Publications by authors named "Nicole Kerlero de Rosbo"

40 Publications

Neuroinflammation induces synaptic scaling through IL-1β-mediated activation of the transcriptional repressor REST/NRSF.

Cell Death Dis 2021 Feb 15;12(2):180. Epub 2021 Feb 15.

Center for Synaptic Neuroscience and Technology, Istituto Italiano di Tecnologia, Largo Rosanna Benzi 10, 16132, Genova, Italy.

Neuroinflammation is associated with synapse dysfunction and cognitive decline in patients and animal models. One candidate for translating the inflammatory stress into structural and functional changes in neural networks is the transcriptional repressor RE1-silencing transcription factor (REST) that regulates the expression of a wide cluster of neuron-specific genes during neurogenesis and in mature neurons. To study the cellular and molecular pathways activated under inflammatory conditions mimicking the experimental autoimmune encephalomyelitis (EAE) environment, we analyzed REST activity in neuroblastoma cells and mouse cortical neurons treated with activated T cell or microglia supernatant and distinct pro-inflammatory cytokines. We found that REST is activated by a variety of neuroinflammatory stimuli in both neuroblastoma cells and primary neurons, indicating that a vast transcriptional change is triggered during neuroinflammation. While a dual activation of REST and its dominant-negative splicing isoform REST4 was observed in N2a neuroblastoma cells, primary neurons responded with a pure full-length REST upregulation in the absence of changes in REST4 expression. In both cases, REST upregulation was associated with activation of Wnt signaling and increased nuclear translocation of β-catenin, a well-known intracellular transduction pathway in neuroinflammation. Among single cytokines, IL-1β caused a potent and prompt increase in REST transcription and translation in neurons, which promoted a delayed and strong synaptic downscaling specific for excitatory synapses, with decreased frequency and amplitude of spontaneous synaptic currents, decreased density of excitatory synaptic connections, and decreased frequency of action potential-evoked Ca transients. Most important, the IL-1β effects on excitatory transmission were strictly REST dependent, as conditional deletion of REST completely occluded the effects of IL-1β activation on synaptic transmission and network excitability. Our results demonstrate that REST upregulation represents a new pathogenic mechanism for the synaptic dysfunctions observed under neuroinflammatory conditions and identify the REST pathway as therapeutic target for EAE and, potentially, for multiple sclerosis.
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http://dx.doi.org/10.1038/s41419-021-03465-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7884694PMC
February 2021

Role of miRNAs shuttled by mesenchymal stem cell-derived small extracellular vesicles in modulating neuroinflammation.

Sci Rep 2021 Jan 18;11(1):1740. Epub 2021 Jan 18.

IRCCS Ospedale Policlinico San Martino, Genoa, Italy.

Mesenchymal stromal/stem cells (MSCs) are characterized by neuroprotective, immunomodulatory, and neuroregenerative properties, which support their therapeutic potential for inflammatory/neurodegenerative diseases, including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). One mode of action through which MSCs exert their immunomodulatory effects is release of extracellular vesicles that carry proteins, mRNAs, and microRNAs (miRNAs), which, once transferred, modify the function of target cells. We identified nine miRNAs significantly dysregulated in IFN-γ-primed MSCs, but present at different levels in their derived small extracellular vesicles (s-EV). We show that miR-467f and miR-466q modulate the pro-inflammatory phenotype of activated N9 microglia cells and of primary microglia acutely isolated from late symptomatic SOD1 mice, a murine ALS model, by downregulating Tnf and Il1b expression. Further analysis of the mode of action of miR-467f and miR-466q indicated that they dampen the pro-inflammatory phenotype of microglia by modulating p38 MAPK signaling pathway via inhibition of expression of their target genes, Map3k8 and Mk2. Finally, we demonstrated that in vivo administration of s-EV leads to decreased expression of neuroinflammation markers in the spinal cord of EAE-affected mice, albeit without affecting disease course. Overall, our data suggest that MSC-derived exosomes could affect neuroinflammation possibly through specific immunomodulatory miRNAs acting on microglia.
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http://dx.doi.org/10.1038/s41598-021-81039-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7814007PMC
January 2021

Mesenchymal stem cells instruct a beneficial phenotype in reactive astrocytes.

Glia 2020 Dec 31. Epub 2020 Dec 31.

IRCCS Ospedale Policlinico San Martino, Genoa, Italy.

Transplanted mesenchymal stromal/stem cells (MSC) ameliorate the clinical course of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS), reducing inflammation and demyelination. These effects are mediated by instructive cross-talk between MSC and immune and neural cells. Astroglial reaction to injury is a prominent feature of both EAE and MS. Astrocytes constitute a relevant target to control disease onset and progression and, based on their potential to acquire stem cell properties in situ, to foster recovery in the post-acute phase of pathology. We have assessed how MSC impact astrocytes in vitro and ex vivo in EAE. Expression of astroglial factors implicated in EAE pathogenesis was quantified by real-time PCR in astrocytes co-cultured with MSC or isolated from EAE cerebral cortex; astrocyte morphology and expression of activation markers were analyzed by confocal microscopy. The acquisition of neural stem cell properties by astrocytes was evaluated by neurosphere assay. Our study shows that MSC prevented astrogliosis, reduced mRNA expression of inflammatory cytokines that sustain immune cell infiltration in EAE, as well as protein expression of endothelin-1, an astrocyte-derived factor that inhibits remyelination and contributes to neurodegeneration and disease progression in MS. Moreover, our data reveal that MSC promoted the acquisition of progenitor traits by astrocytes. These data indicate that MSC attenuate detrimental features of reactive astroglia and, based on the reacquisition of stem cell properties, also suggest that astrocytes may be empowered in their protective and reparative actions by MSC.
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http://dx.doi.org/10.1002/glia.23958DOI Listing
December 2020

X-ray Phase Contrast Tomography Serves Preclinical Investigation of Neurodegenerative Diseases.

Front Neurosci 2020 9;14:584161. Epub 2020 Nov 9.

TomaLab, Institute of Nanotechnology, CNR, Rome, Italy.

We report a qualitative study on central nervous system (CNS) damage that demonstrates the ability of X-ray phase contrast tomography (XPCT) to confirm data obtained with standard 2D methodology and permits the description of additional features that are not detected with 2D or other 3D techniques. In contrast to magnetic resonance or computed tomography, XPCT makes possible the high-resolution 3D imaging of soft tissues classically considered "invisible" to X-rays without the use of additional contrast agents, or without the need for intense processing of the tissue required by 2D techniques. Most importantly for studies of CNS diseases, XPCT enables a concomitant multi-scale 3D biomedical imaging of neuronal and vascular networks ranging from cells through to the CNS as a whole. In the last years, we have used XPCT to investigate neurodegenerative diseases, such as Alzheimer's disease (AD) and multiple sclerosis (MS), to shed light on brain damage and extend the observations obtained with standard techniques. Here, we show the cutting-edge ability of XPCT to highlight in 3D, concomitantly, vascular occlusions and damages, close associations between plaques and damaged vessels, as well as dramatic changes induced at neuropathological level by treatment in AD mice. We corroborate data on the well-known blood-brain barrier dysfunction in the animal model of MS, experimental autoimmune encephalomyelitis, and further show its extent throughout the CNS axis and at the level of the single vessel/capillary.
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http://dx.doi.org/10.3389/fnins.2020.584161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7680960PMC
November 2020

Intranasal delivery of mesenchymal stem cell secretome repairs the brain of Alzheimer's mice.

Cell Death Differ 2021 Jan 23;28(1):203-218. Epub 2020 Jul 23.

Department of Neuroscience, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, via Mario Negri 2, 20156, Milan, Italy.

The multiplicity of systems affected in Alzheimer's disease (AD) brains calls for multi-target therapies. Although mesenchymal stem cells (MSC) are promising candidates, their clinical application is limited because of risks related to their direct implantation in the host. This could be overcome by exploiting their paracrine action. We herein demonstrate that in vivo systemic administration of secretome collected from MSC exposed in vitro to AD mouse brain homogenates (MSC-CS), fully replicates the cell-mediated neuroreparative effects in APP/PS1 AD mice. We found a complete but transient memory recovery by 7 days, which vanished by 14 days, after a single MSC-CS intravenous administration in 12-month or 22-24-month-old mice. Treatment significantly reduced plaque load, microglia activation, and expression of cytokines in astrocytes in younger, but not aged, mice at 7 days. To optimize efficacy, we established a sustained treatment protocol in aged mice through intranasal route. Once-weekly intranasal administration of MSC-CS induced persistent memory recovery, with dramatic reduction of plaques surrounded by a lower density of β-amyloid oligomers. Gliosis and the phagocytic marker CD68 were decreased. We found a higher neuronal density in cortex and hippocampus, associated with a reduction in hippocampal shrinkage and a longer lifespan indicating healthier conditions of MSC-CS-treated compared to vehicle-treated APP/PS1 mice. Our data prove that MSC-CS displays a great multi-level therapeutic potential, and lay the foundation for identifying the therapeutic secretome bioreactors leading to the development of an efficacious multi-reparative cocktail drug, towards abrogating the need for MSC implantation and risks related to their direct use.
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http://dx.doi.org/10.1038/s41418-020-0592-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852675PMC
January 2021

Impact of treatment on cellular immunophenotype in MS: A cross-sectional study.

Neurol Neuroimmunol Neuroinflamm 2020 05 5;7(3). Epub 2020 Mar 5.

From the Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health (M.C., F.I., M.P., A.L., C.L., G.N., G.B., E.S., S.P., M.I., N.K.d.R.) and Center of Excellence for Biomedical Research (A.U.), University of Genoa, Italy; BD Biosciences Italy (G.R.), Milan; Institut d'Investigacions Biomediques August Pi Sunyer (G.V., P.V.), Barcelona, Spain; Charité Universitaetsmedizin Berlin and Max Delbrueck Center for Molecular Medicine (P.B.-K., S.A., F.P.), Germany; Department of Research, Innovation and Education (T.B.), Neuroscience Research Unit, Oslo University Hospital; Department of Mechanical Electronics and Chemical Engineering (T.B.), Oslo Metropolitan University, Norway; University of Oslo (E.H., H.F.H.) and Oslo University Hospital (H.F.H.), Norway; Department of Mathematics and Padova Neuroscience Center (C.C.), University of Padua, Italy; Department of Mathematics (M.P.), University of Genoa, Italy; and IRCCS Ospedale Policlinico San Martino (A.L., M.P., M.I., A.U.), Genoa, Italy.

Objective: To establish cytometry profiles associated with disease stages and immunotherapy in MS.

Methods: Demographic/clinical data and peripheral blood samples were collected from 227 patients with MS and 82 sex- and age-matched healthy controls (HCs) enrolled in a cross-sectional study at 4 European MS centers (Spain, Italy, Germany, and Norway). Flow cytometry of isolated peripheral blood mononuclear cells was performed in each center using specifically prepared antibody-cocktail Lyotubes; data analysis was centralized at the Genoa center. Differences in immune cell subsets were assessed between groups of untreated patients with relapsing-remitting or progressive MS (RRMS or PMS) and HCs and between groups of patients with RRMS taking 6 commonly used disease-modifying drugs.

Results: In untreated patients with MS, significantly higher frequencies of Th17 cells in the RRMS population compared with HC and lower frequencies of B-memory/B-regulatory cells as well as higher percentages of B-mature cells in patients with PMS compared with HCs emerged. Overall, the greatest deviation in immunophenotype in MS was observed by treatment rather than disease course, with the strongest impact found in fingolimod-treated patients. Fingolimod induced a decrease in total CD4 T cells and in B-mature and B-memory cells and increases in CD4 and CD8 T-regulatory and B-regulatory cells.

Conclusions: Our highly standardized, multisite cytomics data provide further understanding of treatment impact on MS immunophenotype and could pave the way toward monitoring immune cells to help clinical management of MS individuals.
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http://dx.doi.org/10.1212/NXI.0000000000000693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7136062PMC
May 2020

Ocrelizumab does not impair B- and T-cell responses to primary VZV infection in a patient with MS.

Neurol Neuroimmunol Neuroinflamm 2020 05 25;7(3). Epub 2020 Feb 25.

From the Department of Neuroscience (G.N.), Ospedale Policlinico San Martino-IRCCS, Genoa; Department of Neuroscience, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health (DINOGMI) (F.I., E.S., N.K.d.R.), University of Genoa; Department of Health Sciences (DISSAL) (M.M.), University of Genoa; Ospedale Policlinico San Martino-IRCCS (M.M., G.P., M.I., A.U.), Genoa; Autoimmunity Laboratory Department of Internal Medicine (DiMI) (G.P.), University of Genoa; and Department of Neuroscience, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health (DINOGMI) and Center of Excellence for Biomedical Research (CEBR) (M.I., A.U.), University of Genoa, Italy.

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http://dx.doi.org/10.1212/NXI.0000000000000695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7051194PMC
May 2020

Detrimental and protective action of microglial extracellular vesicles on myelin lesions: astrocyte involvement in remyelination failure.

Acta Neuropathol 2019 12 30;138(6):987-1012. Epub 2019 Jul 30.

CNR Institute of Neuroscience, via Vanvitelli 32, 20129, Milan, Italy.

Microglia are highly plastic immune cells which exist in a continuum of activation states. By shaping the function of oligodendrocyte precursor cells (OPCs), the brain cells which differentiate to myelin-forming cells, microglia participate in both myelin injury and remyelination during multiple sclerosis. However, the mode(s) of action of microglia in supporting or inhibiting myelin repair is still largely unclear. Here, we analysed the effects of extracellular vesicles (EVs) produced in vitro by either pro-inflammatory or pro-regenerative microglia on OPCs at demyelinated lesions caused by lysolecithin injection in the mouse corpus callosum. Immunolabelling for myelin proteins and electron microscopy showed that EVs released by pro-inflammatory microglia blocked remyelination, whereas EVs produced by microglia co-cultured with immunosuppressive mesenchymal stem cells promoted OPC recruitment and myelin repair. The molecular mechanisms responsible for the harmful and beneficial EV actions were dissected in primary OPC cultures. By exposing OPCs, cultured either alone or with astrocytes, to inflammatory EVs, we observed a blockade of OPC maturation only in the presence of astrocytes, implicating these cells in remyelination failure. Biochemical fractionation revealed that astrocytes may be converted into harmful cells by the inflammatory EV cargo, as indicated by immunohistochemical and qPCR analyses, whereas surface lipid components of EVs promote OPC migration and/or differentiation, linking EV lipids to myelin repair. Although the mechanisms through which the lipid species enhance OPC maturation still remain to be fully defined, we provide the first demonstration that vesicular sphingosine 1 phosphate stimulates OPC migration, the first fundamental step in myelin repair. From this study, microglial EVs emerge as multimodal and multitarget signalling mediators able to influence both OPCs and astrocytes around myelin lesions, which may be exploited to develop novel approaches for myelin repair not only in multiple sclerosis, but also in neurological and neuropsychiatric diseases characterized by demyelination.
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http://dx.doi.org/10.1007/s00401-019-02049-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851224PMC
December 2019

Defining the role of NG2-expressing cells in experimental models of multiple sclerosis. A biofunctional analysis of the neurovascular unit in wild type and NG2 null mice.

PLoS One 2019 14;14(3):e0213508. Epub 2019 Mar 14.

Department of Basic Medical Sciences, Neurosciences and Sense Organs, University of Bari School of Medicine, Bari, Italy.

During experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis associated with blood-brain barrier (BBB) disruption, oligodendrocyte precursor cells (OPCs) overexpress proteoglycan nerve/glial antigen 2 (NG2), proliferate, and make contacts with the microvessel wall. To explore whether OPCs may actually be recruited within the neurovascular unit (NVU), de facto intervening in its cellular and molecular composition, we quantified by immunoconfocal morphometry the presence of OPCs in contact with brain microvessels, during postnatal cerebral cortex vascularization at postnatal day 6, in wild-type (WT) and NG2 knock-out (NG2KO) mice, and in the cortex of adult naïve and EAE-affected WT and NG2KO mice. As observed in WT mice during postnatal development, a higher number of juxtavascular and perivascular OPCs was revealed in adult WT mice during EAE compared to adult naïve WT mice. In EAE-affected mice, OPCs were mostly associated with microvessels that showed altered claudin-5 and occludin tight junction (TJ) staining patterns and barrier leakage. In contrast, EAE-affected NG2KO mice, which did not show any significant increase in vessel-associated OPCs, seemed to retain better preserved TJs and BBB integrity. As expected, absence of NG2, in both OPCs and pericytes, led to a reduced content of vessel basal lamina molecules, laminin, collagen VI, and collagen IV. In addition, analysis of the major ligand/receptor systems known to promote OPC proliferation and migration indicated that vascular endothelial growth factor A (VEGF-A), platelet-derived growth factor-AA (PDGF-AA), and the transforming growth factor-β (TGF-β) were the molecules most likely involved in proliferation and recruitment of vascular OPCs during EAE. These results were confirmed by real time-PCR that showed Fgf2, Pdgfa and Tgfb expression on isolated cerebral cortex microvessels and by dual RNAscope-immunohistochemistry/in situ hybridization (IHC/ISH), which detected Vegfa and Vegfr2 transcripts on cerebral cortex sections. Overall, this study suggests that vascular OPCs, in virtue of their developmental arrangement and response to neuroinflammation and growth factors, could be integrated among the classical NVU cell components. Moreover, the synchronized activation of vascular OPCs and pericytes during both BBB development and dysfunction, points to NG2 as a key regulator of vascular interactions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0213508PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6417733PMC
December 2019

Exploring Alzheimer's disease mouse brain through X-ray phase contrast tomography: From the cell to the organ.

Neuroimage 2019 01 18;184:490-495. Epub 2018 Sep 18.

Istituto di Nanotecnologia, Consiglio Nazionale delle Ricerche, Rome, Italy; Dipartimento di Fisica, Università Sapienza, Rome, Italy. Electronic address:

Alzheimer's disease (AD), the most common form of dementia, is a progressive neurodegenerative disorder associated with aberrant production of beta-amyloid (Aβ) peptide depositing in brain as amyloid plaques. While animal models allow investigation of disease progression and therapeutic efficacy, technology to fully dissect the pathological mechanisms of this complex disease at cellular and vascular levels is lacking. X-ray phase contrast tomography (XPCT) is an advanced non-destructive 3D multi-scale direct imaging from the cell through to the whole brain, with exceptional spatial and contrast resolution. We exploit XPCT to simultaneously analyse disease-relevant vascular and neuronal networks in AD mouse brain, without sectioning and staining. The findings clearly show the different typologies and internal structures of Aβ plaques, together with their interaction with patho/physiological cellular and neuro-vascular microenvironment. XPCT enables for the first time a detailed visualization of amyloid-angiopathy at capillary level, which is impossible to achieve with other approaches. XPCT emerges as added-value technology to explore AD mouse brain as a whole, preserving tissue chemistry and structure, enabling the comparison of physiological vs. pathological states at the level of crucial disease targets. In-vivo translation will permit to monitor emerging therapeutic approaches and possibly shed new light on pathological mechanisms of neurodegenerative diseases.
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http://dx.doi.org/10.1016/j.neuroimage.2018.09.044DOI Listing
January 2019

Astrocytes play a crucial role in the formation and evolution of MS lesions - No.

Mult Scler 2019 01 23;25(1):17-19. Epub 2018 Aug 23.

DINOGMI, University of Genoa, Genoa, Italy.

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http://dx.doi.org/10.1177/1352458518795419DOI Listing
January 2019

Teriflunomide treatment reduces B cells in patients with MS.

Neurol Neuroimmunol Neuroinflamm 2017 Nov 23;4(6):e403. Epub 2017 Oct 23.

Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health Unit (I.G., A.L., F.B., G.M., N.K.d.R., A.U.), and Center of Excellence for Biomedical Research (CEBR) (F.I., A.L., G.M., A.U.), University of Genoa, Italy; Ospedale Policlinico San Martino-IRCCS (A.L., G.M., A.U.), Genoa, Italy; and Rehabilitation Unit (C.S.), Centro di Recupero e Rieducazione Funzionale, M.L. Novarese, Moncrivello, Italy.

Objective: To study the immunomodulatory effect of teriflunomide on innate and adaptive immune cell populations through a pilot, open-label, observational study in a cohort of patients with relapsing-remitting MS.

Methods: Blood lymphocytes were isolated from 10 patients with MS before and after 3 or 12 months of treatment. Adaptive and innate immune cell subsets were analyzed by flow cytometry as follows: B cells (memory, regulatory, and mature subsets), T cells (effector and regulatory subsets), and natural killer (NK) cells (CD56 and CD56 subsets).

Results: Our results show that teriflunomide significantly reduces absolute counts of total CD19 B cells and mature and regulatory B-cell subsets. T cells were affected to a lesser extent, with a trend in reduction of absolute counts for both T effector CD4 cells (Th1, Th17 and Th1/17) and T regulatory CD8 and CD4 cells. Teriflunomide had no detectable effect on NK-cell numbers.

Conclusions: In our small cohort, teriflunomide treatment affects mainly and significantly on B-cell numbers, while having a milder effect on T-cell numbers. Larger cohorts are necessary to confirm these findings and understand the effect of teriflunomide on the functionality of these cells.
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http://dx.doi.org/10.1212/NXI.0000000000000403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5656406PMC
November 2017

X-Ray Phase Contrast Tomography Reveals Early Vascular Alterations and Neuronal Loss in a Multiple Sclerosis Model.

Sci Rep 2017 07 19;7(1):5890. Epub 2017 Jul 19.

Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health Unit, University of Genoa & AOU San Martino - IST Istituto Nazionale per la Ricerca sul Cancro Genoa, Genoa, Italy.

The degenerative effects of multiple sclerosis at the level of the vascular and neuronal networks in the central nervous system are currently the object of intensive investigation. Preclinical studies have demonstrated the efficacy of mesenchymal stem cell (MSC) therapy in experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis, but the neuropathology of specific lesions in EAE and the effects of MSC treatment are under debate. Because conventional imaging techniques entail protocols that alter the tissues, limiting the reliability of the results, we have used non-invasive X-ray phase-contrast tomography to obtain an unprecedented direct 3D characterization of EAE lesions at micro-to-nano scales, with simultaneous imaging of the vascular and neuronal networks. We reveal EAE-mediated alterations down to the capillary network. Our findings shed light on how the disease and MSC treatment affect the tissues, and promote X-ray phase-contrast tomography as a powerful tool for studying neurovascular diseases and monitoring advanced therapies.
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http://dx.doi.org/10.1038/s41598-017-06251-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5517657PMC
July 2017

Regulatory Functions of Natural Killer Cells in Multiple Sclerosis.

Front Immunol 2016 19;7:606. Epub 2016 Dec 19.

Centre of Excellence for Biomedical Research, University of Genova, Genova, Italy; Department of Neuroscience, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genova, Genova, Italy; IRCCS San Martino-IST, Genova, Italy.

There is increasing evidence that natural killer (NK) cells exhibit regulatory features. Among them, CD56 NK cells have been suggested to play a major role in controlling T cell responses and maintaining homeostasis. Dysfunction in NK cell-mediated regulatory features has been recently described in untreated multiple sclerosis (MS), suggesting a contribution to MS pathogenesis. Moreover, biological disease-modifying treatments effective in MS apparently enhance the frequencies and/or regulatory function of NK cells, further pointing toward an immunoprotective role of NK cells in MS. Here, we summarize the current knowledge on the regulatory functions of NK cells, based on their interactions with other cells belonging to the innate compartment, as well as with adaptive effector cells. We review the more recent data reporting disruption of NK cell/T cell interactions in MS and discuss how disease-modifying treatments for MS affect NK cells.
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http://dx.doi.org/10.3389/fimmu.2016.00606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5165263PMC
December 2016

In vitro VLA-4 blockade results in an impaired NK cell-mediated immune surveillance against melanoma.

Immunol Lett 2017 01 2;181:109-115. Epub 2016 Dec 2.

Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health Unit, University of Genoa, Genoa, Italy; IRCCS Azienda Ospedaliera Universitaria San Martino - IST, Genoa, Italy.

Natalizumab (NTZ) is a monoclonal antibody targeting the α4β1 integrin (CD49d/CD29), very late antigen-4 (VLA-4), which is approved for treatment of relapsing-remitting multiple sclerosis (RR-MS). A possible association between NTZ treatment and a higher risk of melanoma is under debate. Natural Killer (NK) cells, which express VLA-4, represent an innate barrier limiting spreading of melanoma under steady state conditions. Indeed, because of their expression of activating receptors, they are very efficient in recognizing and killing melanoma cells without the need of a previous priming. For this reason, we aimed at assessing whether NK-cell functions might be impaired by sustained exposure to NTZ. To investigate this possibility we isolated NK cells from healthy donors and tested their cytotoxic and migratory functions against primary melanoma cells derived from subcutaneous and lymph node metastases. Flow cytometry analysis demonstrated expression of CD49d on both freshly isolated NK cells and activated NK cells. Moreover, VLA-4 and its receptor, vascular cell adhesion protein-1 (VCAM-1) were similarly expressed on freshly isolated NK cells. However, upon a short exposure to NTZ, expression of VLA-4 on NK cells decreased. Analysis of NK receptor expression upon exposure of NK cells from three healthy donors to NTZ indicated that DNAM-1 and NKp46 are apparently decreased, while NKG2A is increased. The degranulation of NK cells towards melanoma cells, which express both VLA-4 and VCAM-1, was not affected when NTZ was added to the co-culture or when both NK cells and melanoma cells were each pre-exposed to NTZ for over 12h. In contrast, degranulation was significantly inhibited after 48h of pre-incubation indicating that NTZ can influence NK-cell degranulation towards melanoma cells only after a prolonged exposure. Using a migration chamber assay, we observed that the migration of NK cells towards melanoma cells was dependent upon the concentration of melanoma cells in the lower chamber, and that it was significantly reduced in presence of NTZ. Our results show that upon exposure to NTZ both cytolytic activity and migration toward melanoma cells were affected, suggesting that binding of NTZ to NK cells affects pathways involved in these NK-cell functions. We analyzed the expression of CD49d on NK cells from MS patients treated with NTZ and observed that it decreases with time of treatment. These data suggest that blockade of VLA-4 on NK-cell surface alters some key functions involved in the immune surveillance toward melanoma by NK cells and may provide a mechanistic explanation for the reported occurrence of melanoma in MS patients treated with NTZ.
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http://dx.doi.org/10.1016/j.imlet.2016.11.015DOI Listing
January 2017

Characterization of mouse spinal cord vascular network by means of synchrotron radiation X-ray phase contrast tomography.

Phys Med 2016 Dec 13;32(12):1779-1784. Epub 2016 Oct 13.

Consiglio Nazionale delle Ricerche, Istituto di Nanotecnologia, Rome Unit, I-00195 Rome, Italy.

High resolution Synchrotron-based X-ray Phase Contrast Tomography (XPCT) allows the simultaneous detection of three dimensional neuronal and vascular networks without using contrast agents or invasive casting preparation. We show and discuss the different features observed in reconstructed XPCT volumes of the ex vivo mouse spinal cord in the lumbo-sacral region, including motor neurons and blood vessels. We report the application of an intensity-based segmentation method to detect and quantitatively characterize the modification in the vascular networks in terms of reduction in experimental visibility. In particular, we apply our approach to the case of the experimental autoimmune encephalomyelitis (EAE), i.e. human multiple sclerosis animal model.
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http://dx.doi.org/10.1016/j.ejmp.2016.09.015DOI Listing
December 2016

IFN-γ orchestrates mesenchymal stem cell plasticity through the signal transducer and activator of transcription 1 and 3 and mammalian target of rapamycin pathways.

J Allergy Clin Immunol 2017 May 23;139(5):1667-1676. Epub 2016 Sep 23.

Dipartimento di Neuroscienze, Riabilitazione, Oftalmologia, Genetica e Scienze Materno-infantili, Facoltà di Medicina e Chirurgia, Università di Genova, Genoa, Italy; Center of Excellence for Biomedical Research (CEBR), Genoa, Italy; IRCCS AOU San Martino-IST, Genoa, Italy. Electronic address:

Background: Mesenchymal stem cells (MSCs) display a therapeutic plasticity because of their ability to modulate immunity, foster tissue repair, and differentiate into mesodermal cells. IFN-γ has been described to differently affect human mesenchymal stem cell (hMSC) and mouse mesenchymal stem cell (mMSC) immunomodulation and differentiation, depending on the inflammatory milieu.

Objective: We aimed at dissecting the relevant intracellular pathways through which IFN-γ affects MSC plasticity and the consequence of their manipulation on MSC functions.

Methods: Modification of relevant IFN-γ-dependent pathways in mMSCs was carried out in vitro through gene silencing or chemical inhibition of key components. Functional outcomes were assessed by means of Western blotting, real-time PCR, differentiation, and proliferation assays on MSCs. The effect on T cells was addressed by T-cell proliferation assays; the effect of mammalian target of rapamycin (mTOR) manipulation in MSCs was studied in vivo in a mouse model of delayed-type hypersensitivity assay. To address whether similar mechanisms are involved also in hMSCs on IFN-γ stimulation, the effect of chemical inhibition on the same intracellular pathways was assessed by means of Western blotting, and the final outcome on immunomodulatory properties was evaluated based on real-time PCR and T-cell proliferation.

Results: We revealed that in mMSCs IFN-γ-induced immunoregulation is mediated by early phosphorylation of signal transducer and activator of transcription (STAT) 1 and STAT3, which is significantly enhanced by an extracellular signal-regulated kinase 1/2-dependent mTOR inhibition, thereby promoting pSTAT1 nuclear translocation. Accordingly, after intracellular mTOR inhibition, MSCs augmented their ability to inhibit T-cell proliferation and control delayed-type hypersensitivity in vivo. Similarly, on mTOR blockade, hMSCs also enhanced their immunoregulatory features. A sustained exposure to IFN-γ led to inhibition of STAT3 activity, which in mMSCs resulted in an impaired proliferation and differentiation.

Conclusion: These results provide new insights about MSC intracellular pathways affected by IFN-γ, demonstrating that pharmacologic or genetic manipulation of MSCs can enhance their immunomodulatory functions, which could be translated into novel therapeutic approaches.
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http://dx.doi.org/10.1016/j.jaci.2016.09.004DOI Listing
May 2017

Dysregulation of regulatory CD56(bright) NK cells/T cells interactions in multiple sclerosis.

J Autoimmun 2016 08 4;72:8-18. Epub 2016 May 4.

Department of Neuroscience, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genova, Largo Daneo 3, 16132 Genova, Italy; Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV, 9, 1612 Genova, Italy. Electronic address:

Recent evidence has shown that CD56(bright) NK cells, a subset of NK cells abundant in lymph nodes, may have an immunoregulatory function. In multiple sclerosis (MS), expansion of CD56(bright) NK cells has been associated to successful response to different treatments and to remission of disease during pregnancy; how whether they exert immunoregulation in physiologic conditions and whether this is impaired in MS is not known. We dissected the immunoregulatory role of CD56(bright) NK cells function in healthy subjects (HS) and compared it with that of untreated MS subjects or patients with clinically isolated syndrome suggestive of MS (CIS). We found that CD56(bright) NK cells from HS acquire, upon inflammatory cues, the capability of suppressing autologous CD4+T cell proliferation through direct cytotoxicity requiring engagement of natural cytotoxicity receptors (NCRs) and secretion of granzyme B. CD56(bright) NK cells from patients with MS/CIS did not differ in frequency and share a similar phenotype but displayed a significantly lower ability to inhibit autologous T cell proliferation. This impairment was not related to deficient expression of NCRs or granzyme B by CD56(bright) NK cells, but to increased HLA-E expression on T cells from MS/CIS subjects, which could enhance the inhibitory effect mediated by NKG2A that is homogeneously expressed on CD56(bright) NK cells. The defect in controlling autologous T cells by CD56(bright) NK cells in MS/CIS might contribute to the excess of autoimmune response that is associated to disease development.
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http://dx.doi.org/10.1016/j.jaut.2016.04.003DOI Listing
August 2016

NG2, a common denominator for neuroinflammation, blood-brain barrier alteration, and oligodendrocyte precursor response in EAE, plays a role in dendritic cell activation.

Acta Neuropathol 2016 07 30;132(1):23-42. Epub 2016 Mar 30.

Department of Neurology, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genoa, Genoa, Italy.

In adult CNS, nerve/glial-antigen 2 (NG2) is expressed by oligodendrocyte progenitor cells (OPCs) and is an early marker of pericyte activation in pathological conditions. NG2 could, therefore, play a role in experimental autoimmune encephalomyelitis (EAE), a disease associated with increased blood-brain barrier (BBB) permeability, inflammatory infiltrates, and CNS damage. We induced EAE in NG2 knock-out (NG2KO) mice and used laser confocal microscopy immunofluorescence and morphometry to dissect the effect of NG2 KO on CNS pathology. NG2KO mice developed milder EAE than their wild-type (WT) counterparts, with less intense neuropathology associated with a significant improvement in BBB stability. In contrast to WT mice, OPC numbers did not change in NG2KO mice during EAE. Through FACS and confocal microscopy, we found that NG2 was also expressed by immune cells, including T cells, macrophages, and dendritic cells (DCs). Assessment of recall T cell responses to the encephalitogen by proliferation assays and ELISA showed that, while WT and NG2KO T cells proliferated equally to the encephalitogenic peptide MOG35-55, NG2KO T cells were skewed towards a Th2-type response. Because DCs could be responsible for this effect, we assessed their expression of IL-12 by PCR and intracellular FACS. IL-12-expressing CD11c+ cells were significantly decreased in MOG35-55-primed NG2KO lymph node cells. Importantly, in WT mice, the proportion of IL-12-expressing cells was significantly lower in CD11c+ NG2- cells than in CD11c+ NG2+ cells. To assess the relevance of NG2 at immune system and CNS levels, we induced EAE in bone-marrow chimeric mice, generated with WT recipients of NG2KO bone-marrow cells and vice versa. Regardless of their original phenotype, mice receiving NG2KO bone marrow developed milder EAE than those receiving WT bone marrow. Our data suggest that NG2 plays a role in EAE not only at CNS/BBB level, but also at immune response level, impacting on DC activation and thereby their stimulation of reactive T cells, through controlling IL-12 expression.
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http://dx.doi.org/10.1007/s00401-016-1563-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4911384PMC
July 2016

Mesenchymal stem cells for the treatment of neurological diseases: Immunoregulation beyond neuroprotection.

Immunol Lett 2015 Dec 18;168(2):183-90. Epub 2015 Aug 18.

Department of Neurology, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genoa, Largo Daneo 3, 16132 Genoa, Italy; Centre of Excellence for Biomedical Research, University of Genoa, Viale Benedetto XV, 5, 16132 Genoa, Italy. Electronic address:

An inflammatory response is often observed in neurological diseases, being characterized sometimes by activation of adaptive cells (T and B lymphocytes) and, almost inexorably, of cells of the innate immunity (microglial cells, macrophages). Mesenchymal stromal/stem cells represent a promising therapeutic approach for the treatment of intractable neurological diseases given the possibility that they affect neurodegeneration both directly and indirectly, through their potent immunomodulatory effect. Here we will review the evidence, mostly deriving from preclinical studies, that MSC, beyond their ability to foster neurorepair, can ameliorate neurodegenerative diseases through their effect on associated immune responses.
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http://dx.doi.org/10.1016/j.imlet.2015.08.007DOI Listing
December 2015

The immunomodulatory function of mesenchymal stem cells: mode of action and pathways.

Ann N Y Acad Sci 2015 Sep 6;1351:114-26. Epub 2015 Jul 6.

Department of Neurology, Rehabilitation, Ophthalmology, Genetics, and Maternal and Child Health (DINOGMI).

Mesenchymal stem cells (MSCs) are being increasingly investigated as a therapeutic alternative, not only for their possible regenerative potential but also for their immunomodulatory action, which is being exploited for controlling diseases associated with inflammation. Understanding their direct and indirect target cells, as well as their mode of action and relevant pathways, is a prerequisite for the appropriate and optimal use of MSCs in therapy. Here, we review recent findings on the effects of MSCs on adaptive and innate immune cells. We also consider the impact of the environment on MSC profile, both anti- and proinflammatory, and the mechanisms and molecular pathways through which their effects are mediated, both at the MSC and target cell levels.
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http://dx.doi.org/10.1111/nyas.12815DOI Listing
September 2015

Fumarates modulate microglia activation through a novel HCAR2 signaling pathway and rescue synaptic dysregulation in inflamed CNS.

Acta Neuropathol 2015 Aug 29;130(2):279-95. Epub 2015 Apr 29.

Department of Neurology, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genoa, Genoa, Italy.

Dimethyl fumarate (DMF), recently approved as an oral immunomodulatory treatment for relapsing-remitting multiple sclerosis (MS), metabolizes to monomethyl fumarate (MMF) which crosses the blood-brain barrier and has demonstrated neuroprotective effects in experimental studies. We postulated that MMF exerts neuroprotective effects through modulation of microglia activation, a critical component of the neuroinflammatory cascade that occurs in neurodegenerative diseases such as MS. To ascertain our hypothesis and define the mechanistic pathways involved in the modulating effect of fumarates, we used real-time PCR and biochemical assays to assess changes in the molecular and functional phenotype of microglia, quantitative Western blotting to monitor activation of postulated pathway components, and ex vivo whole-cell patch clamp recording of excitatory post-synaptic currents in corticostriatal slices from mice with experimental autoimmune encephalomyelitis (EAE), a model for MS, to study synaptic transmission. We show that exposure to MMF switches the molecular and functional phenotype of activated microglia from classically activated, pro-inflammatory type to alternatively activated, neuroprotective one, through activation of the hydroxycarboxylic acid receptor 2 (HCAR2). We validate a downstream pathway mediated through the AMPK-Sirt1 axis resulting in deacetylation, and thereby inhibition, of NF-κB and, consequently, of secretion of pro-inflammatory molecules. We demonstrate through ex vivo monitoring of spontaneous glutamate-mediated excitatory post-synaptic currents of single neurons in corticostriatal slices from EAE mice that the neuroprotective effect of DMF was exerted on neurons at pre-synaptic terminals by modulating glutamate release. By exposing control slices to untreated and MMF-treated activated microglia, we confirm the modulating effect of MMF on microglia function and, thereby, its indirect neuroprotective effect at post-synaptic level. These findings, whereby DMF-induced activation of a new HCAR2-dependent pathway on microglia leads to the modulation of neuroinflammation and restores synaptic alterations occurring in EAE, represent a possible novel mechanism of action for DMF in MS.
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http://dx.doi.org/10.1007/s00401-015-1422-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4503882PMC
August 2015

Altered miRNA expression is associated with neuronal fate in G93A-SOD1 ependymal stem progenitor cells.

Exp Neurol 2014 Mar 21;253:91-101. Epub 2013 Dec 21.

Neurology IV-Neuromuscular Diseases and Neuroimmunology Unit, Fondazione Istituto Neurologico "Carlo Besta" (INNCB), Via Celoria 11, 20133 Milan, Italy. Electronic address:

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive motoneuron loss in the CNS. In G93A-SOD1 mice, motoneuron degeneration is associated with proliferative restorative attempts of ependymal stem progenitor cells (epSPCs), usually quiescent in the spinal cord. The aims of the study were to demonstrate that epSPCs isolated from the spinal cord of G93A-SOD1 mice express neurogenic potential in vitro, and thus gain a better understanding of epSPC neural differentiation properties. For this purpose, we compared the ability of epSPCs from asymptomatic and symptomatic G93A-SOD1 and WT SOD1 transgenic mice to proliferate and differentiate into neural cells. Compared to control cells, G93A-SOD1 epSPCs differentiated more into neurons than into astrocytes, whereas oligodendrocyte proportions were similar in the two populations. G93A-SOD1 neurons were small and astrocytes had an activated phenotype. Evaluation of microRNAs, specific for neural cell fate and cell-cycle regulation, in G93A-SOD1 epSPCs showed that miR-9, miR-124a, miR-19a and miR-19b were differentially expressed. Expression analysis of the predicted miRNA targets allowed identification of a functional network in which Hes1, Pten, Socs1, and Stat3 genes were important for controlling epSPC fate. Our findings demonstrate that G93A-SOD1 epSPCs are a source of multipotent cells that have neurogenic potential in vitro, and might be a useful tool to investigate the mechanisms of neural differentiation in relation to miRNA expression whose modulation might constitute new targeted therapeutic approaches to ALS.
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http://dx.doi.org/10.1016/j.expneurol.2013.12.007DOI Listing
March 2014

Can we switch microglia's phenotype to foster neuroprotection? Focus on multiple sclerosis.

Immunology 2014 Mar;141(3):328-39

Department of Neurosciences, Ophthalmology, Genetics, Rehabilitation and Child Health, University of Genoa, Genoa, Italy.

Microglia cells, the resident innate immune cells in the brain, are highly active, extending and retracting highly motile processes through which they continuously survey their microenvironment for 'danger signals' and interact dynamically with surrounding cells. Upon sensing changes in their central nervous system microenvironment, microglia become activated, undergoing morphological and functional changes. Microglia activation is not an 'all-or-none' process, but rather a continuum depending on encountered stimuli, which is expressed through a spectrum of molecular and functional phenotypes ranging from so-called 'classically activated', with a highly pro-inflammatory profile, to 'alternatively activated' associated with a beneficial, less inflammatory, neuroprotective profile. Microglia activation has been demonstrated in most neurological diseases of diverse aetiology and has been implicated as a contributor to neurodegeneration. The possibility to promote microglia's neuroprotective phenotype has therefore become a therapeutic goal. We have focused our discussion on the role of microglia in multiple sclerosis, a prototype of inflammatory, demyelinating, neurodegenerative disease, and on the effect of currently approved or on-trial anti-inflammatory therapeutic strategies that might mediate neuroprotection at least in part through their effect on microglia by modifying their behaviour via a switch of their functional phenotype from a detrimental to a protective one. In addition to pharmaceutical approaches, such as treatment with glatiramer acetate, interferon-β, fingolimod or dimethyl fumarate, we address the alternative therapeutic approach of treatment with mesenchymal stem cells and their potential role in neuroprotection through their 'calming' effect on microglia.
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http://dx.doi.org/10.1111/imm.12177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3930371PMC
March 2014

A large cohort of myotonia congenita probands: novel mutations and a high-frequency mutation region in exons 4 and 5 of the CLCN1 gene.

J Hum Genet 2013 Sep 6;58(9):581-7. Epub 2013 Jun 6.

Neurology IV-Neuromuscular Diseases and Neuroimmunology Unit, Foundation IRCCS Neurological Institute Carlo Besta, Milan, Italy.

Myotonia congenita is a genetic disease characterized by impaired muscle relaxation after forceful contraction (myotonia) and caused by mutations in the chloride channel voltage-sensitive 1 (CLCN1) gene, encoding the voltage-gated chloride channel of skeletal muscle (ClC-1). In a large cohort of clinically diagnosed unrelated probands, we identified 75 different CLCN1 mutations in 106 individuals, among which 29 were novel mutations and 46 had already been reported. Despite the newly described mutations being scattered throughout the gene, in our patients, mutations were mostly found in exons 4 and 5. Most of the novel mutations located in the region comprising the intramembrane helices are involved in the ion-conducting pathway and predicted to affect channel function. We report for the first time that two mutations, inherited on the same allele as a heterozygous trait, abrogate disease expression, although when inherited singularly they were pathogenic. Such a mode of inheritance might explain the incomplete penetrance reported for autosomal dominant mutations in particular families.
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http://dx.doi.org/10.1038/jhg.2013.58DOI Listing
September 2013

Towards clinical application of mesenchymal stem cells for treatment of neurological diseases of the central nervous system.

J Neuroimmune Pharmacol 2013 Dec 12;8(5):1062-76. Epub 2013 Apr 12.

Department of Neurosciences Ophthalmology, Genetics, Rehabilitation and Child Health, University of Genoa, Genoa, Italy.

The diagnosis of a neurological disease of the central nervous system (CNS) is often associated with the anticipation of an irreversible and untreatable disability. This is the case also of multiple sclerosis (MS) where approved treatments effectively modulate the autoimmune attack to myelin antigens, but poorly affect neurodegeneration and do not promote tissue repair. Thus, stem cell-based therapies are increasingly being considered a possible strategy for diseases of the CNS. Mesenchymal stem cells (MSC), the safety of which has been demonstrated in the last 20 years through clinical trials and case studies, are of particular interest in view not only of their neuroprotective, but also of their immunomodulatory properties. Here, we review the therapeutic features of MSC that make them relevant in the treatment of CNS illnesses and discuss the pioneer clinical experience with MSC-based therapy in neurological diseases.
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http://dx.doi.org/10.1007/s11481-013-9456-6DOI Listing
December 2013

A new thiopurine s-methyltransferase haplotype associated with intolerance to azathioprine.

J Clin Pharmacol 2013 Jan 24;53(1):67-74. Epub 2013 Jan 24.

Neurology IV, Foundation IRCCS Neurological Institute Carlo Besta, Milan, Italy.

The authors have analyzed single nucleotide polymorphisms in the thiopurine S-methyltransferase (TPMT) gene in the context of efficacy and toxicity of azathioprine (AZA) to determine possible genotype-phenotype correlations between TPMT allelic variants and response to AZA treatment in 76 Italian patients with myasthenia gravis. They confirm known intronic and exonic TPMT polymorphisms that do not correlate with AZA responses and demonstrate a novel intronic polymorphism in a patient intolerant to AZA. Most importantly, they show that of the 22 AZA-intolerant patients, all 5 who carried mutations of the intolerance-linked haplotype TPMT*3A also carried the intronic T140+114A (rs3931660), all 3 mutations being part of a new haplotype designated TMPT*3E. TPMT*3E was not observed in unresponsive or responsive patients. The association of TPMT*3E with AZA intolerance and its frequency must be ascertained in larger, ethnically different cohorts. Nevertheless, in view of the highly significant association (Psim = 0.0026) between TPMT*3E and AZA intolerance in the study, this new haplotype should be taken into consideration in pharmacogenetic profiling for AZA.
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http://dx.doi.org/10.1177/0091270011435989DOI Listing
January 2013

AMDA 2.13: A major update for automated cross-platform microarray data analysis.

Biotechniques 2012 Jul;53(1):33-40

Genopolis-Consortium for Functional Genomics, Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy.

Microarray platforms require analytical pipelines with modules for data pre-processing including data normalization, statistical analysis for identification of differentially expressed genes, cluster analysis, and functional annotation. We previously developed the Automated Microarray Data Analysis (AMDA, version 2.3.5) pipeline to process Affymetrix 3' IVT GeneChips. The availability of newer technologies that demand open-source tools for microarray data analysis has impelled us to develop an updated multi-platform version, AMDA 2.13. It includes additional quality control metrics, annotation-driven (annotation grade of Affymetrix NetAffx) and signal-driven (Inter-Quartile Range) gene filtering, and approaches to experimental design. To enhance understanding of biological data, differentially expressed genes have been mapped into KEGG pathways. Finally, a more stable and user-friendly interface was designed to integrate the requirements for different platforms. AMDA 2.13 allows the analysis of Affymetrix (cartridges and plates) and whole transcript probe design (Gene 1.0/1.1 ST and Exon 1.0 ST GeneChips), Illumina Bead Arrays, and one-channel Agilent 4×44 arrays. Relative to early versions, it supports various experimental designs and delivers more insightful biological understanding and up-to-date annotations.
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http://dx.doi.org/10.2144/0000113889DOI Listing
July 2012

'Multi-epitope-targeted' immune-specific therapy for a multiple sclerosis-like disease via engineered multi-epitope protein is superior to peptides.

PLoS One 2011 29;6(11):e27860. Epub 2011 Nov 29.

Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel.

Antigen-induced peripheral tolerance is potentially one of the most efficient and specific therapeutic approaches for autoimmune diseases. Although highly effective in animal models, antigen-based strategies have not yet been translated into practicable human therapy, and several clinical trials using a single antigen or peptidic-epitope in multiple sclerosis (MS) yielded disappointing results. In these clinical trials, however, the apparent complexity and dynamics of the pathogenic autoimmunity associated with MS, which result from the multiplicity of potential target antigens and "epitope spread", have not been sufficiently considered. Thus, targeting pathogenic T-cells reactive against a single antigen/epitope is unlikely to be sufficient; to be effective, immunospecific therapy to MS should logically neutralize concomitantly T-cells reactive against as many major target antigens/epitopes as possible. We investigated such "multi-epitope-targeting" approach in murine experimental autoimmune encephalomyelitis (EAE) associated with a single ("classical") or multiple ("complex") anti-myelin autoreactivities, using cocktail of different encephalitogenic peptides vis-a-vis artificial multi-epitope-protein (designated Y-MSPc) encompassing rationally selected MS-relevant epitopes of five major myelin antigens, as "multi-epitope-targeting" agents. Y-MSPc was superior to peptide(s) in concomitantly downregulating pathogenic T-cells reactive against multiple myelin antigens/epitopes, via inducing more effective, longer lasting peripheral regulatory mechanisms (cytokine shift, anergy, and Foxp3+ CTLA4+ regulatory T-cells). Y-MSPc was also consistently more effective than the disease-inducing single peptide or peptide cocktail, not only in suppressing the development of "classical" or "complex EAE" or ameliorating ongoing disease, but most importantly, in reversing chronic EAE. Overall, our data emphasize that a "multi-epitope-targeting" strategy is required for effective immune-specific therapy of organ-specific autoimmune diseases associated with complex and dynamic pathogenic autoimmunity, such as MS; our data further demonstrate that the "multi-epitope-targeting" approach to therapy is optimized through specifically designed multi-epitope-proteins, rather than myelin peptide cocktails, as "multi-epitope-targeting" agents. Such artificial multi-epitope proteins can be tailored to other organ-specific autoimmune diseases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0027860PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3226621PMC
April 2012

Hind limb muscle atrophy precedes cerebral neuronal degeneration in G93A-SOD1 mouse model of amyotrophic lateral sclerosis: a longitudinal MRI study.

Exp Neurol 2011 Sep 14;231(1):30-7. Epub 2011 May 14.

Department of Neurology IV, Neuromuscular Diseases and Neuroimmunology, Fondazione Istituto Neurologico Carlo Besta, Milan, 20133, Italy.

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, neurodegenerative disorder caused by the degeneration of motor neurons in the CNS, which results in complete paralysis of skeletal muscles. Recent experimental studies have suggested that the disease could initiate in skeletal muscle, rather than in the motor neurons. To establish the timeframe of motor neuron degeneration in relation to muscle atrophy in motor neuron disease, we have used MRI to monitor changes throughout disease in brain and skeletal muscle of G93A-SOD1 mice, a purported model of ALS. Longitudinal MRI examination of the same animals indicated that muscle volume in the G93A-SOD1 mice was significantly reduced from as early as week 8 of life, 4 weeks prior to clinical onset. Progressive muscle atrophy from week 8 onwards was confirmed by histological analysis. In contrast, brain MRI indicated that neurodegeneration occurs later in G93A-SOD1 mice, with hyperintensity MRI signals detected only at weeks 10-18. Neurodegenerative changes were observed only in the motor nuclei areas of the brainstem; MRI changes indicative of neurodegeneration were not detected in the motor cortex where first motor neurons originate, even at the late disease stage. This longitudinal MRI study establishes unequivocally that, in the experimental murine model of ALS, muscle degeneration occurs before any evidence of neurodegeneration and clinical signs, supporting the postulate that motor neuron disease can initiate from muscle damage and result from retrograde dying-back of the motor neurons.
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http://dx.doi.org/10.1016/j.expneurol.2011.05.007DOI Listing
September 2011