Biomedical subjects
Publications and source records attributed to E Bartoccioni.
The authors measured anti-muscle-specific tyrosine kinase (anti-MuSK) antibodies (Abs) in 83 serum samples from 40 patients and evaluated their correlation with myasthenia gravis severity and treatment response. Ab concentrations were often reduced by immunosuppression but not after thymectomy. Both in individual cases and in the whole population, a correlation between Ab levels and disease severity was found.
OBJECTIVE: To compare the electrophysiological and histopathological features of immunological myasthenia gravis (MG) subtypes. METHODS: Fifty MG patients underwent clinical examination, MuSK-Ab and AChR-Ab analysis. The majority underwent quantitative and single-fiber electromyography (QEMG, SFEMG), repetitive nerve stimulation and deltoid muscle biopsy. From muscle specimens with histological mitochondrial dysfunction, we amplified mitochondrial DNA (mtDNA). In specimens with mtDNA deletions, the nuclear gene POLG1 was sequenced. RESULTS: Five AChR-Ab seropositive [AChR(+)] and 5 seronegative [AChR(-)] patients were MuSK-Ab seropositive [MuSK(+)]. Five of 7 neurophysiologically examined MuSK(+) patients (71%) had proximal myopathic pattern, compared to 7 of 31 MuSK(-)/AChR(+) patients (23%) (P=0.012). SFEMG was abnormal in all examined MuSK(+) patients. All 7 biopsied MuSK(+) and 32 MuSK(-) patients (89%) had cytochrome c oxidase (COX) negative fibers. Three of five MuSK(+) and 13 of 20 MuSK(-) patients analyzed had multiple mtDNA deletions but no POLG1 mutations. CONCLUSIONS: Similar degree of SFEMG abnormalities was present in proximal muscles among MuSK(+) and AChR(+) patients. Proximal myopathy was over-represented in MuSK(+) patients; however, both MuSK(+) and MuSK(-) patients had mild myopathy with frequent mitochondrial abnormalities. SIGNIFICANCE: The weakness in MuSK(+) patients is most likely due to disturbed neuromuscular transmission. The frequently encountered mitochondrial dysfunction in MG warrants further study.
The aim of this study was to compare the neurophysiological and clinical pictures of a large sample of seronegative myasthenia gravis (SNMG) patients with and without anti-MuSK antibodies. Fifty-two consecutive SNMG patients were retrospectively evaluated. They had undergone an extended neurophysiological evaluation: repetitive nerve stimulation (RNS), single fiber EMG (SFEMG), and electromyography (EMG) with nerve conduction study. A muscle biopsy was performed in 11 of 52 patients, the edrophonium test in 44 of 52 patients and anti-AChR antibodies and anti-MuSK antibodies were tested in all patients. Anti-MuSK antibodies were detected in 25 SNMG patients (48.1%). The number of women in the MuSK+ group was significantly higher (P = 0.01) than in the MuSK- group. Seronegative MuSK+ patients are more severely affected and the deficit often involves the bulbar and the respiratory muscles. No statistically significant differences were observed in the edrophonium test between MuSK+ and MuSK- groups. The RNS test was abnormal in a significantly higher number of MUSK- patients than MUSK+ patients (P < 0.00001). With regard to SFEMG data, MuSK- patients were characterized to have more severe neurophysiological pattern. Our observations showed several differences between the clinical and neurophysiological pictures of MUSK+ and MUSK- patients.
Morphologic findings of thymuses from 32 anti-acetylcholine receptor (AChR)-negative myasthenia gravis patients, 12 with and 20 without antibodies against the muscle-specific kinase (MuSK), were compared with those from 30 AChR-positive subjects. In contrast with the high frequency of thymic hyperplastic changes in AChR-positive patients, in MuSK-positive subjects histologic alterations were minimal, arguing against an intrathymic disease pathogenesis. Since hyperplastic changes were seen in 35% of MuSK-negative patients, the thymus could be involved in some of these cases.
We studied a well-selected population of patients with active rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) without immunosuppressive therapy. Control and patient peripheral blood mononuclear cells (PBMC) were incubated with IL-1beta, IL-10, TGF-beta or LPS for 20 h and the in vitro basal and stimulated secretions of IL-6, TNF-alpha, IL-1beta and IL-1ra were measured by ELISA. We found that in the SLE patients the basal secretion of IL-6 was significantly lower and that of IL-1ra significantly higher than in control subjects, while in the RA group the basal IL-1ra secretion was higher than in healthy subjects. SLE and RA PBMC responded to LPS and IL-1beta reaching higher cytokine secretion values than controls. The in vitro response of SLE and RA PBMC to TGFbeta was normal, while that to IL-10 was defective: IL-10 was able to stimulate the production of IL-6 and IL-1ra in PBMC from normal subjects, but it was unable to enhance IL-6 secretion in RA cells and it was also completely ineffective in inducing IL-1ra secretion in both SLE and RA PBMC. Our work add new data useful for the evaluation of IL-10 and IL-1ra as therapeutic agents in rheumatic diseases.
We studied the expression of MHC-I and MHC-II molecules and ICAM-1 in cultured human myoblasts in response to IL-1beta, IL-4, IL-6, IFN-gamma and LPS. IFN-gamma, LPS and IL-4 greatly increase MHC-I molecule expression. MHC-II molecule expression is induced only by IFN-gamma. Membrane ICAM-1 and mRNA expression are absent under basal conditions, but can be induced by IFN-gamma, IL-1beta, IL-4, LPS and IL-6 with different efficiencies and time-courses. Soluble ICAM-1 secretion can be induced to a different extent by all cytokines. Our study shows that the expression of adhesion-related molecules in muscle is finely regulated by these cytokines.
Transforming growth factor-beta (TGF-beta) is involved in several autoimmune neurological diseases. It is still unclear whether its local action can be pro-inflammatory or anti-inflammatory in the muscle tissue, because of the few reports on this subject. We have previously shown that human myoblasts secrete interleukin-6 (IL-6) when stimulated with inflammatory cytokine such as interleukin-1beta (IL-1beta) or tumor necrosis factor alpha. In the present report, we show that TGF-beta1 can induce IL-6 production; moreover, costimulation or short term pre-incubation with TGF-beta1 increases IL-1beta effect, while a longer incubation inhibits its action.
Juvenile myasthenia gravis (JMG) with prepubertal onset is an uncommon disease. We studied 19 patients with age at onset ranging from 1.5 to 9.2 years and compared their clinical characteristics and response to therapy with 114 cases with MG onset after the prepubertal age, up to 20 years. Neither sex prevalence nor autoimmune diseases other than MG were found in younger patients. Although ocular myasthenia was more frequent than in later-onset JMG, children with generalized symptoms were often severely affected and respiratory involvement was present in 8/19 patients. Anti-acetylcholine receptor antibodies were detected at a lower rate and, in contrast with results in older patients, seronegativity was more frequent among children with generalized disease. Three out of six patients with onset before the age of five showed spontaneous remission. Nine prepubertal patients underwent thymectomy and, as most of them also received immunosuppressive therapy, the influence of surgery on disease outcome remains unclear; in no case was thymoma found. This is in contrast to the good results after thymectomy and the presence of thymoma in the later-onset group. Eleven patients in the prepubertal series were treated with immunosuppressive therapy. At the end of follow-up, most patients were in good condition. The frequency of immunosuppressive therapy and the rate of good therapeutic results did not differ from those observed in older patients.
Muscle fibers are the target of T cell-mediated cytotoxic reactions in polymyositis and inclusion body myositis, while the success of myoblast transplantation depends on the absence of an immune rejection against the myofibers. In order to study the behaviour of muscle cells in an inflammatory milieu, we investigated the production of IL-6 and its modulation, including the second messenger pathways controlling it, in in vitro highly purified human myoblast cultures. We found that IL-1beta, tumor necrosis factor (TNF)-alpha and lipopolysaccharide (LPS) stimulated myoblast IL-6 secretion in a dose- and time-dependent manner, whereas forskolin and cholera toxin did not. HA1004 at 10 microM did not significantly affect the IL-1beta- and TNF-alpha-induced IL-6 secretion, suggesting that cAMP and protein kinase A are not sufficient to stimulate this process. To investigate the role of protein kinase C (PKC) in this signal transduction, we employed the inhibitor calphostin C, and the activators phorbol-12-myristate-13-acetate (PMA) and calcium ionophore A23187. Calphostin C blocked IL-6 secretion, PMA had a small stimulatory effect and A23187 had no effect; moreover, PKC down-regulation by PMA did not inhibit IL-1beta stimulation, while it reduced TNF-alpha stimulation. These data indicate that different PKC isoforms may be involved in TNF-alpha and IL-1beta signal transduction. Such a difference can distinguish the action of two traditionally 'overlapping' inflammatory cytokines. Our data suggest that muscle cells, like myoblasts, satellite cells and in vivo regenerating myofibers, may discriminate between different stimuli and produce IL-6 when activated in response to muscle injury.
Several recent studies have shown that some inflammatory myopathies are autoimmune diseases. It is possible that certain alterations in the muscle-immune cell microenvironment and in the local production of cytokines could take part in the pathogenesis of inflammatory myopathies. In the present study we investigated the effects of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on the secretion of interleukin-6 (IL-6) by myoblasts. Purified human myoblasts from normal subjects and from patients with polymositis were cultured in the presence of TNF-alpha and IFN-gamma at two concentrations (100 and 200 U/ml), alone or in combination, for 12, 24 and 48 h. The supernatants were collected and the IL-6 concentrations tested by ELISA (Genzyme). We found that myoblasts secrete IL-6 constitutively. The secretion of IL-6 was greatly increased by TNF-alpha; the increase was both time- and dose-dependent. IFN-gamma caused a moderate increase in IL-6 secretion, but this effect was not significant, despite a slight positive trend over time. There was no synergism in the effect of IFN-gamma and TNF-alpha. It is known that inflammatory myopathies are characterized by mononuclear cell infiltration and muscle regeneration: myoblasts are present in infiltrated tissues. Thus, the local production of cytokines that characterizes the inflammatory reaction, could stimulate myoblasts to secrete IL-6, which might add to the pro-inflammatory effects of IL-6 produced by activated macrophages and T cells.
We investigated the relationship between the MHC-I, MHC-II and intercellular adhesion molecule-1 (ICAM-1) expression on myofibres and the presence of inflammatory cells in muscle specimens of 18 patients with inflammatory myopathies (nine polymyositis, seven dermatomyositis, two inclusion body myositis). We observed MHC-I expression in muscle fibres, infiltrating mononuclear cells and endothelial cells in every specimen. In seven patients, some muscle fibres were MHC-II-positive for the DR antigen, while the DP and DQ antigens were absent. ICAM-1 expression, detected in seven patients, was found in clusters of myofibres, associated with a marked MHC-I positivity and a widespread mononuclear infiltration. Most of the ICAM-1-positive fibres were regenerating fibres. Furthermore, some fibres expressed both ICAM-1 and DR antigens near infiltrating cells. This finding could support the hypothesis that myofibres may themselves be the site of autosensitization.
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The effects of high-dose intravenous immunoglobulin (i.v.Ig) in 12 MG patients were studied. All patients had severe symptoms. In two cases anti-acetylcholine receptor antibodies (anti-AChR abs) were not detectable. I.v.Ig was administered to 9 patients already on long-term immunosuppressive therapy and to 3 patients at the beginning of azathioprine treatment. 10 patients (83%) improved; the duration of improvement was longer in immunosuppressed patients. Anti-AChR abs generally decreased after infusion but we did not find a constant correlation between reduction in ab titers and clinical improvement. Side effects included one case of severe hemolysis. In our experience i.v.Ig therapy is effective in MG. The chief indication for its use appears to be the treatment of deterioration of the disease in patients already on immunosuppressive therapy.
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We performed ten plasma perfusion (PP) treatments on eight patients affected by Myasthenia Gravis (MG) with high serum levels of autoantibodies against acetylcholine receptors (anti AChR-AB), and one PP treatment on a patient with MG of probable genetic origin and without specific antibodies. All patients (Osserman group III-IV) had undergone thymectomy and immunosuppressive therapy. Each patient received a treatment cycle of six PP sessions. Clinical conditions were assessed before and after the treatment with evaluation of muscular strength, ventilatory function, and electromyographic testing. Immunologic markers were tested before and after each PP. The patient without specific antibodies showed no improvement with PP and was excluded from our study. All the other patients showed continued improvement, with increased muscle strength and improved respiratory function. Four patients (follow-up 16-24 months) still maintain the clinical improvement; two, unexpectedly relapsed 11-12 months after PP, received a new treatment successfully (follow-up 9-10 month).
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The clinical and immunological characteristics of myasthenia gravis (MG) patients without detectable anti-Acetylcholine Receptor (anti-AChR) antibody were studied. Sixty anti-AChR negative MG patients were compared with 287 anti-AChR positive cases. Significant differences were found with respect to the sex incidence, the disease severity and the thymic pathology, while the age at the onset of the disease, the presence of other auto-antibodies and the efficacy of medical treatments were similar in the two groups of patients. Anti-AChR-negative MG could have pathogenetic mechanisms different from the antibody-positive disease.
We investigated the in vitro effects (kinetics and dose-response) of adrenal and sexual steroid hormones on the secretion of thymulin, a thymic hormone, by human thymic epithelial cells in primary cultures as well as in a rat epithelial cell line. We demonstrated that all steroids tested, in a range of physiological doses, stimulated thymulin production to various extents. Progesterone and estradiol, however, were revealed to be the most efficient. Specific steroid antagonists abrogated the steroid-induced stimulation of thymulin production. These findings confirm our previous in vivo results and demonstrate that steroid hormones can act directly on thymic epithelial cells to modulate their endocrine production.