICLE This article is commented on by Gillingwater, pp. 858–860 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01612.x

Objective: Microvesicles (MVs) have been indicated as important mediators of intercellular communication and are emerging as new biomarkers of tissue damage. Our previous data indicate that reactive microglia/macrophages release MVs in vitro. The aim of the study was to evaluate whether MVs are released by microglia/macrophages in vivo and whether their number varies in brain inflammatory conditions, such as multiple sclerosis (MS). Methods: Electron and fluorescence microscopy and flow cytometry were used to detect myeloid MVs in the cerebrospinal fluid (CSF) of healthy controls, MS patients, and rodents affected by experimental autoimmune encephalomyelitis (EAE), the animal model of MS. Results: Myeloid MVs were detected in CSF of healthy controls. In relapsing and remitting EAE mice, the concentration of myeloid MVs in the CSF was significantly increased and closely associated with disease course. Analysis of MVs in the CSF of 28 relapsing patients and 28 patients with clinical isolated syndrome from 2 independent cohorts revealed higher levels of myeloid MVs than in 13 age-matched controls, indicating a clinical value of MVs as a companion tool to capture disease activity. Myeloid MVs were found to spread inflammatory signals both in vitro and in vivo at the site of administration; mice impaired in MV shedding were protected from EAE, suggesting a pathogenic role for MVs in the disease. Finally, FTY720, the first approved oral MS drug, significantly reduced the amount of MVs in the CSF of EAE-treated mice. Interpretation: These findings identify myeloid MVs as a marker and therapeutic target of brain inflammation. ANN NEUROL 2012;72:610–624

Objective: The frequency of inflammatory episodes in the early stages of multiple sclerosis (MS) has been correlated with late neurodegeneration, but the mechanism by which inflammation gives rise to delayed neuronal damage is unknown. Increased activity of the neurotransmitter glutamate is thought to play a role in the inflammation-driven neurodegenerative process of MS, and therefore we tested whether inflammatory cytokines released during acute MS attacks have the property of enhancing glutamate-mediated transmission and excitotoxicity in central neurons. Methods: We compared the effect of cerebrospinal fluid (CSF) from active and quiescent MS patients on glutamate-mediated excitatory postsynaptic currents (EPSCs) and excitotoxic damage in rodent brain slices. We also measured CSF concentrations of tumor necrosis factor-α, of interleukin-1β (IL-1β), and of IL-1 receptor antagonist (IL-1ra), and correlated cytokine levels with cortical excitability assessed in MS patients by means of paired-pulse transcranial magnetic stimulation (TMS). Results: CSF from MS patients with enhanced brain lesions at magnetic resonance imaging was able to increase spontaneous EPSC frequency and glutamate-mediated neuronal swelling in vitro, through a mechanism dependent on enhanced IL-1β signaling and increased glutamate α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor stimulation. Furthermore, IL-1β/IL-1ra ratio was significantly higher in the CSF of active MS subjects, and correlated with intracortical facilitation, an accredited TMS measure of glutamate transmission. Finally, we identified for the first time transient receptor potential vanilloid 1 channels as essential intermediates for the synaptic action of IL-1β on central glutamatergic synapses. Interpretation: Our results provide compelling evidence of the synaptic mechanism linking inflammation and excitotoxic neurodegeneration in MS. ANN NEUROL 2012;71:76–83

The aim of this study was to investigate in vitro the effects of hydroalcoholic extract, infusion and essential oil of Mentha piperita L. on Candida spp. Hydroalcoholic extract and essential oil were evaluated regarding microbial activity against 50 strains of Candida albicans , 10 Candida glabrata, 10 Candida tropicalis , 8 Candida parapsilosis and 2 Candida krusei . The minimum inhibitory concentration (MIC) of the hydroalcoholic extract was determined by the broth dilution method and the essential oil antimicrobial activity was determined by the agar diffusion test. The effect of infusion of Mentha piperita L. on the adherence of standard Candida strains to acrylic resin was also investigated. The results demonstrated that hydroalcoholic extract of Mentha piperita L. showed fungistatic activity against strains of C. albicans , C. tropicalis and C. glabrata . The essential oil showed greater inhibitory activity on strains of C. albicans , C. tropicalis and C. parapsilosis . Infusion of Mentha piperita L. did not inhibit the adherence of Candida to acrylic resin. In conclusion, Mentha piperita L. presented greater antimicrobial activity against strains of C. albicans and C. tropicalis , but showed no effect on the adherence of these microorganisms to acrylic resin.

2D-immunomics may be useful in the identification of autoantigens in neurological autoimmune diseases, but its application may be limited by denaturation of target proteins. Here we compared the capacity of a single or multiple antigens to elicit autoantibodies targeting multiple neural autoantigens by ELISA and 2D-immunomics. We induced experimental autoimmune encephalomyelitis (EAE) with MBP peptide 89–104 , total MBP or spinal cord homogenate. Both techniques showed anti-MBP IgG only after immunization with total MBP. In addition, 2D-immunomics revealed the presence in EAE mice of autoantibodies targeting other neural proteins, some displaying partial sequence homology with MBP. The present finding by 2D-immunomics of multiple neural proteins targeted by autoantibodies generated by a single antigen may help to explain the complex autoimmune response observed in multiple sclerosis.

Neuromyelitis optica (NMO) is a rare demyelinating disease, affecting selectively the optic nerve and the spinal cord. It was previously considered to be a severe variant of multiple sclerosis (MS) due to the similar pathological features and its resemblance to optico-spinal, or Japanese, MS, typical of Asian populations. The finding that most NMO patients have auto-antibodies against aquaporin-4, a water channel particularly abundant on the astrocytes of the glia limitans, has allowed early diagnosis and specific treatment of these patients, and has greatly improved our knowledge of its pathogenesis. When laboratories worldwide can detect anti-aquaporin-4 auto-antibodies with comparable sensitivity and specificity, we will be able to have large multi-centric studies to define better the epidemiological, clinical and pathological features of patients and their responses to treatment. © 2010 Elsevier B.V.

There is a long history of research into body fluid biomarkers in neurodegenerative and neuroinflammatory diseases. However, only a few biomarkers in cerebrospinal fluid (CSF) are being used in clinical practice. Anti-aquaporin-4 antibodies in serum are currently useful for the diagnosis of neuromyelitis optica (NMO), but we could expect novel CSF biomarkers that help define prognosis and response to treatment for this disease. One of the most critical factors in biomarker research is the inadequate powering of studies performed by single centers. Collaboration between investigators is needed to establish large biobanks of well-defined samples. A key issue in collaboration is to establish standardized protocols for biobanking to ensure that the statistical power gained by increasing the numbers of CSF samples is not compromised by pre-analytical factors. Here, consensus guidelines for CSF collection and biobanking are presented, based on the guidelines that have been published by the BioMS-eu network for CSF biomarker research. We focussed on CSF collection procedures, pre-analytical factors and high quality clinical and paraclinical information. Importantly, the biobanking protocols are applicable for CSF biobanks for research targeting any neurological disease.

CD4+Foxp3+ regulatory T cells (Tregs) have been considered crucial in controlling immune system homeostasis, and their derangement is often associated to autoimmunity. Tregs identification is, however, difficult because most markers, including CD25 and Foxp3, are shared by recently activated T cells. We show in this paper that CD4+Foxp3+ T cells are generated in peripheral lymphoid organs on immunization and readily accumulate in the target organ of an autoimmune reaction, together with classical inflammatory cells, constituting up to 50% of infiltrating CD4+ T cells. Most CD4+Foxp3+ T cells are, however, CD25− and express proinflammatory cytokines such as IL-17 and IFN-γ, questioning their suppressive nature. Moreover, in vitro CD4+ T lymphocytes from naive and autoimmune mice, stimulated to differentiate into Th1, Th2, Th17, and induced Tregs, display early mixed expression of lineage-specific markers. These results clearly point to an unprecedented plasticity of naive CD4+ T cells, that integrating inflammatory signals may change their fate from the initial lineage commitment to a different functional phenotype.

Background:Autoimmune activation and deregulated apoptosis of T lymphocytes are involved in multiple sclerosis (MS). c-Jun N-terminal kinase (JNK) plays a role in T-cell survival and apoptosis. Objectives:The aim of this work was to investigate the role of the JNK-dependent apoptosis pathway in relapsing–remitting MS (RRMS). Methods:The immunomodulatory effect of AS602801, a JNK inhibitor, was firstly evaluated on activated peripheral blood mononuclear cells (PBMCs) from healthy volunteers (HVs) and secondly in unstimulated purified CD4+, CD8+ and CD11b+ cells from RRMS patients and HVs. Moreover JNK/inflammation/apoptosis related genes were investigated in RRMS and HV samples. Results:In activated PBMCs from HVs, we showed that AS602801 blocked T-lymphocyte proliferation and induced apoptosis. In RRMS CD4+ and CD8+ cells, AS602801 induced apoptosis genes and expression of surface markers, while in RRMS CD11b+ cells it induced expression of innate immunity receptors and co-stimulatory molecules. Untreated cells from RRMS active-phase patients significantly released interleukin-23 (IL-23) and interferon-gamma (IFN-γ) and expressed less apoptosis markers compared to the cells of HVs. Moreover, gene expression was significantly different in cells from RRMS active-phase patients vs. HVs. By comparing RRMS PBMCs in the active and stable phases, a specific genomic signature for RRMS was indentified. Additionally, CASP8AP2, CD36, ITGAL, NUMB, OLR1, PIAS-1, RNASEL, RTN4RL2 and THBS1 were identified for the first time as being associated to the active phase of RRMS. Conclusions:The analysis of the JNK-dependent apoptosis pathway can provide biomarkers for activated lymphocytes in the active phase of RRMS and a gene expression signature for disease status. The reported results might be useful to stratify patients, thereby supporting the development of novel therapies.

Rapamycin is an oral immunosuppressant drug previously reported to efficiently induce naturally occurring CD4(+)CD25(+)FoxP3(+) regulatory T ((n)T(reg)) cells re-establishing long-term immune self-tolerance in autoimmune diseases. We investigated the effect of rapamycin administration to SJL/j mice affected by PLP(139-151)-induced relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE). We found that oral or intraperitoneal treatment at the peak of disease or at the end of the first clinical attack, dramatically ameliorated the clinical course of RR-EAE. Treatment suspension resulted in early reappearance of disease. Clinical response was associated with reduced central nervous system demyelination and axonal loss. Rapamycin induced suppression of IFN-gamma, and IL-17 release from antigen-specific T cells in peripheral lymphoid organs. While CD4(+)FoxP3(+) cells were unaffected, we observed disappearance of CD4(+)CD45RB(high) effector T (T(eff)) cells and selective expansion of T(reg) cells bearing the CD4(+)CD45RB(low)FoxP3(+)CD25(+)CD103(+) extended phenotype. Finally, the dual action of rapamycin on both T(eff) and T(reg) cells resulted in modulation of their ratio that closely paralleled disease course. Our data show that rapamycin inhibits RR-EAE, provide evidence for the immunological mechanisms, and indicate this compound as a potential candidate for the treatment of multiple sclerosis.

ZusammenfassungDie Erforschung von Biomarkern in Körperflüssigkeiten bei neurodegenerativen und neuroinflammatorischen Erkrankungen blickt auf eine langjährige Geschichte zurück. Dennoch werden nur wenige Liquor cerebrospinalis (Liquor)-Biomarker in der klinischen Praxis verwendet. Einer der problematischen Faktoren in der Liquorbiomarker-Forschung ist die eingeschränkte Aussagekraft von Studien aufgrund einer nicht ausreichend großer Anzahl von Proben, die in Studien von einzelnen Zentren akquiriert werden können. Deshalb ist die Kooperation zwischen mehreren Zentren erforderlich, um große Biobanken von definierten Proben zu etablieren. Standardisierte Protokolle für Biobanking sind unumgänglich, um die durch die größere Anzahl von Liquorproben gewonnene statistische Aussagekraft sicherzustellen und nicht durch mangelhafte Präanalytik einzuschränken. Hier wird ein Konsensusbericht über Leitlinien zu Liquorentnahme und Biobanking durch das BioMS-eu Netzwerk für Liquorbiomarker-Forschung in Multipler Sklerose präsentiert. Schwerpunkte des Berichts sind Liquorentnahme, präanalytische Faktoren und klinische sowie sonstige Informationen. Biobanking-Protokolle sind für Liquor-Biobanken im Rahmen der Erforschung jeder neurologischen Krankheit anwendbar.

In recent years a companies’ need for information about customer requirements, which represent the key to retain actual customers and attract new ones, has steadily increased. Kano et al. [9] proposed an interesting model aiming at the classification of product or service characteristics as dissatisfiers, delighters, performance or indifferent attributes. Unfortunately, the required questionnaire structure is quite complex, consequently the rate of incomplete/inconsistent questionnaires is not negligible at all. In the present manuscript, the authors propose a model which allows allocation of a the customers’ requirements into Kano’s categories in a simple way. The underlying idea is to conduct two distinct analysis on the same attributes. Both analysis aim at investigating the attributes’ relationships with the overall variable (satisfaction, loyalty, etc), but one analysis focuses attention on the bottom part of the scale (low scores), while the other on the top part (high scores).

We have earlier shown that microglia, the immune cells of the CNS, release microparticles from cell plasma membrane after ATP stimulation. These vesicles contain and release IL-1beta, a crucial cytokine in CNS inflammatory events. In this study, we show that microparticles are also released by astrocytes and we get insights into the mechanism of their shedding. We show that, on activation of the ATP receptor P2X7, microparticle shedding is associated with rapid activation of acid sphingomyelinase, which moves to plasma membrane outer leaflet. ATP-induced shedding and IL-1beta release are markedly reduced by the inhibition of acid sphingomyelinase, and completely blocked in glial cultures from acid sphingomyelinase knockout mice. We also show that p38 MAPK cascade is relevant for the whole process, as specific kinase inhibitors strongly reduce acid sphingomyelinase activation, microparticle shedding and IL-1beta release. Our results represent the first demonstration that activation of acid sphingomyelinase is necessary and sufficient for microparticle release from glial cells and define key molecular effectors of microparticle formation and IL-1beta release, thus, opening new strategies for the treatment of neuroinflammatory diseases.

Neurodegeneration is the irremediable pathological event occurring during chronic inflammatory diseases of the CNS. Here we show that, in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, inflammation is capable in enhancing glutamate transmission in the striatum and in promoting synaptic degeneration and dendritic spine loss. These alterations occur early in the disease course, are independent of demyelination, and are strongly associated with massive release of tumor necrosis factor-α from activated microglia. CNS invasion by myelin-specific blood-borne immune cells is the triggering event, and the downregulation of the early geneArc/Arg3.1, leading to the abnormal expression and phosphorylation of AMPA receptors, represents a culminating step in this cascade of neurodegenerative events. Accordingly, EAE-induced synaptopathy subsided during pharmacological blockade of AMPA receptors. Our data establish a link between neuroinflammation and synaptic degeneration and calls for early neuroprotective therapies in chronic inflammatory diseases of the CNS.

Autoantibodies, a hallmark of autoimmune disease, are directed against diverse antigens and epitopes. This diversity aids in characterising disease progression and identifying disease-associated autoantibodies. Here we aimed to develop a sensitive assay to detect and quantify epitope-specific autoantibodies. We generated constructs to integrate known peptide epitopes into the TrxA protein, and used these to in vitro synthesize radio-labelled proteins for a radiobinding assay (RBA). This assay was first validated with an animal model using mice immunized with the MOG(40-55) peptide. Using type 1 diabetes-associated protein tyrosine phosphatase-like autoantigen IA-2 as a human model, we expressed IA-2(609-621), IA-2(619-631), or IA-2(609-631) peptide in the TrxA construct and used the RBA to test sera from 113 patients with type 1 diabetes and 87 controls. Antibodies were detected to the IA-2(609-631) epitope in 37 of 113 patient sera and one control; 17 sera were reactive to the IA-2(609-621) (JM1) epitope, and 16 to the IA-2(619-631) (JM2) epitope. Interestingly, a novel third epitope (JM3) was identified in 7 sera that reacted to IA-2(609-631) but not the JM1 and JM2 sub-specificities. An ELISA using IA-2(609-631) was less sensitive than the RBA, as it detected only some of the JM1 and JM3 antibody positive sera and none of the JM2. Mutagenesis of single IA-2(609-631) amino acids in combination with the RBA showed that antibodies to JM2 and JM3 were highly diverse in their specific residue requirements. Our strategy using in vitro synthesized antigens and the RBA was thus a highly sensitive and versatile method to identify epitopes and quantify epitope-specific antibodies.

The very simplistic view that inflammation in multiple sclerosis (MS) is tout court detrimental has to be profoundly reconsidered. Experimental evidence strongly supports the concept that inflammation in MS is aimed not only at destroying and phagocytosing damaged (infected?) cells (detrimental phase) but also at promoting tissue regeneration or tissue repair via scar formation (resolution or protective phase). Microglia seem to play a crucial role in the different inflammatory phases in MS because they move through multiple functional levels (quantum jumps) either to remove the "danger" signal or to restore the integrity of the central nervous system. The understanding of the molecular signature of any of the quantum microglial states could be useful to better define the cellular and molecular mechanisms underling the different phases of inflammation in MS. This could contribute not only to the design of better therapeutic drugs, but also to the comprehension of the shortcomings of currently used anti-inflammatory drugs.

Os últimos vinte anos caracterizaram-se pela proliferação de tecnologias que tornaram possível decifrar o genoma das espécies, localizar e identificar particularidades na sua seqüência, elucidar as suas funções dentro dos sistemas biológicos e, sobretudo, começar a entender os mecanismos que controlam as interações entre os genótipos e os estímulos ambientais, que são responsáveis pela diversidade fenotípica. Estes estudos sobre as bases moleculares da variabilidade fenotípica abriram uma nova abordagem científica, caracterizada pela multiplicidade das questões envolvidas, que resultou no surgimento de novas áreas de pesquisa, cujos conhecimentos estão sendo aplicados em diversos campos da biologia, inclusive na zootecnia. Tendo em vista o grande impacto que tais conhecimentos estão tendo sobre a compreensão dos fenômenos biológicos, parece ser oportuno fazer uma avaliação das potencialidades de aplicação das abordagens de Genômica Funcional em pesquisas de nutrição e alimentação de ruminantes. Nesse contexto, este artigo está focado na descrição das principais "ferramentas genômicas" disponíveis e na discussão sobre a viabilidade de se utilizar as informações por elas geradas em benefício da produção animal.The last twenty years have been characterized by some remarkable technological advances in the field of genomics which were essential to unveil the genome of varies species. Furthermore, these genomic advances contributed to pinpoint particularities in genome sequences which might be associated with phenotypic variations, therefore, it became possible to study functions associated with specific sequences in a more holistic context within biological systems. More importantly, the blossoming in genomics contributed to a better understanding on the mechanisms that control the interactions between genotype and environmental factors which are responsible for phenotypic variability. Genomic advances also promoted innovative and multi-dimensional scientific perspectives in research fields of biology, such as in animal science. Having in mind the remarkable impacts of genomics on the understanding of biological phenomena, it becomes clear that an assessment of the potential applications of functional genomics in the field ruminant nutrition should be performed. For this reason, this paper reviews several important genomic tools, and discusses the feasibility of using these tools towards an enhancement of animal production.

A tecnologia de microarrays, ou microarranjos de DNA, possibilita a avaliação simultânea da expressão de milhares de genes em diferentes tecidos em determinado organismo, em diferentes estágios de desenvolvimento ou condições ambientais. Microarrays são bastante utilizados em experimentos de genômica funcional com diversas espécies animais e vegetais, e têm sido gradativamente incorporados em diferentes áreas da pesquisa zootécnica, como crescimento e metabolismo, resposta imune a doenças, reprodução e resposta a fatores de estresse não-infecciosos (restrição alimentar, exposição a elementos tóxicos e outras condições ambientais desfavoráveis), bem como melhoramento genético animal. Tais experimentos, entretanto, são ainda consideravelmente caros, como consequência, geralmente são conduzidos com tamanhos amostrais relativamente pequenos. Por outro lado, a realização dos experimentos com microarrays, desde a coleta das amostras, até a obtenção das imagens para análise, envolve uma série de procedimentos laboratoriais de alta complexidade, que frequentemente introduzem variações adicionais aos resultados obtidos. Desta maneira, a condução de ensaios com microarrays requer cuidadoso delineamento experimental e análise estatística dos dados. Nesta apresentação são discutidos princípios básicos do planejamento de ensaios com microarrays, bem como as ferramentas estatísticas e computacionais mais comuns para a análise dos mesmos. São também discutidos alguns exemplos de aplicação de experimentos com microarrays em zootecnia e, numa última seção, são traçadas algumas considerações finais envolvendo os tópicos gerais abordados.Microarray technology allows monitoring thousands of genes simultaneously in a specific tissue of an organism, in different developmental stages or environmental conditions. Microarrays are very common in functional genomics experiments with both animals and plant species, and they have been increasingly used also in different areas of livestock research, such as growth and metabolism, reproduction, immune response to diseases and parasites, response to non-infectious stress factors (such as dietary restriction, exposure to toxic elements and other unfavorable environmental conditions) as well as animal breeding. Such experiments, however, are still considerably expensive and time consuming and, consequently, they are performed with relatively small sample sizes. Nonetheless, microarray experiments are extremely complex, as they involve a number of laboratorial procedures such as sample collection, RNA extraction, reverse transcription and labeling, and the final hybridization. Hence, microarray assays require careful experimental planning and statistical data analysis. In this manuscript, basic principles of experimental design for microarray studies are reviewed, as well as the most common statistical and computational tools used for their analysis. In addition, some examples of application of microarray technology in animal science are discussed, and some concluding remarks are presented afterward.

Roberto Furlan,1 Sangeeta Krishnan,2 Jeffrey Vietri3 1Advanced Methods, Kantar Health, Epsom, Surrey, UK; 2Global Health Economics and Outcomes Research, Biogen, MA, USA; 3Health Outcomes, Kantar Health, Milan, Italy Introduction: New longer-acting factor products will potentially allow for less frequent infusion in prophylactic treatment of hemophilia. However, the role of administration frequency relative to other treatment attributes in determining preferences for prophylactic hemophilia treatment regimens is not well understood. Aim: To identify the relative importance of frequency of administration, efficacy, and other treatment characteristics among candidates for prophylactic treatment for hemophilia A and B. Method: An Internet survey was conducted among hemophilia patients and the parents of pediatric hemophilia patients in Australia, Canada, and the US. A monadic conjoint task was included in the survey, which varied frequency of administration (three, two, or one time per week for hemophilia A; twice weekly, weekly, or biweekly for hemophilia B), efficacy (no bleeding or breakthrough bleeding once every 4months, 6months, or 12months), diluent volume (3mL vs 2.5mL for hemophilia A; 5mL vs 3mL for hemophilia B), vials per infusion (2 vs 1), reconstitution device (assembly required vs not), and manufacturer (established in hemophilia vs not). Respondents were asked their likelihood to switch from their current regimen to the presented treatment. Respondents were told to assume that other aspects of treatment, such as risk of inhibitor development, cost, and method of distribution, would remain the same. Results: A total of 89 patients and/or parents of children with hemophilia A participated; another 32 were included in the exercise for hemophilia B. Relative importance was 47%, 24%, and 18% for frequency of administration, efficacy, and manufacturer, respectively, in hemophilia A; analogous values were 48%, 26%, and 21% in hemophilia B. The remaining attributes had little impact on preferences. Conclusion: Patients who are candidates for prophylaxis and their caregivers indicate a preference for reduced frequency of administration and high efficacy, but preferences were more sensitive to administration frequency than small changes in annual bleeding rate. Keywords: prophylaxis, conjoint analysis, treatment preferences, breakthrough bleeds, frequency of administration

HIV type 1 (HIV-1) infects CD4(+) T lymphocytes and tissue macrophages. Infected macrophages differ from T cells in terms of decreased to absent cytopathicity and for active accumulation of new progeny HIV-1 virions in virus-containing compartments (VCC). For these reasons, infected macrophages are believed to act as "Trojan horses" carrying infectious particles to be released on cell necrosis or functional stimulation. Here we explored the hypothesis that extracellular ATP (eATP) could represent a microenvironmental signal potentially affecting virion release from VCC of infected macrophages. Indeed, eATP triggered the rapid release of infectious HIV-1 from primary human monocyte-derived macrophages (MDM) acutely infected with the CCR5-dependent HIV-1 strain. A similar phenomenon was observed in chronically infected promonocytic U1 cells differentiated to macrophage-like cells (D-U1) by costimulation with phorbol esters and urokinase-type plasminogen activator. Worthy of note, eATP did not cause necrotic, apoptotic, or pyroptotic cell death, and its effect on HIV-1 release was suppressed by Imipramine (an antidepressant agent known to inhibit microvesicle formation by interfering with membrane-associated acid sphingomyelinase). Virion release was not triggered by oxidized ATP, whereas the effect of eATP was inhibited by a specific inhibitor of the P2X7 receptor (P2X7R). Thus, eATP triggered the discharge of virions actively accumulating in VCC of infected macrophages via interaction with the P2X7R in the absence of significant cytopathicity. These findings suggest that the microvesicle pathway and P2X7R could represent exploitable targets for interfering with the VCC-associated reservoir of infectious HIV-1 virions in tissue macrophages.

Made available in DSpace on 2019-09-11T20:58:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2015 RESUMO O óleo volátil da melaleuca (Melaleuca alternifolia Maiden & Betche, Cheel) possui atividade antimicrobiana podendo causar efeitos sobre as plantas. Avaliou-se a inibição do óleo em Cercospora beticolaSacc., e seu efeito no aumento da produção e qualidade de raízes de beterraba. As doses foram de 0,13; 0,67; 0,80 e 1,00% do óleo, além das testemunhas composta pelo meio de cultura Batata Dextrose Ágar (BDA) no experimento in vitro, e água no experimento in vivo. As plantas foram pulverizadas duas vezes por semana. O delineamento foi inteiramente casualizado, com 4 repetições, e as médias foram comparadas pelo teste Tukey a 5% de probabilidade. O índice de infecção das folhas foi determinado por escala diagramática além do peso e diâmetro das raízes. Os resultados de inibição do crescimento micelial para as doses do óleo foram 0; 56; 87; 83 e 99%, e os índices de infecção: 77,08; 35,62; 21,04; 19,37 e 20,00%, respectivamente, para a testemunha e as doses 0,13; 0,67; 0,80 e 1,00% do óleo. Somente na concentração de 0,80% o óleo proporcionou relação positiva entre o ganho de peso e o diâmetro das raízes. O óleo de Melaleuca foi eficaz no controle de C. beticola e, como consequência, houve produção de raízes de beterraba com melhor desenvolvimento. ABSTRACT The volatile oil from Melaleuca (Melaleuca alternifolia Maiden & Betche Cheel.) has antimicrobial properties and can promote several effects on plant cultivation. The aim of this study was to evaluate the inhibition of the oil in Cercospora beticola Sacc. and if it favors the growth and development of beet root. The doses were 0.13, 0.67, 0.8 and 1% of oil, besides the control PDA (potato-dextrose-agar) in vitro (laboratory condition) and with water as treatment control in vivo (field conditions). The plants were sprayed twice a week. The treatments were completely randomized and the averages were compared using the Tukey test at 5%. The infection rate of leaves was measured by diagrammatic scale besides the weight and diameter of tubers. The inhibition results of the radial growth by oil treatments were 0; 56, 87, 83 and 99%, while the infection rate showed: 77.08, 35.62, 21.04, 19.37 and 20% respectively to the control and to the oil concentration of 0,13; 0,67; 0,80 e 1,00%. Only at concentration of 0.8% the tea tree oil showed a positive relationship between tuber´s weight and tuber´s diameter gains. It can be concluded that tea tree oil is effective to controlling C. beticola, and also promotes an increase on development in beet tubers. [SOUZA, A.D.; ROGGERIO, T.U] Faculdade Integral Cantareira, Brazil FURLAN, M.R.] Universidade de Taubaté, Brazil AOYAMA, E.M.] Centro Universitário do Espírito Santo, Brazil

Background and objective: We explored which clinical and biochemical variables predict conversion from clinically isolated syndrome (CIS) to clinically definite multiple sclerosis (CDMS) in a large international cohort. Methods: Thirty-three centres provided serum samples from 1047 CIS cases with at least two years’ follow-up. Age, sex, clinical presentation, T2-hyperintense lesions, cerebrospinal fluid (CSF) oligoclonal bands (OCBs), CSF IgG index, CSF cell count, serum 25-hydroxyvitamin D3 (25-OH-D), cotinine and IgG titres against Epstein-Barr nuclear antigen 1 (EBNA-1) and cytomegalovirus were tested for association with risk of CDMS. Results: At median follow-up of 4.31 years, 623 CIS cases converted to CDMS. Predictors of conversion in multivariable analyses were OCB (HR = 2.18, 95% CI = 1.71–2.77, p < 0.001), number of T2 lesions (two to nine lesions vs 0/1 lesions: HR = 1.97, 95% CI = 1.52–2.55, p < 0.001; >9 lesions vs 0/1 lesions: HR = 2.74, 95% CI = 2.04–3.68, p < 0.001) and age at CIS (HR per year inversely increase = 0.98, 95% CI = 0.98–0.99, p < 0.001). Lower 25-OH-D levels were associated with CDMS in univariable analysis, but this was attenuated in the multivariable model. OCB positivity was associated with higher EBNA-1 IgG titres. Conclusions: We validated MRI lesion load, OCB and age at CIS as the strongest independent predictors of conversion to CDMS in this multicentre setting. A role for vitamin D is suggested but requires further investigation.

Neuroinflammatory disorders such as multiple sclerosis (MS) encountered a therapeutic revolution in the last 20 years and have raised hope also for neurodegenerative diseases affecting especially the elderly population in western countries. Biomarkers, especially those from the field of neuroimaging, were decisive in accelerating drug development and optimization of treatments. Neuroimaging, however, may be not sufficient in the case of neurodegeneration, where structural alterations accumulate at a very low rate over several years. To capture this slow evolution, biomarkers in the cerebrospinal fluid appear as a very plausible candidate. We will review here, in general terms, the state of the art in the field biomarkers in the CSF with a special focus on the application of “omics” platforms.

CSF oligoclonal bands (OCBs) in patients with clinically isolated syndromes (CIS) are a risk factor for clinically definite multiple sclerosis (CDMS). We aimed to address the relevance of the number of OCBs in the prognosis of CIS patients. 219 CIS patients were included in the study, and 42% of them developed the disease during follow-up (median: 5.04 years). Patients with a high number of CSF OCBs (third quartile, 8-12 OCBs) had 2.5-fold increase in CDMS risk, while no further increase in the HR of disease was observed for patients with more than 12 OCBs. The results did not change after adjustment for additional correlates of CDMS development. This association may be due to the epitope-spreading phenomenon and may reflect the stage of the disease at the time of the examination.

Primary proinflammatory cytokines, such as IL-1beta, play a crucial pathogenic role in multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE), and may represent, therefore, a suitable therapeutic target. We have previously established the delivery of anti-inflammatory cytokine genes within the central nervous system (CNS), based on intracisternal (i.c.) injection of non-replicative HSV-1-derived vectors. Here we show the therapeutic efficacy of i.c. administration of an HSV-1-derived vector carrying the interleukin-1receptor antagonist (IL-1ra) gene, the physiological antagonist of the proinflammatory cytokine IL-1, in C57BL/6 mice affected by myelin oligodendrocyte glycoprotein-induced EAE. IL-1ra gene therapy is effective preventively, delaying EAE onset by almost 1 week (22.4+/-1.4 days post-immunization vs 15.9+/-2.1 days in control mice; P=0.0229 log-rank test), and decreasing disease severity. Amelioration of EAE course was associated with a reduced number of macrophages infiltrating the CNS and in a decreased level of proinflammatory cytokine mRNA in the CNS, suggesting an inhibitory activity of IL-1ra on effector cell recruitment, as antigen-specific peripheral T-cell activation and T-cell recruitment to the CNS is unaffected. Thus, local IL-1ra gene therapy may represent a therapeutic alternative for the inhibition of immune-mediated demyelination of the CNS.

The growth hormone receptor (GHR) is the cell surface receptor for growth hormone (GH) and is required for GH to carry out its effects on target tissues. The objectives of the present study were to estimate the allele and genotype frequencies of the GHR/Alu I gene polymorphism located in the regulatory region in beef cattle belonging to different genetic groups and to determine associations between this polymorphism and growth and carcass traits. Genotyping was performed on 384 animals, including 79 Nellore (Zebu), 30 Canchim (5/8 Charolais + 3/8 Zebu), 30 Simmental × Nellore crossbred and 245 Angus × Nellore crossbred cattle. Alleles Alu I(+), Alu I(−) and Alu I(N)–null allele–were evidenced for the GHR/Alu I polymorphism and the frequency of the Alu I(N) allele was significantly higher than the frequency of the Alu I(+) and Alu I(−) alleles in all genetic groups. Genotype Alu I(N/N) of the GHR/Alu I predominated in Nellore animals, while the Alu I(N/+) and Alu I(N/−) predominated in the other genetic groups. In the association studies, traits of interest were analyzed using the General Linear Model (GLM) procedure of the SAS program and least squares means of the genotypes were compared by the Tukey test. Significant associations (P

The secondary progressive phase of multiple sclerosis (MS), which is characterised by a steady accrual of fixed disability after an initial relapsing remitting course, is not clearly understood. Although there is no consensus on the mechanisms underlying such a transition to the progressive phase, epidemiological and neuroimaging studies indicate that it is probably driven by the high prevalence of neurodegenerative compared with inflammatory pathological changes. This notion is lent support by the limited efficacy of available immunomodulating and immunosuppressive treatment strategies, which seems to be further decreased in the late stages of secondary progressive MS. No established clinical or paraclinical predictors of the transition from relapsing remitting to secondary progressive MS have been described. However, the use of quantitative MRI-derived measures is warranted to monitor natural history studies and therapeutic trials of secondary progressive MS with increased reliability. In view of the small effects of immunomodulating and immunosuppressive treatments in preventing the transition to secondary progression, the development of treatments promoting neuroaxonal repair remains an important goal in this disease.

Since the late 1970s conjoint analysis has been applied in estimating consumer preferences in marketing research. This article focuses on paired comparisons of profile design. In particular, it discusses how to reduce the number of pairs to be presented by incomplete comparisons of attributes. First, we present the reductions offered by cyclic designs and by group-divisible designs. Then, we show that the number of pairs to be presented can effectively be reduced by applying a random selection procedure. Copyright © 2006 John Wiley & Sons, Ltd.

Background: Acute optic neuritis is often in association with multiple sclerosis (MS). Proinflammatory cytokines trigger neuronal damage in neuroinflammatory disorders but their role in optic neuritis is poorly investigated. Objective: The objective of this work is to investigate the associations of intrathecal contents of proinflammatory cytokines with transient and persistent dysfunctions after optic neuritis. Methods: In 50 MS patients followed for up to six months, cerebrospinal fluid (CSF) levels of IL-1β, TNF and IL-8 were determined, along with clinical, neurophysiological and morphological measures of optic neuritis severity. Results: Visual impairment, measured by high- and low-contrast visual acuity, and delayed visual-evoked potential (VEP) latencies were significantly correlated to IL-8 levels during optic neuritis. IL-8 at the time of optic neuritis was also associated with persistent demyelination and final axonal loss, inferred by VEP and optical coherence tomography measures, respectively. Contents of IL-8 were correlated to functional visual outcomes, being higher among patients with incomplete recovery. Multivariate analysis confirmed that IL-8 significantly predicted final visual acuity, at equal values of demographics and baseline visual scores. Conclusion: Our study points to IL-8 as the main inflammatory cytokine associated with demyelination and secondary neurodegeneration in the optic nerve after optic neuritis.

Purpose of review Spontaneous remyelination occurs in the central nervous system of patients with multiple sclerosis. However, this process is not robust enough to promote a functional and stable recovery of the myelin architecture. The development of cellbased therapies, aimed at promoting multifocal remyelination, is therefore foreseen. Recent findings Several experimental cell-based strategies aimed at replacing damaged myelin-forming cells have been developed in the last few years. However, most of these therapeutic approaches – although consistently able to form new myelin sheaths at the transplantation site – are unfeasible owing to the mutifocality of the demyelinating process in multiple sclerosis patients and the inability to grow and produce large numbers of differentiated myelin-forming cells in vitro. Stem cell-based therapies that partially overcome these limitations have been proposed recently. Summary Stem cell-based remyelinating therapies can be considered a plausible alternative strategy in immune-mediated demyelinating disorders. However, before any potential applications in patients with multiple sclerosis can be envisaged, it is necessary to confront the following preliminary, and still unsolved, questions: (1) the ideal stem cell source for transplantation; (2) the most appropriate route of stem cell administration; and, last but not least, (3) the best approach for achieving an appropriate, functional and long-lasting integration of transplanted stem cells into the host tissue.

Over 40,000 sugarcane (Saccharum officinarum) consensus sequences assembled from 237,954 expressed sequence tags were compared with the protein and DNA sequences from other angiosperms, including the genomes of Arabidopsis and rice (Oryza sativa). Approximately two-thirds of the sugarcane transcriptome have similar sequences in Arabidopsis. These sequences may represent a core set of proteins or protein domains that are conserved among monocots and eudicots and probably encode for essential angiosperm functions. The remaining sequences represent putative monocot-specific genetic material, one-half of which were found only in sugarcane. These monocot-specific cDNAs represent either novelties or, in many cases, fast-evolving sequences that diverged substantially from their eudicot homologs. The wide comparative genome analysis presented here provides information on the evolutionary changes that underlie the divergence of monocots and eudicots. Our comparative analysis also led to the identification of several not yet annotated putative genes and possible gene loss events in Arabidopsis.

Alpha-lipoic acid (alpha-LA) is a neuroprotective metabolic antioxidant that has been shown to cross the blood brain barrier. We tested whether alpha-LA is capable to prevent MOG35-55-induced experimental autoimmune encephalomyelitis (EAE), an established model of multiple sclerosis (MS). Daily oral administration of alpha-LA, starting at the time of immunization, significantly prevented EAE progression as compared to control mice. This was associated with a reduction of CNS infiltrating T cells and macrophages as well as decreased demyelination. We then tested alpha-LA in a therapeutic protocol aimed at suppressing EAE after its onset. Intraperitoneal (i.p.), but not oral, administration of alpha-LA significantly prevented disease progression when compared to vehicle-treated controls. Similarly, we observed significant reduction of demyelination and inflammatory infiltration. This clinical effect was not due to an impairment of MOG35-55 recognition by encephalitogenic T cells. In contrast, MOG-specific T cells showed a decreased production of IFNgamma and IL-4, suggesting an immunosuppressive activity on both Th1 and Th2 cytokines. In addition, alpha-LA inhibited the proteolytic activity of MMP2 and MMP9 only at very high doses. Our data indicate that alpha-LA can effectively interfere with the autoimmune reaction associated with EAE through mechanisms other than its antioxidant activity and supports further studies on the use of alpha-LA as a potential therapy for MS.

Invariant NKT (inv. NKT) cells co-express an invariant alpha beta T cell receptor and the NK receptor NK1.1 and, upon CD1d-restricted recognition of the glycosphingolipid antigen alpha-galactosyl ceramide (alphaGalCer), secrete large amounts of regulatory cytokines. We investigated whether alphaGalCer-dependent activation of inv. NKT cells protects from experimental autoimmune encephalomyelitis (EAE), an immune-mediated disease of the central nervous system mimicking multiple sclerosis, induced in C57BL/6 mice by the myelin oligodendrocyte glycoprotein (MOG) encephalitogenic peptide aa 35-55. alphaGalCer was administered at the time of immunization s.c., mixed with complete Freund's adjuvant and MOG35-55 peptide, or administered i.p., diluted in PBS. EAE onset was delayed and disease severity was decreased only when alphaGalCer was s.c. administered. The protective effect of s.c. administration of alphaGalCer was associated with a markedly enhanced IFN-gamma production by liver-confined inv. NKT cells which, in turn, suppressed Th1-cytokine production and fostered secretion of IL-10 from MOG35-55-specific T cells. In vivo neutralization of IFN-gamma, but notIL-4, reversed the protective effect induced by s.c. administration of alphaGalCer, further confirming the critical regulatory role exerted by IFN-gamma-producing inv. NKT cells. Our results indicate that alphaGalCer, properly administered, may elicit an inv. NKT-cell-mediated suppressive effect on the effector function of encephalitogenic T cells; this effect is able to ameliorate autoimmunedemyelination.

Gene therapy protocols aimed to deliver therapeutic molecules into the central nervous system may represent an alternative therapeutic strategy in patients affected by inflammatory demyelinating diseases of the central nervous system where systemic therapies have shown limited therapeutic efficacy possibly owing to the blood-brain barrier, a major obstacle for the entry of therapeutic molecules into the central nervous system.Among inflammatory demyelinating diseases of the central nervous system, gene therapy approaches have been so far developed almost exclusively for multiple sclerosis. However, the chronic/relapsing nature of the disease, the restriction to the central nervous system of the pathological process as well as the necessity to inhibit the ongoing inflammatory process but also to foster endogenous remyelinating pathways, have posed several questions which still need to be properly addressed for the development of a successful gene therapy strategy in multiple sclerosis patients.The gene therapy approaches for multiple sclerosis have been so far developed and tested only in rodents and monkeys with experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. The results of these studies clearly indicate that the delivery of therapeutic genes within the central nervous system is superior to the peripheral delivery. In particular, the intracerebral delivery of genes coding for anti-inflammatory and/or neurotrophic molecules, using gene vectors derived from non-replicative viruses, showed to inhibit not only the detrimental function of blood-borne mononuclear effector cells but also to foster proliferation and differentiation of surviving oligodendrocytes within demyelinated areas. Here, we summarize the most recent findings of this novel area of research.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2014-02-26T17:15:29Z No. of bitstreams: 1 WOS000180834300035.pdf: 1288581 bytes, checksum: c80b0ab1625eeba1cd15e9da6e465b26 (MD5) Made available in DSpace on 2014-02-26T17:15:29Z (GMT). No. of bitstreams: 1 WOS000180834300035.pdf: 1288581 bytes, checksum: c80b0ab1625eeba1cd15e9da6e465b26 (MD5) Previous issue date: 2003-02-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T15:25:38Z No. of bitstreams: 1 WOS000180834300035.pdf: 1288581 bytes, checksum: c80b0ab1625eeba1cd15e9da6e465b26 (MD5) Made available in DSpace on 2014-05-20T15:25:38Z (GMT). No. of bitstreams: 1 WOS000180834300035.pdf: 1288581 bytes, checksum: c80b0ab1625eeba1cd15e9da6e465b26 (MD5) Previous issue date: 2003-02-01 Xylella fastidiosa is a xylem-dwelling, insect-transmitted, gamma-proteobacterium that causes diseases in many plants, including grapevine, citrus, periwinkle, almond, oleander, and coffee. X. fastidiosa has an unusually broad host range, has an extensive geographical distribution throughout the American continent, and induces diverse disease phenotypes. Previous molecular analyses indicated three distinct groups of X.fastidiosa isolates that were expected to be genetically divergent. Here we report the genome sequence of X. fastidiosa (Temecula strain), isolated from a naturally infected grapevine with Pierce's disease (PD) in a wine-grape-growing region of California. Comparative analyses with a previously sequenced X.fastidiosa strain responsible for citrus variegated chlorosis (CVC) revealed that 98% of the PD X.fastidiosa Temecula genes are shared with the CVC X. fastidiosa strain 9a5c genes. Furthermore, the average amino acid identity of the open reading frames in the strains is 95.7%. Genomic differences are limited to phage-associated chromosomal rearrangements and deletions that also account for the strain-specific genes present in each genome. Genomic islands, one in each genome, were identified, and their presence in other X.fastidiosa strains was analyzed. We conclude that these two organisms have identical metabolic functions and are likely to use a common set of genes in plant colonization and pathogenesis, permitting convergence of functional genomic strategies. Univ São Paulo, Inst Biociencias, Dept Bot, BR-05508900 São Paulo, Brazil Univ São Paulo, Inst Quim, BR-05508900 São Paulo, Brazil Univ São Paulo, Inst Ciências Biomed, BR-05508900 São Paulo, Brazil Univ São Paulo, Escola Super Agr Luiz de Queiroz, BR-13418900 Piracicaba, Brazil Univ São Paulo, Ctr Energia Nucl Agr, BR-13400970 Piracicaba, Brazil Inst Agron Campinas, Ctr Citricultura Sylio Moreira, BR-13490970 Cordeiropolis, Brazil Univ Estadual Paulista, Fac Ciências Agr & Vet, BR-14884900 Jaboticabal, Brazil Univ Estadual Campinas, Ctr Biol Mol & Engn Genet, BR-13083970 Campinas, SP, Brazil Univ Estadual Campinas, Inst Biol, BR-13083970 Campinas, SP, Brazil Univ Estadual Campinas, Inst Comp, BR-13083970 Campinas, SP, Brazil Univ São Paulo, Fac Ciências Farmaceut, BR-14040901 Ribeirao Preto, Brazil Univ São Paulo, Fac Filosofia Ciências & Letras, BR-14040901 Ribeirao Preto, Brazil Inst Ludwig Pesquisa Canc, BR-01509010 São Paulo, Brazil Univ Mogi das Cruzes, Nucleo Integrado Biotecnol, BR-08780911 Mogi das Cruzes, Brazil Inst Agron Campinas, BR-13001970 Campinas, SP, Brazil Inst Biol, BR-04014002 São Paulo, Brazil Univ Estadual Paulista, Fac Med Vet & Zootecnia, BR-18603970 Botucatu, SP, Brazil Univ Estadual Paulista, Fac Ciências Agron, BR-18603970 Botucatu, SP, Brazil Univ Estadual Paulista, Inst Biociencias, BR-18618000 Botucatu, SP, Brazil Univ Fed Sao Carlos, BR-13600970 São Paulo, Brazil Univ Fed Mato Grosso do Sul, Dept Comp & Estatist, BR-79070900 Campo Grande, MS, Brazil USDA ARS, Parlier, CA 93648 USA Univ Estadual Paulista, Fac Ciências Agr & Vet, BR-14884900 Jaboticabal, Brazil Univ Estadual Paulista, Fac Med Vet & Zootecnia, BR-18603970 Botucatu, SP, Brazil Univ Estadual Paulista, Fac Ciências Agron, BR-18603970 Botucatu, SP, Brazil Univ Estadual Paulista, Inst Biociencias, BR-18618000 Botucatu, SP, Brazil

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T19:06:29Z No. of bitstreams: 1 S1516-35982002000900015.pdf: 49088 bytes, checksum: d1a7fbbf19e30df27d1816356ffd81d0 (MD5) Made available in DSpace on 2013-08-22T19:06:29Z (GMT). No. of bitstreams: 1 S1516-35982002000900015.pdf: 49088 bytes, checksum: d1a7fbbf19e30df27d1816356ffd81d0 (MD5) Previous issue date: 2002-11-01 Made available in DSpace on 2013-09-30T18:27:09Z (GMT). No. of bitstreams: 2 S1516-35982002000900015.pdf: 49088 bytes, checksum: d1a7fbbf19e30df27d1816356ffd81d0 (MD5) S1516-35982002000900015.pdf.txt: 34595 bytes, checksum: 4d431bd7c64547f297e6cea6bcaee7fd (MD5) Previous issue date: 2002-11-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:40:32Z No. of bitstreams: 2 S1516-35982002000900015.pdf: 49088 bytes, checksum: d1a7fbbf19e30df27d1816356ffd81d0 (MD5) S1516-35982002000900015.pdf.txt: 34595 bytes, checksum: 4d431bd7c64547f297e6cea6bcaee7fd (MD5) Made available in DSpace on 2014-05-20T13:40:32Z (GMT). No. of bitstreams: 2 S1516-35982002000900015.pdf: 49088 bytes, checksum: d1a7fbbf19e30df27d1816356ffd81d0 (MD5) S1516-35982002000900015.pdf.txt: 34595 bytes, checksum: 4d431bd7c64547f297e6cea6bcaee7fd (MD5) Previous issue date: 2002-11-01 Este trabalho foi realizado com o objetivo de estudar a influência do período de arraçoamento sobre parâmetros zootécnicos, desenvolvimento visceral e composição da carcaça de frangos de corte. Foram utilizados 630 frangos da linhagem Cobb-500®, divididos em três programas de alimentação, a partir do 8º até o 50º dia de vida: ad libitum (AL - alimento durante 24 horas), arraçoamento diurno (AD - alimento à vontade das 7-19 h e restrito durante o período noturno 19-7 h) e arraçoamento noturno (AN - alimento à vontade das 19-7 h e restrito durante o período diurno 7-19h). A água foi fornecida à vontade para todos os grupos durante o período experimental. O período de arraçoamento (12 horas noturno ou diurno) reduziu significativamente o peso vivo final das aves. No entanto, aves arraçoadas durante o período noturno consumiram menos ração e mostraram melhor índice de conversão alimentar. O uso dos diferentes períodos de arraçoamento não afetou o desenvolvimento do fígado, moela, coração e comprimento do intestino. Aves arraçoadas por 12 horas, tanto no período noturno quanto no diurno, apresentaram maior teor de gordura na carcaça. O teor de cinzas não foi afetado pelo programa alimentar. Os resultados deste experimento sugerem que a adaptação ao novo programa alimentar foi muito lenta. Assim, as aves não foram capazes de manter o consumo de alimento compatível com seu desenvolvimento, ocorrendo com isso prejuízo no desempenho produtivo (peso vivo), bem como na composição da carcaça (gordura). This investigation was carried out to study the feeding period influence on broilers performance, viscera development, and carcass composition. A total of 630 broiler chickens, Cobb-500® strain, were allocated in three feeding programs from 8th day to 50th day of life: ad libitum (AL - food available 24 hours), diurnal feeding (DF - food provided ad libitum from 7 am to 7 pm and fed restricted from 7 pm to 7 am) and nocturnal feeding (NF- food provided ad libitum from 7 pm to 7 am and fed restricted from 7 am to 7 pm). The water was provided ad libitum for all groups during experimental period. The feeding period (12 hours at nocturnal and diurnal) significantly decreased the broilers' final body weight. However, broilers fed at night showed lower feed intake and had better feed conversion index. The different feeding periods did not affect liver, gizzard, heart and intestine length development. Broilers fed for 12 hours during the night and during the day showed higher carcass fat content. Ash content was not affected by feeding program. The results suggested that the adaptation to feeding program was slow, thus, the broilers were not able to keep the feed consumption compatible with their development, with lower productive performance and higher carcass fat. Faculdade de Ciências Agrárias e Veterinárias Depto. de Morfologia e Fisiologia Animal UNESP FCAV curso de Zootecnia UNESP FCAV UNESP FCAV Depto. de Cências Exatas UNESP FMVZ Depto. de Melhoramento e Nutrição Animal UNESP FCAV curso de Zootecnia UNESP FCAV UNESP FCAV Depto. de Cências Exatas UNESP FMVZ Depto. de Melhoramento e Nutrição Animal

Este trabalho foi realizado com o objetivo de estudar a influência do período de arraçoamento sobre parâmetros zootécnicos, desenvolvimento visceral e composição da carcaça de frangos de corte. Foram utilizados 630 frangos da linhagem Cobb-500®, divididos em três programas de alimentação, a partir do 8º até o 50º dia de vida: ad libitum (AL - alimento durante 24 horas), arraçoamento diurno (AD - alimento à vontade das 7-19 h e restrito durante o período noturno 19-7 h) e arraçoamento noturno (AN - alimento à vontade das 19-7 h e restrito durante o período diurno 7-19h). A água foi fornecida à vontade para todos os grupos durante o período experimental. O período de arraçoamento (12 horas noturno ou diurno) reduziu significativamente o peso vivo final das aves. No entanto, aves arraçoadas durante o período noturno consumiram menos ração e mostraram melhor índice de conversão alimentar. O uso dos diferentes períodos de arraçoamento não afetou o desenvolvimento do fígado, moela, coração e comprimento do intestino. Aves arraçoadas por 12 horas, tanto no período noturno quanto no diurno, apresentaram maior teor de gordura na carcaça. O teor de cinzas não foi afetado pelo programa alimentar. Os resultados deste experimento sugerem que a adaptação ao novo programa alimentar foi muito lenta. Assim, as aves não foram capazes de manter o consumo de alimento compatível com seu desenvolvimento, ocorrendo com isso prejuízo no desempenho produtivo (peso vivo), bem como na composição da carcaça (gordura).This investigation was carried out to study the feeding period influence on broilers performance, viscera development, and carcass composition. A total of 630 broiler chickens, Cobb-500® strain, were allocated in three feeding programs from 8th day to 50th day of life: ad libitum (AL - food available 24 hours), diurnal feeding (DF - food provided ad libitum from 7 am to 7 pm and fed restricted from 7 pm to 7 am) and nocturnal feeding (NF- food provided ad libitum from 7 pm to 7 am and fed restricted from 7 am to 7 pm). The water was provided ad libitum for all groups during experimental period. The feeding period (12 hours at nocturnal and diurnal) significantly decreased the broilers' final body weight. However, broilers fed at night showed lower feed intake and had better feed conversion index. The different feeding periods did not affect liver, gizzard, heart and intestine length development. Broilers fed for 12 hours during the night and during the day showed higher carcass fat content. Ash content was not affected by feeding program. The results suggested that the adaptation to feeding program was slow, thus, the broilers were not able to keep the feed consumption compatible with their development, with lower productive performance and higher carcass fat.

Com o objetivo de se avaliar o comportamento fisiológico das aves, bem como, se ocorre a expressão da proteína do choque térmico (Hsp 70) no fígado e cérebro, como conseqüência da exposição ao estresse por calor, em quatro dias sucessivos (2 a 5 dias de vida), 20 pintos de corte de duas linhagens Pescoço pelado (Na/na) e Hubbard-Petersen, foram ou não estressados à temperatura de 36-37°C, durante cinco horas. Foram colhidas amostras de fígado e cérebro, ao final do período de estresse, e analisadas por Western Blotting, para determinação dos níveis de Hsp 70. Os resultados não mostraram associação entre perda de peso corporal e as demais características. Aumentos da variação de temperatura cloacal apresentaram aumentos nos níveis de Hsp70 no tecido hepático e cerebral. Concluiu-se que a concentração de Hsp70 no cérebro, apresentou-se correlacionada com a variação de temperatura cloacal, quando se usou o condicionamento ao calor em pintos de corte, embora não se possa afirmar que seria indicativo de termotolerância de aves.This study was run to evaluate the physiological response and expression of hepatic and brain Hsp70, of 20 Naked neck (Na/na) and Hubbard-Petersen broiler chicks (2 to 5 days of age), exposed or not to 36-37°C. The trial was conducted during four days and the birds were exposed to high temperature during 5 hours. Liver and brain tissue samples were collected at the end of the stress period and analyzed for Western Blotting for determination of Hsp70 level. No association between body weight loss and the other variables were observed. Increasing the variation in cloacal temperature resulted in an increase in Hsp70 hepatic and brain tissue levels. Results indicate that brain Hsp70 levels were correlated with variation in cloacal temperature, concerning the chicks subjected to heat stress, but it can not be stated that it is an indicative of thermotolerance by the birds.

Alzheimer’s disease (AD) is the most common form of dementia, while mild cognitive impairment (MCI) causesa slight but measurable decline in cognitive abilities. A person with MCI has an increased risk of developing AD or anotherdementia. Thus, it is of medical interest to develop predictive tools to assess this risk. A growing awareness exists that pro-oxidative state and neuro-inflammation are both involved in AD. However, the extent of this relationship is still a matterof debate. Due to the expected non-linear correlations between oxidative and inflammatory markers, traditional statistics isunsuitable to dissect their relationship with the disease. Artificial neural networks (ANNs) are computational models inspiredby central nervous system networks, capable of machine learning and pattern recognition. The aim of this work was to disclosethe relationship between immunological and oxidative stress markers in AD and MCI by the application of ANNs. Througha machine learning approach, we were able to construct an algorithm to classify MCI and AD with high accuracy. Such aninstrument, requiring a small amount of immunological and oxidative-stress parameters, would be useful in the clinical practice.Moreover, applying an innovative non-linear mathematical technique, a global immune deficit was shown to be associated withcognitive impairment. Surprisingly, both adaptive and innate immunity were peripherally defective in AD and MCI patients.From this study, new pathogenetic aspects of these diseases could emerge.Keywords: Artificial neural network, machine learning, oxidative-stress, neurodegeneration, pattern recognition, relationships