Neurology -- Correspondence for Kranz et al., 70 (2) 133-136

Correspondence published:

Neutralizing antibodies in dystonic patients who still respond well to botulinum toxin type A: M. Zouhair Atassi, Joseph Jankovic, Behzod Z. Dolimbek (13 March 2008)

Reply from the authors: Gottfried Kranz, Thomas Sycha, Bernhard Voller, Georg S. Kranz, Peter Schnider, Eduard Auff (13 March 2008)

Neutralizing antibodies in dystonic patients who still respond well to botulinum toxin type A 13 March 2008

M. Zouhair Atassi,
Department of Biochemistry and Molecular Biology, Baylor College of Medicine
Houston, Texas 77030,
Joseph Jankovic, Behzod Z. Dolimbek
Send Correspondence to journal:
Re: Neutralizing antibodies in dystonic patients who still respond well to botulinum toxin type A

matassi{at}bcm.edu M. Zouhair Atassi, et al.

Kranz et al. used a ninhydrin sweat test (NST) and mouse diaphragm test (MDT) in 14 dystonic patients and 14 controls and reported subclinical neutralizing antibodies (Abs) in patients with “good clinical responses” to BoNT/A. The authors defined the responses based on: a three-point decrease on modified-Tsui scale; injected-muscle atrophy; or reported BoNT/A-associated adverse events. [1] Only one patient had anti-BoNT/A Abs (1 mU/mL, by MDT) and a marked anhydrotic area reduction (0.26 cm2). Seven other patients had “borderline” titers of either 0.4 or 0.8 mU/mL and reduced anhidrosis area.
The study design, small sample size, and subjective clinical-response assessment precludes extension to incidence value (>40%). Mixing BOTOX®- and Dysport®-treated patients complicates analysis because these products have different immune-response rates. NST employs area, but neglects color density. NST correlation value was missing, as the anhydrotic area could vary with injection method. Hyperhidrosis effect reproducibility was also overlooked. The Results imply that NST was done with both BOTOX® and Dysport® but this contradicts the statement in the Methods that Dysport® was the test agent.
The observation that patients had different Ab levels after similar toxin doses is expected. [2,3] Immune responses mature with time, boosters, and are genetically controlled. [2,3] This means that patients responding to treatment would have different Ab levels, even after similar doses and protocols. [2,3] Antibodies are polyclonal. Titer partially affects blocking as recognized epitopes, immunoglobulin class, isotype and affinity play major roles. [3,4] It is unclear whether Abs blocking palm sweating and those acting at neuromuscular junctions have similar epitope recognition, class, isotype and affinity.
Although NST results vary and tests are not generally accepted, Kranz et al.’s findings do not conflict with mouse protection assay (MPA) results, the “gold standard” for assaying blocking Abs. [5]
MPA typically employs five mice per test serum. [4] If all mice survive the challenge with test serum+LD100 of BoNT then serum has blocking Abs, which are absent if none survive. However, with some test sera not all mice die or conversely survive. If 4/5 to 1/5 mice survive, then the sample has blocking Abs, but at obviously decreasing titers. Operationally, 4/5, 3/5 mice surviving are considered MPA-positive and 2/5 or 1/5 are MPA-negative.
Kranz et al.’s work might draw attention that MPA results, reported as MPA-positive or MPA-negative, should provide titer information (number of mice that die or survive). This could guide treatment and if blocking Abs start to appear, subsequent injection could be appropriately modified (e.g., increasing inter-treatment interval).
References
1. Kranz G, Sycha T, Voller B, Kranz GS, Schnider P, Auff E. Neutralizing antibodies in dystonic patients who still respond well to botulinum toxin type A. Neurology 2008;70:133-136.
2. Atassi MZ, Basic immunological aspects of botulinum toxin therapy. Mov Disord, Suppl 8, 2004;S68–S84.
3. Dolimbek BZ, Aoki KR, Steward LE, Jankovic J, Atassi MZ. 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. Mol Immunol. 2007;44:1029-1041.
4. Atassi MZ, Dolimbek GS, Deitiker PR, Aoki KR, Dolimbek BZ. Submolecular recognition profiles in two mouse strains of non-protective and protective antibodies against botulinum neurotoxin A. Mol. Immunol. 2005;42:1509–1520.
5. Jankovic J, Vuong KD, Ahsan J. Comparison of efficacy and immunogenicity of original versus current botulinum toxin in cervical dystonia. Neurology 2003;60:1186-1188.
Disclosure: M. Z. Atassi has received unrestricted research grants from Allergan; J. Jankovic has received research grants and honoraria from Allergan Inc, Ipsen Limited, and Merz Pharmaceuticals.

Reply from the authors 13 March 2008

Gottfried Kranz,
Medical University of Vienna, Department of Neurology
Waehringer Guertel 18-20, 1090 Vienna, Austria,
Thomas Sycha, Bernhard Voller, Georg S. Kranz, Peter Schnider, Eduard Auff
Send Correspondence to journal:
Re: Reply from the authors

gottfried.kranz{at}meduniwien.ac.at Gottfried Kranz, et al.

Atassi et al. feel that the method used in our study precludes extension to an incidence value of subclinical BoNT/A-AB level of >40% as described.
We randomly selected 14 dystonic patients from a large population (n = 119 well responding patients). Therefore, the AB status of those randomly selected patients should be representative of our population. Earlier data showed a similar number using only the MDT without comparing the results to controls; a prevalence of 36% subclinical BoNT/A-AB levels in well responding dystonic patients. [5]
We obtained our data from patients treated with both Botox® and Dysport®, which underlines the broad validity of the results and reflects the clinical situation in Europe. Atassi et al state that using Dysport® in the NST for patients that were also treated with Botox® contradicts the method. The prevalence of BoNT/A-AB may differ between type A products. However, there is no evidence in the literature that BoNT/A-AB for one type A product would not react with another type A product. On the contrary, switching between type A preparations in patients with blocking BoNT/A-ABs does not overcome therapy failure. [6]
Our results indicate that blocking antibodies occur in a continuum from non-existent to subclinical and clinical levels. We agree that the mouse lethality assay might also be adapted to indicate subclinical AB titers, if correlated to a quantitative AB test. The NST, though detecting AB only indirectly, is a simple, inexpensive and quick bedside test that does not sacrifice animals. It highly correlated with the quantitative MDT in two studies even with a small sample size in one study. [1,7]
In our experience, when using the same injection technique for all patients as described in both studies, the reproducibility of the NST is high. We agree that the level of individual perspiration may influence the anhidrotic area to a small extent. The marginal hypohidrotic edge around the anhidrotic area in the NST may appear anhidrotic in subjects with very little perspiration. However, hypohidrotic areas were small compared to the large difference of anhidrotic areas between subjects with and without subclinical antibodies.
Neutralizing BoNT/A-ABs are targeted against the toxin, not against the neuromuscular or neuroglandular endplates. Whether different subclasses of BoNT/A-AB reduce the neuromuscular or neuroglandular effects to a different extent is theoretically possible but seems unlikely in the light of our results with high autonomous muscular correlations.
References
5. Wittstock M, Benecke R, Bigalke H, Dressler D. Quantitative antibody status of patients with continued sensitivity to long-term botulinum toxin therapy. Neurology 2001;56 (suppl 3):A347. Abstract.
6. Dressler D. Clinical presentation and management of antibody- induced failure of botulinum toxin therapy. Mov Disord 2004;19:92-100.
7. Voller B, Moraru E, Auff E, et al. Ninhydrin sweat test: a simple method for detecting antibodies neutralizing botulinum toxin type A. Mov Disord 2004;19:943–947.
Disclosures: The authors report no conflicts of interest. This work was supported by the Medical University of Vienna, Department of Neurology.