Seven patients, six with cervical dystonia and one with oromandibular dystonia immunoresistant to BTX-A therapy (Ab+), were retested by mouse bioassay for the persistence of neutralizing antibodies. Six patients were women (mean age, 56 years; range, 41-80 years). This bioassay, originally described by Hatheway and Dang9 and others10 detects the ability of the patient's serum to block the effect of a lethal dose of BTX-A. The assay is performed by injecting 5 units (U) of toxin intraperitoneally with 0.4 mL of patient's serum into each of four mice. The assay was positive when three or more mice survived and negative when three or more mice died. A positive assay indicated that the serum contained > 0.052 IU/mL of type A antitoxin.

The response to repeat BTX-A injections was also assessed in some patients by injecting 15 U into the right frontalis muscle or the corrucrator muscle and observing for asymmetry in forehead wrinkles or in frowning, indicative of weakness in the injected muscle and hence a response to BTX-A. In some, the response to repeat BTX-A injections was based on the information provided by the patients, by their daily diary of their symptoms, or by interviews of the spouse or friends. The peakeffect score, defined as the maximum benefit after the injection. was rated on a 0-4 scale based on this information (0 = no effect; 1= mild improvement; 2 = moderate improvement; 3 = moderate improvement in severity and function; and 4 = marked improvement in severity and function).11 Six patients reverted to Ab- status and are included in this report.

CASE REPORTS

Patient 2

This 66-year-old woman was initially evaluated by us in 1987 for a 10-year history of cervical dystonia and essential tremor. In 1980, electrodes were implanted at another institution, presumably for deep brain stimulation in both thalami. Her cervical dystonia improved during the test stimulation, but the procedure apparently did not help when the system was internalized. She was first injected with BTX-A in 1987 and received a total cumulative dose of 1800 U in six sessions over a 20-month period. She then failed to respond to subsequent BTX-A injections and was found to be Ab+ in December 1988. In 1989, she had surgical denervation with excellent benefit postoperatively and could maintain her head in the primary position most of the time. Three months later, however, she developed head tremor, which responded to primidone. She then received BTX-F injections over a period of 2 years and benefited considerably from these treatments, but the improvement after each set of injections lasted only 2 months. In 1995, 78 months after the last BTX-A injection, she was rechecked for antibodies to BTX-A and was found to be Ab-. She was reinjected again with BTX-A in January 1996, with grade 3 improvement in her symptoms, but she had no improvement in her symptoms when reinjected in April 1996. She was reinjected in July and November t996 again with grade 3 improvement according to the previously described scale. She was retested for antibodies again and was found to be Ab+ 15 months after the necative response. Although this result was determined by the Western blot. the patient elected not to be reinjected or retested by the mouse bioassay.

Patient 3

This 80-year-old woman had an onset of cervical dystonia at age 50. She was first treated with BTX-A in 1990. By 1992, she had received a total of 1975 U over 10 visits, which included boosters. She was found to be Ab+, but 10 months later she was found to be Ab-. She subsequently received three series of BTX-A injections at 4-month intervals. The first two were markedly successful and produced clinical improvement in the dystonia, complicated by mild neck weakness and with weakness of the right frontalis muscle injected simultaneously. The third was unsuccessful. She was found to be Ab+, but six months later she was again Ab-. A retreatment with 400 U of BTX-A had a good response, and she continues to benefit from subsequent BTX-A injections even at 7-month intervals. This patient has also been included in other studies.12

Patient 4

This 56-year-old woman had an onset of cervical dystonia at age 48. After nine visits, including booster injections separated by 2 weeks, and a total dose of 1875 U of BTX-A over 43 months, she became unresponsive to further treatments and was found to be Ab+. Surgical denervation provided only limited benefit. Fourteen months after she was found to be Ab+ she was retested and was Ab-. She received 495 U of repeat BTX-A injections with moderate improvement in her pain, but only slight improvement in spasm. She also had focal weakness of her right frontalis muscle. When retested in August 1996, she was again Ab+.

Patient 5

This 60-year-old woman with posttraumatic oromandibular dystonia that began following the replacement of old dentures was initially treated with splints by her dentist without benefit. Treatment with clonazepam, baciofen, and trihexyphenidyl was tried, with only limited benefit. An otolaryngologist administered her first BTX-A injections on October 1991, and she had mild improvement. Subsequent five sets of injections provided limited or no benefit. She was found to be Ab+ in December 1994. but repeat antibody testing in February 1996 was negative. She was reinjected with 200 U BTX-A and had grade 2 benefit with reduced pain at 3 months after injection.

Patient 6

This 49-year-old woman had an onset of cervical dystonia at age 36. She received a total of 1500 U BTX-A over 26 months after 11 visits, including boosters. She became unresponsive to BTX-A treatment and was found to be Ab+ in October 1992. Surgical denervation in November 1993 provided excellent benefit. She was found to be Ab- when retested in October 1996. Reinjection of BTX-A into her right frontalis muscle produced focal weakness and asymmetric frontalis muscle contraction. She has not been reinjected with BTX-A in her neck.

Patient 7

This 41-year-old woman had an onset of cervical dystonia 3 months after a whiplash injury. She was first injected with BTX-A in December 1990. After eight visits, including boosters, and a total dose of 1850 U BTX-A over 26 months, she became unresponsive to further treatments and was found to be Ab+ in February 1993. When she was Ab+, she received a single injection of BTX-B that produced benefit comparable to that of BTX-A. She was found to be Ab- when retested in August 1995, and BTX-A was reinjected into her neck muscles I year later. She had excellent improvement, lasting 3 months, in her cervical dystonia, but also had right frontalis muscle weakness.

RESULTS

The mean age at onset among these seven patients was 40 years (range, 17-55 years); six of the seven had the onset of their illness before age 50. The mean duration of symptoms was 197 months (range, 84-360 months). The mean duration of treatment with BTX-A before the development of immunoresistance was 27 months (range, 15-43 months), and the mean total cumulative dose was 1659 U (range, 810-1975 U). The patients received a mean dose of 207 U (range, 50-400 U) per visit during an average number of eight visits per patient.

All patients reverted from BTX-A Ab+ status to BTX-A Ab- status, which was confirmed by mouse assay performed at a mean interval of 30 months (range, 10-78 months) after the initial antibody test. Six of the seven Ab+ patients were reinjected with BTX-A after they reverted to an Ab- status (see Table 1). All improved after the reinjections, but three lost their response to subsequent injections, which was confirmed by the reemergence of Ab+. In four of these patients, response to repeat BTX-A injections was confirmed by weakness of the right frontalis muscle injected simultaneously.

Some patients received alternative treatments after thev became immunoresistant and before they reverted to Ab- status. Three with cervical dystonia were treated with surgical denervation: two obtained excellent benefit, and the other had only moderate benefit. Two Ab+ patients received BTX-F injections over a 2-vear period; both patients improved, although the effect was briefer (6-8 weeks) than that of BTX-A (12-16 weeks). One Ab+ patient received one session of BTX-B injections with benefit similar to that obtained from BTX-A.

DISCUSSION

Since 1897, when Van Ermengen13 first established that botulism is caused by a bacterial toxin, eight distinct types of botulinum toxins have been isolated (A, B, C, C2, D, E, F, and G).14 All are neurotoxins except C2, which is a cytotoxin.14 All can evoke a distinct immunogenic response in a given individual without crossreactivity, with the exception of minor antigenic overlap between types C and D and types E and F.15 The immunologic response to these toxins can be monitored by detection of antibodies with the mouse protective assay, which detects antibodies capable of neutralizing the biologic activity of a lethal dose of toxin. Whether absence of weakness to unilateral in . ection of frontalis muscle or corrugator muscle correlates with the presence of antibodies requires further study. Our preliminary experience, however, succests that this simple technique (injection of 15 U into one corru-ator muscle and observing for asymmetry in frowning as evidence of a response) correlates well with Ab status.

The immunologic response to botulinum toxoids is well documented in individuals who have received botulinum pentavalent toxoid (A, B, C, D, and E). 16, 17 In one study, the toxoid was administered at 0, I-, and 12 weeks. 16 Of 23 individuals, 21 (91%) had detectable BTX-A antibodies after the primary series at the end of 12 weeks. In the same study, another 21 individuals who had received the primary series of the toxoid were rechecked for antibodies by using the same mouse assays after I year, before they received their annual booster. and only 11 (52%) had detectable antibodies. Of the 21 individuals, 20 (95%) received their first annual booster and all had become Ab+ to BTX-A toxoid after the booster. A similar observation was made by Fiock et al. 17 in studying individuals who received pentavalent toxoid at O-, 2-, and 12-week intervals as a primary series of vaccination. A significant decrease in the BTX-A antibody titer was found when these individuals were retested 52 weeks after the initial toxoid vaccination. All received booster injections at the 52-week interval, and all had elevated titers to all antigens (A, B, C, D, and E), except for one individual who persistently had a negative titer to type B toxoid.17,18

Six of the patients described here received repeat BTX-A injections after they turned Ab-, and three lost their clinical benefit to infrequent injections, presumably because of an anamnestic immunologic response reactivatedby repeat BTX-A injection. This is analogous to receiving a booster dose of pentavalent toxoid. 16,17 Indeed, the reemergence of antibodies was noted in three patients. 16,17

T'he presence of antibodies to BTX-A toxin did not interfere with the response to BTX-F injections. 19 This is consistent with the observation that these neurotoxins are immunologically distinct and evoke a specific antibody response.15 As expected, individuals who received pentavalent toxoid (A, B, C, D, and E) failed to show a detectable antibody titer to BTX-F toxoids.15 The briefer BTX-F effect, however, necessitates frequent injections and thus increases the risk of immunoresistance to this type of toxin.20 .BTX-F may be particularly useful, however, in treating patients previously vaccinated with the pentavalent toxoid, because this toxoid does not evoke antibodies against BTX-F.16 BTX-B offers a useful altemative for the treatment of patients who no loncer respond to BTX-A.21 In addition to its distinct antigenicity, BTX-B has been observed to evoke weaker immunoaenic response in the individuals who received botulinum pentavalent toxoid. 16.17

In conclusion, all of our patients who were Ab+ and later reverted to Ab- had a good response to BTX-A injections. This benefit, however, was not maintained in half of the patients, possibly because the anamnestic immune response was reactivated. Patients immunoresistant to BTX-A may benefit from the use of other BTX serotypes. It is not known whether immunosuppression. plasmapheresis, or intravenous immuno-lobulins would be useful in minimizing the risk of the reemergence of immunoresistance in these patients, as seen in hemophilic patients receiving factor VIII replacement with factor VIII antibodies or in patients with alloimmune thrombocytopenic purpura who develop antibodies to platelets after receiving platelet replacement. 22.23 Once different types of BTX become clinically available. it may be prudent to alternate between the different serotypes to increase the interval between the injections with the same serotype and minimize the risk of developinneutralizing antibodies.

REFERENCES

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18. Wright GG, Duff JT, Fiock MA, Devlin HB, Sonderstrom RL. Studies on immunity to toxins of Clostridium botulinum. V. Detoxification of purified type A & type B toxins and the antigenicity of univalent and bivalent aluminium phosphate adsorbed toxoids. i Immunol t960;84:384-389.

19. Ludlow CL, Hallett M. Rhew K, et al. -Merapeutic use of type F botutinum toxin. N Engi J Med 1992;326:349-350.

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21. Tsui JK, Hayward M, Mak EK, Schulzer M. BoEuiinum toxin type B in the treatment of cervical dystonia: a pilot study. Neurology 1995@45:2109-21 1 0.

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Published in : Movement Disorders Vol. 13, No. 1. 1998, pp. 150-154 © 1998 Movement Disorder Society

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