Publications by authors named "Clara Ballerini"

41 Publications

An innovative and affordable quantitative assessment of human TCR repertoire.

EBioMedicine 2020 Nov 20;61:103021. Epub 2020 Oct 20.

Department of Experimental and Clinical Medicine, University of Florence, Florence Italy. Electronic address:

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http://dx.doi.org/10.1016/j.ebiom.2020.103021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7578669PMC
November 2020

Anti-inflammatory effects of androgens in the human vagina.

J Mol Endocrinol 2020 10;65(3):109-124

Andrology, Women's Endocrinology and Gender Incongruence Unit, Department of Experimental Clinical and Biomedical Sciences 'Mario Serio', University of Florence, Florence, Italy.

Chronic inflammation is involved in the genitourinary syndrome of menopause (GSM) and beneficial effects of androgens in the vagina have been described. We investigated the potential involvement of human vagina smooth muscle cells (hvSMCs) in the inflammatory response and the immunomodulatory effect of androgen receptor (AR) agonist dihydrotestosterone (DHT). HvSMCs isolated from menopausal women were evaluated for sex steroids receptors and toll-like receptors mRNA expression, and left untreated or treated in vitro with lipopolysaccharide (LPS) or IFNγ, in the presence or absence of DHT. We evaluated mRNA expression (by RT-PCR) and secretion in cell culture supernatants (by a bead-based immunoassay) of pro-inflammatory markers. Nuclear translocation of NF-κB (by immunofluorescence) and cell surface HLA-DR expression (by flow cytometry) were also evaluated. Similar experiments were repeated in rat vSMCs (rvSMCs). In hvSMCs and rvSMCs, AR was highly expressed. DHT pre-treatment inhibited LPS-induced mRNA expression of several pro-inflammatory mediators (i.e. COX2, IL-6, IL-12A and IFNγ), effect significantly blunted by AR antagonist bicalutamide. DHT significantly counteracted the secretion of IL-1RA, IL-2, IL-5, IL-15, FGF, VEGF and TNFα. LPS-induced NF-κB nuclear translocation was significantly inhibited by DHT, an effect counteracted by bicalutamide. DHT pre-treatment significantly decreased IFNγ-induced expression of HLA-DR, mRNA expression of iNOS, COX2 and MCP1, and secretion of IL-1, IL-2, IL-5, IL-6, MCP1 and GCSF. Similar effects were observed in rvSMCs. The activation of AR suppresses the inflammatory response in hvSMCs, reducing their potential to be involved in the initiation and maintaining of inflammation, thus representing a therapeutic strategy in conditions, such as the GSM.
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http://dx.doi.org/10.1530/JME-20-0147DOI Listing
October 2020

The anti-inflammatory and immune-modulatory effects of OEA limit DSS-induced colitis in mice.

Biomed Pharmacother 2020 Sep 16;129:110368. Epub 2020 Jun 16.

Dipartimento di Scienze della Salute, Università di Firenze (I), Italy. Electronic address:

Fatty acid ethanolamides acting on proliferator-activated receptor (PPAR)-α are among the endogenous lipid molecules that attenuate inflammatory processes and pain sensitivity. Whereas these properties are well-known for palmitoylethanolamide (PEA), the efficacy of oleoylethanolamide (OEA, first described as a satiety hormone synthesized in the jejunum) has been overlooked. In this study, we aimed to evaluate the effect of OEA administration in a mouse model of colitis. C57BL/6J mice were exposed to 2.5% dextran sodium sulphate (DSS) in drinking water for 5 days. Daily i.p. administration of 10 mg/kg OEA started 3 days before DSS and lasted for 12 days. The DSS-untreated control group received only ultrapure water. DSS mice treated with OEA had a significant improvement of disease score. OEA restored mRNA transcription of PPAR-α, of tight junctions and protective factors of colon integrity disrupted by DSS. The improvement correlated with significant decrease of colonic and systemic levels of pro-inflammatory cytokines compared to the DSS group. OEA antiinflammatory effects were mediated by the selective targeting of the TLR4 axis causing a downstream inhibition of nuclear factor kappa B (NF-κB)- MyD88-dependent and NLRP3 inflammation pathways. OEA treatment also inhibited DSS-induced increase of inflammatory cytokines levels in the mesenteric lymph nodes. CONCLUSIONS AND IMPLICATIONS: These results underscore the validity of OEA as a potent protective and anti-inflammatory agent in ulcerative colitis that may be exploited to broaden the pharmacological strategies against inflammatory bowel disease.
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http://dx.doi.org/10.1016/j.biopha.2020.110368DOI Listing
September 2020

T Cell Delivery of Nanoparticles-Bound Anti-CD20 Monoclonal Antibody: Successful B Cell Depletion in the Spinal Cord during Experimental Autoimmune Encephalomyelitis.

J Neuroimmune Pharmacol 2020 Jun 9. Epub 2020 Jun 9.

Department of Clinical and Experimental Medicine, University of Florence, Viale Gaetano Pieraccini 6, 50125, Florence, Italy.

We developed a nanotechnology based-cell mediated drug delivery system by loading myelin antigen-specific T cells with nanoparticles bound to anti-CD20 monoclonal antibody. Anti-CD20 antibody is a current treatment (ocrelizumab) for multiple sclerosis (MS), a chronic, inflammatory and autoimmune disease of the central nervous system (CNS). CD20-depletion has been associated with efficacy in active relapsing and progressive MS, but may not efficiently target inflammatory cells compartmentalized in the CNS. In our work, the intravenous transfer of T cells containing nanoparticle-anti-CD20 complex in mice causes B cell depletion in the spleen and in the brain, whereas the injection of anti-CD20 alone depletes B cells only in the spleen. Testing this system in Experimental Autoimmune Encephalomyelitis (EAE), animal model of MS, we found that spinal cord B cell depletion ameliorates the disease course and pathology. Graphical Abstract.
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http://dx.doi.org/10.1007/s11481-020-09931-wDOI Listing
June 2020

The TCR Repertoire Reconstitution in Multiple Sclerosis: Comparing One-Shot and Continuous Immunosuppressive Therapies.

Front Immunol 2020 9;11:559. Epub 2020 Apr 9.

Dipartimento di Medicina Sperimentale e Clinica (DMSC), University of Florence, Florence, Italy.

Natalizumab (NTZ) and autologous hematopoietic stem cell transplantation (AHSCT) are two successful treatments for relapsing-remitting multiple sclerosis (RRMS), an autoimmune T-cell-driven disorder affecting the central nervous system that is characterized by relapses interspersed with periods of complete or partial recovery. Both RRMS treatments have been documented to impact T-cell subpopulations and the T-cell receptor (TCR) repertoire in terms of clone frequency, but, so far, the link between T-cell naive and memory populations, autoimmunity, and treatment outcome has not yet been established hindering insight into the post-treatment TCR landscape of MS patients. To address this important knowledge gap, we tracked peripheral T-cell subpopulations (naïve and memory CD4+ and CD8+) across 15 RRMS patients before and after two years of continuous treatment (NTZ) and a single treatment course (AHSCT) by high-throughput TCRß sequencing. We found that the two MS treatments left treatment-specific multidimensional traces in patient TCRß repertoire dynamics with respect to clonal expansion, clonal diversity and repertoire architecture. Comparing MS TCR sequences with published datasets suggested that the majority of public TCRs belonged to virus-associated sequences. In summary, applying multi-dimensional computational immunology to a TCRß dataset of treated MS patients, we show that qualitative changes of TCRß repertoires encode treatment-specific information that may be relevant for future clinical trials monitoring and personalized MS follow-up, diagnosis and treatment regimes. Natalizumab (NTZ) and autologous hematopoietic stem cell transplantation (AHSCT) are two successful treatments for relapsing-remitting multiple sclerosis (RRMS), an autoimmune T-cell-driven disorder affecting the central nervous system that is characterized by relapses interspersed with periods of complete or partial recovery. Both RRMS treatments have been documented to impact T-cell subpopulations and the T-cell receptor (TCR) repertoire in terms of clone frequency, but, so far, the link between T-cell naive and memory populations, autoimmunity, and treatment outcome has not yet been established hindering insight into the posttreatment TCR landscape of MS patients. To address this important knowledge gap, we tracked peripheral T-cell subpopulations (naive and memory CD4+ and CD8+) across 15 RRMS patients before and after 2 years of continuous treatment (NTZ) and a single treatment course (AHSCT) by high-throughput TCRβ sequencing. We found that the two MS treatments left treatment-specific multidimensional traces in patient TCRβ repertoire dynamics with respect to clonal expansion, clonal diversity, and repertoire architecture. Comparing MS TCR sequences with published datasets suggested that the majority of public TCRs belonged to virus-associated sequences. In summary, applying multidimensional computational immunology to a TCRβ dataset of treated MS patients, we show that qualitative changes of TCRβ repertoires encode treatment-specific information that may be relevant for future clinical trials monitoring and personalized MS follow-up, diagnosis, and treatment regimens.
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http://dx.doi.org/10.3389/fimmu.2020.00559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7160336PMC
March 2021

Prognostic role of intrathecal IgM synthesis in multiple sclerosis: Results from a clinical series.

Mult Scler 2021 02 24;27(2):198-207. Epub 2020 Feb 24.

Department of Neurofarba, University of Florence, Florence, Italy/IRCCS Fondazione Don Carlo Gnocchi, Florence, Italy.

Background: There is emerging evidence that intrathecal IgM synthesis (ITMS) is a risk factor for conversion to clinically defined multiple sclerosis (CDMS) in clinically isolated syndrome (CIS) patients.

Objectives: The objective of this study is to verify the prognostic role of ITMS as a risk factor for the second clinical attack in patients after the first demyelinating event.

Methods: Monocentric observational study performed on prospectively acquired clinical data and retrospective evaluation of magnetic resonance imaging (MRI) data. ITMS was assessed according to Reiber's non-linear function. We compared time to the second attack by using Kaplan-Meier curves and performed adjustment by Cox regression analysis.

Results: Demographics and clinical data were collected prospectively in a cohort of 68 patients. ITMS occurred in 40% (27/68) of patients who had a higher T1-hypointense lesion load at brain MRI ( = 0.041). In multivariate Cox regression analysis (adjusted for age, sex, baseline Expanded Disability Status Scale, IgG oligoclonal bands and disease-modifying treatment exposure), relapsing-remitting multiple sclerosis (MS) patients with ITMS were at higher risk to experience a second clinical attack (adjusted hazard ratio (aHR) = 6.3, 95% confidence interval (CI) = 2.1-18.4,  = 0.001).

Conclusion: Together with previous studies, our findings support the role of ITMS as a prognostic biomarker in MS.
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http://dx.doi.org/10.1177/1352458520907913DOI Listing
February 2021

Cytokine inflammatory threat, but not LPS one, shortens GABAergic synaptic currents in the mouse spinal cord organotypic cultures.

J Neuroinflammation 2019 Jun 25;16(1):127. Epub 2019 Jun 25.

International School for Advanced Studies (SISSA/ISAS), 34136, Trieste, Italy.

Background: Synaptic dysfunction, named synaptopathy, due to inflammatory status of the central nervous system (CNS) is a recognized factor potentially underlying both motor and cognitive dysfunctions in neurodegenerative diseases. To gain knowledge on the mechanistic interplay between local inflammation and synapse changes, we compared two diverse inflammatory paradigms, a cytokine cocktail (CKs; IL-1β, TNF-α, and GM-CSF) and LPS, and their ability to tune GABAergic current duration in spinal cord cultured circuits.

Methods: We exploit spinal organotypic cultures, single-cell electrophysiology, immunocytochemistry, and confocal microscopy to explore synaptic currents and resident neuroglia reactivity upon CK or LPS incubation.

Results: Local inflammation in slice cultures induced by CK or LPS stimulations boosts network activity; however, only CKs specifically reduced GABAergic current duration. We pharmacologically investigated the contribution of GABAR α-subunits and suggested that a switch of GABAR α1-subunit might have induced faster GABAR decay time, weakening the inhibitory transmission.

Conclusions: Lower GABAergic current duration could contribute to providing an aberrant excitatory transmission critical for pre-motor circuit tasks and represent a specific feature of a CK cocktail able to mimic an inflammatory reaction that spreads in the CNS. Our results describe a selective mechanism that could be triggered during specific inflammatory stress.
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http://dx.doi.org/10.1186/s12974-019-1519-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6593520PMC
June 2019

A T Cell Suppressive Circuitry Mediated by CD39 and Regulated by ShcC/Rai Is Induced in Astrocytes by Encephalitogenic T Cells.

Front Immunol 2019 10;10:1041. Epub 2019 May 10.

Department of Life Sciences, University of Siena, Siena, Italy.

Multiple sclerosis is an autoimmune disease caused by autoreactive immune cell infiltration into the central nervous system leading to inflammation, demyelination, and neuronal loss. While myelin-reactive Th1 and Th17 are centrally implicated in multiple sclerosis pathogenesis, the local CNS microenvironment, which is shaped by both infiltrated immune cells and central nervous system resident cells, has emerged a key player in disease onset and progression. We have recently demonstrated that ShcC/Rai is as a novel astrocytic adaptor whose loss in mice protects from experimental autoimmune encephalomyelitis. Here, we have explored the mechanisms that underlie the ability of Rai astrocytes to antagonize T cell-dependent neuroinflammation. We show that Rai deficiency enhances the ability of astrocytes to upregulate the expression and activity of the ectonucleotidase CD39, which catalyzes the conversion of extracellular ATP to the immunosuppressive metabolite adenosine, through both contact-dependent and-independent mechanisms. As a result, Rai-deficient astrocytes acquire an enhanced ability to suppress T-cell proliferation, which involves suppression of T cell receptor signaling and upregulation of the inhibitory receptor CTLA-4. Additionally, Rai-deficient astrocytes preferentially polarize to the neuroprotective A2 phenotype. These results identify a new mechanism, to which Rai contributes to a major extent, by which astrocytes modulate the pathogenic potential of autoreactive T cells.
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http://dx.doi.org/10.3389/fimmu.2019.01041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6524536PMC
June 2020

3D Organotypic Spinal Cultures: Exploring Neuron and Neuroglia Responses Upon Prolonged Exposure to Graphene Oxide.

Front Syst Neurosci 2019 24;13. Epub 2019 Jan 24.

Neuron Physiology and Technology Lab, International School for Advanced Studies (SISSA), Trieste, Italy.

Graphene-based nanomaterials are increasingly engineered as components of biosensors, interfaces or drug delivery platforms in neuro-repair strategies. In these developments, the mostly used derivative of graphene is graphene oxide (GO). To tailor the safe development of GO nanosheets, we need to model tissue responses, and in particular the reactivity of microglia, a sub-population of neuroglia that acts as the first active immune response, when challenged by GO. Here, we investigated central nervous system (CNS) tissue reactivity upon long-term exposure to GO nanosheets in 3D culture models. We used the mouse organotypic spinal cord cultures, ideally suited for studying long-term interference with cues delivered at controlled times and concentrations. In cultured spinal segments, the normal presence, distribution and maturation of anatomically distinct classes of neurons and resident neuroglial cells are preserved. Organotypic explants were developed for 2 weeks embedded in fibrin glue alone or presenting GO nanosheets at 10, 25 and 50 μg/mL. We addressed the impact of such treatments on premotor synaptic activity monitored by patch clamp recordings of ventral interneurons. We investigated by immunofluorescence and confocal microscopy the accompanying glial responses to GO exposure, focusing on resident microglia, tested in organotypic spinal slices and in isolated neuroglia cultures. Our results suggest that microglia reactivity to accumulation of GO flakes, maybe due to active phagocytosis, may trim down synaptic activity, although in the absence of an effective activation of inflammatory response and in the absence of neuronal cell death.
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http://dx.doi.org/10.3389/fnsys.2019.00001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6354065PMC
January 2019

Oleoylethanolamide treatment affects gut microbiota composition and the expression of intestinal cytokines in Peyer's patches of mice.

Sci Rep 2018 10 5;8(1):14881. Epub 2018 Oct 5.

Dipartimento di Scienze della Salute, Università di Firenze, Firenze, Italy.

The lipid sensor oleoylethanolamide (OEA), an endogenous high-affinity agonist of peroxisome proliferator-activated receptor-α (PPAR-α) secreted in the proximal intestine, is endowed with several distinctive homeostatic properties, such as control of appetite, anti-inflammatory activity, stimulation of lipolysis and fatty acid oxidation. When administered exogenously, OEA has beneficial effects in several cognitive paradigms; therefore, in all respects, OEA can be considered a hormone of the gut-brain axis. Here we report an unexplored modulatory effect of OEA on the intestinal microbiota and on immune response. Our study shows for the first time that sub-chronic OEA administration to mice fed a normal chow pellet diet, changes the faecal microbiota profile, shifting the Firmicutes:Bacteroidetes ratio in favour of Bacteroidetes (in particular Bacteroides genus) and decreasing Firmicutes (Lactobacillus), and reduces intestinal cytokines expression by immune cells isolated from Peyer's patches. Our results suggest that sub-chronic OEA treatment modulates gut microbiota composition towards a "lean-like phenotype", and polarises gut-specific immune responses mimicking the effect of a diet low in fat and high in polysaccharides content.
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http://dx.doi.org/10.1038/s41598-018-32925-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173739PMC
October 2018

CSF/serum matrix metallopeptidase-9 ratio discriminates neuro Behçet from multiple sclerosis.

Ann Clin Transl Neurol 2018 Apr 10;5(4):493-498. Epub 2018 Mar 10.

Department of Neurosciences, Psychology, Drug and Child Health University of Florence Florence Italy.

In neuro Behçet disease with multiple sclerosis-like features, diagnosis could be challenging. Here, we studied the cerebrospinal fluid and serum inflammatory profile of 11 neuro Behçet and 21 relapsing-remitting multiple sclerosis patients. Between the soluble factors analyzed (MMP9, TNF , IL6, CXCL13, CXCL10, CXCL8, IFN , IL10, IL17, IL23, and others) we found MMP9 increased in neuro Behçet serum compared to multiple sclerosis and decreased in cerebrospinal fluid. Furthermore, neuro Behçet analysis of circulating natural killer CD56 subset suggests their potential involvement in increased MMP9 production. We believe that these findings may have a translational utility in clinical practice.
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http://dx.doi.org/10.1002/acn3.538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5899916PMC
April 2018

Cerebrospinal fluid analysis and the determination of oligoclonal bands.

Neurol Sci 2017 Oct;38(Suppl 2):217-224

Laboratory of Neuroimmunology, C. Mondino National Neurological Institute, Via Mondino 2, 27100, Pavia, Italy.

This document presents the guidelines for the cerebrospinal fluid (CSF) analysis and the determination of oligoclonal bands (OCBs) as pivotal tests in neuroinflammatory pathologies of the central nervous system. The guidelines have been developed following a consensus process built on questionnaire-based surveys, internet contacts, and discussions at workshops of the sponsoring Italian Association of Neuroimmunology (AINI) congresses. Essential clinical information on the pathologies in which the CSF analysis is indicated, and, particularly, on those characterized by the presence of OCBs in the intrathecal compartment, indications and limits of CSF analysis and OCB determination, instructions for result interpretation, and agreed laboratory protocols (Appendix) are reported for the communicative community of neurologists and clinical pathologists.
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http://dx.doi.org/10.1007/s10072-017-3034-2DOI Listing
October 2017

The Adaptor Protein Rai/ShcC Promotes Astrocyte-Dependent Inflammation during Experimental Autoimmune Encephalomyelitis.

J Immunol 2016 07 10;197(2):480-90. Epub 2016 Jun 10.

Department of Life Sciences, University of Siena, 2 53100, Siena, Italy;

Th17 cells have been casually associated to the pathogenesis of autoimmune disease. We have previously demonstrated that Rai/ShcC, a member of the Shc family of adaptor proteins, negatively regulates Th17 cell differentiation and lupus autoimmunity. In this study, we have investigated the pathogenic outcome of the Th17 bias associated with Rai deficiency on multiple sclerosis development, using the experimental autoimmune encephalomyelitis (EAE) mouse model. We found that, unexpectedly, EAE was less severe in Rai(-/-) mice compared with their wild-type counterparts despite an enhanced generation of myelin-specific Th17 cells that infiltrated into the CNS. Nevertheless, when adoptively transferred into immunodeficient Rai(+/+) mice, these cells promoted a more severe disease compared with wild-type encephalitogenic Th17 cells. This paradoxical phenotype was caused by a dampened inflammatory response of astrocytes, which were found to express Rai, to IL-17. The results provide evidence that Rai plays opposite roles in Th17 cell differentiation and astrocyte activation, with the latter dominant over the former in EAE, highlighting this adaptor as a potential novel target for the therapy of multiple sclerosis.
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http://dx.doi.org/10.4049/jimmunol.1502063DOI Listing
July 2016

Histamine Regulates Actin Cytoskeleton in Human Toll-like Receptor 4-activated Monocyte-derived Dendritic Cells Tuning CD4+ T Lymphocyte Response.

J Biol Chem 2016 Jul 13;291(28):14803-14. Epub 2016 May 13.

From the Departments of Neuroscience, Psychology, Drugs and Child Health,

Histamine, a major mediator in allergic diseases, differentially regulates the polarizing ability of dendritic cells after Toll-like receptor (TLR) stimulation, by not completely explained mechanisms. In this study we investigated the effects of histamine on innate immune reaction during the response of human monocyte-derived DCs (mDCs) to different TLR stimuli: LPS, specific for TLR4, and Pam3Cys, specific for heterodimer molecule TLR1/TLR2. We investigated actin remodeling induced by histamine together with mDCs phenotype, cytokine production, and the stimulatory and polarizing ability of Th0. By confocal microscopy and RT-PCR expression of Rac1/CdC42 Rho GTPases, responsible for actin remodeling, we show that histamine selectively modifies actin cytoskeleton organization induced by TLR4, but not TLR2 and this correlates with increased IL4 production and decreased IFNγ by primed T cells. We also demonstrate that histamine-induced cytoskeleton organization is at least in part mediated by down-regulation of small Rho GTPase CdC42 and the protein target PAK1, but not by down-regulation of Rac1. The presence and relative expression of histamine receptors HR1-4 and TLRs were determined as well. Independently of actin remodeling, histamine down-regulates IL12p70 and CXCL10 production in mDCs after TLR2 and TLR4 stimulation. We also observed a trend of IL10 up-regulation that, despite previous reports, did not reach statistical significance.
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http://dx.doi.org/10.1074/jbc.M116.720680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4938197PMC
July 2016

Plant microRNAs as novel immunomodulatory agents.

Sci Rep 2016 05 11;6:25761. Epub 2016 May 11.

Research and Innovation Centre, Fondazione Edmund Mach, via E. Mach 1, 38010 San Michele all'Adige (TN), Italy.

An increasing body of literature is addressing the immuno-modulating functions of miRNAs which include paracrine signaling via exosome-mediated intercellular miRNA. In view of the recent evidence of intake and bioavailability of dietary miRNAs in humans and animals we explored the immuno-modulating capacity of plant derived miRNAs. Here we show that transfection of synthetic miRNAs or native miRNA-enriched fractions obtained from a wide range of plant species and organs modifies dendritic cells ability to respond to inflammatory agents by limiting T cell proliferation and consequently dampening inflammation. This immuno-modulatory effect appears associated with binding of plant miRNA on TLR3 with ensuing impairment of TRIF signaling. Similarly, in vivo, plant small RNAs reduce the onset of severity of Experimental Autoimmune Encephalomyelities by limiting dendritic cell migration and dampening Th1 and Th17 responses in a Treg-independent manner. Our results indicate a potential for therapeutic use of plant miRNAs in the prevention of chronic-inflammation related diseases.
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http://dx.doi.org/10.1038/srep25761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4863160PMC
May 2016

Immune and Epstein-Barr virus gene expression in cerebrospinal fluid and peripheral blood mononuclear cells from patients with relapsing-remitting multiple sclerosis.

J Neuroinflammation 2015 Jul 14;12:132. Epub 2015 Jul 14.

Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy.

Background: Gene expression analyses in paired cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMC) from patients with multiple sclerosis (MS) are restrained by the low RNA amounts from CSF cells and low expression levels of certain genes. Here, we applied a Taqman-based pre-amplification real-time reverse-transcription polymerase chain reaction (RT-PCR) (PreAmp RT-PCR) to cDNA from CSF cells and PBMC of MS patients and analyzed multiple genes related to immune system function and genes expressed by Epstein-Barr virus (EBV), a herpesvirus showing strong association with MS. Using this enhanced RT-PCR method, we aimed at the following: (1) identifying gene signatures potentially useful for patient stratification, (2) understanding whether EBV infection is perturbed in CSF and/or blood, and (3) finding a link between immune and EBV infection status.

Methods: Thirty-one therapy-free patients with relapsing-remitting MS were included in the study. Paired CSF cells and PBMC were collected and expression of 41 immune-related cellular genes and 7 EBV genes associated with latent or lytic viral infection were determined by PreAmp RT-PCR. Clinical, radiological, CSF, and gene expression data were analyzed using univariate and multivariate (cluster analysis, factor analysis) statistical approaches.

Results: Several immune-related genes were differentially expressed between CSF cells and PBMC from the whole MS cohort. By univariate analysis, no or only minor differences in gene expression were found associated with sex, clinical, or radiological condition. Cluster analysis on CSF gene expression data grouped patients into three clusters; clusters 1 and 2 differed by expression of genes that are related mainly to innate immunity, irrespective of sex and disease characteristics. By factor analysis, two factors grouping genes involved in antiviral immunity and immune regulation, respectively, accurately discriminated cluster 1 and cluster 2 patients. Despite the use of an enhanced RT-PCR method, EBV transcripts were detected in a minority of patients (5 of 31), with evidence of viral latency activation in CSF cells or PBMC and of lytic infection in one patient with active disease only.

Conclusions: Analysis of multiple cellular and EBV genes in paired CSF cell and PBMC samples using PreAmp RT-PCR may yield new information on the complex interplay between biological processes underlying MS and help in biomarker identification.
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http://dx.doi.org/10.1186/s12974-015-0353-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4501166PMC
July 2015

Nanomaterial applications in multiple sclerosis inflamed brain.

J Neuroimmune Pharmacol 2015 Mar 24;10(1):1-13. Epub 2015 Jan 24.

Department of Neurofarba, University of Florence, Viale Pieraccini, 6, 50137, Florence, Italy,

In the last years scientific progress in nanomaterials, where size and shape make the difference, has increased their utilization in medicine with the development of a promising new translational science: nanomedicine. Due to their surface and core biophysical properties, nanomaterials hold the promise for medical applications in central nervous system (CNS) diseases: inflammatory, degenerative and tumors. The present review is focused on nanomaterials at the neuro-immune interface, evaluating two aspects: the possible CNS inflammatory response induced by nanomaterials and the developments of nanomaterials to improve treatment and diagnosis of neuroinflammatory diseases, with a focus on multiple sclerosis (MS). Indeed, nanomedicine allows projecting new ways of drug delivery and novel techniques for CNS imaging. Despite the wide field of application in neurological diseases of nanomaterials, our topic here is to review the more recent development of nanomaterials that cross blood brain barrier (BBB) and reach specific target during CNS inflammatory diseases, a crucial strategy for CNS early diagnosis and drug delivery, indeed the main challenges of nanomedicine.
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http://dx.doi.org/10.1007/s11481-015-9588-yDOI Listing
March 2015

Increased CXCL10 expression in MS MSCs and monocytes is unaffected by AHSCT.

Ann Clin Transl Neurol 2014 Sep 29;1(9):650-8. Epub 2014 Aug 29.

Dept. NEUROFARBA, University of Florence Florence, Italy.

Objective: To confirm CXCL10 over production in bone marrow mesenchymal stem cells (MSCs) and circulating monocytes isolated from multiple sclerosis patients (MS) and identify predate cell molecular signature; to extend this analysis after autologous hematopoietic stem cell transplantation (AHSCT) to test if therapy has modifying effects on MSCs and circulating monocytes.

Methods: MSCs and monocytes were isolated from 19 MS patients who undergone AHSCT before and seven of them at least 3 years after transplant. CXCL10 production was detected after LPS/IFN-γ stimulation. TLR4 signaling pathways were investigated by means of transcription factors phosphorylation/activation level. RT-PCR of activated transcription factors was performed to quantify their expression. All experiments were conducted in parallel with 24 matched healthy donors (HD).

Results: CXCL10 expression was significantly increased in both peripheral circulating monocytes and BM MSCs compared to HD. We showed that CXCL10 production is determined by an altered signaling pathway downstream TLR4, with the involvement of STAT-1, NF-κB, p38, JNK, and CREB. All upregulated transcription factors are more phosphorylated in MS patient sample. These features are not modified after AHSCT.

Interpretation: We demonstrated that in MS two different cell lineages are characterized by significantly increased production of CXCL10, due to altered signaling pathways of innate immune reaction mediated by TLR4, probably associated with disease phenotype. This characteristic is not modified by AHSCT, suggesting that when T and B lymphocytes are reset, other possible components of MS pathology, such as CXCL10 over production, do not determine therapy outcome.
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http://dx.doi.org/10.1002/acn3.92DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4241792PMC
September 2014

Human dendritic cell activation induced by a permannosylated dendron containing an antigenic GM3-lactone mimetic.

Beilstein J Org Chem 2014 10;10:1317-1324. Epub 2014 Jun 10.

Department of Chemistry, University of Florence, Via della Lastruccia 13, 50019 Sesto Fiorentino (FI), Italy.

Vaccination strategies based on dendritic cells (DCs) armed with specific tumor antigens have been widely exploited due the properties of these immune cells in coordinating an innate and adaptive response. Here, we describe the convergent synthesis of the bifunctional multivalent glycodendron 5, which contains nine residues of mannose for DC targeting and one residue of an immunogenic mimetic of a carbohydrate melanoma associated antigen. The immunological assays demonstrated that the glycodendron 5 is able to induce human immature DC activation in terms of a phenotype expression of co-stimulatory molecules expression and MHCII. Furthermore, DCs activated by the glycodendron 5 stimulate T lymphocytes to proliferate in a mixed lymphocytes reaction (MLR).
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http://dx.doi.org/10.3762/bjoc.10.133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4077398PMC
July 2014

Carbon nanotube scaffolds instruct human dendritic cells: modulating immune responses by contacts at the nanoscale.

Nano Lett 2013 15;13(12):6098-105. Epub 2013 Nov 15.

Department of NEUROFARBA, University of Florence , 50134 Firenze, Italy.

Nanomaterials interact with cells and modify their function and biology. Manufacturing this ability can provide tissue-engineering scaffolds with nanostructures able to influence tissue growth and performance. Carbon nanotube compatibility with biomolecules motivated ongoing interest in the development of biosensors and devices including such materials. More recently, carbon nanotubes have been applied in several areas of nerve tissue engineering to study cell behavior or to instruct the growth and organization of neural networks. To gather further knowledge on the true potential of future constructs, in particular to assess their immune-modulatory action, we evaluate carbon nanotubes interactions with human dendritic cells (DCs). DCs are professional antigen-presenting cells and their behavior can predict immune responses triggered by adhesion-dependent signaling. Here, we incorporate DC cultures to carbon nanotubes and we show by phenotype, microscopy, and transcriptional analysis that in vitro differentiated and activated DCs show when interfaced to carbon nanotubes a lower immunogenic profile.
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http://dx.doi.org/10.1021/nl403396eDOI Listing
September 2014

Rosiglitazone promotes the differentiation of Langerhans cells and inhibits that of other dendritic cell types from CD133 positive hematopoietic precursors.

Histol Histopathol 2014 03 24;29(3):323-32. Epub 2013 Jul 24.

Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.

Dendritic cells and their precursors express PPAR-gamma, whose stimulation has inhibitory effects on the maturation and function of dendritic cells in vivo. Dendritic cells can differentiate in vitro from CD133+ progenitors; the influence of PPAR-gamma stimulation on this process is unknown. We have addressed the effect of PPAR-gamma agonist rosiglitazone, at a concentration as used in clinics, on the differentiation of dendritic cells from human CD133+ progenitors. Cells were harvested from cord blood by density gradient and immunomagnetic separation, and cultured for 18 days with fetal calf serum, cytokines and 1 μmol/L rosiglitazone. Analyses included flow cytometry, electron microscopy and mixed lymphocyte reaction. As expected, control cells generated without rosiglitazone were dendritic, expressed MHC-II, CD80, CD83 and CD86 and stimulated mixed reaction potently. A minority of cells expressed the Langerhans cell marker CD207/langerin, but none contained Birbeck granules. With rosiglitazone much fewer cells were generated; they were all dendritic, expressed differentiation and maturation-related antigens in higher percentage and were better stimulators of lymphocytes than those generated without the drug. The vast majority of cells expressed CD207/langerin and many contained Birbeck granules, i.e. were full-fledged Langerhans cells. We conclude that stimulation of PPAR-gamma, while negatively affecting the number of generated cells, promotes the maturation of human cord blood CD133 positive precursors into efficient, immunostimulating dendritic cells with a Langerhans cell phenotype.
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http://dx.doi.org/10.14670/HH-29.323DOI Listing
March 2014

Histamine and neuroinflammation: insights from murine experimental autoimmune encephalomyelitis.

Front Syst Neurosci 2012 3;6:32. Epub 2012 May 3.

Dipartimento di Farmacologia Preclinica e Clinica, Universita' di Firenze Firenze, Italy.

Multiple sclerosis (MS) is a chronic inflammatory, neurodegenerative disease of the CNS whose pathogenesis remains largely unknown, and available therapies are rarely successful in reversing neurological deficits or stopping disease progression. Ongoing studies on MS and the widely used murine model of experimental autoimmune encephalomyelitis (EAE) are focused on the many components of this complex and heterogeneous neurodegenerative disease in the hope of providing a mechanism-based characterization of MS that will afford successful strategies to limit and repair the neuronal damage. Recently, histamine has been postulated to have a key regulatory role in EAE and MS pathogenesis. Histamine is a mediator of inflammation and immune responses, exerting its many actions through four G protein-coupled receptors (H(1,2,3,4)R) that signal through distinct intracellular pathways and have different therapeutic potentials as they vary in expression, isoform distribution, signaling properties, and function. Immune cells involved in MS/EAE, including dendritic cells (DCs) and T lymphocytes, express H(1)R, H(2)R and H(4)R, and histamine may have varying and counteracting effects on a particular cell type, depending on the receptor subtypes being activated. Here, we review evidence of the complex and controversial role of histamine in the pathogenesis of MS and EAE and evaluate the therapeutic potential of histaminergic ligands in the treatment of autoimmune diseases.
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http://dx.doi.org/10.3389/fnsys.2012.00032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3342557PMC
October 2012

Exacerbation of experimental autoimmune encephalomyelitis in prion protein (PrPc)-null mice: evidence for a critical role of the central nervous system.

J Neuroinflammation 2012 Jan 26;9:25. Epub 2012 Jan 26.

INSERM, UMR S 938, Centre de Recherche Hôpital Saint-Antoine, Paris, France.

Background: The cellular prion protein (PrPc) is a host-encoded glycoprotein whose transconformation into PrP scrapie (PrPSc) initiates prion diseases. The role of PrPc in health is still obscure, but many candidate functions have been attributed to the protein, both in the immune and the nervous systems. Recent data show that experimental autoimmune encephalomyelitis (EAE) is worsened in mice lacking PrPc. Disease exacerbation has been attributed to T cells that would differentiate into more aggressive effectors when deprived of PrPc. However, alternative interpretations such as reduced resistance of neurons to autoimmune insult and exacerbated gliosis leading to neuronal deficits were not considered.

Method: To better discriminate the contribution of immune cells versus neural cells, reciprocal bone marrow chimeras with differential expression of PrPc in the lymphoid or in the central nervous system (CNS) were generated. Mice were subsequently challenged with MOG35-55 peptide and clinical disease as well as histopathology were compared in both groups. Furthermore, to test directly the T cell hypothesis, we compared the encephalitogenicity of adoptively transferred PrPc-deficient versus PrPc-sufficient, anti-MOG T cells.

Results: First, EAE exacerbation in PrPc-deficient mice was confirmed. Irradiation exacerbated EAE in all the chimeras and controls, but disease was more severe in mice with a PrPc-deleted CNS and a normal immune system than in the reciprocal construction. Moreover, there was no indication that anti-MOG responses were different in PrPc-sufficient and PrPc-deficient mice. Paradoxically, PrPc-deficient anti-MOG 2D2 T cells were less pathogenic than PrPc-expressing 2D2 T cells.

Conclusions: In view of the present data, it can be concluded that the origin of EAE exacerbation in PrPc-ablated mice resides in the absence of the prion protein in the CNS. Furthermore, the absence of PrPc on both neural and immune cells does not synergize for disease worsening. These conclusions highlight the critical role of PrPc in maintaining the integrity of the CNS in situations of stress, especially during a neuroinflammatory insult.
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http://dx.doi.org/10.1186/1742-2094-9-25DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305405PMC
January 2012

PARP-1 inhibition prevents CNS migration of dendritic cells during EAE, suppressing the encephalitogenic response and relapse severity.

Mult Scler 2011 Jul 22;17(7):794-807. Epub 2011 Feb 22.

Department of Pharmacology, University of Florence, Florence, Italy.

Background: Pharmacological inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1) are currently evaluated in clinical trials for various malignancies but, interestingly, also proved of remarkable efficacy in preclinical models of autoimmune disorders including experimental autoimmune encephalomyelitis (EAE).

Objectives: The objectives of the study were to determine molecular mechanisms underlying suppression of the encephalitogenic response by these drugs; likewise, whether clinically-relevant post-treatment paradigms with PARP-1 inhibitors could prevent EAE relapses.

Methods: Adopted both in vitro techniques (bone marrow-derived cultured DC) as well as in vivo models of chronic or relapsing-remitting (RR) EAE.

Results: We report that two structurally unrelated PARP-1 inhibitors negatively regulated NFκB activation, as well as maturation, cytokine production and APC function of cultured mouse bone marrow-derived dendritic cells (DCs). PARP-1 inhibitors also reduced the number and APC function of DCs migrating in the draining lymph nodes of ovalbumin-immunized mice. In C57Bl mice with chronic EAE or SJL mice with RR EAE, pharmacological inhibition of PARP-1 reduced CNS DC migration and demyelination as well as neurological impairment to an extent similar to that achieved with the potent immunosuppressant cyclosporine A. Remarkably, PARP-1 inhibitors injected after the first phase of disease reduced relapse incidence and severity, as well as the spinal cord number of autoreactive Th17 cells. Under this clinically-relevant treatment paradigm, PARP inhibitors also suppressed epitope spreading of the encephalitogenic response.

Conclusions: Overall, data underscore the potential relevance of PARP-1 inhibitors to MS therapy and suppression of autoimmunity.
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http://dx.doi.org/10.1177/1352458511399113DOI Listing
July 2011

Dendritic cells with lymphocyte-stimulating activity differentiate from human CD133 positive precursors.

Blood 2011 Apr 8;117(15):3983-95. Epub 2011 Feb 8.

Department of Anatomy, Histology and Forensic Medicine, University of Florence, Florence, Italy.

CD133 is a hallmark of primitive myeloid progenitors. We have addressed whether human cord blood cells selected for CD133 can generate dendritic cells, and Langerhans cells in particular, in conditions that promote that generation from CD34(+) progenitors. Transforming growth factor-β1 (TGF-β1) and anti-TGF-β1 antibody, respectively, were added in some experiments. With TGF-β, monocytoid cells were recognized after 7 days. Immunophenotypically immature dendritic cells were present at day 14. After 4 more days, the cells expressed CD54, CD80, CD83, and CD86 and were potent stimulators in mixed lymphocyte reaction; part of the cells expressed CD1a and langerin, but not Birbeck granules. Without TGF-β, only a small fraction of cells acquired a dendritic shape and expressed the maturation-related antigens, and lymphocytes were poorly stimulated. With anti-TGF-β, the cell growth was greatly hampered, CD54 and langerin were never expressed, and lymphocytes were stimulated weakly. In conclusion, CD133(+) progenitors can give rise in vitro, through definite steps, to mature, immunostimulatory dendritic cells with molecular features of Langerhans cells, although without Birbeck granules. Addition of TGF-β1 helps to stimulate cell growth and promotes the acquisition of mature immunophenotypical and functional features. Neither langerin nor Birbeck granules proved indispensable for lymphocyte stimulation.
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http://dx.doi.org/10.1182/blood-2010-08-299735DOI Listing
April 2011

Cerebrospinal BAFF and Epstein-Barr virus-specific oligoclonal bands in multiple sclerosis and other inflammatory demyelinating neurological diseases.

J Neuroimmunol 2011 Jan 24;230(1-2):160-3. Epub 2010 Nov 24.

Laboratory of Neuroimmunology, IRCCS National Neurological Institute C Mondino, University of Pavia, Italy.

We measured circulating serum and cerebrospinal fluid (CSF) concentrations of B lymphocyte activating factor of the tumour necrosis factor superfamily (BAFF), and determined total and Epstein-Barr virus (EBV)-specific oligoclonal IgG bands (OCBs) in 43 patients with multiple sclerosis (MS), 23 patients with other inflammatory demyelinating neurological diseases, and 20 patients with non-inflammatory neurological diseases. Serum and CSF BAFF concentrations did not differ in the three studied groups. In MS, the highest BAFF concentrations were found in the CSF samples with more than 6 OCBs (233.1 ± 129.5 vs 79.2 ± 51.6 pg/mL in the samples with less than 7 OCBs, p<0.0001). Irrespectively from BAFF levels, EBV-specific OCBs were detected in MS and in the other non-inflammatory and inflammatory demyelinating neurological diseases, with a similar frequency, and as a 'mirror pattern' in 30 of 33 EBV-specific OCB-positive cases (p<0.0001). These results indicate that circulating CSF BAFF concentrations cannot help differentiate MS from other inflammatory demyelinating neurological diseases, but positively associates with the qualitative expression of elevated intrathecal IgG production in MS, and that the oligoclonal EBV-specific antibody response, when present, is mostly systemic in all the studied neurological patients, and not preferentially restricted to MS.
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http://dx.doi.org/10.1016/j.jneuroim.2010.10.027DOI Listing
January 2011

Inhibition of immune synapse by altered dendritic cell actin distribution: a new pathway of mesenchymal stem cell immune regulation.

J Immunol 2010 Nov 1;185(9):5102-10. Epub 2010 Oct 1.

Department of Neurological Sciences, University of Florence, Florence, Italy.

Immune synapse formation between dendritic cells (DCs) and T cells is one of the key events in immune reaction. In immunogenic synapses, the presence of fully mature DCs is mandatory; consequently, the modulation of DC maturation may promote tolerance and represents a valuable therapeutic approach in autoimmune diseases. In the field of cell therapy, bone marrow mesenchymal stem cells (MSCs) have been extensively studied for their immunoregulatory properties, such as inhibiting DC immunogenicity during in vitro differentiation and ameliorating in vivo models of autoimmune diseases (e.g., experimental allergic encephalomyelitis). MSCs seem to play different roles with regard to DCs, depending on cell concentration, mechanism of stimulation, and accompanying immune cells. The aim of this work was to elucidate the immunogenic effects of MSC/DC interactions during DC activation (LPS stimulation or Ag loading). Human monocyte-derived DCs, bone marrow-derived MSCs, and circulating lymphocytes obtained from healthy donors, as well as the laboratory-generated influenza virus hemagglutinin-derived peptide, aa 306-318 peptide-specific T cell line were used for this study. We demonstrate that MSCs mediate inhibition of DC function only upon cell-cell contact. Despite no modification observed in cell phenotype or cytokine production, MSC-treated DCs were unable to form active immune synapses; they retained endocytic activity and podosome-like structures, typical of immature DCs. The transcriptional program induced by MSC-DC direct interaction supports at the molecular pathway level the phenotypical features observed, indicating the genes involved into contact-induced rearrangement of DC cytoskeleton.
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http://dx.doi.org/10.4049/jimmunol.1001332DOI Listing
November 2010

Modulating dendritic cells (DC) from immunogenic to tolerogenic responses: a novel mechanism of AZA/6-MP.

J Neuroimmunol 2010 Jan 24;218(1-2):28-35. Epub 2009 Nov 24.

Department of Neurological Sciences, University of Florence, Italy.

Azathioprine (Aza), 6-Mercaptopurine (6-MP) and 6-Thioguanine (6-TG) are thiopurine drugs widely used as immunosuppressants/anti-inflammatory agents in organ transplantation and chemotherapy. Aza is well tolerated and effective in modifying the course of MS. Here we investigated the action of 6-MP on human dendritic cells (DCs). We described for the first time that 6-MP impairs in vitro differentiation of DCs, has an inhibitory effect during DC activation processes inducing a functionally less immunogenic phenotype. Moreover, 6-MP significantly reduces DC IL-23 production and CCR7 expression, at the same time induces IL-10 augmentation. All these findings add a novel action mechanism in Aza immune modulation.
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http://dx.doi.org/10.1016/j.jneuroim.2009.11.001DOI Listing
January 2010

Mouse vaccination with dendritic cells loaded with prion protein peptides overcomes tolerance and delays scrapie.

J Gen Virol 2010 Mar 28;91(Pt 3):809-20. Epub 2009 Oct 28.

INSERM UMR S 938, UPMC Univ Paris 06, Hôpital St-Antoine, F-75012 Paris, France.

Prion diseases are presumed to be caused by the accumulation in the brain of a pathological protein called prion protein (PrP) scrapie which results from the transconformation of cellular PrP, a ubiquitous glycoprotein expressed in all mammals. Since all isoforms of PrP are perceived as self by the host immune system, a major problem in designing efficient immunoprophylaxis or immunotherapy is to overcome tolerance. The present study was aimed at investigating whether bone-marrow-derived dendritic cells (DCs) loaded with peptides previously shown to be immunogenic in PrP-deficient mice, can overcome tolerance in PrP-proficient wild-type mice and protect them against scrapie. Results show that, in such mice, peptide-loaded DCs elicit both lymphokine release by T cells and antibody secretion against native cellular PrP. Repeated recalls with peptide-loaded DCs reduces the attack rate of 139A scrapie inoculated intraperitoneally and retards disease duration by 40 days. Most interestingly, survival time in individual mice appears to be correlated with the level of circulating antibody against native cellular PrP.
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http://dx.doi.org/10.1099/vir.0.013417-0DOI Listing
March 2010