CSF filtration is an effective treatment of Guillain-Barré syndrome: a randomized clinical trial.

OBJECTIVE: To compare CSF filtration (CSFF) and plasma exchange (PE) in the treatment of patients with Guillain-Barré syndrome (GBS). METHODS: In a prospective controlled clinical trial, 37 patients with acute GBS were randomized to receive either CSFF or PE. Inclusion criteria were fulfillment of National Institute of Neurological and Communicative Disorders and Stroke criteria and disability to walk >5 m unassisted. RESULTS: With similar baseline features in both groups (initial disability grades on the six-point grading scale of the GBS Study Group) the primary outcome variable (improvement within 28 days after randomization) was almost identical (test for equivalence p = 0.0014), the mean grade values being 0.82 in the CSFF group and 0.80 in the PE group. After 56 days, 56% (9 of 16 patients) of the CSFF group and 37% (7 of 19 patients) of the PE group had reached grade 2 (i.e., ability of unassisted walking >5 m). After 6 months, the probability to reach grade 2 was about 80% in both groups. In the CSFF group, transient pleocytosis occurred without apparent clinical complications. Clinically relevant complications were higher in the PE-treated group. CONCLUSIONS: Although the number of patients was small, the authors found that the treatment of GBS with CSFF is at least as effective as with PE. CSFF might work by removing from the CSF inflammatory mediators, autoantibodies, or other factors.

Optical detection of mitochondrial NADH content in intact human myotubes.

Time-gated fluorescence spectroscopy in combination with non-radiative energy transfer was used on intact human skeletal myotubes for the determination of the mitochondrial NADH content which is considered to be a sensitive indicator of mitochondrial function. To mimic dysfunction of the mitochondrial energy metabolism, complexes I or III of the respiratory chain were inhibited by drugs. In the absence of the fluorescent mitochondrial marker rhodamine (R123), the NADH autofluorescence (i.e. a signal monitoring cytoplasmic plus mitochondrial NADH) remained unchanged upon inhibition of complex I by rotenone, and was increased by a factor of 2 upon inhibition of complex III by antimycin. In the presence of R123, the autofluorescence of NADH was reduced indicating non-radiative energy transfer from NADH to R123. The ratio of the R123 fluorescence signals obtained with the two excitation wavelengths of 355 nm and 488 nm was taken as a measure of mitochondrial NADH. Relative NADH changes were estimated in the presence of the above-mentioned inhibitors. Upon complex I inhibition, mitochondrial NADH was increased by a factor of 1.5. Upon inhibition of complex III, mitochondrial NADH was increased by a factor of 2. We conclude that time-gated spectroscopy combined with non-radiative energy transfer is an appropriate tool for probing mitochondrial enzyme complex deficiencies.

Cellular uptake and efficacy of antisense oligonucleotides against RNAs of two Na(+) channel isoforms.

The effects of 15-mer phosphorothioate antisense oligodeoxynucleotides (aODNs) specifically designed against the RNAs of either of two closely related Na(+) channel isoforms, hSkM1 or hH1, were tested in human myotubes. Fluorescence (3'-fluorescein isothiocyanate) labeling showed that mere incubation of cultures with aODNs did not result in aODN uptake, but liposome-mediated transfer was successful and resulted in cytoplasmic and nuclear localization of ODNs. Intracellular fluorescence was stable for at least 3 days. At 5 microM, the hH1-specific aODN was effective in suppressing ion channel function, but the hSkM1-specific aODN was not. Reverse transcription-polymerase chain reaction gave corresponding results on the mRNA level. However, in HEK-293 cells stably expressing hSkM1, the same hSkM1-specific aODN was able to reduce Na(+) currents (2.4 +/- 0.5 nA, n = 11; controls: 6.5 +/- 1.0 nA, n = 14). We conclude that cellular uptake of aODNs and intracellular access to the RNA target are necessary, but not always sufficient conditions for an effective block of mRNA translation in intact cells.

Anti-GM1 antibodies can block neuronal voltage-gated sodium channels.

Anti-GM1 antibodies, frequently found in the serum of patients with Guillain-Barré syndrome (GBS), have been suggested to interfere with axonal function. We report that IgG anti-GM1 antibodies, raised in rabbits, can reversibly block the voltage-gated Na(+) channels of nerve cells, thus causing a reduction of the excitatory Na(+) current. The block was, however, only substantial when the antibodies were applied together with rabbit complement factors. A solution containing anti-GM1 sera (dilution 1:100) and complement (1:50) reduced the Na(+) current to 0.5 +/- 0.2 times control (mean value +/- SD). Applications of the antibody by itself, complement by itself, or anti-GM2 or anti-GM4 antibodies (1:100) plus complement had little effect. The complexes of anti-GM1 antibodies and complement factors block the ion-conducting pore of the channel directly. In addition, they increase the fraction of channels that are inactivated at the resting potential and alter channel function by changing the membrane surface charge. The described effects may be responsible for conduction slowing and reversible conduction failure in some GBS patients.

Calcium ion in skeletal muscle: its crucial role for muscle function, plasticity, and disease.

Mammalian skeletal muscle shows an enormous variability in its functional features such as rate of force production, resistance to fatigue, and energy metabolism, with a wide spectrum from slow aerobic to fast anaerobic physiology. In addition, skeletal muscle exhibits high plasticity that is based on the potential of the muscle fibers to undergo changes of their cytoarchitecture and composition of specific muscle protein isoforms. Adaptive changes of the muscle fibers occur in response to a variety of stimuli such as, e.g., growth and differentition factors, hormones, nerve signals, or exercise. Additionally, the muscle fibers are arranged in compartments that often function as largely independent muscular subunits. All muscle fibers use Ca(2+) as their main regulatory and signaling molecule. Therefore, contractile properties of muscle fibers are dependent on the variable expression of proteins involved in Ca(2+) signaling and handling. Molecular diversity of the main proteins in the Ca(2+) signaling apparatus (the calcium cycle) largely determines the contraction and relaxation properties of a muscle fiber. The Ca(2+) signaling apparatus includes 1) the ryanodine receptor that is the sarcoplasmic reticulum Ca(2+) release channel, 2) the troponin protein complex that mediates the Ca(2+) effect to the myofibrillar structures leading to contraction, 3) the Ca(2+) pump responsible for Ca(2+) reuptake into the sarcoplasmic reticulum, and 4) calsequestrin, the Ca(2+) storage protein in the sarcoplasmic reticulum. In addition, a multitude of Ca(2+)-binding proteins is present in muscle tissue including parvalbumin, calmodulin, S100 proteins, annexins, sorcin, myosin light chains, beta-actinin, calcineurin, and calpain. These Ca(2+)-binding proteins may either exert an important role in Ca(2+)-triggered muscle contraction under certain conditions or modulate other muscle activities such as protein metabolism, differentiation, and growth. Recently, several Ca(2+) signaling and handling molecules have been shown to be altered in muscle diseases. Functional alterations of Ca(2+) handling seem to be responsible for the pathophysiological conditions seen in dystrophinopathies, Brody's disease, and malignant hyperthermia. These also underline the importance of the affected molecules for correct muscle performance.

An endogenous pentapeptide acting as a sodium channel blocker in inflammatory autoimmune disorders of the central nervous system.

Reversible blockade of sodium channels by endogenous substances has been claimed to account for the fast exacerbations and relapses commonly seen in demyelinating autoimmune diseases. Evidence has been provided that in the cerebrospinal fluid of patients with multiple sclerosis or Guillain-Barré syndrome, a sodium-channel-blocking factor exists that has properties of local anesthetic agents. This factor could contribute to the nerve conduction block and paresis seen in these disorders. We describe here a previously unknown endogenous substance in human cerebrospinal fluid with distinct channel-blocking properties even at very low (0.00001 M) concentrations. The pentapeptide with the sequence Gln-Tyr-Asn-Ala-Asp exerted its blocking action by shifting the steady-state inactivation curve of the sodium channels to more-negative potentials, as most local anesthetics do. In the cerebrospinal fluid of healthy individuals, its concentration was about 3 microM, whereas in patients with multiple sclerosis and Guillain-Barré syndrome, it increased 300-1,400%. At these concentrations, the peptide's blocking efficacy was higher than that of 50 microM lidocaine. At a concentration of 10 microM, lidocaine is able to 'unmask' subclinical lesions in multiple sclerosis; thus, the endogenous pentapeptide may well contribute to the fast changes of symptoms. Furthermore, it may become valuable as a marker of disease activity.

The small sodium-channel blocking factor in the cerebrospinal fluid of multiple sclerosis patients is probably an oligopeptide.

An endogenous factor that is able to reduce the fast transient sodium current of excitable cells has been reported to exist in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients. This was confirmed with nine clinically definite MS patients in the acute relapse. In order to purify and chemically identify the factor, microconcentration and gel filtration high-performance liquid chromatography (HPLC) were applied. After each purification step the activity-containing fraction was determined using a biological assay. With all CSFs the activity was contained in the fraction corresponding to 600-800 Da molecular weight, indicating that the factor is chemically homogeneous. The biological activity of the CSF specimens was not correlated to the laboratory CSF data; however, it was correlated to the area under the 210 nm UV light absorption peak in the corresponding chromatogram, i.e. the 600-800 Da MW fraction. As the factor was degradable by acid hydrolysis and a carboxypeptidase, it is suggested that it might be a small peptide.

A small sodium channel blocking factor in the cerebrospinal fluid is preferentially found in Guillain-Barré syndrome: a combined cell physiological and HPLC study.

The cerebrospinal fluid (CSF) of patients with Guillain-Barré syndrome (GBS) contains a low molecular weight factor with sodium channel blocking activity. This study investigated whether such activity also exists in the CSF of patients with other neurological diseases. Further, using high-performance liquid chromatography (HPLC) we tested whether the electrophysiological effect of the CSF is correlated with the size of the corresponding peak in the chromatograms. The existence of sodium channel blocking activity was tested in 27 native CSF samples of three groups of patients (group 1: GBS, n = 13; group 2: other inflammatory diseases, n = 8; group 3: controls, n = 6). NH15-CA2 neuroblastoma x glioma cells in the whole-cell recording configuration was used as a system for assaying the sodium channel blocking activity of CSF specimens. CSF shifted the steady-state inactivation curve of the sodium channels reversibly by -10.2 +/- 4.4 mV in group 1, -6.7 +/- 3.9 mV in group 2, and - 3.5 +/- 2.8 mV in group 3 (P < 0.01). The shift was greater in demyelinating (9.3 +/- 4.7 mV) than in nondemyelinating (5.6 +/- 3.9 mV) diseases (P < 0.04). HPLC analysis of CSFs showed a well separated peak containing the substance responsible for the electrophysiological effect at about 41 min elution time. The peak covered the molecular weight range of 600-800 Da. Sodium channel blocking activity of CSFs and areas of the corresponding peak in the chromatograms were well correlated. We conclude that sodium current inhibition by a low molecular weight factor is generally present but increased in GBS.

A transient and a persistent calcium release are induced by chlorocresol in cultivated mouse myotubes.

The effect of 4-chloro-m-cresol (4-CmC), a stabilizing agent used in commercial preparations of the muscle relaxant succinylcholine, on intracellular free calcium levels in cultivated mouse myotubes was studied. Calcium signals were monitored with an inverted microscope equipped for fluorescence photometry using fura-2 as the calcium indicator. Upon bath application of 500 microM 4-CmC for 90 s, two separate calcium signals, a transient and a sustained one, could be regularly discriminated. First, with a delay of 2 s, the intracellular calcium concentration increased from 41+/-13 to 541+/-319 nM, peaked after 2-5 s and declined within 10 s to nearly resting values (n=36). Then, after a delay of up to 20 s, intracellular calcium rose quickly again to almost the same value and stayed elevated as long as the drug was applied. Upon drug removal, intracellular calcium rapidly decreased to a new level that was always slightly higher than the original base line. At 250 microM 4-CmC, the response was small, whereas at 500 microM it was at its maximum. Thus, the concentration-response curve was very steep. Replacement of extracellular calcium by EGTA and application of calcium channel blockers revealed that, for both the transient and the sustained response, calcium was released from intracellular stores. Pre-treatment with thapsigargin (0.1 microM) or ryanodine (10 microM) abolished both signal components. Repeated short-term applications of 4-CmC suggest that the two components may arise from different systems.

Increased calcium entry into dystrophin-deficient muscle fibres of MDX and ADR-MDX mice is reduced by ion channel blockers.

1. Single fibres were enzymatically isolated from interosseus muscles of dystrophic MDX mice, myotonic-dystrophic double mutant ADR-MDX mice and C57BL/10 controls. The fibres were kept in cell culture for up to 2 weeks for the study of Ca2+ homeostasis and sarcolemmal Ca2+ permeability. 2. Resting levels of intracellular free Ca2+, determined with the fluorescent Ca2+ indicator fura-2, were slightly higher in MDX (63 +/- 20 nM; means +/- s.d.; n = 454 analysed fibres) and ADR-MDX (65 +/- 12 nM; n = 87) fibres than in controls (51 +/- 20 nM; n = 265). 3. The amplitudes of electrically induced Ca2+ transients did not differ between MDX fibres and controls. Decay time constants of Ca2+ transients ranged between 10 and 55 ms in both genotypes. In 50 % of MDX fibres (n = 68), but in only 20 % of controls (n = 54), the decay time constants were > 35 ms. 4. Bath application of Mn2+ resulted in a progressive quench of fura-2 fluorescence emitted from the fibres. The quench rate was about 2 times higher in MDX fibres (3.98 +/- 1.9 % min-1; n = 275) than in controls (2.03 +/- 1.4 % min-1; n = 204). The quench rate in ADR-MDX fibres (2.49 +/- 1.4 % min-1; n = 87) was closer to that of controls. 5. The Mn2+ influx into MDX fibres was reduced to 10 % by Gd3+, to 19 % by La3+ and to 47 % by Ni2+ (all at 50 microM). Bath application of 50 microM amiloride inhibited the Mn2+ influx to 37 %. 6. We conclude that in isolated, resting MDX muscle fibres the membrane permeability for divalent cations is increased. The presumed additional influx of Ca2+ occurs through ion channels, but is well compensated for by effective cellular Ca2+ transport systems. The milder dystrophic phenotype of ADR-MDX mice is correlated with a smaller increase of their sarcolemmal Ca2+ permeability.

Malignant hyperthermia causing Gly2435Arg mutation of the ryanodine receptor facilitates ryanodine-induced calcium release in myotubes.

We have investigated if cultivated muscle cells from malignant hyperthermia (MH) patients can be distinguished pharmacologically from controls. Muscle specimens from four individuals carrying the Gly2435Arg mutation of the skeletal muscle ryanodine receptor protein (RYR1) and from four controls were used to culture myotubes. Resting intracellular calcium concentration ([Ca2+]i) of MH myotubes was similar to controls. However, when ryanodine 0.5 mumol litre-1 was added, the kinetics of the increase in the calcium signals in MH and control cells were significantly different; the time for half maximum increase was mean 197 (SD 131) s for MH cells and 474 (61) s for controls (n = 80 cells each). On average, the area under the MH response curves was twice the control value. These results give rise to hopes that the phenotype of MH can be characterized using cultured human muscle and that a culture-based test for MH susceptibility may eventually be developed.

Myotonic ADR-MDX mutant mice show less severe muscular dystrophy than MDX mice.

In Duchenne muscular dystrophy (DMD) and its murine model, the dystrophic mouse (MDX), the skeletal musculature lacks dystrophin. The presumed function of this cytoskeletal protein is to protect the sarcolemma against mechanical stress during muscle activity. To test this hypothesis in vivo, we bred a double mutant mouse that combines two genetic defects: the dystrophin-deficiency of the MDX mouse and the Cl- channel myotonia of the arrested development of righting response (ADR) mouse. We hypothesized that high mechanical muscle activity would aggravate muscular dystrophy in double mutant ADR-MDX mice. On the contrary, ADR-MDX mice showed fewer signs of muscle fiber necrosis and fibrosis than MDX mice at all ages. Plasma creatine kinase levels were slightly increased in ADR-MDX, but significantly lower when compared to MDX mice. Sections of ADR-MDX muscle showed a uniform pattern of oxidative muscle fibers. Similar findings have been obtained in dystrophin-positive ADR mice, they result from a complete fiber-type IIB to IIA transformation in myotonic muscle. Our results suggest that small, oxidative fibers of myotonic mice are less sensitive to dystrophin deficiency. Therefore, ADR-MDX mice develop less severe muscular dystrophy than MDX mice do, although their muscles are continually stressed. The new ADR-MDX double mutant mouse is the first animal model combining both a dystrophinopathy and a channelopathy. The results presented here give new insights into the pathomechanism of muscular dystrophy and may be helpful for the therapeutic management of DMD.

Intracellular calcium chelator BAPTA protects cells against toxic calcium overload but also alters physiological calcium responses.

The effect of the membrane-permeant calcium chelator 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM) on ionomycin-induced cellular calcium overload was studied in single differentiated NH15-CA2 neuroblastoma x glioma hybrid cells. To monitor [Ca2+]i we used the fluorescent indicator Fura-2. Preincubation of the cells with 3 microM BAPTA/AM reduced the number of cells showing deregulation of [Ca2+]i during ionomycin-induced calcium influx. The calcium transients elicited by application of KCl were also severely affected by the chelator. These transients, although varying from cell to cell in shape, amplitude and duration, are well reproducible in individual cells. After incubation of cells for 1 h with 0.3-30 microM BAPTA/AM the time course of these cellular transients was markedly slowed. At 1 microM BAPTA/AM, the time constant of decline of [Ca2+]i was increased by a factor of 4.1 +/- 2.4 (n = 14) and the amplitude was reduced to about 50%. With 30 microM BAPTA/AM, the K(+)-induced calcium transients were almost completely inhibited. We conclude that intracellularly loaded calcium chelators may be used for the prevention of [Ca2+]i-induced cell damage, however, at the expense of a disturbed calcium signalling.

Antisense oligonucleotides discriminating between two muscular Na+ channel isoforms.

Various 15-mer antisense oligodeoxynucleotides (aODNs) were constructed against RNAs coding for two closely related isoforms of the voltage-dependent Na+ channel, i.e. those of human heart (hH1) and skeletal (hSkM1) muscle. When translated in vitro, either RNA yielded a 220 kDa band on polyacrylamide gels, indicating that the translation product had full length. Of six different aODN constructs developed against hH1 RNA, two each inhibited translation completely, moderately or not at all, depending on the target position. The specificity of the effect (no cross reaction at 10 microM) was confirmed by incubation with 15-mer aODNs against hSkM1 RNA. The most effective aODNs were those hybridizing between bases 3840 and 3880 of hSkM1 RNA and the homologous segment of hH1 RNA. When either of the RNAs was co-injected with its most effective (phospho rothioate-capped) aODN into Xenopus oocytes, the production of Na+ channels was strongly suppressed (relative INa for hSkM1: 0.08 +/- 0.05 times control, n = 14; for hH1: 0.11 +/- 0.08, n = 11). We conclude that aODNs are able to discriminate between closely related RNAs. The efficacy of an aODN depends strongly on its RNA target position.

Excitatory sodium currents of NH15-CA2 neuroblastoma x glioma hybrid cells are differently affected by interleukin-2 and interleukin-1beta.

The neuroblastoma x glioma hybrid cell line NH15-CA2 was used to test the effects of two interleukins on neuronal Na+ channels. The cells were cultured in the presence of dibutyryl cAMP and retinoic acid, which yielded a high expression of Na+ channels so that the cells were excitable. Na+ currents were triggered and recorded in the whole-cell recording mode. Comparison of the effects of tetrodotoxin and mu-conotoxin established that the expressed Na+ channels were of the neuronal type. Bath-applied recombinant human interleukin-2 (rIL-2) had a reversible inhibitory effect on the Na+ currents, although to a lesser extent than on muscular Na+ currents (more than 1000 U/ml required for 50% block in NH15-CA2 cells vs 500 U/ml in myoballs). The current/voltage relationship was not affected by the presence of rIL-2, but the steady-state inactivation curve was shifted by -7.7 +/- 4.8 mV (mean +/- SD, n=18). Recombinant human interleukin-1beta (rIL-1beta), applied at 1000 U/ml, showed an inhibitory effect on the Na+ currents in about one-third of the cells tested. The mechanism of inhibition was different from that of rIL-2, as rIL-1beta seemed to cause a block without affecting voltage dependence or kinetics of the channels.

The human endogenous local anesthetic-like factor (ELLF) is functionally neutralized by serum albumin.

The cerebrospinal fluid (CSF) of patients with multiple sclerosis or Guillain-Barré syndrome contains a factor that inhibits excitation of nerve and muscle cells like local anesthetics. CSF samples containing the endogenous local anesthetic-like factor (ELLF) were analyzed by gel filtration chromatography and ultraviolet (UV) absorption at 210 nm. The active component was in a single peak corresponding to a molecular weight of 600-800 Da. This peak was decreased and the Na+ channel blocking activity was neutralized by the addition of 40 g/l human serum albumin to the CSF. When the albumin was separated from the CSF/albumin mixture by acetonitrile treatment, the Na+ channel blocking activity reappeared. The ELLF and its neutralization may be of relevance for the clinical fluctuations known with these diseases.

Ether lipid-induced cell damage of neuroblastoma cells is only weakly correlated with increased intracellular Ca2+ levels.

The cell-damaging action of the ether lipid ET-18-OCH3 was studied in single NH15-CA2 neuroblastoma x glioma hybrid cells using light microscopy, and correlated with changes of the free intracellular calcium concentration, [Ca2+]i, as measured with the fluorescent Ca2+ indicator Fura-2. Addition of 3-100 microM ET-18-OCH3 to the cultures caused disintegration of neurites, cell rounding and detachment of the cells from the bottom of the culture dish. The effects occurred within 30-240 min, the faster, the higher the ET-18-OCH3 concentration. Also [Ca2+]i increased in a concentration-dependent manner, however, within seconds, and stayed high during the recording time of 20 min. The presence of 50 microM lanthanum or gadolinium ions prevented the ET-18-OCH3-induced increases of [Ca2+]i, but had no effect on neurite destruction and cell rounding. Preincubation with 1 mM diisopropylfluorphosphate or 100 microM leupeptin, both membrane-permeant inhibitors of intracellular proteases, did not prevent the effects. Nor was neurite destruction prohibited in the presence of 10 microM of the actin-stabilizing agent phalloidin or 2 microM taxol, a microtubule-stabilizer. We conclude that [Ca2+]i, although being increased during ET-18-OCH3-induced cell damage, is not the key factor of cell destruction.