Pia Bernasconi

Transforming growth factor-β1 and fibrosis in congenital muscular dystrophies

We evaluated transforming growth factor-beta1 (TGF-beta1) expression in the muscle of four laminin alpha2-negative, four laminin alpha2-positive and seven partial laminin alpha2-deficient congenital muscular dystrophy (CMD) patients, and compared it to Duchenne muscular dystrophy (DMD) patients and controls. TGF-beta1 mRNA levels in skeletal muscle from laminin alpha2-negative and laminin alpha2-positive CMD patients were significantly greater than in controls (P < 0.05 and P < 0.005, respectively), while in partial laminin alpha2-deficient muscular dystrophy patients the amount was not significantly higher than in controls (P > 0.1). The TGF-beta1 values were lower than those found in DMD, although the extent of fibrosis was greater in CMD than in DMD and controls. Our findings suggest that TGF-beta1 is involved in CMD muscle fibrosis, but differently from what we observed in DMD muscles as it seems not to be the major player in connective tissue proliferation.

Myasthenia Gravis (MG): Epidemiological Data and Prognostic Factors

Abstract: Data from 756 myasthenic patients were analyzed for diagnostic criteria, clinical aspects, and therapeutic approaches. The patients were followed up at our institution from 1981 to 2001. Clinical evaluation was performed according to the myasthenia gravis score adopted at our clinic. Clinical features of each patient (comprising demographic, clinical, neurophysiological, immunological, radiological, and surgical data, as well as serial myasthenia gravis scores) were filed in a relational database containing more than 7000 records. Clinical efficacy and variables influencing outcome were assessed by life‐table methods and Cox proportional hazards regression analysis. Complete stable remission, as defined by the Task Force of the Medical Scientific Advisory Board of the Myasthenia Gravis Foundation of America, was the end point for good prognosis. Four hundred and ninety‐nine patients (66%) were female and 257 (34%) were male. Mean follow‐up was 55.1 ± 48.1 months. Onset of symptoms peaked in the third decade in females, whereas the male distribution was bimodal with peaks in the third and sixth decades. Modality of myasthenia gravis presentation was as follows: ocular, 39.3%; generalized, 28.5%; bulbar, 31.3%; and respiratory, 0.8%. Thymectomy was carried out on 63.7% of our patients by different approaches: (1) transcervical; (2) transsternal; (3) video‐thoracoscopic mini‐invasive surgery. The last approach has been preferentially used in more recent years and accounted for 62.4% of the thymectomized myasthenia gravis population. Univariate analysis and Kaplan‐Meier analysis showed that variables such as sex (female), age at onset (below 40 years), thymectomy, and histological diagnosis of thymic hyperplasia were significantly associated with complete stable remission, whereas on multivariate analysis only age at onset below 40 years and thymectomy were confirmed.

Video-assisted thoracoscopic extended thymectomy and extended transsternal thymectomy (T-3b) in non-thymomatous myasthenia gravis patients: remission after 6 years of follow-up

The aims of this study were to assess the efficacy of video-assisted thoracoscopic extended thymectomy (VATET) as a treatment for myasthenia gravis (MG) and to identify prognostic factors for thymectomy success. Clinical efficacy and variables influencing outcome were assessed by life-table and Cox proportional hazards regression analysis. Complete stable remission (CSR), as defined by the MGFA Medical Task Force, was the end point for efficacy. VATET was performed in 159 MG patients and T-3b in 47 MG patients. At 6 years of follow-up, CSR, assessed by life-table analysis, was 50.6% in non-thymomatous VATET patients and 48.7% in non-thymomatous T-3b surgery. By univariate analysis, the presence of thymic hyperplasia (P=0.0002) and treatment only with anticholinesterases (P<0.0001) were positively associated with the probability of CSR. By multivariate analysis, the chance of complete remission was significantly increased by the use of anticholinesterases (odds ratio [OR] 2.45; 95% confidence interval [CI] 1.44-4.17; P=0.001) and the presence of thymic hyperplasia (OR 1.96; 95% CI 1.05-3.68; P=0.036). VATET seems to be effective in inducing CSR in MG with an efficiency similar to that of the T-3b transsternal (TS) approach; it is easy to perform in experienced hands and is associated with low morbidity and negligible esthetic sequelae.

Rasmussen's encephalitis: Early characteristics allow diagnosis

Objective: To identify early manifestations of Rasmussen encephalitis (RE) that can prompt early and reasonably secure diagnosis, allowing medical or surgical therapies at an early stage when they may be more effective in slowing the disease. Methods: The authors studied 12 patients with clinical and neuropathologic diagnosis of RE, followed from disease onset, assessing clinical history, imaging, and EEG and focusing on early characteristics. Anti-GluR3 antibody assays were also considered in 11 patients. Results: By 4 months from first symptoms, all cases had 1) refractory focal seizures with a predominant motor component, 2) slow focal activity on EEG contralateral to the motor manifestations, and 3) focal contralateral white matter hyperintensity with insular cortical atrophy on neuroimaging. Less constant or later findings were epilepsia partialis continua, oligoclonal bands, and serum anti-GluR3 antibodies. Conclusions: The association of partial seizures with focal EEG and neuroimaging changes allows a tentative diagnosis of RE 4 to 6 months after first symptoms.

Immunomodulation of TGF-beta1 in mdx mouse inhibits connective tissue proliferation in diaphragm but increases inflammatory response: Implications for antifibrotic therapy

Irreversible connective tissue proliferation in muscle is a pathological hallmark of Duchenne muscular dystrophy (DMD), a genetic degenerative muscle disease due to lack of the sarcolemmal protein dystrophin. Focal release of transforming growth factor-beta1 (TGF-beta1) is involved in fibrosis development. Murine muscular dystrophy (mdx) is genetically homologous to DMD and histopathological alterations comparable to those in DMD muscles occur in diaphragm of older mdx mice. To investigate the early development of fibrosis and TGF-beta1 involvement, we assessed diaphragms in 6-36-week-old mdx and C57/BL6 (control) mice for fibrosis, and used real-time PCR and ELISA to determine TGF-beta1 expression. Significantly greater fibrosis and TGF-beta1 expression were found in mdx from the 6th week. Mice treated with neutralizing antibody against TGF-beta1 had lower levels of TGF-beta1 protein, reduced fibrosis, unchanged muscles fiber degeneration/regeneration, but increased inflammatory cells (CD4+lymphocytes). These data demonstrate early and progressive fibrosis in mdx diaphragm accompanied by TGF-beta1 upregulation. Reduction of TGF-beta1 appears promising as a therapeutic approach to muscle fibrosis, but further studies are required to evaluate long term effects of TGF-beta1 immunomodulation on the immune system.

Epstein‐Barr virus persistence and reactivation in myasthenia gravis thymus

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.

Type I interferon and Toll-like receptor expression characterizes inflammatory myopathies

Objectives: Juvenile dermatomyositis (JDM), adult dermatomyositis, and polymyositis (PM) are idiopathic inflammatory myopathies (IIMs) characterized by muscle infiltration and specific muscle fiber alterations. They are thought to have an autoimmune etiology, but triggering factors, and how immunologic attack induces muscle weakness, remain unknown. Recent evidence suggests a key role for type I interferon (IFN)-mediated innate immunity in dermatomyositis, which we explored in JDM, dermatomyositis, and PM by gene expression profiling, and other methods. Methods: Ten IIM and 5 control muscle biopsies were assessed for expression of approximately 16,000 genes by microarray; 37 additional IIM, 10 dystrophinopathic, and 14 nonmyopathic control muscles were studied for type I IFN-dependent genes, and Toll-like receptor (TLR) expression by immunochemistry and PCR. Results: Type I IFN-dependent transcripts were significantly upregulated in IIM muscles compared to controls; in JDM the most expressed were ISG15 (408-fold), IFIT3 (261-fold), MX1 (99-fold), and IRF7 (37-fold). IFN-β (but not IFN-α) transcripts were upregulated in PM as well as dermatomyositis/JDM. TLR3 was upregulated particularly in JDM, being localized on vascular endothelial cells, muscle infiltrating cells (mainly myeloid dendritic cells), and regenerating myofibers; TLR7 and TLR9 proteins were present in IIM (prominently in PM), mainly on cell infiltrates, particularly plasma cells, and on some injured myofibers. Conclusions: IFN-β and type I IFN-induced molecules are involved in PM as well as JDM/dermatomyositis. Endosomal TLRs (effectors of innate immunity) are also involved (but differently) in the 3 conditions, further suggesting viral involvement, although TLR activation could be secondary to tissue damage.

Transforming Growth Factor-β1 in Polymyositis and Dermatomyositis Correlates with Fibrosis but not with Mononuclear Cell Infiltrate

The idiopathic inflammatory myopathies are diseases of unknown etiology characterized by T cell-mediated myocytotoxicity in polymyositis and complement-mediated angiopathy of muscle fibers in dermatomyositis. A variable degree of fibrosis is present in muscles in these conditions both perimysially and endomysially. We evaluated the expression of TGF-β1, a pleiotropic cytokine with fibrogenic and immunomodulating activity, by means of quantitative-polymerase chain reaction and immunocytochemistry in DM and PM muscle biopsies. TGF-β1 mRNA was significantly higher in DM compared with controls, whereas in PM the values were not significantly different when compared with controls and DM. TGF-β1 was localized in connective tissue but did not correspond with mononuclear cell infiltrates. These findings suggest a correlation between TGF-β1 and connective tissue proliferation in inflammatory myopathy, while its immunomodulatory role remains to be elucidated.

Lack of mRNA and dystrophin expression in DMD patients three months after myoblast transfer

We report our experience on myoblast transplantation in three Duchenne muscular dystrophy patients. Pure myoblasts (55 x 10(6) per patient) from HLA-matched donors, were injected into a tibialis anterior and the controlateral muscle was sham injected. Three months after transplantation, biopsies from the injected muscles were negative for dystrophin expression by immunocytochemistry. Reverse transcriptase-PCR (RT-PCR) failed to amplify any fragments of the deleted regions. This result confirms that myoblast transplantation is feasible, although the efficacy of this therapeutic approach is poor.

Increased Toll-Like Receptor 4 Expression in Thymus of Myasthenic Patients with Thymitis and Thymic Involution

Thymic abnormalities are present in approximately 80% of myasthenia gravis (MG) patients, and the thymus seems to be the main site of autosensitization to the acetylcholine receptor. In view of findings that the innate immune system can generate an autoimmune response, we studied the expression of Toll-like receptors (TLRs) 2 to 5, key components of innate immunity signaling pathways, in 37 thymuses from patients with autoimmune MG. TLR4 mRNA levels were significantly greater in thymitis (hyperplasia with diffuse B-cell infiltration) and involuted thymus than in germinal center hyperplasia and thymoma. By immunohistochemistry and confocal microscopy, cells positive for TLR4 protein were rarely detected in thymoma. However, in thymitis TLR4 protein was mostly found on epitheliomorphic (cytokeratin-positive) cells located in close association with clusters of acetylcholine receptor-positive myoid cells in thymic medulla and also at the borders between cortical and medullary areas. B cells were never TLR4-positive. TLR4 protein was also present in remnant tissue of involuted thymus. This is the first finding of a possible link between innate immunity and MG. We speculate that in a subgroup of MG patients, an exogenous or endogenous danger signal may activate the innate immune system and give rise to TLR4-mediated mechanisms contributing to autoimmunity.