Erythropoietin concentration in serum and cerebrospinal fluid of patients with amyotrophic lateral sclerosis.

Erythropoietin (EPO) acts as a neuroprotective factor and is upregulated after neuronal injury. It has been reported that in cerebrospinal fluid (CSF) of amyotrophic lateral sclerosis (ALS) patients, the EPO concentration is decreased. In this study, EPO levels in serum and CSF of 30 patients with ALS and in 15 controls, using an ELISA technique, were estimated. EPO level in serum was decreased, especially in patients with bulbar onset ALS. A trend toward a progressive EPO decline with the duration of the disease in the mild + moderate ALS cases was observed. In severe cases, a tendency towards a positive correlation of EPO and duration of the disease was present. Serum EPO values were age related only in mild + moderate ALS in patients below 40 years of age. In CSF, the EPO levels were significantly decreased. Lower EPO values in the bulbar onset ALS when compared with the spinal onset ALS were present. The EPO decrease did not correlate with the severity and duration of the disease. Age relation of the EPO level only in the mild + moderate ALS cases more than 40 years was present. Lack of differences in EPO levels between patients with ALS of rapid and slow progression indicates that EPO concentration cannot be used as a prognostic factor. Nevertheless, the decreased serum and CSF EPO concentration and the known EPO neuroprotective action may indicate that EPO administration can be a new promising therapeutic approach in ALS.

Matrix metalloproteinases and their tissue inhibitors in serum and cerebrospinal fluid of patients with amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: Matrix metalloproteinases (MMPs) are implicated in the pathogenesis of motor neuron degeneration in amyotrophic lateral sclerosis (ALS). We investigated the expression of MMPs and tissue inhibitors of matrix metalloproteinases (TIMPs) in serum and cerebrospinal fluid (CSF) correlating the results with age, disease duration and the clinical course. METHODS: The material consisted of 30 ALS patients and 15 age-matched healthy controls. ELISA method to determine the expression of MT-MMP-1, MMP-2, MMP-9, TIMP-1 and TIMP-2 in serum and CSF was used. MMP-2 and MMP-9 by zymography was also tested. RESULTS: In serum MT-MMP-1, MMP-2, MMP-9 and TIMP-1 expression was increased, especially in mild ALS cases. TIMP-2 values were normal. In CSF MT-MMP-1, MMP-2 and TIMP-1 level was either increased or normal, that of MMP-9 was decreased. TIMP-2 did not change. No correlation of MMPs and TIMP-1 expression in serum and CSF and the age of the patients was found. A correlation was observed between MMPs and TIMPs and disease duration. CONCLUSIONS: Increased level of MMPs and TIMP-1 of ALS patients may reflect the degeneration process of motor neurons and skeletal muscles and/or is associated with tissues remodeling. The low level of MMP-9 in CSF may result from impaired balance between MMP-9 and TIMP-1 and/or its increased intrathecal degradation and physical clearance. Although the role of changed MMPs/TIMPs level in the pathogenesis of ALS is not clear their analysis in serum may be used as prognostic factor and a potential marker for monitoring treatment effects.

X-linked Emery-Dreifuss muscular dystrophy with lamin A deficiency and IBM inclusions.

The study demonstrates a 12-year-old patient with progressive proximal muscle weakness, joint contractures, rigidity of the neck, and absence of emerin and lamin A in the muscle nuclei, which is caused by intronic mutation IVS3-27del18 (c.266-27del18) in the emerin gene. The most surprising finding was the appearance of IBM-like inclusions in euchromatin, as well as aberrant nuclei. It may be speculated that altered expression of the emerin-lamin complex and modification of the nuclear matrix leads to formation of tubulofilamentous structures in the presented case.

Circulating autoantibodies to troponin I in Emery-Dreifuss muscular dystrophy.

The pathogenesis of dilated cardiomyopathy in Emery- Dreifuss muscular dystrophy (EDMD) is still unknown. Autoimmune mechanisms have recently been taken into account. The aim of this investigation was to determine whether the level of circulating antibodies to heart proteins which were previously detected, correlates with disease progression. Troponin I was chosen as the target. Ten patients with EDMD and 10 age-matched normal controls were tested. An enzyme linked immunoassay (ELISA) technique was used to determine the possible relation between the level of anti-troponin I antibodies at diagnosis and at followup. Autoantibodies against troponin I were detected in all EDMD patients. At diagnosis the level was higher in the X-linked EDMD form (X-EDMD), as compared to the autosomal dominant form (AD-EDMD). At follow-up the elevated level of the autoantibodies persisted in all the EDMD cases. However, in the AD-EDMD form, the level was found to be significantly rising with disease progression, in the X-EDMD form, on the other hand, it was declining. No clear-cut relationship between the level of the circulating antibodies and cardiac symptomatology was present. Detection of anti-troponin I antibodies may provide a non-invasive marker of early stages of dilated cardiomyopathy in EDMD.

Altered distribution of lamin and emerin in muscle nuclei of sIBM patients.

OBJECTIVE: Sporadic inclusion body myositis (sIBM) is a chronic acquired inflammatory myopathy. The cause of sIBM remains unknown and its pathogenesis is controversial. There is a hypothesis [Karpati and Carpenter 1993] that the rimmed vacuoles result from nuclear breakdown, and IBM filaments are formed from components of the nuclear matrix. MATERIAL AND METHODS: For nuclear membrane protein detection, six IBM patients were studied using immunohistochemical and immunochemical techniques. RESULTS: It was demonstrated that in the interior of 10-15% myonuclei emerin and lamin A/C inclusions appeared constantly. This finding indicated an abberant localization of lamin A/C epitopes, the presence of presumptive lamin A (67 KDu) and emerin as in the affected nuclei. CONCLUSION: We support the suggestion that truncated, changed lamin A protein takes part in nuclear disintegration and rimmed vacuole formation.

Evidence for autoimmunity to heart-specific antigens in patients with Emery-Dreifuss muscular dystrophy.

Dilated cardiomyopathy is one of the leading abnormalities in Emery-Dreifuss Muscular Dystrophy (EDMD). The pathogenesis of heart involvement in EDMD is, however, unknown. Autoimmune mechanisms have also to be taken into account. The aim of this study was to search for the presence of anti-heart antibodies in EDMD patients. The anti-heart auto-antibodies were detected in serum of 14 EDMD patients (the X-linked and the AD-Autosomal Dominant form). The control groups comprised 10 patients with Dilated Cardiomyopathy (DCM) and 10 healthy subjects. To screen serum for anti-heart antibodies against ventricular muscle proteins, they were separated by polyacrylamide gel electrophoresis, followed by Western blotting. In EDMD and DCM, IgG anti-heart antibodies against heart ventricular proteins were detected. In both diseases, 85 kD protein appeared to be the most immunogenic. Anti-troponin I (24 kD), anti-tropomyosin (35 kD) and anti-actin (43 kD) reactivity was less intense. There were significant differences in the reactivity of auto-antibodies between both EDMD forms, and also between EDMD and the DCM patients. No clear-cut correlation between the reactivity and frequency of the antibodies and clinical parameters of the EDMD patients was detected. The anti-heart proteins are reliable markers of immune involvement in dilated cardiomyopathy in the course of EDMD. Short- and long-term follow-up may define the role of anti-heart antibodies in predicting the susceptibility at risk of dilated cardiomyopathy in EDMD patients.

[Na+ + K+] Mg2+-ATPase of muscle plasma membranes in Duchenne muscular dystrophy.

The activity of [Na+ + K+] Mg2+-ATPase of muscle surface membrane was investigated in 20 cases of the Duchenne type of progressive muscular dystrophy; it was found to be diminished and to have a changed reactivity to ouabain. There was nothing like it in cases of limb-girdle dystrophy and neurogenic muscular atrophies investigated for the purpose of comparisons, whereas in some cases of myotonic dystrophy and myotonia congenita the activity of the ATPase was indeed depressed, but the response to ouabain invariably remained normal.

Elevated calmodulin levels and reduced calmodulin-stimulated calcium-ATPase in Duchenne progressive muscular dystrophy.

We determined the calmodulin concentration and Ca2+-ATPase activity in subcellular fractions recovered from samples of vastus lateralis muscle obtained from 18 patients with Duchenne muscular dystrophy, 10 patients with other primary myopathies, 5 with spinal muscular atrophy, and 16 age-matched controls. Calmodulin levels were increased in the cytosol, plasmalemma, and heavy sarcoplasmic reticulum fractions from Duchenne dystrophy patients; the greatest increases occurred at early stages of disease or in mildly progressive cases. The total Ca2+-ATPase activities were decreased in the Duchenne dystrophy muscles; calmodulin caused a minimal stimulation of the activity in calmodulin-depleted membranes from Duchenne dystrophy compared with control membranes. The changes in calmodulin concentration and Ca2+-ATPase activity complement previous observations of reduced calsequestrin and dystrophin concentrations in Duchenne dystrophy muscles and suggest that these muscles lose calcium regulatory functions at early stages of the disease process.

Lipid storage myopathy in Kearns-Sayre syndrome.

A 7-year-old girl had external ophthalmoplegia, limb weakness, short stature, hearing loss, pigmentary degeneration of the retina, and increased CSF protein content. Muscle biopsy revealed vacuolar myopathy with accumulation of lipids. Electronmicroscopy showed abnormalities of shape, size, and internal structure of muscle mitochondria. Muscle activity of palmitoyl-CoA synthetase was decreased, and the content of lipids was increased. Serum and muscle carnitine levels were normal, as were muscle carnitine palmitoyltransferase and carnitine acetyltransferase.

Fluorescent probe analysis of muscle plasmalemma in Duchenne's progressive muscular dystrophy.

The relationship between fluorescence intensity and binding of 1-anilino-naphthalene-8-sulphonate (ANS) to muscle plasmalemma in patients with Duchenne's muscular dystrophy (DD) and controls was studied. The fluorescence of ANS was markedly enhanced in DD as compared with controls. The magnitude of this enhancement was increased by monovalent and divalent cations; treatment of DD plasmalemma with trypsin caused an opposite effect. Treatment with phospholipase A and C altered the ANS fluorescence in DD and controls equally. These findings may indicate an increase of the hydrophobic character in the apolar-polar interface of DD plasmalemma. The relationship of these changes to a lack of dystrophin in DD remains to be established.

Erythrocyte ghosts (Na+ + K+) ATPase activity in Duchenne's dystrophy and myotonia.

In Duchenne muscular dystrophy the activity of (Na+ + K+)ATPase in erythrocyte ghosts is reduced and its reaction to ouabain is paradoxical both in low sodium and high sodium systems. No such changes were seen in a case of Becker dystrophy, in limb-girdle dystrophy, and in neurogenic atrophy of muscles. In myotonic dystrophy and congenital myotonia the activity of ATPase and its inhibition by ouabain were depressed.

The distribution of inside-out and right-side-out erythrocyte membrane vesicles in Duchenne progressive muscular dystrophy.

In ten cases of Duchenne muscular dystrophy, the distribution of erythrocyte ghost vesicles in dextran 110 gradient was examined. When compared with controls a greater number of inside-out vesicles was observed. It is suggested that the tendency to form abnormally oriented vesicles could result from structural abnormalities of the erythrocyte membranes.

Immunoblot analysis of sarcoplasmic calcium binding proteins in Duchenne muscular dystrophy.

The Western blotting technique was used to detect parvalbumin and S-100 protein in muscles from 10 Duchenne muscular dystrophy (DD) patients, 13 patients with other muscle diseases and 5 age-matched healthy subjects. DD muscles were found to contain decreased amounts of parvalbumin and the S-100 protein. The parvalbumin level did not relate to the age of the patients and the stage of the disease. The S-100 protein decreased progressively with the age of the patients. In a very advanced DD case the S-100 protein was present in trace amounts. In other primary myopathies, including Becker dystrophy, and neurogenic muscular atrophy both parvalbumin and S-100 protein levels were similar to that observed in healthy subjects. The decrease in the amount of both calcium binding proteins may contribute to the elevation of free intracellular Ca2+ level in the sarcoplasm of dystrophic muscle and would result in abnormalities in processes regulated by these proteins. The mechanism(s) responsible for the decrease of parvalbumin and S-100 protein in DD muscles are discussed.

Plasma membranes of muscle in experimental myotonia in rats.

Determinations of protein and phospholipid composition, as well as enzymatic activity, were carried out in plasma membranes isolated from the muscle of rats, after different periods of 20,25-diazacholesterol administration. A decrease in the level of phospholipids, and in the total amount of plasma membrane proteins, connected with a relative reduction in the amount of protein of a molecular weight of 100000 daltons, was found. The activity of (Na+ + K+)-ATP-ase gradually decreased while a reverse tendency was observed in the case of 5'-nucleotidase. Changes in ATP-ase and phospholipids appeared even prior to electrophysiologically recorded signs of the myotonia. The mechanism of these changes and their possible role in myotonia are discussed.

Sarcoplasmic reticulum in experimental myotonia in rats.

Determinations of enzymatic activity, protein structure and phospholipid composition of sarcoplasmic reticulum isolated from the soleus muscle (S), extensor digitorum longus muscle (EDL) and gastrocnemius muscle (G) in rats were carried out after various periods of 20,25-diazacholesterol administration. The sarcoplasmic reticulum from G and EDL of myotonic rats exhibited a rise in basal ATP-ase activity and a fall of total phospholipids. The protein of molecular weight of 100000 daltons in G and EDL was slightly more pronounced.

Is central core disease with structural core a fetal defect?

Morphological and biochemical studies were performed in three cases of congenital non-progressive myopathy in two generations of the same family. In the muscle biopsy nearly all the fibres were uniform in enzyme activity and belonged to type 2 C. Typical structural central cores were observed in 90% of the muscle fibres. Some ultrastructural characteristics of the core area, as well as disturbances of the myofibrillar proteins pattern, seen in the examined cases suggest that core formation may be a result of protein synthesis disturbances in an early stage of myogenesis.

Myofibrillar protein pattern in experimental myotonia in rats.

Experimental myotonia was induced in rats by long-term administration of 20,25-diazacholesterol. Electrophysiological, morphological and biochemical investigations were carried out on m. soleus, m. extensor digitorum longus and m. gastrocnemius. The effect of 20,25-diazacholesterol administration on myofibrillar proteins was studied and a significant rise in the concentration of a protein presumed to be alpha-actinin was demonstrated in m. gastrocnemius. A change of the same character, not statistically significant, was observed in the m. extensor digitorum longus.

Application of an exponential curve equation for calculation of serum creatine kinase activity.

A four-point scale for determination of serum creatine kinase activity is suggested and the application of an exponential curve equation is proposed for cases with this enzyme activity exceeding 177 I.U. The introduction of this modification permits the activity of creatine kinase to be determined in high-activity serum without diluting the serum before determination since this is a source of error in certain muscular diseases.

Expression of emerin and lamins in muscle of patients with different forms of Emery-Dreifuss muscular dystrophy.

Emerin and lamins are nuclear proteins, which are missing or defective in Emery-Dreifuss muscular dystrophy (EDMD). The aim of this study was to test the expression of these proteins in skeletal muscles in the X-linked (X-EDMD) and autosomal dominant (AD-EDMD) form. The study group consisted of 11 patients with X-EDMD, 11 patients of the AD-EDMD and 20 age-matched normal subjects. Expression of emerin and lamins in muscles were analyzed by Western blotting and the immunocytochemical technique. Using the Western blotting procedure emerin was detected in traces in the X-linked form. In the majority of these cases (6/11) it was connected with a decreased concentration of lamin A, in four patients a lowered concentration of lamin C was present. Lamin B2 was either normal (8/11), or decreased (3/11). Deficit of lamin A was a characteristic feature for AD-EDMD in the majority of these cases (9/11), while in two of these patients a decrease of lamin C, in four cases a lowered level of emerin was also present. In one AD-EDMD patient of a decrease of lamin C, but normal lamin A was present. Following the immunocytochemical examination the decrease of lamin A/C in X-EDMD and of emerin in AD-EDMD was also observed. The above mentioned data demonstrated that in X-EDMD and AD-EDMD the deficit of the appropriate proteins is not restricted either to emerin or lamins. The defect is more widespread and results in disruption of several nuclear proteins. This study also indicated that for the diagnostic EDMD purposes the immunocytochemical detection of emerin/lamins has to be accomplished by quantitative immunochemical analyses of the above mentioned proteins.

Cytotoxic activity of serum and cerebrospinal fluid of amyotrophic lateral sclerosis (ALS) patients against acetylcholinesterase.

The activity of acetylcholinesterase (AChE) was tested in serum of 20 cases of amyotrophic lateral sclerosis (ALS), 4 "disease controls" and 20 age-matched healthy normals. The AChE activity has been tested also in cerebrospinal fluid (CSF) of 20 ALS patients, 2 "disease controls" and 10 normal subjects. An increase in serum AChE was present in the majority of ALS patients with a mild course of the disease, in the severe ALS group elevated serum AChE activity was a rare finding. Serum ACHE was also increased in multifocal motor neuropathy (MMN). In the majority of mild and severe ALS the CSF AChE activity was decreased. No AChE changes were found in CSF of the "disease controls". Serum and CSF ultrafiltrates of ALS patients and "disease controls" were modifying in vitro the spinal cord AChE activity. In the mild ALS group serum and CSF ultrafiltrates with high molecular weight compounds were decreasing the AChE activity. On the other hand in the severe ALS group serum and CSF ultrafiltrates with low molecular weight compounds were increasing the AChE activity. AChE was modified also in some of the "disease controls", especially in MMN and Guillain-Barré syndrome (GBS) by serum ultrafiltrates containing high molecular weight compounds. The AChE activity in serum and CSF is the consequence of the enzyme leakage from brain, degenerating cholinergic neurons and neuromuscular junctions. We suggest that because of the evoked peripherally divergent changes of the enzyme activity, the AChE values in serum and CSF in ALS do not equal to the degree of the changes in the affected tissues and cannot be taken into account in the prognosis of the disease in particular ALS cases.