Francesca Andreetta

Analysis of T cell receptor repertoire of muscle-infiltrating T lymphocytes in polymyositis. Restricted V alpha/beta rearrangements may indicate antigen-driven selection.

Polymyositis is an inflammatory myopathy characterized by mononuclear cell infiltration of muscle tissue. Myocytotoxic T lymphocytes have been recognized in the infiltrates, but the muscle antigen, target of the immune attack, has not been identified. Molecular characterization of the variable regions of T cell receptors (TCRs) on the infiltrating lymphocytes can be expected to provide insights into the pathogenic process. The V alpha/beta TCR repertoire was investigated by RNA-PCR in muscle biopsies from 15 polymyositis patients and 16 controls (6 Duchenne muscular dystrophy and 10 with no inflammatory or dystrophic myopathy). A variety of rearranged variable TCR genes was found in polymyositis, V alpha 1, V alpha 5, V beta 1, and V beta 15 being the most common (present in 60-100% of patients). In Duchenne muscular dystrophy patients TCR V alpha or beta rearrangements were found although no restriction was observed; no rearrangements were found in muscles from the other controls. Sequence analysis revealed the presence of the J beta 2.1 region in 90% of the V beta 15 clones studied, no random N additions in the diversity region, and a common motif within the CDR3 region. These results suggest that selection of muscle-infiltrating T lymphocytes is antigen driven in polymyositis.

A Multidisciplinary Evaluation of the Effectiveness of Cyclosporine A in Dystrophic Mdx Mice

Chronic inflammation is a secondary reaction of Duchenne muscular dystrophy and may contribute to disease progression. To examine whether immunosuppressant therapies could benefit dystrophic patients, we analyzed the effects of cyclosporine A (CsA) on a dystrophic mouse model. Mdx mice were treated with 10 mg/kg of CsA for 4 to 8 weeks throughout a period of exercise on treadmill, a protocol that worsens the dystrophic condition. The CsA treatment fully prevented the 60% drop of forelimb strength induced by exercise. A significant amelioration (P < 0.05) was observed in histological profile of CsA-treated gastrocnemius muscle with reductions of nonmuscle area (20%), centronucleated fibers (12%), and degenerating area (50%) compared to untreated exercised mdx mice. Consequently, the percentage of normal fibers increased from 26 to 35% in CsA-treated mice. Decreases in creatine kinase and markers of fibrosis were also observed. By electrophysiological recordings ex vivo, we found that CsA counteracted the decrease in chloride conductance (gCl), a functional index of degeneration in diaphragm and extensor digitorum longus muscle fibers. However, electrophysiology and fura-2 calcium imaging did not show any amelioration of calcium homeostasis in extensor digitorum longus muscle fibers. No significant effect was observed on utrophin levels in diaphragm muscle. Our data show that the CsA treatment significantly normalized many functional, histological, and biochemical endpoints by acting on events that are independent or downstream of calcium homeostasis. The beneficial effect of CsA may involve different targets, reinforcing the usefulness of immunosuppressant drugs in muscular dystrophy.

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.

Muscle inflammation and MHC class I up-regulation in muscular dystrophy with lack of dysferlin: an immunopathological study

Muscle inflammation is characteristic of inflammatory myopathies but also occurs in muscular dystrophy with lack of the sarcolemmal protein dysferlin. We quantified inflammatory cells and major histocompatibility complex (MHC) expression in muscle from 10 patients with dysferlinopathy. Infiltrating cells were always present although numbers varied considerably; macrophages were more common than T cells, T cytotoxicity was absent, and MHC class I was overexpressed on muscle fibers. These findings differ from polymyositis (PM) but are closely similar to those in SJL/J mice (which lack dysferlin) and emphasize the relationship between absence of dysferlin and immune system abnormalities in muscle.

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.

Role of tumour necrosis factor α, but not of cyclo-oxygenase-2-derived eicosanoids, on functional and morphological indices of dystrophic progression in mdx mice : a pharmacological approach

The role of tumour necrosis factor (TNF)‐α or cyclo‐oxygenase‐2 (COX‐2) eicosanoids in dystrophinopathies has been evaluated by chronically treating (4–8 weeks) adult dystrophic mdx mice with the anti‐TNF‐α etanercept (0.5 mg/kg) or the COX‐2 inhibitor meloxicam (0.2 mg/kg). Throughout the treatment period the mdx mice underwent a protocol of exercise on treadmill in order to worsen the pathology progression; gastrocnemious muscles from exercised mdx mice showed an intense staining for TNF‐α by immunohistochemistry. In vivo, etanercept, but not meloxicam, contrasted the exercise‐induced forelimb force drop. Electrophysiological recordings ex vivo, showed that etanercept counteracted the decrease in chloride channel function (gCl), a functional index of myofibre damage, in both diaphragm and extensor digitorum longus (EDL) muscle, meloxicam being effective only in EDL muscle. None of the drugs ameliorated calcium homeostasis detected by electrophysiology and/or spectrofluorimetry. Etanercept, more than meloxicam, effectively reduced plasma creatine kinase (CK). Etanercept‐treated muscles showed a reduction of connective tissue area and of pro‐fibrotic cytokine TGF‐β1 vs. untreated ones; however, the histological profile was weakly ameliorated. In order to better evaluate the impact of etanercept treatment on histology, a 4‐week treatment was performed on 2‐week‐old mdx mice, so to match the first spontaneous degeneration cycle. The histology profile of gastrocnemious was significantly improved with a reduction of degenerating area; however, CK levels were only slightly lower. The present results support a key role of TNF‐α, but not of COX‐2 products, in different phases of dystrophic progression. Anti‐TNF‐α drugs may be useful in combined therapies for Duchenne patients.

Overactivity of exercise-sensitive cation channels and their impaired modulation by IGF-1 in mdx native muscle fibers: beneficial effect of pentoxifylline.

Cell-attached patch-clamp recordings on native striated myofibers from adult dystrophic mdx mice revealed a higher occurrence and open probability compared to non-dystrophic wild-type myofibers of a 30 pS voltage-insensitive Ca2+-permeable channel, inhibited by Gd3+, streptomycin and ruthenium red. Myofibers from in vivo exercised animals had higher channel occurrence and/or open probability. Insulin-like growth factor 1 (3.3 nM) induced and/or enhanced channel activity, via PI3 kinase, in wild-type but not in mdx myofibers. Interestingly, in both genotypes the current was silenced by db-cAMP or pentoxifylline, a phosphodiesterase inhibitor. The channel activity/occurrence in pentoxifylline-treated exercised mdx (50 mg/kg/day i.p. for 4-8 weeks) overlapped that of exercised wild-type mice. Thus, a growth factor-sensitive current, likely due to a TRP channel, is activated in vivo by exercise in native striated fibers; its deregulation in the absence of dystrophin may contribute to Ca2+ homeostasis alteration. The possibility to pharmacologically counteract abnormal channel activity discloses important therapeutic application.

Oral administration of an immunodominant T-cell epitope downregulates Th1/Th2 cytokines and prevents experimental myasthenia gravis

The mucosal administration of the native antigen or peptide fragments corresponding to immunodominant regions is effective in preventing or treating several T cell-dependent models of autoimmune disease. No data are yet available on oral tolerance with immunodominant T-cell peptides in experimental autoimmune myasthenia gravis (EAMG), an animal model of B cell-dependent disease. We report that oral administration of the T-cell epitope alpha146-162 of the Torpedo californica acetylcholine receptor (TAChR) alpha-subunit suppressed T-cell responses to AChR and ameliorated the disease in C57Bl/6 (B6) mice. Protection from EAMG was associated with reduced serum Abs to mouse AChR and reduced AChR loss in muscle. The effect of Talpha146-162 feeding was specific; treatment with a control peptide did not affect EAMG manifestations. The protective effect induced by peptide Talpha146-162 was mediated by reduced production of IFN-gamma, IL-2, and IL-10 by TAChR-reactive cells, suggesting T-cell anergy. TGF-beta-secreting Th3 cells did not seem to be involved in tolerance induction. We therefore demonstrate that feeding a single immunodominant epitope can prevent an Ab-mediated experimental model of autoimmune disease.

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

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.

First evaluation of the potential effectiveness in muscular dystrophy of a novel chimeric compound, BN 82270, acting as calpain-inhibitor and anti-oxidant

BN 82270 is a membrane-permeable prodrug of a chimeric compound (BN 82204) dually acting as calpain inhibitor and anti-oxidant. Acute in vivo injection of dystrophic mdx mice (30 mg/kg, s.c.) fully counteracted calpain overactivity in diaphragm. A chronic 4-6 weeks administration significantly prevented in vivo the fore limb force drop occurring in mdx mice exercised on treadmill. Ex vivo electrophysiological recordings showed that BN 82270 treatment contrasted the decrease in chloride channel function (gCl) in diaphragm, an index of spontaneous degeneration, while it was less effective on both exercise-impaired gCl and calcium-dependent mechanical threshold of the hind limb extensor digitorum longus (EDL) muscle fibres. The BN 82270 treated mdx mice showed a marked reduction of plasma creatine kinase and of the pro-fibrotic cytokine TGF-beta1 in both hind limb muscles and diaphragm; however, the histopathological profile of gastrocnemious muscle was poorly ameliorated. In hind limb muscles of treated mice, the active form was detected by HPLC in the low therapeutic concentration range. In vitro exposure to 100 microM BN 82270 led to higher active form in diaphragm than in EDL muscle. This is the first demonstration that this class of chimeric compounds, dually targeting pathology-related events, exerts beneficial effects in muscular dystrophy. The drug/prodrug system may require posology adjustment to produce wider beneficial effects on all muscle types.