Publications by authors named "Fulvio Baggi"

55 Publications

7-T MRI tracking of mesenchymal stromal cells after lung injection in a rat model.

Eur Radiol Exp 2020 10 8;4(1):54. Epub 2020 Oct 8.

Department of Thoracic Surgery, IRCCS European Institute of Oncology, Milan, Italy.

Background: Mesenchymal stromal cells (MSCs) are able to migrate and engraft at sites of inflammation, injuries, and tumours, but little is known about their fate after local injection. The purpose of this study is to perform MSC tracking, combining in vivo 7-T magnetic resonance imaging (MRI) and histological assessment, following lung injection in a rat model.

Methods: Five lungs were injected with ferumoxide-labelled MSCs and five with perfluorocarbon-labelled MSCs and underwent 7-T MRI. MRI acquisitions were recorded immediately (T), at 24 h (T) and/or 48 h (T) after injection. For each rat, labelled cells were assessed in the main organs by MRI. Target organs were harvested under sterile conditions from rats sacrificed 0, 24, or 48 h after injection and fixed for histological analysis via confocal and structured illumination microscopy.

Results: Ferumoxide-labelled MSCs were not detectable in the lungs, whereas they were not visible in the distant sites. Perfluorocarbon-labelled MSCs were seen in 5/5 injected lungs at T, in 1/2 at T, and in 1/3 at T. The fluorine signal in the liver was seen in 3/5 at T, in 1/2 at T24, and in 2/3 at T. Post-mortem histology confirmed the presence of MSCs in the injected lung.

Conclusions: Ferumoxide-labelled cells were not seen at distant sites; a linear decay of injected perfluorocarbon-labelled MSCs was observed at T, T, and T in the lung. In more than half of the experiments, perfluorocarbon-labelled MSCs scattering to the liver was observed, with a similar decay over time as observed in the lung.
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http://dx.doi.org/10.1186/s41747-020-00183-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541802PMC
October 2020

Halogenation of the -Terminus Tyrosine 10 Promotes Supramolecular Stabilization of the Amyloid-β Sequence 7-12.

ChemistryOpen 2020 02 25;9(2):253-260. Epub 2020 Feb 25.

Dept. Chem., Mater., and Chem. Eng. "Giulio Natta" Politecnico di Milano Via L. Mancinelli 7 20131 Milano Italy.

Here, we demonstrate that introduction of halogen atoms at the tyrosine 10 phenol ring of the DSGYEV sequence derived from the flexible amyloid-β -terminus, promotes its self-assembly in the solid state. In particular, we report the crystal structures of two halogen-modified sequences, which we found to be stabilized in the solid state by halogen-mediated interactions. The structural study is corroborated by Non-Covalent Interaction (NCI) analysis. Our results prove that selective halogenation of an amino acid enhances the supramolecular organization of otherwise unstructured biologically-relevant sequences. This method may develop as a general strategy for stabilizing highly polymorphic peptide regions.
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http://dx.doi.org/10.1002/open.201900350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7041548PMC
February 2020

Autoantibody Diagnostics in Neuroimmunology: Experience From the 2018 Italian Neuroimmunology Association External Quality Assessment Program.

Front Neurol 2019 14;10:1385. Epub 2020 Jan 14.

Neuroimmunology Laboratory, IRCCS Mondino Foundation, Pavia, Italy.

Neuroimmunology has impressively expanded in the past decade. Novel assays, especially cell-based assays (CBAs) can detect conformational antibodies (Abs) recognizing antigens in their native conformation. Generally, the availability of in-house and of commercial tests has improved the diagnostics, but introduced demanding laboratory tasks. Hence, standardization and quality controls represent a key step to promote accuracy. We report on the results of the 2018 external quality assessment program (EQAP) organized by the Italian Neuroimmunology Association. EQAP regarded 10 schemes, including oligoclonal bands (OCBs), intracellular-neuronal (ICN)-Abs, neuronal-surface (NS)-Abs, aquaporin-4 (AQP4)-Abs, myelin oligodendrocyte glycoprotein (MOG)-Abs, myelin-associated glycoprotein (MAG)-Abs, ganglioside-Abs, acetylcholine-receptor (AChR)-Abs, and muscle-specific-kinase (MuSK)-Abs, and 34 laboratories. Assays were classified as tissue-based assays (TBAs), solid-phase assays (SPAs), liquid-phase assays (LPAs), and CBAs. Thirty-three samples were provided. Three-quarter of the tests were commercial. Median accuracy for the laboratories was 75% (range 50-100). In 8/10 schemes, at least one sample provided discrepant results. Inter-laboratory "substantial agreement" was found in 6/10 schemes (AChR, MuSK, MAG, AQP4, MOG, and NS-Abs), whereas the worst agreements regarded OCBs and ganglioside-Abs. Both commercial and in-house assays performed better in experienced laboratories. Assays could be divided in (a) robust commercial tests with substantial inter-laboratory agreement (MAG-Abs; AChR- and MuSK-Abs); commercial/"in-house" tests with (b) partial inter-laboratory agreement (AQP4-Abs, MOG-Abs, NS-Abs, ICN-Abs), and (c) with large inter-laboratory disagreement (OCBs, ganglioside-Abs). This real-life snapshot of the neuroimmunology test performances highlights shortcomings attributable to technician-dependent performances, assay structural limitations, and errors in test interpretations.
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http://dx.doi.org/10.3389/fneur.2019.01385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6971200PMC
January 2020

Therapeutic Effect of Bifidobacterium Administration on Experimental Autoimmune Myasthenia Gravis in Lewis Rats.

Front Immunol 2019 19;10:2949. Epub 2019 Dec 19.

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

Beneficial effects of probiotics on gut microbiota homeostasis and inflammatory immune responses suggested the investigation of their potential clinical efficacy in experimental models of autoimmune diseases. Indeed, administration of two bifidobacteria and lactobacilli probiotic strains prevented disease manifestations in the Lewis rat model of Myasthenia Gravis (EAMG). Here, we demonstrate the clinical efficacy of therapeutic administration of vital bifidobacteria (i.e., from EAMG onset). The mechanisms involved in immunomodulation were investigated with and experiments. Improvement of EAMG symptoms was associated to decreased anti-rat AChR antibody levels, and differential expression of TGFβ and FoxP3 immunoregulatory transcripts in draining lymph nodes and spleen of treated-EAMG rats. Exposure of rat bone marrow-derived dendritic cells to bifidobacteria or lactobacilli strains upregulated toll-like receptor 2 mRNA expression, a key molecule involved in bacterium recognition via lipotheicoic acid. Live imaging experiments of AChR-specific effector T cells, co-cultured with BMDCs pre-exposed to bifidobacteria, demonstrated increased percentages of motile effector T cells, suggesting a hindered formation of TCR-peptide-MHC complex. Composition of gut microbiota was studied by 16S rRNA gene sequencing, and α and β diversity were determined in probiotic treated EAMG rats, with altered ratios between Tenericutes and Verrucomicrobia (phylum level), and Ruminococcaceae and Lachnospiraceae (family level). Moreover, the relative abundance of Akkermansia genus was found increased compared to healthy and probiotic treated EAMG rats. In conclusion, our findings confirms that the administration of vital bifidobacteria at EAMG onset has beneficial effects on disease progression; this study further supports preclinical research in human MG to evaluate probiotic efficacy as supplementary therapy in MG.
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http://dx.doi.org/10.3389/fimmu.2019.02949DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6951413PMC
November 2020

Administration of bifidobacterium and lactobacillus strains modulates experimental myasthenia gravis and experimental encephalomyelitis in Lewis rats.

Oncotarget 2018 Apr 27;9(32):22269-22287. Epub 2018 Apr 27.

Neuroimmunology and Neuromuscular Diseases Unit, Neurological Institute 'Carlo Besta', Milan, Italy.

Probiotics beneficial effects on the host are associated with regulation of the intestinal microbial homeostasis and with modulation of inflammatory immune responses in the gut and in periphery. In this study, we investigated the clinical efficacy of two lactobacillus and two bifidobacterium probiotic strains in experimental autoimmune myasthenia gravis (EAMG) and experimental autoimmune encephalomyelitis (EAE) models, induced in Lewis rats. Treatment with probiotics led to less severe disease manifestation in both models; analyses showed preservation of neuromuscular junction in EAMG and myelin content in EAE spinal cord. Immunoregulatory transcripts were found differentially expressed in gut associated lymphoid tissue and in peripheral immunocompetent organs. Feeding EAMG animals with probiotics resulted in increased levels of Transforming Growth Factor-β (TGFβ) in serum, and increased percentages of regulatory T cells (Treg) in peripheral blood leukocyte. Exposure of immature dendritic cells to probiotics induced their maturation toward an immunomodulatory phenotype, and secretion of TGFβ. Our data showed that bifidobacteria and lactobacilli treatment effectively modulates disease symptoms in EAMG and EAE models, and support further investigations to evaluate their use in autoimmune diseases.
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http://dx.doi.org/10.18632/oncotarget.25170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5976463PMC
April 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

Gut microbiota and probiotics: novel immune system modulators in myasthenia gravis?

Ann N Y Acad Sci 2018 02 17;1413(1):49-58. Epub 2018 Jan 17.

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

Gut microorganisms (microbiota) live in symbiosis with the host and influence human nutrition, metabolism, physiology, and immune development and function. The microbiota prevents pathogen infection to the host, and in turn the host provides a niche for survival. The alteration of gut bacteria composition (dysbiosis) could contribute to the development of immune-mediated diseases by influencing the immune system activation and driving the pro- and anti-inflammatory responses in order to promote or counteract immune reactions. Probiotics are nonpathogenic microorganisms able to interact with the gut microbiota and provide health benefits; their use has recently been exploited to dampen immunological response in several experimental models of autoimmune diseases. Here, we focus on the relationships among commensal bacteria, probiotics, and the gut, describing the main interactions occurring with the immune system and recent data supporting the clinical efficacy of probiotic administration in rheumatoid arthritis, multiple sclerosis, and myasthenia gravis (MG) animal models. The encouraging results suggest that selected strains of probiotics should be evaluated in clinical trials as adjuvant therapy to restore the disrupted tolerance in myasthenia gravis.
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http://dx.doi.org/10.1111/nyas.13567DOI 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

LYVE-1 is 'on stage' now: an emerging player in dendritic cell docking to lymphatic endothelial cells.

Cell Mol Immunol 2018 07 27;15(7):663-665. Epub 2017 Nov 27.

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

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http://dx.doi.org/10.1038/cmi.2017.126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6123476PMC
July 2018

Diagnostics of myasthenic syndromes: detection of anti-AChR and anti-MuSK antibodies.

Neurol Sci 2017 Oct;38(Suppl 2):253-257

UO Neurologia IV, Istituto Neurologico Carlo Besta, Milan, Italy.

This paper presents the Italian guidelines for autoantibody testing in myasthenia gravis that have been developed following a consensus process built on questionnaire-based surveys, internet contacts and discussions during dedicated workshops of the sponsoring Italian Association of Neuroimmunology (AINI). Essential clinical information on myasthenic syndromes, indications and limits of antibody testing, instructions for result interpretation and an agreed laboratory protocol (Appendix) are reported for the communicative community of neurologists and clinical pathologists.
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http://dx.doi.org/10.1007/s10072-017-3026-2DOI Listing
October 2017

A Novel Approach to Reinstating Tolerance in Experimental Autoimmune Myasthenia Gravis Using a Targeted Fusion Protein, mCTA1-T146.

Front Immunol 2017 13;8:1133. Epub 2017 Sep 13.

Neurology IV, Fondazione I.R.C.C.S. Istituto Neurologico Carlo Besta, Milan, Italy.

Reinstating tissue-specific tolerance has attracted much attention as a means to treat autoimmune diseases. However, despite promising results in rodent models of autoimmune diseases, no established tolerogenic therapy is clinically available yet. In the experimental autoimmune myasthenia gravis (EAMG) model several protocols have been reported that induce tolerance against the prime disease-associated antigen, the acetylcholine receptor (AChR) at the neuromuscular junction. Using the whole AChR, the extracellular part or peptides derived from the receptor, investigators have reported variable success with their treatments, though, usually relatively large amounts of antigen has been required. Hence, there is a need for better formulations and strategies to improve on the efficacy of the tolerance-inducing therapies. Here, we report on a novel targeted fusion protein carrying the immunodominant peptide from AChR, mCTA1-T146, which given intranasally in repeated microgram doses strongly suppressed induction as well as ongoing EAMG disease in mice. The results corroborate our previous findings, using the same fusion protein approach, in the collagen-induced arthritis model showing dramatic suppressive effects on Th1 and Th17 autoaggressive CD4 T cells and upregulated regulatory T cell activities with enhanced IL10 production. A suppressive gene signature with upregulated expression of mRNA for TGFβ, IL10, IL27, and Foxp3 was clearly detectable in lymph node and spleen following intranasal treatment with mCTA1-T146. Amelioration of EAMG disease was accompanied by reduced loss of muscle AChR and lower levels of anti-AChR serum antibodies. We believe this targeted highly effective fusion protein mCTA1-T146 is a promising candidate for clinical evaluation in myasthenia gravis patients.
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http://dx.doi.org/10.3389/fimmu.2017.01133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5604076PMC
September 2017

Suppression of CHRN endocytosis by carbonic anhydrase CAR3 in the pathogenesis of myasthenia gravis.

Autophagy 2017 29;13(11):1981-1994. Epub 2017 Sep 29.

b Shanghai Institute of Immunology, Institutes of Medical Sciences , Shanghai Jiao Tong University School of Medicine , Shanghai , China.

Myasthenia gravis is an autoimmune disorder of the neuromuscular junction manifested as fatigable muscle weakness, which is typically caused by pathogenic autoantibodies against postsynaptic CHRN/AChR (cholinergic receptor nicotinic) in the endplate of skeletal muscle. Our previous studies have identified CA3 (carbonic anhydrase 3) as a specific protein insufficient in skeletal muscle from myasthenia gravis patients. In this study, we investigated the underlying mechanism of how CA3 insufficiency might contribute to myasthenia gravis. Using an experimental autoimmune myasthenia gravis animal model and the skeletal muscle cell C2C12, we find that inhibition of CAR3 (the mouse homolog of CA3) promotes CHRN internalization via a lipid raft-mediated pathway, leading to accelerated degradation of postsynaptic CHRN. Activation of CAR3 reduces CHRN degradation by suppressing receptor endocytosis. CAR3 exerts this effect by suppressing chaperone-assisted selective autophagy via interaction with BAG3 (BCL2-associated athanogene 3) and by dampening endoplasmic reticulum stress. Collectively, our study illustrates that skeletal muscle cell CAR3 is critical for CHRN homeostasis in the neuromuscular junction, and its deficiency leads to accelerated degradation of CHRN and development of myasthenia gravis, potentially revealing a novel therapeutic approach for this disorder.
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http://dx.doi.org/10.1080/15548627.2017.1375633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5788490PMC
June 2019

Validity, reliability, and sensitivity to change of the myasthenia gravis activities of daily living profile in a sample of Italian myasthenic patients.

Neurol Sci 2017 Nov 5;38(11):1927-1931. Epub 2017 Aug 5.

Department of Neuroimmunology and Neuromuscular Disorders, Neurological Institute "C. Besta" IRCCS Foundation, Milan, Italy.

The purpose of this study is to report on the validity, reliability, and sensitivity of the myasthenia gravis activities of daily living profile (MG-ADL) in a sample of Italian patients. Patients with myasthenia gravis (MG) completed a protocol that included the MG-ADL, the WHO Disability Assessment Schedule (WHODAS 2.0), the Besta Neurological Institute rating scale for myasthenia gravis, and the MG-composite. Cronbach's alpha was used to test reliability, Spearman's correlation and intra-class correlation coefficient (ICC) to test short-term test-retest, Kruskal-Wallis test to assess differences in MG-ADL between patients with different disease severity, and Wilcoxon signed-rank test to assess sensitivity to change. In total, 58 patients were enrolled: 44 were females, mean MG duration 10.5 ± 10.4 years, mean MG-ADL 3.98 ± 3.07. The MG-ADL showed good internal consistency (alpha = .774), stability (test-retest correlation = .98, ICC = .97). It was superior to the WHODAS 2.0 in differentiating patients with different MG type and severity (P < .001), it showed higher sensitivity to change (P = .001 for improved and P = .007 for worsened patients) and higher correlation with the MG-composite (RHO = .625). Our analysis shows that the Italian version of the MG-ADL is valid, reliable, stable, and sensitive to change.
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http://dx.doi.org/10.1007/s10072-017-3083-6DOI Listing
November 2017

A propensity score analysis for comparison of T-3b and VATET in myasthenia gravis.

Neurology 2017 Jul 7;89(2):189-195. Epub 2017 Jun 7.

From the Department of Neuroimmunology and Neuromuscular Diseases (G.B., C.A., F.B., R.M.), Fondazione Istituto Neurologico "Carlo Besta," Milan; and Dipartimento di Sanità Pubblica (C.M.), Medicina Sperimentale e Forense, Unità di Biostatistica e Epidemiologia Clinica, Università di Pavia, Italy.

Objective: We performed propensity score (PS) models to compare the outcome of patients with myasthenia gravis (MG) submitted to 2 different surgical approaches: extended transsternal (T-3b) or thoracoscopic extended thymectomy (VATET).

Methods: Patients' clinical data were retrieved from the MG database of the C. Besta Neurologic Institute Foundation. In the PS analysis, a matching ratio of 1:1 of the main clinical variables was obtained for the 2 groups of patients and treatment effect was estimated by comparing their outcome.

Results: A total of 210 patients met the inclusion criteria, by having a complete set of clinical data, and were included in the PS model; a matched dataset of 122 participants (61 per group) showed an adequate balance of all the covariates. Our analysis demonstrated that 68.9% of patients who had thymectomy by the VATET technique reached the pharmacologic remission/remission status at 2 years from thymectomy compared to 34.4% of those operated on by the T-3b technique ( < 0.001), had a lower INCB-MG score ( < 0.001), and had less muscle fatigability ( = 0.004). Similar results were found considering only nonthymomatous patients with MG. Results were also confirmed by paired statistical tests.

Conclusions: Our PS matching analysis showed that VATET is a reliable and effective surgical approach alternative to T-3b in patients with MG who are candidates for thymectomy.

Classification Of Evidence: This study provides Class IV evidence that for patients with MG, VATET is more effective than T-3b thymectomy.
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http://dx.doi.org/10.1212/WNL.0000000000004082DOI Listing
July 2017

In vitro labelling and detection of mesenchymal stromal cells: a comparison between magnetic resonance imaging of iron-labelled cells and magnetic resonance spectroscopy of fluorine-labelled cells.

Eur Radiol Exp 2017 29;1(1). Epub 2017 Jun 29.

Department of Neuroradiology, Neurological Institute IRCCS "Carlo Besta", Milan, Italy.

Background: Among the various stem cell populations used for cell therapy, adult mesenchymal stromal cells (MSCs) have emerged as a major new cell technology. These cells must be tracked after transplantation to monitor their migration within the body and quantify their accumulation at the target site. This study assessed whether rat bone marrow MSCs can be labelled with superparamagnetic iron oxide (SPIO) nanoparticles and perfluorocarbon (PFC) nanoemulsion formulations without altering cell viability and compared magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) results from iron-labelled and fluorine-labelled MSCs, respectively.

Methods: Of MSCs, 2 × 10 were labelled with Molday ION Rhodamine-B (MIRB) and 2 × 10 were labelled with Cell Sense. Cell viability was evaluated by trypan blue exclusion method. Labelled MSCs were divided into four samples containing increasing cell numbers (0.125 × 10, 0.25 × 10, 0.5 × 10, 1 × 10) and scanned on a 7T MRI: for MIRB-labelled cells, phantoms and cells negative control, T1, T2 and T2* maps were acquired; for Cell Sense labelled cells, phantoms and unlabelled cells, a F non-localised single-pulse MRS sequence was acquired.

Results: In total, 86.8% and 83.6% of MIRB-labelled cells and Cell Sense-labelled cells were viable, respectively. MIRB-labelled cells were visible in all samples with different cell numbers; pellets containing 0.5 × 10 and 1 × 10 of Cell Sense-labelled cells showed a detectable F signal.

Conclusions: Our data support the use of both types of contrast material (SPIO and PFC) for MSCs labelling, although further efforts should be dedicated to improve the efficiency of PFC labelling.
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http://dx.doi.org/10.1186/s41747-017-0010-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5909334PMC
June 2017

Validation of the italian version of the 15-item Myasthenia Gravis Quality-of-Life questionnaire.

Muscle Nerve 2017 Oct 27;56(4):716-720. Epub 2017 Apr 27.

Department of Neuroimmunology and Neuromuscular Disorders, Neurological Institute C. Besta IRCCS Foundation, Milan, Italy.

Introduction: In this study we assess the Italian version of the 15-item Myasthenia Gravis Quality-of-Life questionnaire (MG-QOL15).

Methods: The validation protocol included the MG-QOL15, the 36-item Short Form (SF-36), the Besta Neurological Institute Rating Scale for Myasthenia Gravis, and the MG-Composite. We used the Cronbach α to test reliability, the Spearman correlation to test short-term test-retest, the Kruskal-Wallis test to assess differences in MG-QOL15 between patients with different disease severity, and the Wilcoxon signed-rank test to assess sensitivity to change.

Results: Seventy-two patients were enrolled in the study. The mean MG-QOL15 score was 15.2 ± 12.2, with α = 0.93 and test-retest correlation = 0.93. Compared with the SF-36, the MG-QOL15 was superior in differentiating patients with different MG types (P = 0.041) and severity (P = 0.004), showed higher sensitivity to change (P = 0.003 for improved and P = 0.024 for worsened patients), and had higher correlations with the MG-Composite (rho = 0.367 vs. -0.213 and -0.154).

Conclusion: The Italian version of the MG-QOL15 is valid, reliable, stable, and sensitive to changes. Muscle Nerve 56: 716-720, 2017.
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http://dx.doi.org/10.1002/mus.25545DOI Listing
October 2017

Identification of a gene expression signature in peripheral blood of multiple sclerosis patients treated with disease-modifying therapies.

Clin Immunol 2016 Dec 5;173:133-146. Epub 2016 Oct 5.

Neurology IV - Neuroimmunology and Neuromuscular Diseases Unit, Neurological Institute "Carlo Besta", Milan, Italy. Electronic address:

Multiple Sclerosis (MS) is an inflammatory disease with neurodegenerative alterations, ultimately progressing to neurological handicap. Therapies are effective in counteracting inflammation but not neurodegeneration. Biomarkers predicting disease course or treatment response are lacking. We investigated whether altered gene and protein expression profiles were detectable in the peripheral blood of 78 relapsing remitting MS (RR-MS) patients treated by disease-modifying therapies. A discovery/validation study on RR-MS responsive to glatiramer acetate identified 8 differentially expressed genes: ITGA2B, ITGB3, CD177, IGJ, IL5RA, MMP8, P2RY12, and S100β. A longitudinal study on glatiramer acetate, Interferon-β, or Fingolimod treated RR-MS patients confirmed that 7 out of 8 genes were downregulated with reference to the different therapies, whereas S100β was always upregulated. Thus, we identified a peripheral gene signature associated with positive response in RR-MS which may also explain drug immunomodulatory effects. The usefulness of this signature as a biomarker needs confirmation on larger series of patients.
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http://dx.doi.org/10.1016/j.clim.2016.10.002DOI Listing
December 2016

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

Animal models of myasthenia gravis: utility and limitations.

Int J Gen Med 2016 4;9:53-64. Epub 2016 Mar 4.

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

Myasthenia gravis (MG) is a chronic autoimmune disease caused by the immune attack of the neuromuscular junction. Antibodies directed against the acetylcholine receptor (AChR) induce receptor degradation, complement cascade activation, and postsynaptic membrane destruction, resulting in functional reduction in AChR availability. Besides anti-AChR antibodies, other autoantibodies are known to play pathogenic roles in MG. The experimental autoimmune MG (EAMG) models have been of great help over the years in understanding the pathophysiological role of specific autoantibodies and T helper lymphocytes and in suggesting new therapies for prevention and modulation of the ongoing disease. EAMG can be induced in mice and rats of susceptible strains that show clinical symptoms mimicking the human disease. EAMG models are helpful for studying both the muscle and the immune compartments to evaluate new treatment perspectives. In this review, we concentrate on recent findings on EAMG models, focusing on their utility and limitations.
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http://dx.doi.org/10.2147/IJGM.S88552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4786081PMC
March 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

Validation of the Besta Neurological Institute rating scale for myasthenia gravis.

Muscle Nerve 2016 Jan 10;53(1):32-7. Epub 2015 Oct 10.

U.O. Neurologia IV - Neuroimmunologia e Malattie Neuromuscolari, Fondazione IRCCS Istituto Nazionale Neurologico C. Besta, Via Celoria 11, 20133, Milano.

Introduction: We validated the scale for myasthenia gravis (MG) developed at the Neurological Institute Foundation of Milan (INCB-MG scale).

Methods: A total of 174 patients were evaluated with the INCB-MG and compared with the MG Composite (MGC) as the gold standard. Dimensionality, reliability, and validity of the INCB-MG scale were studied by principal component factor analysis, Cronbach alpha, and Pearson correlation coefficients; interobserver reliability was calculated by the weighted Cohen K coefficient.

Results: Generalized and bulbar INCB-MG subscales were unidimensional with excellent consistency; the INCB-MG and MGC scales were strongly correlated. Fatigability assessment was correlated with the INCB-MG generalized, bulbar, and respiratory subscales.

Conclusions: The INCB-MG scale is a reliable tool to assess MG and is strongly correlated with the MGC. The INCB-MG scale is a valid tool for every day practice and should be further investigated for its application in clinical trials.
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http://dx.doi.org/10.1002/mus.24911DOI Listing
January 2016

In vivo quantitative magnetization transfer imaging correlates with histology during de- and remyelination in cuprizone-treated mice.

NMR Biomed 2015 Mar 9;28(3):327-37. Epub 2015 Jan 9.

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

The pool size ratio measured by quantitative magnetization transfer MRI is hypothesized to closely reflect myelin density, but their relationship has so far been confirmed mostly in ex vivo conditions. We investigate the correspondence between this parameter measured in vivo at 7.0 T, with Black Gold II staining for myelin fibres, and with myelin basic protein and beta-tubulin immunofluorescence in a hybrid longitudinal study of C57BL/6 and SJL/J mice treated with cuprizone, a neurotoxicant causing relatively selective myelin loss followed by spontaneous remyelination upon treatment suspension. Our results confirm that pool size ratio measurements correlate with myelin content, with the correlation coefficient depending on strain and staining method, and demonstrate the in vivo applicability of this MRI technique to experimental mouse models of multiple sclerosis.
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http://dx.doi.org/10.1002/nbm.3253DOI Listing
March 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

European database for myasthenia gravis: a model for an international disease registry.

Neurology 2014 Jul;83(2):189-91

From the Neurology IV Unit, Neurological Institute "Carlo Besta," Milan, Italy.

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http://dx.doi.org/10.1212/WNL.0000000000000563DOI Listing
July 2014

A superfluorinated molecular probe for highly sensitive in vivo(19)F-MRI.

J Am Chem Soc 2014 Jun 6;136(24):8524-7. Epub 2014 Jun 6.

Laboratory of Nanostructured Fluorinated Materials (NFMLab), Department of Chemistry, Materials, and Chemical Engineering "Giulio Natta", ‡Fondazione Centro Europeo Nanomedicina, and ∥Department of Electronics, Information, and Bioengineering, Politecnico di Milano , Milan, Italy.

(19)F-MRI offers unique opportunities to image diseases and track cells and therapeutic agents in vivo. Herein we report a superfluorinated molecular probe, herein called PERFECTA, possessing excellent cellular compatibility, and whose spectral properties, relaxation times, and sensitivity are promising for in vivo (19)F-MRI applications. The molecule, which bears 36 equivalent (19)F atoms and shows a single intense resonance peak, is easily synthesized via a simple one-step reaction and is formulated in water with high stability using trivial reagents and methods.
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http://dx.doi.org/10.1021/ja503270nDOI Listing
June 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

Development of the MG-DIS: an ICF-based disability assessment instrument for myasthenia gravis.

Disabil Rehabil 2014 19;36(7):546-55. Epub 2013 Jun 19.

Neurology, Public Health and Disability Unit and.

Purpose: To develop a preliminary version of a disease-specific, patient-reported disability assessment instrument for myasthenia gravis (MG) based on the International Classification of Functioning, Disability and Health (ICF): the MG-DIS.

Methods: Five consecutive steps were taken: literature review and selection of outcome measures; linking of measures' concepts to ICF categories and selection of those reported by 30% of the instruments; comparison of linking results with a previous selection of MG-relevant ICF categories; patient interview; development of questions based on retained ICF categories.

Results: Thirty-one papers containing 21 different outcome measures were found: 13 ICF categories were linked to them. Fifty-five items were retained after the comparison with the list of MG-specific categories, and were used for patient interview. Thirteen interviews were conducted before saturation of data was reached and the final list was composed of 42 categories: based upon them, 44 questions were developed.

Conclusions: The preliminary version of the MG-DIS contains more information than each single MG-specific tool, in particular, for the component of environmental factors. Further research is needed to test its psychometric properties.

Implications For Rehabilitation: It is important that patient-reported outcome is incorporated in MG patient's assessment. MG features can be evaluated with ICF-based methods. An MG-specific patient-reported disability assessment instrument can be used to monitor changes of functioning in patients on MG-specific treatments, and can be used in clinical trials as outcome measure.
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http://dx.doi.org/10.3109/09638288.2013.804591DOI Listing
December 2014

Differential targeting of immune-cells by Pixantrone in experimental myasthenia gravis.

J Neuroimmunol 2013 May 21;258(1-2):41-50. Epub 2013 Mar 21.

Preclinical Neuroimmunology Laboratory, Neurology IV, Neurological Institute Carlo Besta, Milan, Italy.

Pixantrone was shown to reduce the severity of clinical manifestation in experimental myasthenia gravis. In the present work we further studied its therapeutic effect. Our results demonstrate that a single administration suppressed AChR-specific immune-responses in primed rats. However, clinical symptoms could be improved only by repeated drug administrations (q7dx6 protocol-8.12 mg/kg); this treatment allowed stable serum drug levels for at least 7 days, as assessed by a functional T-cell bioassay. Pixantrone exerted strong in vitro inhibitory effect only on proliferating T-cells without impairing dendritic cell differentiation and B-cell viability. Our data further demonstrate that Pixantrone is a promising immunosuppressant drug that should be investigated in myasthenia gravis.
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http://dx.doi.org/10.1016/j.jneuroim.2013.02.021DOI Listing
May 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