ORIGINAL ARTICLES

Myelin Basic Protein Treatment of Experimental Allergic Encephalomyelitis in Monkeys Ellsworth C. Alvord, Jr, MD, Cheng-Mei Shaw, MD, and Sarka Hruby, MS ~~

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Treatment of experimental allergic encephalomyelitis (EAE) in two strains of monkeys with large amounts of myelin basic protein (BP) fails unless an adjunct is also used. In both strains the adjunct by itself is more effective than BP by itself, but in the one strain which could be investigated sufficiently, the combination can be made almost totally effective in reversing EAE. The adjunct varies with the strain of monkey, an antibiotic inMacaca mulatta and a steroid in Macaca fascictllaris. Similar adjunctive treatments should be considered in the management of multiple sclerosis, for which EAE remains one of the best studied models. Alvord EC Jr, Shaw C-M, Hruby S: Myelin basic protein treatment of experimental allergic encephalomyelitis in monkeys. Ann Neurol 6:469-473, 1979

[4, 8-10]. O n the assumption that humans with MS have a much more complicated immunological reactivity than inbred guinea pigs with EAE, it seems likely that the development of a protocol for adequate treatment of EAE in monkeys would be a much more revealing model on which to base potential treatments of MS in humans [ 111.

Experimental allergic encephalomyelitis (EAE) is one of the models being exploited in attempts to understand the cause, pathogenesis, prevention, and therapy of multiple sclerosis (MS). Since none of these aspects of MS is understood, and since each has been rather successfully analyzed in EAE [3, 5,28,30,431, clinical investigators may be led to believe that the reports [13, 141 of failure of myelin basic protein (BP) treatments in acute or chronic MS represent the demise of the hypothesis that EAE and MS could be related. Such failures, however, are exactly what would happen if EAE and MS were identical! How can such a surprising contradiction occur? It is well known that induction of delayed-type hypersensitivity to homologous B P also induces EAE [ 191 and that BP can specifically interfere with this hypersensitivity in situations which also either prevent the onset of EAE [ 7 ] or treat the disease after it appears [181. The popular concept that BP should be completely successful in the therapeutic situation in either EAE or MS stems from a lack of critical thinking, however, because only in highly inbred strain 13 guinea pigs is BP by itself a completely effective treatment [18]. In noninbred guinea pigs BP is much less effective [18], and in monkeys B P by itself is almost completely ineffective in treating EAE [34, 351. We have been studying monkeys with EAE for several years, trying to understand the species differences in responses to apparently the same stimulus

Monkeys were obtained by and housed in the Regional Primate Research Center at the University of Washington, Seattle. They were housed individually in cages and studied in groups of 12 maintained in isolation from any other animals. EAE was induced by intradermal injection of a total dose of 0.1 ml of a water-in-oil emulsion containing 5 mg of monkey BP and 0.5 mg of heat-killed Mycobacterium tuberculosis [34, 351 into the hindfoot pads. BP was prepared by the batch tecgnique of Deibler et al [17]. A blind experimental design was maintained as follows: The onset of EAE was determined by one of us (C.-M. S.) according to criteria previously published [ 3 51, and one or another treatment was then instituted according to a randomized schedule maintained by the two other investigators (S. H. and E. C. A,). The clinical evaluations of the diagnostician (C.-M. S.) were recorded at least twice daily, and he also evaluated the therapy as either successful or unsuccessful and decided when to terminate it, remaining uninformed as to just what treatments were being investigated in either the group as a whole or individual animals. Following successful treatments, any relapse in a particular animal was treated in the same way with the same

From the Laboratory of Neuropathology, Department of Pathology, University of Washington School of Medicine, Seattle, WA .

Address reprint requests to Dr Alvord, RJ-05, Laboratory of Neuropathology, Department of Pathology, University of Washington School of Medicine, Seattle, WA 98195.

Methods

Accepted for publication May 6, 1979.

0364-5134/79/120469-05$01.25 @ 1978 by Ellsworth C. Alvord, Jr

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therapy. At varying intervals after apparent recovery, or if moribund following an unsuccessful treatment, the animals were sacrificed with an overdose of Nembutal followed by perfusion-fixation and autopsy. Animals that died were also autopsied. Following fixation the brain and spinal cord were examined carefully for gross and microscopic lesions, hematoxylin and eosin being routinely supplemented by gallocyanine and Darrow red preparations. Holzer astroglial preparations were made frequently on sections from paraffin-embedded blocks, and sections from frozen blocks were occasionally stained with oil red 0 and hematoxylin. Treatments included monkey B P in Freund’s incomplete adjuvants (IFA, or water-in-oil emulsion without mycobacteria) o r in physiological saline, procaine penicillin G (200,000 units daily intramuscularly), Keflin (125 mg every 12 hours intramuscularly), Decadron (0.7 mg every 12 hours intramuscularly), or combinations of these agents. Cop I, a synthetic copolymer of the four amino acids alanine, glutamic acid, lysine, and tyrosine with a molecular weight of 23,000 daltons, was generously supplied by Drs Ruth Arnon and Michael Sela [42]and was substituted for the BP in some experiments. Trasylol was obtained in concentrations of 10,000 KIU per milliliter from FBA Pharmaceuticals (New York, NY) and 20,000 KIU per milliliter from Calbiochem (La Jolla, CA). For ease of presentation, the particular amounts of B P and the timings of injections are given with the results.

Results Our results in rhesus monkeys (Maraca mulatta) are summarized in Table 1, in which the results of 16 treatments utilizing Keflin instead of penicillin are added to the 2 5 penicillin or saline combinations previously reported by Shaw et a1 [35]. It is clear from Table 1 that BP by itself (10 mg daily in 0.5 ml of IFA injected intramuscularly every day) is totally ineffective, none of 5 monkeys showing any improvement. If the 3 monkeys receiving IFA alone are included with these, a “spontaneous” remission rate of about 12% can be expected in this type of experiment. By contrast, if penicillin or Keflin is given, a considerable increase in successful treatments occurs, 40% without BP and 56% if BP is also given. Although these results are not statistically different, we were unable to continue the experiments because of a sharp drop in the supply of M . mulatta. We shifted our experiments to the very closely related Macura fascicularis. The experimental design was exactly the same except that Cop I was substituted for BP in some of the animals at the same dose level (10 mg per day in IFA); but the results were quite different (Table 2). No more than 20% of the animals in any of the subgroups of this strain improved. The only immediate explanation for the markedly different results in M . fasrirduris compared with M. nzzllatta was the frequent appearance of sterile

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Table 1. Comparison of Effectiveness of Various Combinations of Treatments for E A E in Macaca mulatta”

Antigen Daily Adjunct

NaCl/IFA

NaCl Penicillin

1/3

Keflin Total

BP/IFA

Total

Ol 5

1’4) 40% 316 5 / 1 3 (38%)

118 (12%)

56% 13/28 (46%)

52%

18/41 (44%)

aThe ratios represent the number of improvements versus number of treatments since some animals were treated repeatedly.

Table 2. Comparison o f Ineffectiveness i n Macaca fascicularis of Similar Combinations of Treatments for EAE as Those Used i n Macaca mulatta” Antieen Dailv in IFA Adjunct

NaCl

BP

Cop I

Total

NaCl Keflin

0/5 0/3

113

O/ 3 0/10

1 / 1 1 (9%) 1/20 ( 5 % )

Total

117

0/8 ( 0 % ) 2/10 (20%) 0113 (0%) 2/31 (6%)

“The ratios represent the number of animals improved versus number of animals treated.

abscesses at the sites of the IFA injections, a phenomenon never seen in M. mulatta. This observation suggested that absorption of BP or Cop I might have been impaired in M . fasricdaris. Accordingly, we decided to shift to saline solutions of BP (2 mg every 2 hours), which we thought should be injected every 2 hours because of the rapid destruction of BP by proteolytic enzymes in plasma. We had previously shown [lOa] that BP is degraded so rapidly (initially about 5% per minute) by plasma in vitro that no B P was detectable after 2 hours of incubation at 37°C. The addition of Trasylol-an antiproteolytic, highly basic peptide effective against many trypsinlike enzymes, including kallikrein and plasmin but not thrombin [25, 401-at 10 KIU per milliliter decreased this exponential rate in vitro to about 3% per minute, so that at least small amounts (about 5 % ) of BP persisted for 2 hours. As a corollary of these around-the-clock injections, we observed that nocturnal epileptic seizures could be fatal. We speculated that edema in and around acute EAE lesions involving or impinging upon the cerebral cortex might be initiating these seizures and that steroids might be more effective than anticonvulsants. Whether these speculations are correct or not, Table 3 indicates that the combination of Keflin and Decadron without BP gave distinct improvement

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Table 3. Comparison of Effectiveness of Various Combinations of Treatments for E A E in Macaca fascicularis”

Table 5 . Summary of Therapeutic Results Obtained i n Macaca fascicularis”

Antigen Every 2 Hours

Antigen

Adiunctb

OlNaCl

BP/NaCI

Total

Adjunct

NaCl Trasylol Tot a1

3/5 2/4 5/9 (56%)

4/5 1/ 1 5/6 (83%)

7/10 (70%) 3/5 (60%) l o l l 5 (67%)

NaCld 0/10 3/14 (21%) 0113 Decadrond 7/12 ( 5 8 % ) 11/12 (92%) . . . 7/22 14/26 0/13 Total

NaCl

BPb

Cop Ic Total 3/37 18/24 21/61

aThe ratios represent the number of improvements versus number of treatments since some animals were treated repeatedly. addition to Decadron (0.7 mg) and Keflin (125 mg) every 12 hours.

aThe ratios represent the number of improvements versus number of treatments since some animals were treated repeatedly. bDose: 10 mg in IFA daily or 2 mg in saline every 2 hours. “Dose: 10 mg in IFA daily. dWith or without Keflin (125 mg every 12 hours).

Table 4. Comparison of Effectiveness of Various Cornbinations of Treatments for E A E in Macaca fascicularis Which Were Given No Other Drugs“

75% positive results with B P and penicillin as compared to no spontaneous recoveries in M . mzclatta; however, their experiments [2 11 were not conducted blind, in contrast to the present experiments, in which great efforts were made to prevent any personal bias being introduced. Furthermore, Eylar et a1 [ 2 11 did not compare B P and penicillin individually. We cannot confirm the positive results of Arnon et a1 [12, 39,411, who studied Cop I without Decadron in small numbers of rhesus monkeys (M. mzllattu) and baboons. However, it should be noted that Cop I has been tested only with Keflin (see Table 2) and has not yet received an adequate trial combined with Decadron in M. fasciczclaris. It should be emphasized that neither M . mzllatta nor M . fasciczllaris responds to B P by itself and that both strains require an adjunct. However, the adjunct is quite different, an antibiotic for M . mulattu and a steroid for M. fasciczllaris. An effective treatment protocol in an available species provides the vehicle for investigating the mechanisms underlying successful treatment of EAE. We have noted synchronization of many variables (increase in total white blood cells, decrease in lymphocytes, inhibition of migration and clumping of buffy coat leukocytes in the presence of BP or tuberculin, increase in C-reactive protein, and development of antibodies to BP) as EAE develops in M . mzllatta [4, 8-10a1, but only one of these seems to correlate with improvement: reversal of the lymphocyte count toward normal. Our data in M . fasciczllaris tend to confirm this impression. From all these observations we suspect that a reduction simply in the continued production of specifically sensitized lymphocytes in peripheral lymph nodes (and possibly in other portions of the reticuloendothelial system) is not a sufficient requirement for improvement, and that a concomitant reduction in other leukocytes must also occur before the lesions in the central nervous system can begin to heal. But just where this effect occurs-in the lymph nodes, in the circulation, or in the nervous system-is not known.

Antigen Every 2 Hours Adjunct

OlNaCl

BP/NaCI

Total

NaCl Decadron Total

012

1/4

213 2/5 (40%)

616 7/10 (70%)

1/6 (17%) 819 ( 8 9 % ) 9/15 (605%)

T h e ratios represent the number of improvements versus number of treatments since some animals were treated repeatedly.

(56%), and that still further improvement (83%) occurred if BP was also given. Trasylol (10,000 KlU/kg every 2 hours intramuscularly) did not appear to have any beneficial effect (Table 3). Therefore we eliminated Trasylol in the next experiment (Table 4), which was designed to see whether the antibiotic Keflin was necessary or not. Table 4 clearly indicates that, in contrast to the results in M . mzllatta (see Table l), an antibiotic such as Keflin is not necessary for effective treatment of EAE in M. fasciczllaris. Discussion Since each of the experiments in the present report was performed in identical fashion, all the results obtained in M .fasciczllaris are summarized in Table 5 . Decadron by itself or combined with Keflin produced improvement 58% of the time and was almost three times as effective as BP, which by itself or combined with Keflin produced improvement only 2 1% of the time. The combination of Decadron and BP was still more effective, so that about 90% successful treatments of M . fasciczllaris were obtained. It is noteworthy that the results in M . mzllatta were quite different and that treatments which were reasonably effective in M . mulutta (see Table l) were not at all effective in M . fasciczllaris (see Table 5). From Table 1 it is clear that we can only partially confirm the results of Eylar et a1 [21], who reported

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Since so little is understood about the pathogenesis of MS [2, 6, 22, 26, 29, 381, it would be fruitless to speculate on the location of the therapeutic attack against the disease. Should it be directed toward the peripheral reticuloendothelial system, toward the possibly sensitized lymphocytes in the circulation [14, 15, 16, 24, 27, 36, 371 or about the veins in the MS plaques [2, 20, 441, toward the macrophages at the circumference of the radially expanding lesion [6, 291, or toward other cells [31, 321 and other sites? Regardless of where the effective site of action may prove to be, it is clear that the therapeutic trials in MS with BP [13, 231 or Cop I [l] have not yet been conducted on a sound basis of animal experiments. Neither the amounts of BP that might be required in humans nor the possibility of an adjunct has been considered [ 111. In view of the results of the monkey experiments reported here, it is not at all surprising that MS patients have failed to respond to the previously administered doses of BP-too small by a factor of about 1,000 [ 111-given without an antibiotic, a steroid [33], or some other adjunct that human beings may be found to require. Supported in part by Research Grant RG-805-C-18 from the National Multiple Sclerosis Society and N I H Grant RR00166 to the Regional Primate Research Center at the University of Washington, Seattle.

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