Dopaminergic and serotonergic alterations in plasma in three groups of dystonia patients.

INTRODUCTION: In dystonia, dopaminergic alterations are considered to be responsible for the motor symptoms. Recent attention for the highly prevalent non-motor symptoms suggest also a role for serotonin in the pathophysiology. In this study we investigated the dopaminergic, serotonergic and noradrenergic metabolism in blood samples of dystonia patients and its relation with (non-)motor manifestations. METHODS: Concentrations of metabolites of dopaminergic, serotonergic and noradrenergic pathways were measured in platelet-rich plasma in 41 myoclonus-dystonia (M-D), 25 dopa-responsive dystonia (DRD), 50 cervical dystonia (CD) patients and 55 healthy individuals. (Non-)motor symptoms were assessed using validated instruments, and correlated with concentrations of metabolites. RESULTS: A significantly higher concentration of 3-methoxytyramine (0.03 vs. 0.02 nmol/L, p < 0.01), a metabolite of dopamine, and a reduced concentration of tryptophan (50 vs. 53 µmol/L, p = 0.03), the precursor of serotonin was found in dystonia patients compared to controls. The dopamine/levodopa ratio was higher in CD patients compared to other dystonia groups (p < 0.01). Surprisingly, relatively high concentrations of levodopa were found in the untreated DRD patients. Low concentrations of levodopa were associated with severity of dystonia (rs = -0.3, p < 0.01), depression (rs = -0.3, p < 0.01) and fatigue (rs = -0.2, p = 0.04). CONCLUSION: This study shows alterations in the dopaminergic and serotonergic metabolism of patients with dystonia, with dystonia subtype specific changes. Low concentrations of levodopa, but not of serotonergic metabolites, were associated with both motor and non-motor symptoms. Further insight into the dopaminergic and serotonergic systems in dystonia with a special attention to the kinetics of enzymes involved in these pathways, might lead to better treatment options.

A metabolomic study of cervical dystonia.

INTRODUCTION: Cervical dystonia is the most common of the adult-onset focal dystonias. Most cases are idiopathic. The current view is that cervical dystonia may be caused by some combination of genetic and environmental factors. Genetic contributions have been studied extensively, but there are few studies of other factors. We conducted an exploratory metabolomics analysis of cervical dystonia to identify potentially abnormal metabolites or altered biological pathways. METHODS: Plasma samples from 100 cases with idiopathic cervical dystonia and 100 controls were compared using liquid chromatography coupled with mass spectrometry-based metabolomics. RESULTS: A total of 7346 metabolic features remained after quality control, and up to 289 demonstrated significant differences between cases and controls, depending on statistical criteria chosen. Pathway analysis revealed 9 biological processes to be significantly associated at p < 0.05, 5 pathways were related to carbohydrate metabolism, 3 pathways were related to lipid metabolism. CONCLUSION: This is the first large scale metabolomics study for any type of dystonia. The results may provide potential novel insights into the biology of cervical dystonia.

Clinical Implications of Difference in Antigenicity of Different Botulinum Neurotoxin Type A Preparations: Clinical Take-Home Messages from Our Research Pool and Literature.

The three different botulinum toxin type A (BoNT/A) preparations being licensed in Europe and the U.S. differ in protein content, which seems to be a major factor influencing the antigenicity of BoNT/A. In the present study, several arguments out of our research pool were collected to demonstrate that the clinical response and antigenicity were different for the three BoNT/A preparations: some results of (1) a cross-sectional study on clinical outcome and antibody formation of 212 patients with cervical dystonia (CD) being treated between 2 and 22 years; 2) another cross-sectional study on the clinical aspects and neutralizing antibody (NAB) induction of 63 patients having developed partial secondary treatment under abobotulinum (aboBoNT/A) onabotulinumtoxin (onaBoNT/A) who were switched to incobotulinumtoxin (incoBoNT/A) in comparison to 32 patients being exclusively treated with incoBoNT/A. These results imply that (1) the presence of NAB cannot be concluded from the course of treatment, that (2) an increase in the dose and variability of outcome with treatment duration indicates the ongoing induction of NABs over time, that (3) the higher protein load of BoNT/A goes along with a higher incidence and prevalence of NAB induction and that (4) the best response to a BoNT/A is also dependent on the protein load of the preparation.

High prevalence of neutralizing antibodies after long-term botulinum neurotoxin therapy.

OBJECTIVE: To investigate the prevalence of neutralizing antibodies (NAbs) against botulinum neurotoxin type A (BoNT/A) during long-term BoNT/A treatment in different neurologic indications. METHODS: In this monocentric, observational cross-sectional study, 596 outpatients treated with BoNT/A for different indications were tested for BoNT/A binding antibodies by ELISA. Positive samples were investigated for NAbs with the mouse hemidiaphragm test. The prevalence of NAbs was analyzed for different indications: facial hemispasm, blepharospasm, cervical dystonia, other dystonia, and spasticity. Besides the rate of NAb-positive patients overall and per patient subgroup, a Kaplan-Meier analysis of the probability of remaining NAb negative with duration of treatment is provided, and a stepwise binary logistic regression analysis is performed to identify factors significantly contributing to the induction of NAbs. RESULTS: Overall, 83 of 596 patients (13.9%) had measurable NAbs. The probability of developing NAbs increased with the single and cumulative dose of treatment and was influenced by the BoNT/A formulation, while all other factors analyzed, including disease entity and treatment duration, had no additional influence. CONCLUSIONS: We present the largest study to date of the prevalence of BoNT/A NAbs in a large unbiased cohort of patients including the relevant neurologic indications. Repeated injections of BoNT/A inevitably bear the risk of developing NAbs. However, in addition to avoiding booster injections and providing short intervals between injections, reducing the individual injected doses may diminish the risk of NAb induction independently of the indication for which BoNT/A is used.

[The efficacy of botulinotherapy in the correction of the pain syndrome and quality of life of patients with cervical dystonia]. / Éffektivnost' botulinoterapii v korrektsii stepeni bolevogo sindroma i kachestva zhizni patsientov s tservikal'noi distoniei.

AIM: To evaluate the severity of pain, emotional status and humoral serotonin in patients with cervical dystonia (CD) before and after the botulinotherapy. MATERIAL AND METHODS: A simple, open, comparative study of clinical characteristics of hyperkinesis, pain and emotional status, quality of life and contents of serum and blood platelet serotonin in 48 patients (32 women and 16 men) with CD, in age from 37 to 53 years, before and one month after the botulinotherapy with disport in dose of 500--1000 U was carried out. A control group included 15 healthy people. RESULTS: All patients (100%) complained of involuntary movements and pain in the neck. The overall score on a scale of dystonic movements in the group of patients was 16,7±7,7 points, on TWSTRS - 46,48±6,2 points, on the Visual Analogue Scale, the average level of pain was 6,4±1,08 points. The degree of depression according to the Hamilton scale was significantly higher (p<0.05) compared to the control group. The level of trait and state anxiety measured with the Spielberger-Khanin scale was significantly higher (p<0.005) in patients with CD than in the controls. The correlation analysis revealed a direct dependence of the intensity of pain subscale TWSTRS with the degree of anxiety on the Hamilton scale and the amount of final points of dystonic movements. The level of serotonin in the serum was significantly lower in patients compared to the controls. After botulinotherapy, pain scores, anxiety and depression have significantly decreased and the level of blood platelet serotonin has increased. CONCLUSION: Botulinotherapy with dysport in CD patients reduces the degree of pain, depression, improves quality of life and stimulates the serotoninergic system.

Torticollis in a haemophilic infant with inhibitor: a case of spinal epidural haematoma.

Central nervous system bleeding, which can be a life-threatening complication, is seen in 2.7% of patients with haemophilia. Spinal epidural haematomas represent about one-tenth of such cases. Here, we report on a 10-month-old boy with severe haemophilia A, who presented with torticollis. Although administration of factor VIII at a dose of 50 U/kg, the patient developed flaccid paralysis of the upper extremities. Factor VIII inhibitor screen was positive. Magnetic resonance imaging of the spine revealed spinal epidural haematomas, extending from C-1 to the cauda equina. Treatment was continued with recombinant activated factor VIIa without surgery. After 1 month, complete neurological recovery was achieved and fully resolved haematomas were detected on spinal MRI. A prompt radiological evaluation of the cervical spine with MRI should be made in patients with haemophilia presenting with torticollis. In addition, in the case of life-threatening bleeding in patients with haemophilia, the possibility of an inhibitor should be kept in mind.

Molecular immune recognition of botulinum neurotoxin B. The light chain regions that bind human blocking antibodies from toxin-treated cervical dystonia patients. Antigenic structure of the entire BoNT/B molecule.

We recently mapped the regions on the heavy (H) chain of botulinum neurotoxin, type B (BoNT/B) recognized by blocking antibodies (Abs) from cervical dystonia (CD) patients who develop immunoresistance during toxin treatment. Since blocking could also be effected by Abs directed against regions on the light (L) chain, we have mapped here the L chain, using the same 30 CD antisera. We synthesized, purified and characterized 32 19-residue L chain peptides that overlapped successively by 5 residues (peptide L32 overlapped with peptide N1 of the H chain by 12 residues). In a given patient, Abs against the L chain seemed less intense than those against H chain. Most sera recognized a limited set of L chain peptides. The levels of Abs against a given region varied with the patient, consistent with immune responses to each epitope being under separate MHC control. The peptides most frequently recognized were: L13, by 30 of 30 antisera (100%); L22, by 23 of 30 (76.67%); L19, by 15 of 30 (50.00%); L26, by 11 of 30 (36.70%); and L14, by 12 of 30 (40.00%). The activity of L14 probably derives from its overlap with L13. The levels of Ab binding decreased in the following order: L13 (residues 169-187), L22 (295-313), L19 (253-271), and L26 (351-369). Peptides L12 (155-173), L18 (239-257), L15 (197-215), L1 (1-19) and L23 (309-327) exhibited very low Ab binding. The remaining peptides had little or no Ab-binding activity. The antigenic regions are analyzed in terms of their three-dimensional locations and the enzyme active site. With the previous localization of the antigenic regions on the BoNT/B H chain, the human Ab recognition of the entire BoNT/B molecule is presented and compared to the recognition of BoNT/A by human blocking Abs.

A peptide-based immunoassay for antibodies against botulinum neurotoxin A.

Cervical dystonia (CD) is due to neck-muscle spasms that cause pain and involuntary contractions resulting in abnormal neck movements and posture. Symptoms can be relieved by injecting the affected muscle with a botulinum neurotoxin (BoNT, usually type A or type B). The therapeutic benefits are impermanent and toxin injections need to be repeated every 3-6 months. In a very small percentage of patients (less with BoNT/A than with BoNT/B) the treatment elicits blocking anti-toxin antibodies (Abs), which reduce or terminate the patient's responsiveness to further treatment. We have recently mapped (Dolimbek et al., 2006) the CD sera Ab-binding profile using a panel of 60, 19-residue peptides that encompassed the entire H chain sequence 449-1296 and overlapped consecutively by 5 residues. Abs in CD sera bound to one or more of the peptides N25, C10, C15, C20, and C31. This suggested the possibility that binding to these peptides could be used for assay of Abs in CD sera. Data analysis reported here found that Ab binding to these regions showed very significant deviations from the control responses. Of these four peptides, C10 showed the most significant level of separation between patient and control groups (p = 5 x 10(-7)) and the theoretical resolution (i.e., ability to distinguish CD patients from control, see full definition under 'Statistical analysis' in Methods), 84%, was about 4% higher than the least resolved response, C31 (p = 6 x 10(-6), resolution 80%). Since the amounts of Abs bound to a given peptide varied with the patient and not all the patients necessarily recognized all four peptides, there was the possibility that binding to combinations of two or more peptides might give a better discriminatory capability. Using two peptides, C10 plus C31, the resolution improved to 87% (p = 4 x 10(-8)). These two peptides appeared to compliment each other and negate the lower resolution of C31. Combination of three peptides gave resolutions that ranged from 85 (N25 + C15 + C31; p = 2 x 10(-7)) to 88% (C10 + C15 + C31; p = 1 x 10(-8)). Finally, using the data of all four peptides, N25 + C10 + C15 + C31, gave a resolution of 86% (p = 1 x 10(-7)). Although these levels of resolution are somewhat lower than that obtained with whole BoNT/A (resolution 97%; p = 6 x 10(-12)), it may be concluded that the two-peptide combination C10 + C31, or the three-peptide combination C10 + C15 + C31 (affording resolutions of 87 and 88%, respectively) provide a good diagnostic, toxin-free procedure for assay of total specific anti-toxin Abs in BoNT/A-treated CD patients.

Mapping of the regions on the heavy chain of botulinum neurotoxin A (BoNT/A) recognized by antibodies of cervical dystonia patients with immunoresistance to BoNT/A.

The purpose of this work was to map the entire recognition profile of the H chain of botulinum neurotoxin A (BoNT/A) by Abs in sera that have protective anti-BoNT/A Abs by the mouse protection assay (MPA) from cervical dystonia (CD) patients who had been treated with botulinum neurotoxin, serotype A (BOTOX). In previous studies we found that human anti-tetanus neurotoxin (TeNT) Abs cross-react with BoNT/A and BoNT/B. In the present work we devised an assay procedure for measuring specific anti-BoNT/A Abs in human sera by absorbing out or inhibiting the anti-TeNT Abs with TeNT before analyzing the sera for the anti-BoNT/A Abs. The sera were obtained from 28 CD patients who had become unresponsive to treatment with BoNT/A and the sera were found to protect mice against a lethal dose of BoNT/A. For localization of the Ab-binding regions on the H chain we employed a set of sixty, 19-residue synthetic peptides (except for peptide C31 which was 22 residues) that encompassed the entire H chain sequence 449-1296 and overlapped consecutively by five residues. The pattern of Ab recognition varied from patient to patient, but a very limited set of peptides were recognized by most of the patients. These were, in decreasing amounts of Ab binding, peptide N25 (H chain residues 785-803), C9/C10 (967-985/981-999), C31 (1275-1296), C15 (1051-1069), C20 (1121-1139), N16 (659-677), N22 (743-761), and N4 (491-509). But not every serum recognized all these peptides. The finding that the binding profile was not the same for all the patients is consistent with previous observations that immune responses to protein antigens are under genetic control and that the response to each epitope within a protein is under separate genetic control. Except for the region within C9/C10, the other regions either coincided (N16 and C31), or overlapped (N4, N22, N25, C15 and C20), with the recently mapped synaptosomes (snps)-binding regions on the H chain. The molecular and clinical implications of these findings are discussed.

Mouse diaphragm assay for detection of antibodies against botulinum toxin type B.

With the advent of a commercial preparation of botulinum toxin type B (BT-B) for treatment of cervical dystonia detection of antibodies against BT-B (BT-B-AB) becomes necessary. For this purpose, we carried out a mouse diaphragm assay (MDA) by continuous measurement of the twitch force of a mouse hemidiaphragm preparation elicited by electric stimulation of its phrenic nerve. After exposing the preparation to BT-B 3 ng/ml the time to half-maximal twitch force reduction (paralysis time [PT]) was 69 +/- 4 min (n = 25). Addition of sera from patients with antibodies against BT-A produced a PT of 68 +/- 5 min (n = 24), whereas addition of sera from controls with antibodies against tetanus toxoid produced a PT of 67 +/- 6 min (n = 30). When defined amounts of BT-B-AB were added to the MDA, PT was prolonged. This prolongation was correlated closely to the amount of BT-B-AB added, thus producing a calibration curve. The threshold for BT-B-AB detection was 0.4 mU/ml. When sera from 7 patients (4 women, 3 men; age 50.6 +/- 14.2 years) with cervical dystonia (Toronto Western Spasmodic Torticollis Rating Scale score, 18.9 +/- 2.9) and complete secondary failure of BT-B therapy (NeuroBloc; Elan Pharmaceuticals, Shannon, Ireland; 12,229 +/- 2,601 MU/injection series, 1.86 +/- 0.69 injection series before complete secondary therapy failure; 100.4 +/- 15.8 days between injection series with normal therapeutic effect) were tested, BT-B-AB titers of more than 10 mU/ml were found in all of them. The MDA can be used to measure neutralizing BT-B-AB titers quantitatively and with adequate sensitivity and specificity. Further studies are necessary to understand the role of intermediate BT-B-AB titers in partial BT-B therapy failure.

Comparison of efficacy and immunogenicity of original versus current botulinum toxin in cervical dystonia.

The authors compared 130 patients treated for cervical dystonia with original botulinum toxin (BTX) type A (Botox; Allergan, Inc., Irvine, CA), 42 of whom were exposed only to the original BTX type A used before 1998 (25 ng protein/100 units), and 119 treated only with the current BTX type A (5 ng of protein/100 units). Blocking antibodies were detected in 4 of 42 (9.5%) patients treated only with original BTX type A but in none of the 119 patients treated exclusively with current BTX type A (p < 0.004). The current preparation decreased the risk of antibody formation by a factor of six. The authors conclude that the low risk of antibody formation after current BTX type A treatment is related to lower protein load.

Sudomotor testing predicts the presence of neutralizing botulinum A toxin antibodies.

The increasing number of patients being treated with botulinum toxin A complex (BoNT/A) has led to a higher incidence of neutralizing anti-BoNT/A antibodies (ABAs). Because BoNT/A is known to inhibit sweating, here we report sudometry as a possibility for predicting the presence of ABA. Sixteen patients suffering from spasmodic torticollis were selected: in 2 patients, BoNT/A treatment continued to be effective, in 9 patients, the treatment effect was impaired, and in 5 patients, secondary treatment failure developed. BoNT/A (100 mouse units, Dysport; Ipsen Pharma, Berkshire, United Kingdom) was injected subcutaneously into the lateral calves. Sweating was visualized with iodine starch staining. In addition, quantitative sudomotor axon reflex testing was performed at the injection site. Individual ABA titers were determined with a mouse bioassay. Results of sudometry significantly correlated with the BoNT/A treatment success. The quantitative sudomotor axon reflex testing was 0.58 +/- 0.63 fraction of the normal mean in patients with treatment failure, 0.18 +/- 0.13 fraction of the normal mean in those who responded partially, and 0 in responders (p < 0.01). Accordingly, the areas of the anhidrotic skin after subcutaneous injections were 4.5 +/- 10.3 cm(2), 32.7 +/- 16.5 cm(2), and 62 cm(2) (p < 0.01). Discrimination analysis indicated that the presence of ABA (6 ABA-positive and 10 ABA-negative) could be predicted correctly in all patients from the results of sudometry. Therefore, sudometry is a useful tool for identifying patients with neutralizing ABAs and might be helpful for identifying reasons for BoNT/A treatment failure.

Neutralizing botulinum toxin type a antibodies: clinical observations in patients with cervical dystonia.

Neutralization of antibodies poses a problem for a substantial number of cervical dystonia (CD) patients treated with botulinum toxin type A (BoNT/A). Presence of these antibodies may lead to a secondary nonresponse to BoNT/A treatment. In this study, we compared 6 antibody-positive (Ab+) with 12 antibody- negative (Ab-) CD patients treated with BoNT/A (Dysport) and matched for du- ration of treatment, number of BoNT/A injections, and severity of clinical symptoms. The two groups differed in cumulative BoNT/A dose (Ab+, 5984 mouse units [MU ], SD = 3151 MU; Ab-, 3143 MU, SD =1294 MU; P <.05), in addition, ab+ patients were significantly younger (ab+ mean age = 41.3 y, sd =5.9 y; ab - mean age = 56.8 y, sd = 15.3 y; p <.05), in or- der to avoid formation of neutralizing antibodies, doses of bont/a should be kept as low as possible, the risk of antibody formation seems to be higher in younger patients.

Red cell choline in spasmodic torticollis and in a monozygotic twin pair with Tourette's syndrome.

1. Alterations in cholinergic function may play a role in the pathophysiology of idiopathic spasmodic torticollis (ST) and Gilles de la Tourette's syndrome (GTS). We measured red blood cell (RBC) choline in (i) ST (n = 24) and paired controls matched for age and gender (ii) a 20-year old pair of monozygotic twins with GTS, one of whom was moderately affected (CV) and the other virtually recovered (DV) (iii) both parents of the GTS twins, using gas chromatography-mass spectrometry. 2. RBC choline decreased with age in control men (r = -0.76; p less than 0.01) but not in control women. 3. RBC choline (nmol/ml) was higher in control men (18.3 +/- 4.8, X +/- SD) vs control women (13.1 +/- 4.3) (p = 0.025). 4. There was no significant difference in RBC choline (nmol/ml) between ST patients (16.6 +/- 5.0) and controls (15.5 +/- 5.2). 5. The RBC choline values (nmol/ml) in the twins and parents were: 56.6 (CV), 58.3 (DV), 89.8 (father), 38.3 (mother) and in the controls (age 20-24) (n = 5) 18.2 +/- 3.6. 6. These data suggest (i) RBC choline is affected by age and gender (ii) RBC choline is unchanged in ST (iii) the regulation of RBC choline is under genetic control (iv) elevated RBC choline is not a state marker for GTS.

Lymphocyte subset abnormalities in patients with spasmodic torticollis.

The aetiology of spasmodic torticollis is unknown but the patients form a heterogeneous group among which are cases apparently precipitated by a viral illness and others associated with autoimmune disease. We therefore decided to investigate the immunoregulatory lymphocyte subsets in our 11 cases. A significant decrease of both helper and suppressor lymphocytes was identified in the group, together with in vitro evidence of depressed suppressor cell function. Disturbance of the immune response may play a role in this puzzling disorder.

Bromocriptine treatment of spasmodic torticollis. A double-blind crossover study.

A double-blind crossover study (12 weeks and 12 weeks) was performed to evaluate the effects of bromocriptine on the symptoms and serum prolactin levels of 14 women with spasmodic torticollis. While the serum prolactin level decreased in response to bromocriptine, no difference was found between the effects of placebo and bromocriptine on the symptoms of spasmodic torticollis. Our findings suggest that dopaminergic neurotransmission is not involved in the pathogenesis of this syndrome.