Publications by authors named "Paola Cavalcante"

33 Publications

Dysregulation of Muscle-Specific MicroRNAs as Common Pathogenic Feature Associated with Muscle Atrophy in ALS, SMA and SBMA: Evidence from Animal Models and Human Patients.

Int J Mol Sci 2021 May 26;22(11). Epub 2021 May 26.

Neurology IV-Neuroimmunology and Neuromuscular Diseases Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, Via Celoria 11, 20133 Milan, Italy.

Motor neuron diseases (MNDs) are neurodegenerative disorders characterized by upper and/or lower MN loss. MNDs include amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and spinal and bulbar muscular atrophy (SBMA). Despite variability in onset, progression, and genetics, they share a common skeletal muscle involvement, suggesting that it could be a primary site for MND pathogenesis. Due to the key role of muscle-specific microRNAs (myomiRs) in skeletal muscle development, by real-time PCR we investigated the expression of miR-206, miR-133a, miR-133b, and miR-1, and their target genes, in G93A-SOD1 ALS, Δ7SMA, and KI-SBMA mouse muscle during disease progression. Further, we analyzed their expression in serum of -mutated ALS, SMA, and SBMA patients, to demonstrate myomiR role as noninvasive biomarkers. Our data showed a dysregulation of myomiRs and their targets, in ALS, SMA, and SBMA mice, revealing a common pathogenic feature associated with muscle impairment. A similar myomiR signature was observed in patients' sera. In particular, an up-regulation of miR-206 was identified in both mouse muscle and serum of human patients. Our overall findings highlight the role of myomiRs as promising biomarkers in ALS, SMA, and SBMA. Further investigations are needed to explore the potential of myomiRs as therapeutic targets for MND treatment.
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http://dx.doi.org/10.3390/ijms22115673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8198536PMC
May 2021

Post-infectious neurologic complications in Covid-2019: a complex case report.

J Nucl Med 2021 May 20. Epub 2021 May 20.

Neurology 5 - Neuropathology Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, Italy.

A 40-year-old woman with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection developed neurologic manifestations (confusion, agitation, seizures, dyskinesias, and parkinsonism) few weeks after SARS onset. Magnetic resonance imaging (MRI) and cerebrospinal fluid (CSF) analyses were unremarkable, but an F-Fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) showed limbic and extra-limbic hypermetabolism. A full recovery, alongside FDG normalization in previously hypermetabolic areas, was observed after intravenous immunoglobulin (IVIg) administration.
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http://dx.doi.org/10.2967/jnumed.120.256099DOI Listing
May 2021

Complement Inhibition for the Treatment of Myasthenia Gravis.

Immunotargets Ther 2020 15;9:317-331. Epub 2020 Dec 15.

Neurology IV - Neuroimmunology and Neuromuscular Diseases Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy.

Generalized myasthenia gravis (gMG) is a rare autoimmune disorder affecting the neuromuscular junction (NMJ). Approximately 80-90% of patients display antibodies directed against the nicotinic acetylcholine receptor (AChR). A major drive of AChR antibody-positive MG pathology is represented by complement activation. The role of the complement cascade has been largely demonstrated in patients and in MG animal models. Complement activation at the NMJ leads to focal lysis of the post-synaptic membrane, disruption of the characteristic folds, and reduction of AChR. Given that the complement system works as an activation cascade, there are many potential targets that can be considered for therapeutic intervention. Preclinical studies have confirmed the efficacy of complement inhibition in ameliorating MG symptoms. Eculizumab, an antibody directed towards C5, has recently been approved for the treatment of AChR antibody-positive gMG. Other complement inhibitors, targeting C5 as well, are currently under phase III study. Complement inhibitors, however, may present prohibitive costs. Therefore, the identification of a subset of patients more or less prone to respond to such therapies would be beneficial. For such purpose, there is a critical need to identify possible biomarkers predictive of therapeutic response, a field not yet sufficiently explored in MG. This review aims to give an overview of the complement cascade involvement in MG, the evolution of complement-inhibiting therapies and possible biomarkers useful to tailor and monitor complement-directed therapies.
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http://dx.doi.org/10.2147/ITT.S261414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7751298PMC
December 2020

Eculizumab for the treatment of myasthenia gravis.

Expert Opin Biol Ther 2020 09 30;20(9):991-998. Epub 2020 Jun 30.

Neurology IV Unit ‒ Neuroimmunology and Neuromuscular Diseases, Fondazione I.R.C.C.S. Istituto Neurologico Carlo Besta , Milan, Italy.

Introduction: Acetylcholine receptor antibody-positive generalized myasthenia gravis (gMG) is effectively treated with symptomatic and immunosuppressive drugs but a proportion of patients has a persistent disease and severe adverse events (AEs). The unmet medical needs are specific immunosuppression and AE lowering. Eculizumab blocks C5 protecting neuromuscular junction from the destructive autoantibody effects. Phase II (Study C08-001) and III (ECU-MG-301) studies, with the open-label extension (ECU-MG-302), demonstrated eculizumab efficacy and safety in refractory gMG patients.

Areas Covered: We provide an overview of eculizumab biological features, clinical efficacy, and safety in gMG patients, highlighting our perspective on the drug positioning in the MG treatment algorithm.

Expert Opinion: Eculizumab has the potential to significantly change the immunosuppressive approach in gMG offering the opportunity to avoid or delay corticosteroids' use due to its speed and selective mechanism of action. Eculizumab prescription will depend on: 1. ability to modify the natural disease course; 2. sustainability in the clinical practice (cost/effectiveness ratio); 3. drug-induced AE reduction. At present we are missing a controlled study on its use as a first-line treatment. We think that immunosuppression in MG will change significantly in the next years by adopting more focused 'Precision Medicine' approaches, and Eculizumab seems to satisfy such a promise.
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http://dx.doi.org/10.1080/14712598.2020.1786530DOI Listing
September 2020

Cytokine Profile in Striated Muscle Laminopathies: New Promising Biomarkers for Disease Prediction.

Cells 2020 06 23;9(6). Epub 2020 Jun 23.

Neurology IV-Neuroimmunology and Neuromuscular Diseases Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, 20133 Milan, Italy.

Laminopathies are a wide and heterogeneous group of rare human diseases caused by mutations of the LMNA gene or related nuclear envelope genes. The variety of clinical phenotypes and the wide spectrum of histopathological changes among patients carrying an identical mutation in the LMNA gene make the prognostic process rather difficult, and classical genetic screens appear to have limited predictive value for disease development. The aim of this study was to evaluate whether a comprehensive profile of circulating cytokines may be a useful tool to differentiate and stratify disease subgroups, support clinical follow-ups and contribute to new therapeutic approaches. Serum levels of 51 pro- and anti-inflammatory molecules, including cytokines, chemokines and growth factors, were quantified by a Luminex multiple immune-assay in 53 patients with muscular laminopathy (Musc-LMNA), 10 with non-muscular laminopathy, 22 with other muscular disorders and in 35 healthy controls. Interleukin-17 (IL-17), granulocyte colony-stimulating factor (G-CSF) and transforming growth factor beta (TGF-β2) levels significantly discriminated Musc-LMNA from controls; interleukin-1β (IL-1β), interleukin-4 (IL-4) and interleukin-8 (IL-8) were differentially expressed in Musc-LMNA patients compared to those with non-muscular laminopathies, whereas IL-17 was significantly higher in Musc-LMNA patients with muscular and cardiac involvement. These findings support the hypothesis of a key role of the immune system in Musc-LMNA and emphasize the potential use of cytokines as biomarkers for these disorders.
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http://dx.doi.org/10.3390/cells9061532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7348753PMC
June 2020

miR-146a in Myasthenia Gravis Thymus Bridges Innate Immunity With Autoimmunity and Is Linked to Therapeutic Effects of Corticosteroids.

Front Immunol 2020 10;11:142. Epub 2020 Mar 10.

Neurology IV-Neuroimmunology and Neuromuscular Diseases Unit, Fondazione I.R.C.C.S. Istituto Neurologico Carlo Besta, Milan, Italy.

Toll-like receptor (TLR)-mediated innate immune responses are critically involved in the pathogenesis of myasthenia gravis (MG), an autoimmune disorder affecting neuromuscular junction mainly mediated by antiacetylcholine receptor antibodies. Considerable evidence indicate that uncontrolled TLR activation and chronic inflammation significantly contribute to hyperplastic changes and germinal center (GC) formation in the MG thymus, ultimately leading to autoantibody production and autoimmunity. miR-146a is a key modulator of innate immunity, whose dysregulation has been associated with autoimmune diseases. It acts as inhibitor of TLR pathways, mainly by targeting the nuclear factor kappa B (NF-κB) signaling transducers, interleukin 1 receptor associated kinase 1 (IRAK1) and tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6); miR-146a is also able to target c-REL, inducible T-cell costimulator (ICOS), and Fas cell surface death receptor (FAS), known to regulate B-cell function and GC response. Herein, we investigated the miR-146a contribution to the intrathymic MG pathogenesis. By real-time PCR, we found that miR-146a expression was significantly downregulated in hyperplastic MG compared to control thymuses; contrariwise, IRAK1, TRAF6, c-REL, and ICOS messenger RNA (mRNA) levels were upregulated and negatively correlated with miR-146a levels. Microdissection experiments revealed that miR-146a deficiency in hyperplastic MG thymuses was not due to GCs, but restricted to the GC-surrounding medulla, characterized by IRAK1 overexpression. We also showed higher c-REL and ICOS mRNA levels, and lower FAS mRNA levels, in GCs than in the remaining medulla, according to the contribution of these molecules in GC formation. By double immunofluorescence, an increased proportion of IRAK1-expressing dendritic cells and macrophages was found in hyperplastic MG compared to control thymuses, along with GC immunoreactivity for c-REL. Interestingly, in corticosteroid-treated MG patients intrathymic miR-146a and mRNA target levels were comparable to those of controls, suggesting that immunosuppressive therapy may restore the microRNA (miRNA) levels. Indeed, an effect of prednisone on miR-146a expression was demonstrated on peripheral blood cells. Serum miR-146a levels were lower in MG patients compared to controls, indicating dysregulation of the circulating miRNA. Our overall findings strongly suggest that defective miR-146a expression could contribute to persistent TLR activation, lack of inflammation resolution, and hyperplastic changes in MG thymuses, thus linking TLR-mediated innate immunity to B-cell-mediated autoimmunity. Furthermore, they unraveled a new mechanism of action of corticosteroids in inducing control of autoimmunity in MG via miR-146a.
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http://dx.doi.org/10.3389/fimmu.2020.00142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075812PMC
March 2021

MicroRNA signature associated with treatment response in myasthenia gravis: A further step towards precision medicine.

Pharmacol Res 2019 10 8;148:104388. Epub 2019 Aug 8.

Neurology IV - Neuroimmunology and Neuromuscular Diseases Unit, Fondazione I.R.C.C.S. Istituto Neurologico Carlo Besta, Via Celoria 11, 20133 Milan, Italy. Electronic address:

Myasthenia gravis (MG) is an autoimmune disorder affecting neuromuscular transmission currently treated with chronic immunosuppression. Inter-subject variation in treatment response and side effects highlight the need for personalized therapies by identification of biomarkers predictive of drug efficacy in individual patients, still lacking in MG. MicroRNAs (miRNAs) play a key role in immune response and drug metabolism modulation. This study, part of an Italian-Israeli collaborative project, aimed to identify specific miRNAs as biomarkers associated with immunosuppressive treatment response in MG patients. Whole miRNome sequencing, followed by miRNA validation by real-time PCR, was performed in peripheral blood from Italian MG patients (n = 40) classified as responder and non-responder to immunosuppressive therapies. MiRNA sequencing identified 41 miRNAs differentially expressed in non-responder compared to responder Italian MG patients. Validation phase pointed out three miRNAs, miR-323b-3p, -409-3p, and -485-3p, clustered on chromosome 14q32.31, the levels of which were significantly decreased in non-responder versus responder patients, whereas miR-181d-5p and -340-3p showed an opposite trend. ROC curve analysis showed sensitivity and specificity performance results indicative of miR-323b-3p, -409-3p, and -485-3p predictive value for responsiveness to immunosuppressive drugs in MG. Validated miRNAs were further analyzed in blood from responder and non-responder MG patients of the Israeli population (n = 33), confirming a role for miR-323b-3p, -409-3p, -485-3p, -181d-5p and -340-3p as biomarkers of drug efficacy. Gene Ontology enrichment analysis, mRNA target prediction, and in silico modeling for function of the identified miRNAs disclosed functional involvement of the five miRNAs, and their putative target genes, in both immune (i.e. neurotrophin TRK and Fc-epsilon receptor signaling pathways) and drug metabolism processes. Our overall findings thus revealed a blood "miR-323b-3p, -409-3p, -485-3p, -181d-5p, and -340-3p" signature associated with drug responsiveness in MG patients. Its identification sets the basis for precision medicine approaches based on "pharmacomiRs" as biomarkers of drug responsiveness in MG, promising to improve therapeutic success in a cost/effective manner.
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http://dx.doi.org/10.1016/j.phrs.2019.104388DOI Listing
October 2019

Hyperexcitability in Cultured Cortical Neuron Networks from the G93A-SOD1 Amyotrophic Lateral Sclerosis Model Mouse and its Molecular Correlates.

Neuroscience 2019 09 7;416:88-99. Epub 2019 Aug 7.

World Research Hub Initiative (WRHI), Institute of Innovative Research, Tokyo Institute of Technology, Yokohama 226-8503, Japan; Complex Systems Theory Department, Institute of Nuclear Physics, Polish Academy of Sciences (IFJ-PAN), 31-342 Kraków, Poland; Center for Mind/Brain Sciences (CIMeC), University of Trento, 38123 Trento, Italy.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting the corticospinal tract and leading to motor neuron death. According to a recent study, magnetic resonance imaging-visible changes suggestive of neurodegeneration seem absent in the motor cortex of G93A-SOD1 ALS mice. However, it has not yet been ascertained whether the cortical neural activity is intact, or alterations are present, perhaps even from an early stage. Here, cortical neurons from this model were isolated at post-natal day 1 and cultured on multielectrode arrays. Their activity was studied with a comprehensive pool of neurophysiological analyses probing excitability, criticality and network architecture, alongside immunocytochemistry and molecular investigations. Significant hyperexcitability was visible through increased network firing rate and bursting, whereas topological changes in the synchronization patterns were apparently absent. The number of dendritic spines was increased, accompanied by elevated transcriptional levels of the DLG4 gene, NMDA receptor 1 and the early pro-apoptotic APAF1 gene. The extracellular Na, Ca, K and Cl concentrations were elevated, pointing to perturbations in the culture micro-environment. Our findings highlight remarkable early changes in ALS cortical neuron activity and physiology. These changes suggest that the causative factors of hyperexcitability and associated toxicity could become established much earlier than the appearance of disease symptoms, with implications for the discovery of new hypothetical therapeutic targets.
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http://dx.doi.org/10.1016/j.neuroscience.2019.07.041DOI Listing
September 2019

Diagnosis and treatment of myasthenia gravis.

Curr Opin Rheumatol 2019 11;31(6):623-633

Neurology IV, Neuroimmunology and Neuromuscular Diseases Unit, Fondazione I.R.C.C.S. Istituto Neurologico Carlo Besta, Milan, Italy.

Purpose Of Review: This article provides an update on the most recent advances in diagnostic procedures and therapeutic approaches for myasthenia gravis, spanning from autoantibody and neuroelectrophysiological tests as diagnostic tools, to innovative and promising treatments based on biological drugs.

Recent Findings: Novel studies performed by cell-based assays (CBAs) indicate an improvement in the chance of identifying serum autoantibodies in myasthenic patients. Clinical trials on the use of biological drugs were recently concluded, providing important data on safety and efficacy of eculizumab, efgartigimod and amifampridine phosphate: the first, a complement blocker, showed long-term safety and efficacy in acetylcholine receptor (AChR)-positive myasthenic patients with refractory generalized disease; the second, the neonatal Fc receptor blocker, was well tolerated and clinically effective in both AChR-specific and muscle-specific kinase receptor (MuSK)-positive patients; the third, a blocker of presynaptic potassium channels, was found to be well tolerated and effective in MuSK-positive patients.

Summary: CBAs can lead to a significant reduction of seronegative patients, improving myasthenia gravis diagnostic process. New biological drugs offer innovative approaches to treat myasthenic patients with generalized disease, promising to change the paradigm of treatment and to significantly enhance therapeutic success within a precision medicine framework.
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http://dx.doi.org/10.1097/BOR.0000000000000647DOI Listing
November 2019

FM19G11-Loaded Gold Nanoparticles Enhance the Proliferation and Self-Renewal of Ependymal Stem Progenitor Cells Derived from ALS Mice.

Cells 2019 03 23;8(3). Epub 2019 Mar 23.

Neurology IV-Neuroimmunology and Neuromuscular Diseases Unit, Fondazione IRCCS Istituto Neurologico "Carlo Besta", 20133 Milan, Italy.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons. In ALS mice, neurodegeneration is associated with the proliferative restorative attempts of ependymal stem progenitor cells (epSPCs) that normally lie in a quiescent in the spinal cord. Thus, modulation of the proliferation of epSPCs may represent a potential strategy to counteract neurodegeneration. Recent studies demonstrated that FM19G11, a hypoxia-inducible factor modulator, induces epSPC self-renewal and proliferation. The aim of the study was to investigate whether FM19G11-loaded gold nanoparticles (NPs) can affect self-renewal and proliferation processes in epSPCs isolated from G93A-SOD1 mice at disease onset. We discovered elevated levels of SOX2, OCT4, AKT1, and AKT3, key genes associated with pluripotency, self-renewal, and proliferation, in G93A-SOD1 epSPCs at the transcriptional and protein levels after treatment with FM19G11-loaded NPs. We also observed an increase in the levels of the mitochondrial uncoupling protein () gene in treated cells. FM19G11-loaded NPs treatment also affected the expression of the cell cycle-related microRNA (miR)-19a, along with its target gene PTEN, in G93A-SOD1 epSPCs. Overall our findings establish the significant impact of FM19G11-loaded NPs on the cellular pathways involved in self-renewal and proliferation in G93A-SOD1 epSPCs, thus providing an impetus to the design of novel tailored approaches to delay ALS disease progression.
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http://dx.doi.org/10.3390/cells8030279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468696PMC
March 2019

Myasthenia gravis: from autoantibodies to therapy.

Curr Opin Neurol 2018 10;31(5):517-525

Neurology IV, Neuroimmunology and Neuromuscular Diseases Unit, Fondazione Istituto Neurologico 'Carlo Besta,' Milan, Italy.

Purpose Of Review: The current article reviews the recent advances in the field of myasthenia gravis, which span from autoantibody profiling and pathogenic mechanisms to therapy innovation. The overview is highlighting specifically the data and the needs of targeted treatments in the light of precision medicine in myasthenia gravis.

Recent Findings: Novel data published recently further increased our knowledge on myasthenia gravis. The use of cell-based assays has greatly improved autoantibody detection in myasthenia gravis patients, and the mechanisms of action of these antibodies have been described. The role of Toll-like receptor activation in the generation of thymic alterations and anti-acetylcholine receptor autosensitization has been further investigated implementing our understanding on the relationships between innate immunity and autoimmunity. Additional studies have been focused on the alterations of T-cell/B-cell regulatory mechanisms in thymus and peripheral blood of myasthenia gravis patients. microRNAs and genetic factors are also emerging as key biomarkers in myasthenia gravis pathogenesis and prediction of drug efficacy in individual patients.

Summary: The recent immunological and pathological findings in myasthenia gravis promise to improve myasthenia gravis treatment, via the development of more precise and personalized therapies.
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http://dx.doi.org/10.1097/WCO.0000000000000596DOI Listing
October 2018

Elevated TGF β2 serum levels in Emery-Dreifuss Muscular Dystrophy: Implications for myocyte and tenocyte differentiation and fibrogenic processes.

Nucleus 2018 01;9(1):292-304

j Institute of Molecular Genetics (IGM)-CNR, Unit of Bologna , Bologna , Italy.

Among rare diseases caused by mutations in LMNA gene, Emery-Dreifuss Muscular Dystrophy type 2 and Limb-Girdle muscular Dystrophy 1B are characterized by muscle weakness and wasting, joint contractures, cardiomyopathy with conduction system disorders. Circulating biomarkers for these pathologies have not been identified. Here, we analyzed the secretome of a cohort of patients affected by these muscular laminopathies in the attempt to identify a common signature. Multiplex cytokine assay showed that transforming growth factor beta 2 (TGF β2) and interleukin 17 serum levels are consistently elevated in the vast majority of examined patients, while interleukin 6 and basic fibroblast growth factor are altered in subgroups of patients. Levels of TGF β2 are also increased in fibroblast and myoblast cultures established from patient biopsies as well as in serum from mice bearing the H222P Lmna mutation causing Emery-Dreifuss Muscular Dystrophy in humans. Both patient serum and fibroblast conditioned media activated a TGF β2-dependent fibrogenic program in normal human myoblasts and tenocytes and inhibited myoblast differentiation. Consistent with these results, a TGF β2 neutralizing antibody avoided fibrogenic marker activation and myogenesis impairment. Cell intrinsic TGF β2-dependent mechanisms were also determined in laminopathic cells, where TGF β2 activated AKT/mTOR phosphorylation. These data show that TGF β2 contributes to the pathogenesis of Emery-Dreifuss Muscular Dystrophy type 2 and Limb-Girdle muscular Dystrophy 1B and can be considered a potential biomarker of those diseases. Further, the evidence of TGF β2 pathogenetic effects in tenocytes provides the first mechanistic insight into occurrence of joint contractures in muscular laminopathies.
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http://dx.doi.org/10.1080/19491034.2018.1467722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5973167PMC
January 2018

Toll-like receptors 7 and 9 in myasthenia gravis thymus: amplifiers of autoimmunity?

Ann N Y Acad Sci 2018 02 24;1413(1):11-24. Epub 2018 Jan 24.

Neurology IV, Neuroimmunology and Neuromuscular Diseases Unit, Fondazione Istituto Neurologico "Carlo Besta", Milan, Italy.

Pathogen infections and dysregulated Toll-like receptor (TLR)-mediated innate immune responses are suspected to play key roles in autoimmunity. Among TLRs, TLR7 and TLR9 have been implicated in several autoimmune conditions, mainly because of their ability to promote abnormal B cell activation and survival. Recently, we provided evidence of Epstein-Barr virus (EBV) persistence and reactivation in the thymus of myasthenia gravis (MG) patients, suggesting an involvement of EBV in the intrathymic pathogenesis of the disease. Considerable data highlight the existence of pathogenic crosstalk among EBV, TLR7, and TLR9: EBV elicits TLR7/9 signaling, which in turn can enhance B cell dysfunction and autoimmunity. In this article, after a brief summary of data demonstrating TLR activation in MG thymus, we provide an overview on the contribution of TLR7 and TLR9 to autoimmune diseases and discuss our recent findings indicating a pivotal role for these two receptors, along with EBV, in driving, perpetuating, and/or amplifying intrathymic B cell dysregulation and autoimmune responses in MG. Development of therapeutic approaches targeting TLR7 and TLR9 signaling could be a novel strategy for treating the chronic inflammatory autoimmune process in myasthenia gravis.
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http://dx.doi.org/10.1111/nyas.13534DOI Listing
February 2018

Epstein-Barr virus in tumor-infiltrating B cells of myasthenia gravis thymoma: an innocent bystander or an autoimmunity mediator?

Oncotarget 2017 Nov 8;8(56):95432-95449. Epub 2017 Sep 8.

Neurology IV - Neuroimmunology and Neuromuscular Diseases Unit, Fondazione Istituto Neurologico "Carlo Besta", 20133 Milan, Italy.

The thymus plays a key role in myasthenia gravis (MG), a B cell-mediated autoimmune disorder affecting neuromuscular junction. Most MG patients have thymic abnormalities, including hyperplasia and thymoma, a neoplasm of thymic epithelial cells. Epstein-Barr virus (EBV) is associated with autoimmune diseases and tumors. Recently, we showed EBV persistence and reactivation in hyperplastic MG thymuses, suggesting that EBV might contribute to intra-thymic B cell dysregulation in MG patients. Here, we investigated EBV involvement in thymoma-associated MG, by searching for EBV markers in MG (n=26) and non-MG (n=14) thymomas. EBV DNA and EBV-encoded small nuclear RNA (EBER) 1 transcript were detected in 14/26 (53.8%) and 22/26 (84.6%) MG thymomas, and only in 3 of 14 (21.4%) non-MG thymomas. Latent EBNA2 and late gp350/220 lytic transcripts were undetectable in all, but one, thymomas, and early lytic BZLF1 transcript was absent in all samples, suggesting that early infection events and EBV reactivation were very rare in thymomas. EBER1 and 2-positive cells were detected in MG, but not in non-MG, thymomas, as well as cells expressing EBV latency proteins (EBNA1, LMP1, LMP2A), that were mainly of B cell phenotype, indicating EBV association with MG rather than with thymoma. Toll-like receptor (TLR) 3 transcriptional levels were higher in MG than non-MG thymomas and positively correlated with EBER1 levels, suggesting a role for EBERs in TLR3 activation. Our findings show that EBV is commonly present in thymoma-infiltrating B cells of myasthenic patients, indicating a contribution of EBV to B cell-mediated autoreactivity in MG associated with thymic tumor.
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http://dx.doi.org/10.18632/oncotarget.20731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5707033PMC
November 2017

A novel infection- and inflammation-associated molecular signature in peripheral blood of myasthenia gravis patients.

Immunobiology 2016 11 15;221(11):1227-36. Epub 2016 Jun 15.

Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Biopolis, 138648 Singapore. Electronic address:

Myasthenia gravis (MG) is a T-cell dependent autoimmune disorder of the neuromuscular junction, characterised by muscle weakness and fatigability. Autoimmunity is thought to initiate in the thymus of acetylcholine receptor (AChR)-positive MG patients; however, the molecular mechanisms linking intra-thymic MG pathogenesis with autoreactivity via the circulation to the muscle target organ are poorly understood. Using whole-transcriptome sequencing, we compared the transcriptional profile of peripheral blood mononuclear cells from AChR-early onset MG (AChR-EOMG) patients with healthy controls: 178 coding transcripts and 229 long non-coding RNAs, including 11 pre-miRNAs, were differentially expressed. Among the 178 coding transcripts, 128 were annotated of which 17% were associated with the 'infectious disease' functional category and 46% with 'inflammatory disease' and 'inflammatory response-associated' categories. Validation of selected transcripts by qPCR indicated that of the infectious disease-related transcripts, ETF1, NFKB2, PLK3, and PPP1R15A were upregulated, whereas CLC and IL4 were downregulated in AChR-EOMG patients; in the 'inflammatory' categories, ABCA1, FUS, and RELB were upregulated, suggesting a contribution of these molecules to immunological dysfunctions in MG. Data selection and validation were also based on predicted microRNA-mRNA interactions. We found that miR-612, miR-3654, and miR-3651 were increased, whereas miR-612-putative AKAp12 and HRH4 targets and the miR-3651-putative CRISP3 target were downregulated in AChR-EOMG, also suggesting altered immunoregulation. Our findings reveal a novel peripheral molecular signature in AChR-EOMG, reflecting a critical involvement of inflammatory- and infectious disease-related immune responses in disease pathogenesis.
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http://dx.doi.org/10.1016/j.imbio.2016.06.012DOI Listing
November 2016

Increased expression of Toll-like receptors 7 and 9 in myasthenia gravis thymus characterized by active Epstein-Barr virus infection.

Immunobiology 2016 Apr 12;221(4):516-27. Epub 2015 Dec 12.

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

Considerable data implicate the thymus as the main site of autosensitization to the acetylcholine receptor in myasthenia gravis (MG), a B-cell-mediated autoimmune disease affecting the neuromuscular junction. We recently demonstrated an active Epstein-Barr virus (EBV) infection in the thymus of MG patients, suggesting that EBV might contribute to the onset or maintenance of the autoimmune response within MG thymus, because of its ability to activate and immortalize autoreactive B cells. EBV has been reported to elicit and modulate Toll-like receptor (TLR) 7- and TLR9-mediated innate immune responses, which are known to favor B-cell dysfunction and autoimmunity. Aim of this study was to investigate whether EBV infection is associated with altered expression of TLR7 and TLR9 in MG thymus. By real-time PCR, we found that TLR7 and TLR9 mRNA levels were significantly higher in EBV-positive MG compared to EBV-negative normal thymuses. By confocal microscopy, high expression levels of TLR7 and TLR9 proteins were observed in B cells and plasma cells of MG thymic germinal centers (GCs) and lymphoid infiltrates, where the two receptors co-localized with EBV antigens. An increased frequency of Ki67-positive proliferating B cells was found in MG thymuses, where we also detected proliferating cells expressing TLR7, TLR9 and EBV antigens, thus supporting the idea that EBV-associated TLR7/9 signaling may promote abnormal B-cell activation and proliferation. Along with B cells and plasma cells, thymic epithelium, plasmacytoid dendritic cells and macrophages exhibited enhanced TLR7 and TLR9 expression in MG thymus; TLR7 was also increased in thymic myeloid dendritic cells and its transcriptional levels positively correlated with those of interferon (IFN)-β. We suggested that TLR7/9 signaling may be involved in antiviral type I IFN production and long-term inflammation in EBV-infected MG thymuses. Our overall findings indicate that EBV-driven TLR7- and TLR9-mediated innate immune responses may participate in the intra-thymic pathogenesis of MG.
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http://dx.doi.org/10.1016/j.imbio.2015.12.007DOI Listing
April 2016

Modulation of TGFbeta 2 levels by lamin A in U2-OS osteoblast-like cells: understanding the osteolytic process triggered by altered lamins.

Oncotarget 2015 Apr;6(10):7424-37

Rizzoli Orthopedic Institute, Laboratory of Musculoskeletal Cell Biology, CNR Institute for Molecular Genetics, Unit of Bologna, Bologna, Italy.

Transforming growth factor beta (TGFbeta) plays an essential role in bone homeostasis and deregulation of TGFbeta occurs in bone pathologies. Patients affected by Mandibuloacral Dysplasia (MADA), a progeroid disease linked to LMNA mutations, suffer from an osteolytic process. Our previous work showed that MADA osteoblasts secrete excess amount of TGFbeta 2, which in turn elicits differentiation of human blood precursors into osteoclasts. Here, we sought to determine how altered lamin A affects TGFbeta signaling. Our results show that wild-type lamin A negatively modulates TGFbeta 2 levels in osteoblast-like U2-OS cells, while the R527H mutated prelamin A as well as farnesylated prelamin A do not, ultimately leading to increased secretion of TGFbeta 2. TGFbeta 2 in turn, triggers the Akt/mTOR pathway and upregulates osteoprotegerin and cathepsin K. TGFbeta 2 neutralization rescues Akt/mTOR activation and the downstream transcriptional effects, an effect also obtained by statins or RAD001 treatment. Our results unravel an unexpected role of lamin A in TGFbeta 2 regulation and indicate rapamycin analogs and neutralizing antibodies to TGFbeta 2 as new potential therapeutic tools for MADA.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4480690PMC
http://dx.doi.org/10.18632/oncotarget.3232DOI Listing
April 2015

Up-regulation of neural and cell cycle-related microRNAs in brain of amyotrophic lateral sclerosis mice at late disease stage.

Mol Brain 2015 Jan 28;8. Epub 2015 Jan 28.

Neurology IV - Neuromuscular Diseases and Neuroimmunology Unit, Fondazione Istituto Neurologico "Carlo Besta", Via Celoria 11, Milan, 20133, Italy.

Background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective motor neuron degeneration in motor cortex, brainstem and spinal cord. microRNAs (miRNAs) are small non-coding RNAs that bind complementary target sequences and modulate gene expression; they are key molecules for establishing a neuronal phenotype, and in neurodegeneration. Here we investigated neural miR-9, miR-124a, miR-125b, miR-219, miR-134, and cell cycle-related miR-19a and -19b, in G93A-SOD1 mouse brain in pre-symptomatic and late stage disease.

Results: Expression of miR-9, miR-124a, miR-19a and -19b was significantly increased in G93A-SOD1 whole brain at late stage disease compared to B6.SJL and Wt-SOD1 control brains. These miRNAs were then analyzed in manually dissected SVZ, hippocampus, primary motor cortex and brainstem motor nuclei in 18-week-old ALS mice compared to same age controls. In SVZ and hippocampus miR-124a was up-regulated, miR-219 was down-regulated, and numbers of neural stem progenitor cells (NSPCs) were significantly increased. In G93A-SOD1 brainstem motor nuclei and primary motor cortex, miR-9 and miR-124a were significantly up-regulated, miR-125b expression was also increased. miR-19a and -19b were up-regulated in primary motor cortex and hippocampus, respectively. Expression analysis of predicted miRNA targets identified miRNA/target gene pairs differentially expressed in G93A-SOD1 brain regions compared to controls.

Conclusions: Hierarchical clustering analysis, identifying two clusters of miRNA/target genes, one characterizing brainstem motor nuclei and primary motor cortex, the other hippocampus and SVZ, suggests that altered expression of neural and cell cycle-related miRNAs in these brain regions might contribute to ALS pathogenesis in G93A-SOD1 mice. Re-establishing their expression to normal levels could be a new therapeutic approach to ALS.
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http://dx.doi.org/10.1186/s13041-015-0095-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4318136PMC
January 2015

VAV1 and BAFF, via NFκB pathway, are genetic risk factors for myasthenia gravis.

Ann Clin Transl Neurol 2014 May 11;1(5):329-39. Epub 2014 Apr 11.

Pharmacogenetics and Translational Genetics Center, The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology Haifa, Israel ; Division of Neuroimmunology, Carmel Medical Center Haifa, Israel.

Objective: To identify novel genetic loci that predispose to early-onset myasthenia gravis (EOMG) applying a two-stage association study, exploration, and replication strategy.

Methods: Thirty-four loci and one confirmation loci, human leukocyte antigen (HLA)-DRA, were selected as candidate genes by team members of groups involved in different research aspects of MG. In the exploration step, these candidate genes were genotyped in 384 EOMG and 384 matched controls and significant difference in allele frequency were found in eight genes. In the replication step, eight candidate genes and one confirmation loci were genotyped in 1177 EOMG patients and 814 controls, from nine European centres.

Results: ALLELE FREQUENCY DIFFERENCES WERE FOUND IN FOUR NOVEL LOCI: CD86, AKAP12, VAV1, B-cell activating factor (BAFF), and tumor necrosis factor-alpha (TNF-α), and these differences were consistent in all nine cohorts. Haplotype trend test supported the differences in allele frequencies between cases and controls. In addition, allele frequency difference in female versus male patients at HLA-DRA and TNF-α loci were observed.

Interpretation: The genetic associations to EOMG outside the HLA complex are novel and of interest as VAV1 is a key signal transducer essential for T- and B-cell activation, and BAFF is a cytokine that plays important roles in the proliferation and differentiation of B-cells. Moreover, we noted striking epistasis between the predisposing VAV1 and BAFF haplotypes; they conferred a greater risk in combination than alone. These, and CD86, share the same signaling pathway, namely nuclear factor-kappaB (NFκB), thus implicating dysregulation of proinflammatory signaling in predisposition to EOMG.
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http://dx.doi.org/10.1002/acn3.51DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4184684PMC
May 2014

Innate immunity in myasthenia gravis thymus: pathogenic effects of Toll-like receptor 4 signaling on autoimmunity.

J Autoimmun 2014 Aug 4;52:74-89. Epub 2014 Jan 4.

Neurology IV Unit, Neurological Institute 'Carlo Besta', Via Celoria 11, 20133 Milan, Italy. Electronic address:

The thymus is the main site of immune sensitization to AChR in myasthenia gravis (MG). In our previous studies we demonstrated that Toll-like receptor (TLR) 4 is over-expressed in MG thymuses, suggesting its involvement in altering the thymic microenvironment and favoring autosensitization and autoimmunity maintenance processes, via an effect on local chemokine/cytokine network. Here, we investigated whether TLR4 signaling may favor abnormal cell recruitment in MG thymus via CCL17 and CCL22, two chemokines known to dictate immune cell trafficking in inflamed organs by binding CCR4. We also investigated whether TLR4 activation may contribute to immunodysregulation, via the production of Th17-related cytokines, known to alter effector T cell (Teff)/regulatory T cell (Treg) balance. We found that CCL17, CCL22 and CCR4 were expressed at higher levels in MG compared to normal thymuses. The two chemokines were mainly detected around medullary Hassall's corpuscles (HCs), co-localizing with TLR4(+) thymic epithelial cells (TECs) and CCR4(+) dendritic cells (DCs), that were present in higher number in MG thymuses compared to controls. TLR4 stimulation in MG TECs increased CCL17 and CCL22 expression and induced the production of Th17-related cytokines. Then, to study the effect of TLR4-stimulated TECs on immune cell interactions and Teff activation, we generated an in-vitro imaging model by co-culturing CD4(+) Th1/Th17 AChR-specific T cells, naïve CD4(+)CD25(+) Tregs, DCs and TECs from Lewis rats. We observed that TLR4 stimulation led to a more pronounced Teff activatory status, suggesting that TLR4 signaling in MG thymic milieu may affect cell-to-cell interactions, favoring autoreactive T-cell activation. Altogether our findings suggest a role for TLR4 signaling in driving DC recruitment in MG thymus via CCL17 and CCL22, and in generating an inflammatory response that might compromise Treg function, favoring autoreactive T-cell pathogenic responses.
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http://dx.doi.org/10.1016/j.jaut.2013.12.013DOI Listing
August 2014

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

Etiology of myasthenia gravis: innate immunity signature in pathological thymus.

Autoimmun Rev 2013 Jul 25;12(9):863-74. Epub 2013 Mar 25.

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

Myasthenia gravis (MG) is an autoimmune disease affecting the neuromuscular junction (NMJ), whose clinical hallmark is muscle weakness and early fatigability. The main target of autoimmunity in MG is the acetylcholine receptor (AChR) located in the NMJ. It is now widely accepted that the thymus is probably the prime site of autoimmunity development and maintenance in AChR-positive MG patients; however, the exact mechanisms triggering and perpetuating the intra-thymic autoimmune response to AChR are still unknown. As with many autoimmune diseases, MG has a multifactorial etiology, resulting from complex interactions between genetic and environmental factors, as fully described in this review. Among environmental factors, viral infections could play a central role in autoimmunity, mainly through the induction of dysregulated Toll-like receptor (TLR)-mediated innate immune responses, which can lead to inflammation and adaptive autoimmune response. Growing evidence of chronic inflammation, TLR activation, and persistent viral infections in the thymus of MG patients, strongly supports the hypothesis that, in the context of a genetic susceptible background, the intrathymic innate immune responses to pathogen infections might contribute to MG etiology.
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http://dx.doi.org/10.1016/j.autrev.2013.03.010DOI Listing
July 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

Autoimmune mechanisms in myasthenia gravis.

Curr Opin Neurol 2012 Oct;25(5):621-9

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

Purpose Of Review: This article reviews recent findings on factors and mechanisms implicated in the pathogenesis of myasthenia gravis and briefly summarizes data on therapies acting at various stages of the autoimmune process.

Recent Findings: Data published over the last year promise to improve understanding of pathogenic mechanisms underlying myasthenia gravis. Animal studies have at last shown that antimuscle-specific kinase (MuSK) autoantibodies, like antiacetylcholine receptor (AChR) autoantibodies, are myasthenogenic. A new autoantigen, the low-density lipoprotein receptor-related protein 4 (LRP4), has been identified in variable proportions of otherwise seronegative patients. Anti-LRP4 antibodies may define a new myasthenia gravis subtype, supporting the concept that myasthenia gravis is not a single disease entity, and that different subtypes can differ in aetiology. Genetic and environmental factors are implicated in myasthenia gravis. The finding of persisting viral infection in the thymus of AChR-myasthenia gravis patients, combined with data on chronic inflammation, suggest that pathogens may favour intrathymic AChR-specific autosensitization and maintenance of autoimmunity in genetically susceptible individuals. Defective immunoregulatory mechanisms, involving pathogenic Th17 and regulatory T cells, contribute to tolerance loss and perpetuation of the autoimmune response in myasthenia gravis patients.

Summary: The recent identification of mechanisms initiating and perpetuating autoimmunity in myasthenia gravis may stimulate the development of more effective therapies.
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http://dx.doi.org/10.1097/WCO.0b013e328357a829DOI Listing
October 2012

Inflammation and epstein-barr virus infection are common features of myasthenia gravis thymus: possible roles in pathogenesis.

Autoimmune Dis 2011 26;2011:213092. Epub 2011 Sep 26.

Department of Neurology IV, Neuromuscular Diseases and Neuroimmunology, Neurological Institute C. Besta Foundation, 20133 Milan, Italy.

The thymus plays a major role in myasthenia gravis (MG). Our recent finding of a persistent Epstein-Barr (EBV) virus infection in some MG thymuses, combined with data showing that the thymus is in a proinflammatory state in most patients, supports a viral contribution to the pathogenesis of MG. Aim of this study was to gain further evidence for intrathymic chronic inflammation and EBV infection in MG patients. Transcriptional profiling by low density array and real-time PCR showed overexpression of genes involved in inflammatory and immune response in MG thymuses. Real-time PCR for EBV genome, latent (EBER1, EBNA1, LMP1) and lytic (BZLF1) transcripts, and immunohistochemistry for LMP1 and BZLF1 proteins confirmed an active intrathymic EBV infection, further supporting the hypothesis that EBV might contribute to onset or perpetuation of the autoimmune response in MG. Altogether, our results support a role of inflammation and EBV infection as pathogenic features of MG thymus.
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http://dx.doi.org/10.4061/2011/213092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3180177PMC
November 2011

The thymus in myasthenia gravis: Site of "innate autoimmunity"?

Muscle Nerve 2011 Oct;44(4):467-84

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

Myasthenia gravis (MG) is an autoimmune disorder caused, in most cases, by autoantibodies against components of the neuromuscular junction, frequently the acetylcholine receptor (AChR), and less often the muscle-specific kinase receptor. The thymus plays a major role in the pathogenesis of MG with anti-AChR antibodies: it shows marked pathologic alterations (hyperplastic or tumoral) in most AChR-positive patients and contains the elements required to initiate and sustain an autoimmune reaction (AChR autoantigen, AChR-specific T cells, and autoantibody-secreting plasma cells). In this study we review early and more recent findings implicating the thymus as site of AChR autosensitization in MG and briefly discuss the therapeutic role of thymectomy. We also summarize data showing that the MG thymus is in a state of chronic inflammation, and we review emerging evidence of a viral contribution to the onset and maintenance of the thymic autoimmune response.
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http://dx.doi.org/10.1002/mus.22103DOI Listing
October 2011

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

Epstein-Barr virus persistence and reactivation in myasthenia gravis thymus.

Ann Neurol 2010 Jun;67(6):726-38

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

Objective: Increasing evidence supports a link between Epstein-Barr virus (EBV), a ubiquitous B-lymphotropic human herpesvirus, and common B-cell-related autoimmune diseases. We sought evidence of EBV infection in thymuses from patients with myasthenia gravis (MG), an autoimmune disease characterized by intrathymic B-cell activation.

Methods: Seventeen MG thymuses (6 follicular hyperplastic, 6 diffuse hyperplastic, 5 involuted) and 6 control thymuses were analyzed using in situ hybridization for EBV-encoded small RNAs (EBERs), immunohistochemistry for EBV latent and lytic proteins, and polymerase chain reaction for EBV DNA and mRNA.

Results: All 17 MG thymuses showed evidence of active EBV infection, whereas none of the control thymuses were infected. Cells expressing EBERs (12 of 17) and EBV latency proteins (EBNA2, LMP1, and LMP2A) (16 of 17) were detected in medullary infiltrates and in germinal centers. Cells expressing early (BFRF1, BMRF1) and late (p160, gp350/220) lytic phase EBV proteins were present in 16 MG thymuses. Latency (EBNA1, LMP2A) or lytic (BZLF1) transcripts (often both) were present in all MG thymuses, and EBV DNA (LMP1 gene) was detected in 13 MG thymuses. We also found CD8+ T cells, CD56 + CD3-natural killer cells, and BDCA-2+ plasmacytoid dendritic cells in immune infiltrates of MG thymuses, but not germinal centers, suggesting an attempt of the immune system to counteract EBV infection.

Interpretation: Dysregulated EBV infection in the pathological thymus appears common in MG and may contribute to the immunological alterations initiating and/or perpetuating the disease.
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http://dx.doi.org/10.1002/ana.21902DOI Listing
June 2010

Thymoma-associated myasthenia gravis: outcome, clinical and pathological correlations in 197 patients on a 20-year experience.

J Neuroimmunol 2008 Sep 22;201-202:237-44. Epub 2008 Aug 22.

Divisione Malattie Neuromuscolari e Neuroimmunologia, Fondazione Istituto Neurologico "Carlo Besta", Milan, Italy.

We studied 197 patients with thymoma-associated myasthenia gravis (T-MG) to identify variables that can influence the natural history of the disease and the therapeutical approaches. Multivariate analysis showed that neither clinical nor pathological variables were associated with a better chance to reach complete stable remission. The video-assisted thoracoscopic extended thymectomy (VATET) was not significantly correlated with a lower chance of achieving complete stable remission compared with the classical transsternal approach (T-3b) (p=0.1090). Thymoma recurrence was not correlated with surgery by VATET or T-3b. VATET was safe and reliable for removal of thymoma. The low chance of achieving remission (9.64%) in T-MG underlines the importance of an early diagnosis as well as the need for more aggressive therapeutic strategies.
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http://dx.doi.org/10.1016/j.jneuroim.2008.07.012DOI Listing
September 2008