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Response and irnrnunoresistance to

botulinurn toxin injections
Joseph Jankovic, MD, and Kenneth Schwartz, PA

~ ~~ ~~

Article abstract-Botulinum toxin antibodies (ABS)may be a reason why occasionally patients do not have a re-
sponse to injections with botulinum toxin type A (BTX). We tested 86 patients with cervical or oromandibular dystonia
for the presence of BTX ABS; 20 were positive and 66 were negative. All patients who tested positive had no response
t o BTX injections on at least two consecutive treatment sessions. When compared with 22 randomly selected patients
with negative BTX ABS results, the patients with positive BTX ABS tests had an earlier age at onset (mean age: 31.8 *
16.7 years versus 43.4 * 10.5;p < 0.05), higher mean close per visit (249.2 * 32.5 U versus 180.8 i 68.7, p < 0.0005),
and higher total cumulative close (mean dose: 1,709 * 638 U versus 1,066 2 938; p < 0.01). Four out of five patients
with positive ABS tests later had a response to botulinum toxin type F injections. Of 26 patients with negative BTX
ABS results who were tested because of poor response on at least one visit, 21 had good response after subsequent in-
jection and five had no effect. Except for young age at onset and higher dosages, there were no other factors that could
reliably predict which patients would become immunoresistant to BTX type A injections. Treatment with alternate
serotypes may offer clinical benefit to this group of patients. Absence of detectable BTX ABS may occur in patients
with poor response to BTX injections because of inadequate dosage, injections of inappropriate muscles, or poor sensi-
tivity of the BTX ABS bioassay.
NEUROLOGY 1995;45:1743-1746

Injections of botulinum toxin type A (BTX) provide
effective, symptomatic relief for several disorders
characterized by abnormal muscular contractions,
such as dystonia, spasticity, tremors, tics, and
other motor disorders.' BTX injections are consid-
ered the treatment of choice for many of the focal
dystonias, particularly blepharospasm, cervical
dystonia (torticollis), laryngeal dystonia (spasmodic
dysphonia), and task-specific dystonias (eg, writer's
cramp). Although the benefits persist in the vast
majority of patients after repeated injectioq2 in
some patients the condition becomes unresponsive
to subsequent treatment~.3,~ One reason for the de-
velopment of resistance to BTX treatment is the de-
velopment of blocking antibodies (ABS). In this
study, we sought to determine the causes of resis-
tance and to identify risk factors for the formation
of ABS.
Methods. Eighty-six of 1,321 patients who received BTX
injections on 6,549 visits at the Baylor College of Medi-
cine Movement Disorder Clinic were tested for BTX ABS.
Twenty-two were randomly selected for this study, which
was approved by the Baylor Institutional Board for
Human Research. The remainder were tested because of
lack of or inadequate response t o BTX injections (60 pa-
tients) or at the request of the patients (four patients),
even though they had had a response t o the treatment.

From the Department of Neurology, Baylor College of Medicine, Houston, TX.

Received November 8,1994. Accepted in final form February 3,1995.
Address correspondence and reprint requests to Dr. Joseph Jankovic, Baylor College of Medicine, Department of Neurology, 6550 Fannin #1801,Houston,
TX 77030.
September 1996 NEUROLOGY 45 1743
 The response to treatment was rated on a 0 to 4 “peak Table. Clinical correlates in patients tested for
effect” scale (0: no effect; 1: mild effect, no functional im- botulinum toxin antibodies
provement; 2: moderate improvement, no change in func-
tional disability; 3: moderate change i n severity and Clinical + Antibodies -Antibodies
function; and 4: marked improvement in severity and variables (range) (range)
function). “Lack of or inadequate response” was defined
as peak effect of 0 or 1.This study used the Allergan, Inc, N 20 22
preparation of BTX (BOTOX). Reason for test Poor response Random selection
The presence of ABS was tested using a bioassay orig- Gender (MIF) 5115 10/12
inally described by Hatheway et a1596 at the Center for Mean age (yr) 46.2 * 13.5 50.4 i 10.9
Disease Control and performed by the Northview Pacific (15-77) (30-75)
Laboratories, Berkeley, CA. Results of this qualitative Mean age a t 31.8 2 16.7” 43.4 * 10.5”
bioassay are reported a s either positive or negative. A onset (yr) (1-54) (24-59)
positive result, indicating the presence of ABS in the pa- Mean duration of 172.6 ? 165.9 108.1 + 155.5
tient’s serum, occurs when the patient’s serum neutral- symptoms (mo) (15-552) (6-728)
izes the effects of BTX and prevents the death of mice in- Mean peak effect 0.78 2 1.2$ 2.95 & 1.46$
oculated with the toxin. In a negative result, mice inocu- (immediately (0-3) (0-4)
lated with BTX die, presumably because they are not before testing)
protected by circulating ABS. Mean total 1,709 i 638* 1,066 i 938$’
cumulative dose (500-3,000) (100-3,850)
(U)
Results. Of the 86 patients who were tested for Total number 148 140
BTX ABS, 20 had positive results and 66 had nega- of visits
tive; 22 who had negative results were randomly Average number 7.3 ? 2.7 6.4 ? 5.6
tested. Five patients were tested more than once: of visits (2-12) (1-23)
in one, results turned negative 1% years after ini- Mean 125.5 i 29.7 116.3 i 56.6
tial positive test results; in the remainder results inter-injection (72-181) (0-225)
were initially negative and subsequently became interval (d)
positive. These latter patients were included only Mean dose per 249.2 ? 32.51 180.8 f 68.7t
in the ABS-positive group. All patients who had visit (U) (189-310) (29-290)
Duration of 2.53 2 1.28 2.36 * 2.23
positive tests had no response to BTX (peak effect
treatment (yr) (0.5-5.0) (0-8.3)
of 0 or 1on at least two consecutive treatment ses-
sions). All these patients were receiving BTX injec- “ p < 0.01.
tions for cervical dystonia, except for one who had ip < 0.0005.
$ p < 0.00001.
oromandibular dystonia manifested by forceful,
spasmodic jaw closure. That patient’s condition
eventually failed to improve even with botulinum
toxin type F. The other four patients treated with my: two subsequently received BTX injections and
botulinum toxin type F obtained satisfactory but had good responses, two had only slight improve-
short-lasting improvement. None of the randomly ment, and two were lost to follow-up.
tested patients had a positive BTX ABS test. This
group was composed of 16 patients with cervical Discussion. This study shows a 100% correlation
dystonia, five with cranial dystonia, and one with between the presence of BTX ABS, as determined
h a d a r m dystonia. by the mouse bioassay, and complete failure of re-
Although there was a marked overlap between sponse to BTX injections. The patients with posi-
the two groups, the patients with positive BTX tive ABS results had absolutely no response and no
ABS results had an earlier age at onset (mean age: atrophy after BTX injections although all but one
31.8 2 16.7 years versus 43.4 k 10.5; p < 0.05), had had responses in the past. The one patient
higher mean dose per visit (249.2 k 32.5 U versus with oromandibular dystonia, who did not have an
180.8 k 68.7, p c 0.0005), and higher total cumula- improvement even with dosages as high as 400 U
tive dose (mean dose: 1,709 k 638 U versus 1,066 per treatment visit, later also did not have a re-
938; p < 0.01) as compared with the randomly se- sponse to botulinum toxin type F. Our study also
lected patient group (table). There was no statisti- establishes a link among the mean dose per treat-
cal difference between patients with negative and ment session, total cumulative dose, and the devel-
positive BTX ABS results for any other variable, opment of BTX antibodies (table). Since we had al-
including gender, age, duration of symptoms, total ways waited at least l month between injections,
visits, number of visits, inter-injection interval, and we did not observe the effects of “boosters” on ABS
duration of treatment. formation. There was no difference in the fre-
Of 26 patients tested because of poor response to quency of treatment sessions between our patients
at least one treatment session and who received ad- with and without ABS. This contrasted with an-
ditional injections, 21 had good response (peak ef- other study4 that showed a direct correlation be-
fect 22) after subsequent injection and five had tween the presence of ABS and “booster”injections,
minimal or no effect (peak effect 51). Six patients usually administered within 2 to 3 weeks after the
were later treated surgically with cervical rhizoto- initial treatment. In that study, ABS were detected
1744 NEUROLOGY 45 September 1995
in 24 of 559 patients (4.3%),and 10.5% of the 559
were estimated t o have developed resistance to
BTX. Since the investigators did not indicate how
many patients were actually tested, the frequency
of ABS could not be determined from that s t ~ d yA. ~
sub-analysis of their results on patients with BTX
resistance identified three potential risk factors for
the development of BTX resistance: (1)frequent in-
jections, (2) “booster” injections, and (3) high doses
of BTX per treatment.
The frequency of BTX ABS has been reported to
be in the 0 to 10% range, based on the results of
the in vivo mouse b i o a ~ s a y . ~ ,In~ ,contrast,
“~ Siat-
kowski et a19 reported positive ABS in 57% of 42
patients treated with BTX for blepharospasm,
hemifacial spasm, and cervical dystonia. This fig-
ure was based on a new test using a sphere-linked
immunodiagnostic assay (SLIDA). Although this
assay apparently has a four-fold greater sensitivity
than ELISA, the presence of ABS detected by the
SLIDA technique failed to correlate with the pa-
tient’s clinical r e s p ~ n s eSince
.~ the method fails to
detect neutralizing ABS and the nature of the ABS
is unknown, the SLIDA method has limited value
in assessing lack of clinical response to BTX treat-
ment. ELISA has been used for the detection of
BTX ABS, but clinical correlation between the
presence of such ABS and a lack of response t o
BTX injections has not been established.6J0J1In
one study,12 only three of 96 patients with focal
dystonia who received repeated injections of the
preparation (Porton Down, UK) had ABS detected
by an in vivo toxin neutralization test. The inter-in-
jection interval was shorter in the patients with
positive ABS tests, but there was no correlation
with dosage or with clinical response. Again, the
assay apparently did not detect blocking, clinically
relevant ABS. In addition to lack of atrophy of the
muscles that underwent injection, other physical
examination techniques can be used to detect BTX
resistance. For example, the lack of depression of
transverse lines in the forehead after a unilateral
injection of 10 to 15 U of BTX in the frontalis mus-
cle, a site remote from that of the therapeutic injec-
tion, has been suggested as evidence for BTX resis-
tance.13 The validity of this observation, however,
has not yet been tested. We are currently evaluat-
ing the effects of unilateral injection into the corru-
gator muscle. In patients who are responsive t o
BTX, such injection produces asymmetrical con-
traction, whereas in immunoresistant patients the
symmetry of eyebrow contractions is preserved.
We believe that the chief reasons for the marked
differences in the frequencies of ABS are: (1)hetero-
geneous patient population with various conditions
requiring different dosages of BTX and (2) utiliza-
tion of different techniques for detecting ABS. ABS
can be directed against different components of the
BTX preparation; while some are directed against
the BTX molecule, others are directed against the
associated proteins. Only those ABS that effectively
block the biologic activity of the toxin are clinically
relevant. Although cumbersome and expensive, the
mouse neutralization bioassay is currently consid-
ered the most reliable assay for biologically relevant
immunoresistance.6 None of our patients with posi-
tive ABS results, measured by the mouse bioassay,
had responses to subsequent BTX injections. In
some of those with no response, however, the mouse
bioassay failed to detect BTX ABS. This may indi-
cate lack of sensitivity of the assay or other reasons
for poor response, such as selection of wrong mus-
cles or inadequate dose.
Patients who develop ABS to BTX type A, and
thus become resistant to this type of toxin, usually
experience benefits with other types of toxins, such
as botulinum toxins B and F.14-17Although the
magnitude of improvement with these toxins is
similar to that with type A, the duration of the ben-
efit tends t o be shorter. Ten of 15 torticollis pa-
t i e n t ~who
~ ~became resistant to BTX A improved
with botulinum toxin F, but the benefits lasted only
1month rather than the 3 months of benefits expe-
rienced before they became resistant to BTX type
A. While the duration of benefit from botulinum
toxin type B appears longer than from type F, only
long-term studies will determine whether the alter-
native types of botulinum toxin offer any advan-
tages other than their clinical benefits in patients
who become immunoresistant to BTX type A. Alter-
nating type A with one of the other types of bo-
tulinum toxin might reduce the long-term risk of
immunoresistance.
In summary, BTX ABS formation is one expla-
nation for the occasional lack of response to BTX
injections. Treatment with alternate serotypes may
offer clinical benefit to this group of patients. Ab-
sence of detectable BTX ABS may occur in patients
with poor response t o BTX injections because of in-
adequate dosage or selection of inappropriate mus-
cles; this condition warrants reinjection at higher
dosages or at alternative sites.
References
1. Jankovic J , Hallett M, eds. Therapy with botulinum toxin.
New York: Marcel Dekker, 1994.
2. Jankovic J, Schwartz KS. Longitudinal experience with bot-
ulinum toxin injections for treatment of blepharospasm and
cervical dystonia. Neurology 1993;43:834-836.
3. Jankovic J , Schwartz KS. Clinical correlates of response to
botulinum toxin injections. Arch Neurol 1991;48:1253-1256.
4. Greene P, Fahn S, Diamond B. Development of resistance to
botulinum toxin type A in patients with torticollis. Mov Dis-
ord 1994;9:213-217.
5. Hatheway CH, Snyder JD, Seals J E , Edell TA, Lewis GE.
Antitoxin levels in botulism patients treated with trivalent
equine botulism antitoxin to toxin types A, B, and E. J Infect
Dis 1984;150:407-412.
6. Hatheway CL, Dang C. Immunogenicity of the neurotoxins
of Clostridium botulinum. In: Jankovic J , Hallett M, eds.
Therapy with botulinum toxin. New York Marcel Dekker,
1994~93-108.
7. Biglan AW, Gonnering R, Lockhart LB, Rabin B, Fuerste
FH. Absence of antibody production in patients treated with
botulinum A toxin. Am J Ophthalmol 1986;101:232-235.
8. Gonnering RS. Negative antibody response to long-term
September 1996 NEUROLOGY 45 1745
 treatment of facial spasm with botulinum toxin. Am J Oph-
thalmol 1988;105:313-315.
9. Siatkowski RM, Tyutyunikow A, Biglan AW. Serum anti-
body production to botulinum A toxin. Ophthalmology 1993;
100:1861-1866.
10. Dezfulian M, Hatheway C, Yolken R, Bartlett J . Enzyme-
linked immunosorbent assay for detection of Clostridium
botulinum type A and type B toxins in stool samples of in-
fants with botulism. J Clin Microbiol 1984;20:379-383.
11. Tsui JK, Wong NLM, Wong E, Calne DB. Production of cir-
culating antibodies to botulinum-A toxin in patients receiv-
ing repeated injections for dystonia [abstract]. Ann Neurol
1988;23:181.
12. Zuber M, Sebald M, Bathien N, de Recondo J , Rondot P. Bot-
ulinum antibodies in dystonic patients treated with type A
botulinum toxin: frequency and significance. Neurology
1993;43:1715-1718.
13. Borodic GE, Pearce B, Duane D, Johnson E. Antibodies to
botulinum toxin [letter]. Neurology 1995;45:204.
14. Moyer E, Settler PE. Botulinum toxin type B: experimental and
clinical experience. In: Jankovic J , Hallett M, eds. Therapy
with botulinum toxin. New York Marcel Dekker, 1994:71-86.
15. Borodic GE, Pearce LB, Smith KL, e t al. Botulinum B toxin
as an alternative to botulinum A toxin: a histologic study.
Ophthal Plast Reconstr Surg 1993;9:182-190.
16. Ludlow CL, Hallett M, Rhew K, e t al. Therapeutic use of
type F botulinum toxin. N Engl J Med 1992;326:349-350.
17. Greene P, Fahn S. Use of botulinum toxin type F injections
to treat torticollis in patients with immunity to botulinum
toxin type A. Mov Disord 1993;8:479-483.

Motion of the cerebellar tonsils in

Chiari type I malformation studied
by cine phase-contrast MRI
J. Pujol, MD; C. Roig, MD; A. Capdevila, MD; A. Pou, MD; J.L. Marti-Vilalta, MD;
J. Kulisevsky, MD; A. Escartin, MD; and G. Zannoli, PhD

Article abstract-We studied the effects on CSF dynamics at the foramen magnum and the clinical significance of
the abnormal tonsillar motion in 14 patients with Chiari type I malformation and 14 control subjects using cine phase-
contrast MRI. Dynamic MRI consisted of axial and sagittal cine phase-contrast sequences. CSF and tonsillar motion
were qualitatively and quantitatively evaluated, and the subarachnoid space at the foramen magnum measured. In
Chiari patients, cine phase-contrast MRI detected the abnormal pulsatile motion of the cerebellar tonsils, which pro-
duced a selective obstruction of CSF flow from the cranial cavity to the spine. The amplitude of the tonsillar pulsation
and the severity of the arachnoid space reduction were associated with the symptom of cough-strain headache, but not
with the presence of syringomyelia. The finding of abnormal valve dynamics of the cerebellar hernia revealed by cine
phase-contrast MRI conforms to the pathophysiologic mechanisms suggested in pressure register studies and opens a
new possibility in the presurgical assessment of Chiari patients with exertional symptoms.
NEUROLOGY 1995;45:1746-1753

The systolic arterial pulsation within the cranial
cavity produces a slight pulsatile movement of the
brain, particularly of the diencephalon and brain
stem, and a marked CSF wave that normally con-
tinues down from the basal cisterns into the cervi-
cal subarachnoid space.1-6The presence of cerebel-
lar herniation in Chiari malformation distorts the
normal dynamics in the craniospinal junction ele-
ments. The CSF pulsation is altered,7,8and the
brain motion transmitted from the arterial pulse
changes appreciably. Oldfield et al,9using intraop-
erative ultrasonography, and Terae et al,1° with an
MRI sequence based on presaturation bolus track-
ing, demonstrated an increase of tonsillar motion
in Chiari malformation. They studied the possible
consequences on syringomyelia and proposed new
pathophysiologic mechanisms. Nevertheless, the
dynamic repercussions and t h e clinical conse-
quences of the tonsillar motion were not fully es-
tablished.
We used cine phase-contrast MRI, a method that
allows direct anatomic depiction of moving struc-
t u r e ~ to ~ ~ the dynamic effects on the CSF
, ~study
flow through the foramen magnum and the clinical

From the Magnetic Resonance Center of Pedralbes (Drs. Pujol and Capdevila), Barcelona; the Department of Neurology (Drs. Roig, Marti-Vilalta, Kuli-
sevsky, and Escartin), Santa Creu i Sant Pau Hospital, Autonomous University of Barcelona; the Department of Neurology (Dr. Pou), Ntra. Sra. del Mar
Hospital, Autonomous University of Barcelona, Spain; and General Electric (Dr. Zannoli), Buc, France.
Supported in part by the Conselleria de Sanitat de la Generalitat de Catalunya (Autonomous Government of Catalonia, Spain) and the Institut de Re-
cerca de YHospital de la Santa Creu i Sant Pau of Barcelona.
Received November 1, 1994. Accepted in final form February 3, 1995.
Address correspondence and reprint requests to Dr. Jesus Pujol, Magnetic Resonance Center of Pedralbes, Monestir, 3,08034 Barcelona, Spain.

1746 NEUROLOGY 45 S e p t e m b e r 1995

 Response and immunoresistance to botulinum toxin injections
Joseph Jankovic and Kenneth Schwartz
Neurology 1995;45;1743-1746
DOI 10.1212/WNL.45.9.1743

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