Therapeutic targeting of "undruggable" MYC.

c-MYC controls global gene expression and regulates cell proliferation, cell differentiation, cell cycle, metabolism and apoptosis. According to some estimates, MYC is dysregulated in ≈70% of human cancers and strong evidence implicates aberrantly expressed MYC in both tumor initiation and maintenance. In vivo studies show that MYC inhibition elicits a prominent anti-proliferative effect and sustained tumor regression while any alteration on healthy tissue remains reversible. This opens an exploitable window for treatment that makes MYC one of the most appealing therapeutic targets for cancer drug development. This review describes the main functional and structural features of the protein structure of MYC and provides a general overview of the most relevant or recently identified interactors that modulate MYC oncogenic activity. This review also summarizes the different approaches aiming to abrogate MYC oncogenic function, with a particular focus on the prototype inhibitors designed for the direct and indirect targeting of MYC.

Integrative Multi-omics Analysis to Characterize Human Brain Ischemia.

Stroke is a major cause of death and disability. A better comprehension of stroke pathophysiology is fundamental to reduce its dramatic outcome. The use of high-throughput unbiased omics approaches and the integration of these data might deepen the knowledge of stroke at the molecular level, depicting the interaction between different molecular units. We aimed to identify protein and gene expression changes in the human brain after ischemia through an integrative approach to join the information of both omics analyses. The translational potential of our results was explored in a pilot study with blood samples from ischemic stroke patients. Proteomics and transcriptomics discovery studies were performed in human brain samples from six deceased stroke patients, comparing the infarct core with the corresponding contralateral brain region, unveiling 128 proteins and 2716 genes significantly dysregulated after stroke. Integrative bioinformatics analyses joining both datasets exposed canonical pathways altered in the ischemic area, highlighting the most influential molecules. Among the molecules with the highest fold-change, 28 genes and 9 proteins were selected to be validated in five independent human brain samples using orthogonal techniques. Our results were confirmed for NCDN, RAB3C, ST4A1, DNM1L, A1AG1, A1AT, JAM3, VTDB, ANXA1, ANXA2, and IL8. Finally, circulating levels of the validated proteins were explored in ischemic stroke patients. Fluctuations of A1AG1 and A1AT, both up-regulated in the ischemic brain, were detected in blood along the first week after onset. In summary, our results expand the knowledge of ischemic stroke pathology, revealing key molecules to be further explored as biomarkers and/or therapeutic targets.

Blood Biomarkers to Differentiate Ischemic and Hemorrhagic Strokes.

OBJECTIVE: To validate a panel of blood biomarkers to differentiate between ischemic stroke (IS) and intracerebral hemorrhage (ICH) in patients with suspected stroke. METHODS: Patients with suspected stroke admitted within 4.5 hours after onset were enrolled. Blood samples were collected at hospital admission. Glial fibrillary acid protein (GFAP), retinol binding protein 4 (RBP-4), N-terminal proB-type natriuretic peptide (NT-proBNP), and endostatin were measured by immunoassays. Cutoff points were obtained for 100% specificity for IS. A high-sensitivity assay to measure GFAP and rapid point-of-care tests (POCTs) to measure RBP-4 and NT-proBNP were used in subsets of patients. Biomarker panels were evaluated in another cohort of 62 stroke mimics. RESULTS: A total of 189 patients (154 IS and 35 ICH) were enrolled. Patients with IS had higher RBP-4, NT-proBNP, and endostatin and lower GFAP levels than patients with ICH. The best biomarker combination for the identification of IS was RBP-4+NT-proBNP, which was able to identify 29.7% of patients with IS with 100% specificity. In the subset of patients for whom GFAP was measured with the high-sensitivity assay, RBP-4, NT-proBNP, and GFAP identified 51.5% of patients with IS with 100% specificity. When stroke mimics were included, specificities were reduced to 98.4 and 96.8%, respectively. POCTs of RBP-4 and NT-proBNP showed results similar results to those of conventional ELISAs. CONCLUSIONS: A biomarker panel including RBP-4, NT-proBNP, and GFAP provided moderate but potentially useful sensitivity rates at 100% specificity for IS diagnosis. If confirmed in future studies, this strategy might allow prehospital treatment in selected patients. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that a biomarker panel including RBP-4, NT-proBNP, and GFAP distinguishes IS from ICH with moderate accuracy.

Discriminative value of glial fibrillar acidic protein (GFAP) as a diagnostic tool in acute stroke. Individual patient data meta-analysis.

Glial fibrillar acidic protein (GFAP) in serum has been evaluated as a promising biomarker to differentiate between intracerebral hemorrhage (ICH) and acute ischemic stroke (AIS). We assessed its value as diagnostic and prognostic tool for ICH through a literature systematic review and individual patient data (IPD) meta-analysis.We performed a systematic search in PubMed database until November 2018 for publications that evaluated GFAP to differentiate AIS and ICH within 4.5 hours after symptoms onset. Thereafter, we invited authors of selected studies to participate in this work by providing IPD from their cohorts. We used standardized individual subject's data to evaluate the association of GFAP concentrations with stroke subtype, demographics, stroke characteristics and factors related with GFAP measurement.From 4 selected studies, we collected data of 340 patients (236 AIS and 104 ICH). Standardized GFAP blood levels were significantly elevated in ICH compared with those with AIS (median and IQR: 0.84 (0.781-1.24), 0.79 (0.74-0.81); p<0.0001). In both stroke types, GFAP concentrations correlated with baseline stroke severity (r=0.27, p<0.0001; r=0.36, p<0.001; for AIS and ICH, respectively) but no correlation was found regarding time to sampling. Limited data precluded the evaluation of GFAP levels and functional outcome.These findings demonstrate substantially different levels of GFAP in the blood of patients with ICH compared with patients with AIS soon after the event, while no association was found with outcome. In summary, GFAP could be a valuable diagnostic tool to assist in medical decision-making and to optimize management of stroke in the acute setting.

Single Cell Immuno-Laser Microdissection Coupled to Label-Free Proteomics to Reveal the Proteotypes of Human Brain Cells After Ischemia.

Cerebral ischemia entails rapid tissue damage in the affected brain area causing devastating neurological dysfunction. How each component of the neurovascular unit contributes or responds to the ischemic insult in the context of the human brain has not been solved yet. Thus, the analysis of the proteome is a straightforward approach to unraveling these cell proteotypes. In this study, post-mortem brain slices from ischemic stroke patients were obtained corresponding to infarcted (IC) and contralateral (CL) areas. By means of laser microdissection, neurons and blood brain barrier structures (BBB) were isolated and analyzed using label-free quantification. MS data are available via ProteomeXchange with identifier PXD003519. Ninety proteins were identified only in neurons, 260 proteins only in the BBB and 261 proteins in both cell types. Bioinformatics analyses revealed that repair processes, mainly related to synaptic plasticity, are outlined in microdissected neurons, with nonexclusive important functions found in the BBB. A total of 30 proteins showing p < 0.05 and fold-change> 2 between IC and CL areas were considered meaningful in this study: 13 in neurons, 14 in the BBB and 3 in both cell types. Twelve of these proteins were selected as candidates and analyzed by immunohistofluorescence in independent brains. The MS findings were completely verified for neuronal SAHH2 and SRSF1 whereas the presence in both cell types of GABT and EAA2 was only validated in neurons. In addition, SAHH2 showed its potential as a prognostic biomarker of neurological improvement when analyzed early in the plasma of ischemic stroke patients. Therefore, the quantitative proteomes of neurons and the BBB (or proteotypes) after human brain ischemia presented here contribute to increasing the knowledge regarding the molecular mechanisms of ischemic stroke pathology and highlight new proteins that might represent putative biomarkers of brain ischemia or therapeutic targets.

Blood markers of inflammation and endothelial dysfunction in cardioembolic stroke: systematic review and meta-analysis.

CONTEXT: Various processes including inflammation and endothelial dysfunction have been implicated in the pathogenesis of cardioembolic (CE) strokes. OBJECTIVE: To review the evidence and investigate the association between immune-inflammatory biomarkers and CE strokes versus other stroke subtypes. METHODS: We systematically reviewed the literature (sources: MEDLINE, web-based register http://stroke-biomarkers.com , reference lists) with quality assessment and meta-analysis of selected articles. RESULTS: The most consistent association was found between C-reactive protein (CRP) and CE strokes when compared to other stroke subtypes (standardized mean difference 0.223 (0.116, 0.343); p < 0.001) Conclusions: Our findings confirm a possible association between selected inflammatory biomarkers and CE stroke.

Profiling and identification of new proteins involved in brain ischemia using MALDI-imaging-mass-spectrometry.

The identification of proteins involved in brain ischemia might allow the discovery of putative biomarkers or therapeutic targets for ischemic stroke. Our aim is to study the distribution of proteins within mouse brain after an ischemic insult using MALDI imaging-mass-spectrometry and to identify relevant proteins involved in brain damage. We occluded the middle cerebral artery of C57BL/6J mice. Brain slices were analyzed by MALDI-TOF and infarct (IC) and contralateral (CL) regions were compared using ClinProTools. The ion distribution maps of relevant m/z values were obtained by FlexImagin3.0. Protein identification was conducted through a bottom-up approach consisting on complementary sample fractionation methods. Some identifications were confirmed by immunohistochemistry and western blot. We identified 102 m/z values with different abundances between IC and CL (p<0.05), from which 21 m/z peaks were selected as more relevant. Thirteen of them were found increased in the infarct region and 4 m/z values showed AUC>90% between IC and CL. Identification analyses confirmed altered expressions of ATP5i, COX6C and UMP-CMP kinase in IC compared to CL. BIOLOGICAL SIGNIFICANCE: Using MALDI-IMS we identified for the first time new proteins that might be involved in brain ischemia representing potential diagnostic biomarkers or target molecules for neurological recovery.

Ischemic stroke outcome: A review of the influence of post-stroke complications within the different scenarios of stroke care.

Stroke remains one of the main causes of death and disability worldwide. The challenge of predicting stroke outcome has been traditionally assessed from a general point of view, where baseline non-modifiable factors such as age or stroke severity are considered the most relevant factors. However, after stroke occurrence, some specific complications such as hemorrhagic transformations or post stroke infections, which lead to a poor outcome, could be developed. An early prediction or identification of these circumstances, based on predictive models including clinical information, could be useful for physicians to individualize and improve stroke care. Furthermore, the addition of biological information such as blood biomarkers or genetic polymorphisms over these predictive models could improve their prognostic value. In this review, we focus on describing the different post-stroke complications that have an impact in short and long-term outcome across different time points in its natural history and on the clinical-biological information that might be useful in their prediction.

Characterization of secretomes from a human blood brain barrier endothelial cells in-vitro model after ischemia by stable isotope labeling with aminoacids in cell culture (SILAC).

UNLABELLED: The human immortalized brain endothelial cell line hCMEC/D3 is considered a simple in-vitro model of the blood-brain-barrier. Our aim was to characterize changes in the secretome of hCMEC/D3 subjected to oxygen and glucose deprivation (OGD) to identify new proteins altered after ischemia and that might trigger blood-brain-barrier disruption and test their potential as blood biomarkers for ischemic stroke. Using a quantitative proteomic approach based on SILAC, 19 proteins were found differentially secreted between OGD and normoxia/normoglycemia conditions. Among the OGD-secreted proteins, protein folding was the main molecular function identified and for the main canonical pathways there was an enrichment in epithelial adherens junctions and aldosterone signaling. Western blot was used to verify the MS results in a set of 9 differentially secreted proteins and 5 of these were analyzed in serum samples of 38 ischemic stroke patients, 18 stroke-mimicking conditions and 18 healthy controls. SIGNIFICANCE: "We characterized changes in the secretome of hCMEC/D3 cells after an ischemic insult by SILAC and identified proteins associated with ischemia that might be involved in the disruption of the blood-brain barrier. Besides we analyzed the putative potential of the candidate proteins to become biomarkers for the diagnosis of ischemic stroke.

Plasmatic retinol-binding protein 4 and glial fibrillary acidic protein as biomarkers to differentiate ischemic stroke and intracerebral hemorrhage.

A rapid differentiation of acute ischemic stroke and intracerebral hemorrhage (ICH) is essential for an adequate treatment and to promote a better outcome. Our aim was to identify new plasma biomarkers to differentiate stroke subtypes and to combine their diagnostic ability with other biomarkers already described for this clinical indication. Plasma samples of ischemic stroke patients (36) and ICH patients (10) were screened using a 177 antibodies library, and 11 showed different concentrations among stroke subtypes (p < 0.05), mainly chemokines, growth factors and angiogenic factors. Five proteins were selected for replication in 16 ischemic stroke patients and 16 ICH patients, and retinol-binding protein 4 (RPB4), apolipoprotein B100 and pigment epithelial-derived factor were replicated (p < 0.05). These proteins, together with glial fibrillary acidic protein (GFAP) and receptor for advanced glycation end product, were tested in 38 ischemic stroke and 28 ICH samples. Finally, RBP4 >61 µg/mL and GFAP <0.07 ng/mL showed a specificity of 100% for both subtypes. Moreover, after multivariate logistic regression analysis, RBP4 >48.75 µg/mL (ORadj : 6.09 (1.3-28.57), p = 0.02) and GFAP <0.07 ng/mL (ORadj : 0.03 (0.003-0.31), p = 0.003) resulted in independent predictors of stroke subtype, improving discrimination by 29% (p < 0.0001). Both biomarkers might be useful as diagnostic biomarkers to differentiate ischemic stroke and ICH. A rapid differentiation of ischemic stroke from intracerebral hemorrhage is essential to provide the appropriate treatment. We describe the discovery and subsequent replications of RBP4 and its combination with circulating GFAP as plasmatic biomarkers for hyperacute stroke subtype differentiation. The combination of these biomarkers and others might aid to speed up the discrimination of both stroke subtypes improving the outcome of patients.

B-type natriuretic peptides help in cardioembolic stroke diagnosis: pooled data meta-analysis.

BACKGROUND AND PURPOSE: Determining the underlying cause of stroke is important to optimize secondary prevention treatment. Increased blood levels of natriuretic peptides (B-type natriuretic peptide/N-terminal pro-BNP [BNP/NT-proBNP]) have been repeatedly associated with cardioembolic stroke. Here, we evaluate their clinical value as pathogenic biomarkers for stroke through a literature systematic review and individual participants' data meta-analysis. METHODS: We searched publications in PubMed database until November 2013 that compared BNP and NT-proBNP circulating levels among stroke causes. Standardized individual participants' data were collected to estimate predictive values of BNP/NT-proBNP for cardioembolic stroke. Dichotomized BNP/NT-proBNP levels were included in logistic regression models together with clinical variables to assess the sensitivity and specificity to identify cardioembolic strokes and the additional value of biomarkers using area under the curve and integrated discrimination improvement index. RESULTS: From 23 selected articles, we collected information of 2834 patients with a defined cause. BNP/NT-proBNP levels were significantly elevated in cardioembolic stroke until 72 hours from symptoms onset. Predictive models showed a sensitivity >90% and specificity >80% when BNP/NT-proBNP were added considering the lowest and the highest quartile, respectively. Both peptides also increased significantly the area under the curve and integrated discrimination improvement index compared with clinical models. Sensitivity, specificity, and precision of the models were validated in 197 patients with initially undetermined stroke with final pathogenic diagnosis after ancillary follow-up. CONCLUSIONS: Natriuretic peptides are strongly increased in cardioembolic strokes. Future multicentre prospective studies comparing BNP and NT-proBNP might aid in finding the optimal biomarker, the best time point, and the optimal cutoff points for cardioembolic stroke identification.

Neuroendocrine hormones as prognostic biomarkers in the setting of acute stroke: overcoming the major hurdles.

Stroke represents one of the major causes of disability and mortality worldwide and prediction of outcome represents a challenge for both clinicians and researchers. In the past years, many blood markers have been associated with stroke outcome but despite this evidence, no biomarker is routinely used in stroke management. In this review, we focus on markers of the neuroendocrine system, which represent potential candidates to be implemented in clinical practice. Moreover, we present a systematic review and literature-based meta-analysis for copeptin, a new biomarker of the hypothalamo-pituitary-adrenal axis that has shown additional predictive value over clinical information in a large prospective study. The meta-analysis of the included 7 studies, with more than 2000 patients, reinforced its association with poor outcome (pooled odds ratio: 2.474 [1.678-3.268]) and mortality (pooled OR: 2.569 [1.642-3.495]). We further review the current situation of the topic and next steps to implement these tools by clinicians.

Prognostic value of blood interleukin-6 in the prediction of functional outcome after stroke: a systematic review and meta-analysis.

We aimed to quantify the association of blood interleukin-6 (IL-6) concentrations with poor outcome after stroke and its added predictive value over clinical information. Meta-analysis of 24 studies confirmed this association with a weighted mean difference of 3.443 (1.592-5.294) pg/mL, despite high heterogeneity and publication bias. Individual participant data including 4112 stroke patients showed standardized IL-6 levels in the 4th quartile were independently associated with poor outcome (OR=2.346 (1.814-3.033), p<0.0001). However, the additional predictive value of IL-6 was moderate (IDI=1.5%, NRI=5.35%). Overall these results indicate an unlikely translation of IL-6 into clinical practice for this purpose.

Role of beta-defensin 2 and interleukin-4 receptor as stroke outcome biomarkers.

Acute ischemic stroke is a complex disease with huge interindividual evolution variability that makes challenging the prediction of an adverse outcome. Our aim was to study the association of bloodstream signatures to early neurological outcome after stroke, by combining a subpooling of samples strategy with protein array discovery approach. Plasma samples from 36 acute stroke patients (< 4.5 h from onset) were equally pooled within outcome groups: worsening, stability, and improvement (n = 3 pools of four patients each, for each outcome group). These nine pools were screened using a 177 antibodies library, and 35 proteins were found altered regarding outcome classification (p < 0.1). Processes of inflammation, immune response, coagulation, and apoptosis were regulated by these proteins. Ten representative candidates, mainly cytokines and chemokines, were assayed for replication in individual baseline plasma samples from 80 new stroke patients: ß-defensin2, MIP-3b, plasminogen activator inhibitor 1 active, ß-cell-attracting chemokine 1, Exodus-2, interleukin-4 receptor (IL-4R), IL-12p40, leukemia inhibitor factor, MIP-1b, and tumor necrosis factor-related weak inducer of apoptosis. Multivariate logistic regression analysis showed ß-defensin 2 (ORadj 4.87 [1.13-20.91] p = 0.033) and IL-4R (ORadj 3.52 [1.03-12.08] p = 0.045) as independent predictors of worsening at 24 h after adjustment by clinical variables. Both biomarkers improve the prediction by 19% as compared to clinical information, suggesting a potential role for risk stratification in acute thrombolyzed stroke patients. Early neurological deterioration after stroke is not easily predictable. The use of blood biomarkers might help in decision-making processes regarding this complication. By combining a sub-pooling of samples strategy with protein array discovery approach, we have found two new biomarkers: beta-defensin-2 and interleukin-4 receptor. Both biomarkers improve the prediction of poor-outcome over clinical variables in the acute phase of stroke.

Fluorescent molecular peroxidation products: a prognostic biomarker of early neurologic deterioration after thrombolysis.

BACKGROUND AND PURPOSE: Fluorescent molecular peroxidation products (FMPPs) are considered potential markers of molecular oxidative damage and may provoke increased permeability and disruption of the blood-brain barrier. This study aimed to determine the value of FMPPs as a biomarker to predict neurological worsening related to early hemorrhagic transformation. METHODS: Baseline FMPP levels were measured in 186 consecutive acute ischemic stroke patients before tissue plasminogen activator treatment was administered. A serial FMPP profile (baseline before tissue plasminogen activator treatment, and 1, 2, 12, and 24 hours from treatment) was determined in a subset of 100 patients. Computed tomographic scans were performed at admission and repeated at 24 to 48 hours or after neurological worsening occurred. Symptomatic intracranial hemorrhage was defined as blood at any site in the brain associated with neurological deterioration. RESULTS: Patients who worsened had higher median FMPP levels compared with those who did not (59.68 [48.63-85.73] versus 44.87 [36.37-58.90] Uf/mL; P=0.035) at baseline. After logistic regression multivariate analysis, FMPP >48.2 Uf/mL together with age, hypertension, and systolic blood pressure remained baseline predictors of worsening at 48 hours. Moreover, baseline FMPP determination helped to distinguish between patients who worsened and those who did not (Integrated Discrimination Improvement index, 5.7%; P=0.0004). Finally, within patients who had worsened at 48 hours, those with symptomatic intracranial hemorrhage had higher FMPP levels (P=0.038). CONCLUSIONS: FMPPs might be a valuable biomarker of poor early neurological outcome and be related to the appearance of symptomatic intracranial hemorrhage in tissue plasminogen activator-treated patients, one of the most feared neurological complications after thrombolytic treatment of acute ischemic stroke.

Prognostic value of plasma chitotriosidase activity in acute stroke patients.

BACKGROUND AND AIMS: Chitotriosidase, a component of innate immunity, constitutes a sensitive parameter of macrophage activation and its elevated plasma activity reflects an inflammatory response. Given the deleterious effects of inflammation in brain ischemia, we aimed to assess the prognostic value of chitotriosidase activity in acute stroke patients. METHODS: The study comprised 159 acute stroke patients and 51 age-matched controls. Plasma chitotriosidase activity was serially determined by fluorometric assay. Short-term neurological outcome was determined at 48 h and functional outcome at three-months. Predictors of neurological and functional outcome were determined via multivariate analysis, and the additional predictive value of chitotriosidase was tested with the Integrated Discrimination Index and the Net Reclassification Improvement. RESULTS: Stroke patients showed increased levels of baseline chitotriosidase activity compared to controls [114·2 (74·65-182·95) nmol/ml/h vs. 54·4 (32·7-76·4); P < 0·0001]. Chitotriosidase activity (<118·75) was found to be an independent predictor of neurological improvement at 48 h (odds ratio: 3·25; 95% confidence interval: 1·54-6·85; P=0·002), and the addition of plasma chitotriosidase activity showed a better prediction of improvement at 48 h (Integrated Discrimination Index=5·7%, Net Reclassification Improvement=11·6%, P<0·05) over the predictive model constituted only with clinical information. Although patients disabled at three-months showed higher baseline chitotriosidase levels, it was not an independent predictor of long-term disability. CONCLUSIONS: Baseline chitotriosidase activity in acute stroke patients treated with tissue plasminogen activator (tPA) may constitute a prognostic predictor of short-term outcome, adding a moderate additional predictive value. Our results underline the deleterious role of inflammation in acute stroke patients.

Cardioembolic stroke diagnosis using blood biomarkers.

Stroke is one of the main causes of death and disability in the world. Cardioembolic etiology accounts for approximately one fifth of all ischemic strokes whereas 25-30% remains undetermined even after an advanced diagnostic workup. Despite there is not any biomarker currently approved to distinguish cardioembolic stroke among other etiologies in clinical practice the use of biomarkers represents a promising valuable complement to determine stroke etiology reducing the number of cryptogenic strokes and aiding in the prescription of the most appropriated primary and secondary treatments in order to minimize therapeutic risks and to avoid recurrences. In this review we present an update about specific cardioembolic stroke-related biomarkers at a protein, transcriptomic and genetic level. Finally, we also focused on reported biomarkers associated with atrial fibrillation (a cardiac illness strongly related with cardioembolic stroke subtype) thus with a potential to become biomarkers to detect cardioembolic stroke in the future.

Treatment with MOG-DNA vaccines induces CD4+CD25+FoxP3+ regulatory T cells and up-regulates genes with neuroprotective functions in experimental autoimmune encephalomyelitis.

BACKGROUND: DNA vaccines represent promising therapeutic strategies in autoimmune disorders such as multiple sclerosis (MS). However, the precise mechanisms by which DNA vaccines induce immune regulation remain largely unknown. Here, we aimed to expand previous knowledge existing on the mechanisms of action of DNA vaccines in the animal model of MS, experimental autoimmune encephalomyelitis (EAE), by treating EAE mice with a DNA vaccine encoding the myelin oligodendrocyte glycoprotein (MOG), and exploring the therapeutic effects on the disease-induced inflammatory and neurodegenerative changes. METHODS: EAE was induced in C57BL6/J mice by immunization with MOG35₋55 peptide. Mice were intramuscularly treated with a MOG-DNA vaccine or vehicle in prophylactic and therapeutic approaches. Histological studies were performed in central nervous system (CNS) tissue. Cytokine production and regulatory T cell (Treg) quantification were achieved by flow cytometry. Gene expression patterns were determined using microarrays, and the main findings were validated by real-time PCR. RESULTS: MOG-DNA treatment reduced the clinical and histopathological signs of EAE when administered in both prophylactic and therapeutic settings. Suppression of clinical EAE was associated with dampening of antigen (Ag)-specific proinflammatory Th1 and Th17 immune responses and, interestingly, expansion of Treg in the periphery and upregulation in the CNS of genes encoding neurotrophic factors and proteins involved in remyelination. CONCLUSIONS: These results suggest for the first time that the beneficial effects of DNA vaccines in EAE are not limited to anti-inflammatory mechanisms, and DNA vaccines may also exert positive effects through hitherto unknown neuroprotective mechanisms.