[show abstract][hide abstract] ABSTRACT: We investigated the molecular mechanisms involved in the pathogenesis of three inflammatory myopathies, dermatomyositis (DM), polymyositis (PM) and inclusion body myositis (IBM). We performed microarray experiments using microdissected pathological muscle fibers from 15 patients with these disorders and 5 controls. Differentially expressed candidate genes were validated by immunohistochemistry on muscle biopsies, and the altered pathways were analysed in human myotube cultures. Upregulation of genes involved in viral and nucleic acid recognition were found in the three myopathies but not in controls. In DM, retinoic acid-inducible gene 1 (RIG-I, DDX58) and the novel antiviral factor DDX60, that promotes RIG-I- mediated signaling, were significantly upregulated, followed by IFIH1 (MDA5) and TLR3. Immunohistochemistry confirmed overexpression of RIG-I in pathological muscle fibers in 5/5 DM, 0/5 PM, and 0/5 IBM patients, and in 0/5 controls. Stimulation of human myotubes with a ligand of RIG-I produced a significant secretion of interferon β (p<0.05) and upregulation of Class I MHC, RIG-I and TLR3 (p<0.05) by interferon β-dependent and TLR3-independent mechanisms. RIG-I- mediated innate immunity, triggered by a viral or damage signal, plays a significant role in the pathogenesis of DM but not in that of PM or IBM.
The Journal of Pathology 03/2014; · 7.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: To describe the frequency of antibodies against neurofascin in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and the associated clinical features.
Immunocytochemistry was used to identify antibodies to neurofascin 155 (NF155) and 186. Serum reactivity with paranodes and brain tissue was tested with immunohistochemistry of teased-nerve fibers and rat brain. Antibody titers and immunoglobulin (Ig) G isotypes were determined using ELISA. Clinical information was obtained retrospectively.
Two of 53 patients, but none of 204 controls, had antibodies to NF155 (p = 0.041). The 2 patients with NF155 antibodies developed severe polyradiculoneuropathy with predominant distal weakness that was refractory to IVIg. Eight additional patients with IVIg-refractory CIDP were then identified from a national database; 2 of them with the same clinical features also had NF155 antibodies. Overall, 3 of the 4 patients with NF155 antibodies had a disabling and characteristic tremor (high amplitude, low frequency, postural, and intention). Patients' antibodies reacted with the paranodes in teased-nerve fibers and with the neuropil of rat cerebellum, brain, and brainstem. Anti-NF155 antibodies were predominantly of the IgG4 isotype in all patients.
Patients with CIDP positive for IgG4 NF155 antibodies constitute a specific subgroup with a severe phenotype, poor response to IVIg, and disabling tremor. Autoantibodies against paranodal structures associate with distinct clinical features in CIDP and their identification has diagnostic, prognostic, and therapeutic implications.
This study provides Class IV evidence that autoantibodies to NF155 identify a CIDP subtype characterized by severe neuropathy, poor response to IVIg, and disabling tremor.
[show abstract][hide abstract] ABSTRACT: Cell adhesion molecules (CAMs) play a crucial role in the formation of the nodes of Ranvier and in the rapid propagation of the nerve impulses along myelinated axons. These CAMs are the targets of autoimmunity in inflammatory neuropathies. We recently showed that a subgroup of patients with aggressive chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) shows autoantibodies to Contactin (1). The complex of Contactin/Caspr/Neurofascin-155 (NF155) enables the formation of paranodal junctions suggesting that antibody attack against paranodes may participate in the severity of CIDP. In the present study, we mapped the molecular determinants of Contactin targeted by the autoantibodies. In three patients, immunoreactivity was directed against the Ig domains of Contactin and was dependent on N-glycans. The serum of one patient was selectively directed against Contactin bearing mannose-rich N-glycans. Strikingly, the oligomannose type sugars of Contactin are required for association with its glial partner NF155 (2). To investigate precisely the role of Contactin N-glycans, we have mutated each of the 9 consensus N-glycosylation sites independently. We found that the mutation of 3 sites (N467,473,494Q) in the Ig domain 5 of Contactin prevented soluble NF155-Fc binding. In contrast, these mutations did not abolish cis-association with Caspr. Next, we showed that the cluster of N-glycosylation sites (N467, N473, N494) was required for immunoreactivity in one patient. Using cell aggregation assays, we showed that the IgG from the four CIDP patients prevented adhesive interaction between Contactin/Caspr and NF155. Importantly, we showed the anti-Contactin autoantibodies induced alteration of paranodal junctions in myelinated neuronal culture. These results strongly support that antibodies to CAMs may be pathogenic and induce demyelination via functional blocking activity.
Journal of Biological Chemistry 02/2014; · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: THIS IS NOT THE FINAL POST-PRINT VERSION. To get full post-print version, please, go to http://journals.lww.com/co-neurology/pages/articleviewer.aspx?year=2013&issue=10000&article=00003&type=abstract
Myasthenic disorders are a well characterized group of diseases of the neuromuscular junction. Their pathogenesis is diverse, including genetic and autoimmune mechanisms. We review recent findings on risk factors, pathogenesis and treatment of autoimmune myasthenia gravis.
Better knowledge of congenital myasthenia has led to the development of efficient diagnostic algorithms that have therapeutic implications. New epidemiological and genetic risk factors have been identified and are considered to play a role in the development of myasthenia gravis. The study of the role of innate immunity in myasthenia gravis has identified relevant pathways to explain myasthenia gravis causes. The description of the pathogenic role of IgG4 anti-MuSK antibodies has revealed heterogeneous immune mechanisms that should lead to more specific therapies. Rituximab seems to be particularly effective in MuSK myasthenia gravis, and eculizumab arises as an option in refractory AChR myasthenia gravis. Therapeutic algorithms need to be tailored to each myasthenia subtype.
Increasing knowledge about the environmental and genetic risk factors and basic immunopathogenesis of myasthenia gravis, including the role of innate immunity, regulatory T cell impairment and autoantibody heterogeneity, is providing a rationale for treatment with new biological agents. Current immunotherapies in myasthenia gravis undoubtedly provide benefits, but also cause side-effects. Controlled trials are, therefore, needed to confirm initial results from pilot studies.
Current opinion in neurology 08/2013; · 5.43 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dysferlinopathies are a group of progressive muscular dystrophies characterized by mutations in the gene DYSF. These mutations cause scarcity or complete absence of dysferlin, a protein that is expressed in skeletal muscle and plays a role in membrane repair. Our objective was to unravel the proteins that constitute the dysferlin complex and their interaction within the complex using immunoprecipitation assays (IP), blue native gel electrophoresis (BN) in healthy adult skeletal muscle and healthy cultured myotubes, and fluorescence lifetime imaging- fluorescence resonance energy transfer (FLIM-FRET) analysis in healthy myotubes. The combination of immunoprecipitations and blue native electrophoresis allowed us to identify previously reported partners of dysferlin -- such as caveolin-3, AHNAK, annexins, or Trim72/MG53 -- and new interacting partners. Fluorescence lifetime imaging showed a direct interaction of dysferlin with Trim72/MG53, AHNAK, cytoplasmic dynein, myomesin-2 and calsequestrin-1, but not with caveolin-3 or dystrophin. In conclusion, although IP and BN are useful tools to identify the proteins in a complex, techniques such as fluorescence lifetime imaging analysis are needed to determine the direct and indirect interactions of these proteins within the complex. This knowledge may help us to better understand the roles of dysferlin in muscle tissue and identify new genes involved in muscular dystrophies in which the responsible gene is unknown.
The international journal of biochemistry & cell biology 06/2013; · 4.89 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dysferlinopathies are caused by mutations in the DYSF gene. Dysferlin is a protein mainly expressed in skeletal muscle and monocytes. Cell therapy constitutes a promising tool for the treatment of muscular dystrophies. The aim of our study was to evaluate the effect of bone marrow transplantation (BMT) using the A/J Dysfprmd mouse model of dysferlinopathy. For that purpose we studied dysferlin expression by Western blot and/or immunohistochemistry in transplanted mice and controls. Computerized analysis of locomotion and electrophysiological techniques were also performed to test functional improvement. We observed dysferlin expression in splenocytes but not in the skeletal muscle of the transplanted mice. However, locomotion test, electromyography studies and muscle histology showed an improvement in all transplanted mice that was more significant in the animals transplanted with dysferlin+/+ cells. In conclusion, although BMT restores dysferlin expression in monocytes but not in skeletal muscle, muscle function was partially recovered. We propose that the slight improvement observed in the functional studies could be related with factors, such as hepatocyte growth factor, released after BMT that prevented muscle degeneration.
Stem cells and development 06/2013; · 4.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: The term paraproteinemic neuropathy refers to a heterogeneous group of neuropathies, which share the common feature of a homogeneous immunoglobulin in the serum. The presence of a monoclonal gammopathy indicates an underlying clonal B-cell expansion, which may appear in the context of a lymphoproliferative disorder. If a neoplastic origin of the gammopathy is identified, the treatment should be targeted to the neoplasm. In most patients, however, the monoclonal gammopathy is not associated with malignant haematological disorders, and is defined as monoclonal gammopathy of undetermined significance.
[show abstract][hide abstract] ABSTRACT: Dysferlin is mutated in a group of muscular dystrophies commonly referred to as dysferlinopathies. It is highly expressed in skeletal muscle, where it is important for sarcolemmal maintenance. Recent studies show that dysferlin is also expressed in monocytes. Moreover, muscle of dysferlinopathy patients is characterized by massive immune cell infiltrates, and dysferlin negative monocytes were shown to be more aggressive and phagocytose more particles. This suggests that dysferlin deregulation in monocytes might contribute to disease progression, but the molecular mechanism is unclear. Here we show that dysferlin expression is increased with differentiation in human monocytes and the THP1 monocyte cell model. Freshly isolated monocytes of dysferlinopathy patients show deregulated expression of fibronectin and fibronectin binding integrins, which is recapitulated by transient knockdown of dysferlin in THP1 cells. Dysferlin forms a protein complex with these integrins at the cell membrane, and its depletion impairs cell adhesion. Moreover, patient macrophages show altered adhesion and motility. These findings suggest that dysferlin is involved in regulating cellular interactions and provide new insight into dysferlin function in inflammatory cells.
Journal of Biological Chemistry 04/2013; · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: OBJECTIVE: Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a frequent autoimmune neuropathy with a heterogeneous clinical spectrum. Clinical and experimental evidence suggests that autoantibodies may be involved in its pathogenesis, but the target antigens are unknown. Axoglial junction proteins have been proposed as candidate antigens. We examined the reactivity of CIDP patients' sera against neuronal antigens and used immunoprecipitation for antigen unraveling. METHODS: Primary cultures of hippocampal neurons were used to select patients' sera that showed robust reactivity with the cell surface of neurons. The identity of the antigens was established by immunoprecipitation and mass spectrometry, and subsequently confirmed with cell-based assays, immunohistochemistry with teased rat sciatic nerve, and immunoabsorption experiments. RESULTS: Four of 46 sera from patients with CIDP reacted strongly against hippocampal neurons (8.6%) and paranodal structures on peripheral nerve. Two patients' sera precipitated contactin-1 (CNTN1), and 1 precipitated both CNTN1 and contactin-associated protein 1 (CASPR1). Reactivity against CNTN1 was confirmed in 2 cases, whereas the third reacted only when CNTN1 and CASPR1 were cotransfected. No other CIDP patient or any of the 104 controls with other neurological diseases tested positive. All 3 patients shared common clinical features, including advanced age, predominantly motor involvement, aggressive symptom onset, early axonal involvement, and poor response to intravenous immunoglobulin. INTERPRETATION: Antibodies against the CNTN1/CASPR1 complex occur in a subset of patients with CIDP who share common clinical features. The finding of this biomarker may help to explain the symptoms of these patients and the heterogeneous response to therapy in CIDP. ANN NEUROL 2012.
Annals of Neurology 10/2012; · 11.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: Late-onset Pompe disease is characterized by progressive weakness involving proximal limb and respiratory muscles. Recently, treatment with enzyme replacement therapy (ERT) has been introduced partially improving patients' prognosis, but a standard consensus on when to start ERT is still lacking. There is also a lack of biomarkers related to the clinical progression of the disease. Here we used muscle magnetic resonance imaging (MRI) or computed tomography (CT) to study the abdominal and paravertebral muscles of 30 late-onset Pompe patients at different stages of disease. We observed a selective pattern of muscle damage, with early involvement of the Multifidus muscle, followed by the Obliquus internus abdominis and Longissimus muscle. Some degree of trunk involvement on MRI occurred even in asymptomatic patients. Severity of muscle involvement in MRI correlated with patients' functional stage. We suggest that: (a) the combination of paravertebral and abdominal muscle involvement may serve as a useful tool in the diagnostic work-up of patients with a clinical suspicion of Pompe disease; (b) trunk abnormalities appear at very early stages of disease and even in asymptomatic patients, possibly "announcing" the onset of the disease and thus the need for a closer clinical follow-up.
[show abstract][hide abstract] ABSTRACT: Dysferlinopathies are a heterogenous group of autosomal recessive inherited muscular dystrophies caused by mutations in DYSF gene. Dysferlin is expressed mainly in skeletal muscle and in monocytes and patients display a severe reduction or absence of protein in both tissues. Vitamin D3 promotes differentiation of the promyelocytic leukemia HL60 cells. We analyzed the effect of vitamin D3 on dysferlin expression in vitro using HL60 cells, monocytes and myotubes from controls and carriers of a single mutation in DYSF. We also performed an observational study with oral vitamin D3 in a cohort of 21 carriers. Fifteen subjects were treated for 1 year and dysferlin expression in monocytes was analysed before and after treatment. Treatment with vitamin D3 increased expression of dysferlin in vitro. The effect of vitamin D3 was mediated by both a nongenomic pathway through MEK/ERK and a genomic pathway involving binding of vitamin D3 receptor to the dysferlin promoter. Carriers treated with vitamin D3 had significantly increased expression of dysferlin in monocytes compared with nontreated carriers (P < 0.05). These findings will have important therapeutic implications since a combination of different molecular strategies together with vitamin D3 uptake could increase dysferlin expression to nonpathological protein levels.
[show abstract][hide abstract] ABSTRACT: INTRODUCTION: New treatments for immune mediated diseases have increased notably in the last 10 years. Monoclonal antibodies directed against different components of the immune system have appeared, along with new drugs from the haematology field. In the case of myasthenia gravis (MG), many of these new treatments have been used in experimental animal models and also in patients. AREAS COVERED: This manuscript reviews the progress in the field of MG treatment achieved in the last 5 years. Firstly, our current treatment protocol is introduced. Secondly, new data from recent randomized trials and case series of patients treated with methotrexate, cyclophosphamide, rituximab or improved systems of apheresis is reported. Finally, all future treatments are discussed that are currently under evaluation in preclinical animal models of experimental autoimmune MG. EXPERT OPINION: Evidence supporting the use of methotrexate and rituximab in MG has been published recently, in addition to conflicting randomized trials that were not successful, evaluating the use of tacrolimus as a steroid sparing agent. New promising treatments are currently under evaluation in clinical trials, such as belimumab and eculizumab.
Expert Opinion on Pharmacotherapy 07/2012; 13(13):1873-83. · 2.86 Impact Factor
[show abstract][hide abstract] ABSTRACT: There is as yet no consensus for considering pure acute sensory ataxic neuropathy (ASAN) as a variant of Guillain-Barré syndrome (GBS). Reactivity against gangliosides sharing disialosyl epitopes has been reported in these patients. The aim of this study was to determine the spectrum of reactivity against gangliosides in ASAN and to define the clinical pattern. From our database we identified patients with suspicion of ASAN. We defined ASAN as the presence of ataxia of peripheral origin with loss of proprioception, and areflexia, absence of ophthalmoplegia and no or minimal muscle weakness. Patients who met these criteria were retrospectively reviewed for their spectrum of reactivity against gangliosides and clinical features. We identified 12 patients fulfilling pre-defined criteria for ASAN. Reactivity against gangliosides containing disialosyl epitopes was present in seven patients. Concomitant reactivity against other gangliosides was present in 6/7 patients. All patients presented good prognosis and an antecedent illness was present in nine. Our results support the previously described clinico-immunological association between ASAN and disialosyl specificity, and widen the spectrum of reactivity against gangliosides. The acute presentation with a monophasic course, and good prognosis in all cases, together with transient immunoglobulin G antiganglioside antibodies and infectious antecedent in 7/12 patients support the inclusion of ASAN as a GBS variant.
Journal of the Peripheral Nervous System 06/2012; 17(2):158-68. · 2.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: Before 2006, Pompe disease or glycogenosis storage disease type II was an incurable disease whose treatment was merely palliative. The development of a recombinant human alpha-glucosidase enzymatic replacement therapy has become the first specific treatment for this illness. The aim of this guide is to serve as reference for the management of the late-onset Pompe disease, the type of Pompe disease that develops after one year of age. In the guide a group of Spanish experts make specific recommendations about diagnosis, follow-up and treatment of this illness. With regard to diagnosis, the dried blood spots method is essential as the first step for the diagnosis of Pompe disease. The confirmation of the diagnosis of Pompe disease must be made by means of an study of enzymatic activity in isolated lymphocytes or a mutation analysis of the alpha-glucosidase gene. With regard to treatment with enzymatic replacement therapy, the experts say that is effective improving or stabilizating the motor function and the respiratory function and it must be introduced when the first symptoms attributable to Pompe disease appear.
Revista de neurologia 04/2012; 54(8):497-507. · 1.18 Impact Factor
[show abstract][hide abstract] ABSTRACT: Rituximab has emerged as an efficacious option for drug-resistant myasthenia gravis (MG). However, reports published only describe the short-term follow-up of patients treated and little is known about their long-term clinical and immunologic evolution. Our objective was to report the clinical and immunologic long-term follow-up of 17 patients (6 MuSK+MG and 11 AChR+MG) and compare the response between AChR+MG and MuSK+MG patients.
Myasthenia Gravis Foundation America postintervention status and changes in treatment and antibody titers were periodically determined. Lymphocyte subpopulations, total immunoglobulin, immunoglobulin G (IgG) anti-MuSK subclasses, and anti-tetanus toxoid IgG before and after treatment were also studied.
After a mean post-treatment period of 31 months, 10 of the AChR+MG patients improved but 6 of them needed reinfusions. In contrast, all MuSK+MG patients achieved a remission (4/6) or minimal manifestations (2/6) status and no reinfusions were needed. Consequently, in the MuSK+MG group, prednisone doses were significantly reduced and concomitant immunosuppressants could be withdrawn. Clinical improvement was associated with a significant decrease in the antibody titers only in the 6 MuSK+MG patients. At last follow-up MuSK antibodies were negative in 3 of these patients and showed a decrease of over 80% in the other 3.
In view of the long-lasting benefit observed in MuSK+MG patients, we recommend to use rituximab as an early therapeutic option in this group of patients with MG if they do not respond to prednisone. Classification of evidence: This study provides Class IV evidence that IV rituximab improves the clinical and immunologic status of patients with MuSK+MG.
[show abstract][hide abstract] ABSTRACT: Growth hormone (GH) is the main regulator of longitudinal growth before puberty, and treatment with human recombinant (rh) GH can increase muscle strength. Nevertheless, molecular mechanisms responsible remain mostly unknown. Many physiological effects of GH require hormone-mediated changes in gene expression. In an attempt to gain insight into the mechanism of GH action in muscle cells we evaluated the effects of rhGH on gene expression profile in a murine skeletal muscle cell line C2C12. The objective of the work was to identify changes in gene expression in the murine skeletal muscle cell line C2C12 after rGH treatment using microarray assays. C2C12 murine skeletal muscle cell cultures were differentiated during 4 days. After 16 h growing in serum-free medium, C2C12 myotubes were stimulated during 6 h with 500 ng/ml rhGH. Four independent sets of experiments were performed to identify GH-regulated genes. Total RNA was isolated and subjected to analysis. To validate changes candidate genes were analyzed by real-time quantitative polymerase chain reaction. One hundred and fifty-four differentially expressed genes were identified; 90 upregulated and 64 downregulated. Many had not been previously identified as GH-responsive. Real-time PCR in biological replicates confirmed the effect of rGH on 15 genes: Cish, Serpina3g, Socs2, Bmp4, Tnfrsf11b, Rgs2, Tgfbr3, Ugdh, Npy1r, Gbp6, Tgfbi, Tgtp, Btc, Clec3b, and Bcl6. This study shows modifications in the gene expression profile of the C2C12 cell line after rhGH exposure. In vitro and gene function analysis revealed genes involved in skeletal and muscle system as well as cardiovascular system development and function.
Hormone and Metabolic Research 11/2011; 43(13):919-30. · 2.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: Charcot-Marie-Tooth disease (CMT) is the most frequent form of inherited neuropathy. In accordance with the inheritance pattern and degree of slowing of motor conduction velocity (MCV) of the median nerve, CMT encompasses five main forms: CMT1 (autosomal dominant [AD] or X-linked transmission and MCV < 38 m/s); CMT2 (AD or X-linked transmission and MCV > 38 m/s); CMT4 (autosomal recessive [AR] and severe slowing of MCV); AR-CMT2 (AR transmission and MCV > 38 m/s); and DI-CMT (intermediate form with AD transmission and MCV between 30 and 40 m/s). In spite of its stereotyped semiological repertoire (basically, symptoms and signs of sensory-motor polyneuropathy and pes cavus), CMT seems to be one of the most complex hereditary neurodegenerative syndromes, 31 causative genes having been cloned.
This paper is aimed at performing a nosological review of the disease, emphasising the guidelines for its molecular diagnosis. Genetic epidemiological studies and genotypes reported in Spanish patients are revised.
In the great majority of CMT cases, mutations involve a reduced number of genes, namely: for CMT1, PMP22, GJB1 and MPZ; for CMT2, MFN2 and GJB1; for CMT4, GDAP1, and NDRG1, HK1 and SH3TC2 (gypsies); for AR-CMT2, GDAP1; and for DI-CMT, GJB1 and MPZ. Given their low prevalence, mutations in other pathogenic genes should be investigated after discarding the previous ones. There is no place for the indiscriminate use of diagnostic CMT genetic panels.