Clin. exp. Immunol (1976) 26, 302-309.

The effect of levamisole on cellular immunity in multiple sclerosis P. C. DAU, K. P. JOHNSON & LYNN E. SPITLER Departments of Medicine and Neurology, University of California, San Francisco; Cellular Immunology Laboratory, Children's Hospital ofSan Francisco; and Department ofNeurology, San Francisco Veterans Administration Hospital, San Francisco, California, U.S.A.

(Received 19 March 1975)

SUMMARY

The lymphocyte stimulation test has been standardized in a normal human population using four virus cell-associated antigens (VCAA): human embryonic lung cells infected with the LEC and Norrby strains of measles virus, mumps virus, and vaccinia virus. Following 1 week of treatment with the immunopotentiating drug levamisole, a group of multiple sclerosis (MS) patients was found to have increased lymphocyte stimulation responses towards VCAA and increased delayed hypersensitivity responses towards a battery of skin test antigens. No change in the percentage of short- or long-incubation E rosettes occurred. Measles haemagglutinin inhibition (HI) antibody titres measured before and after the entire course of levamisole therapy (12 weeks) did not change. The neurological status of five out of seven MS patients deteriorated while they were taking levamisole.

INTRODUCTION In a previous study (Dau & Peterson, 1970) we found intact cellular immune responses from MS patients towards two paramyxoviruses, the Schwarz strain of measles virus and respiratory syncytial virus; Knowles & Saunders (1970) also found normal responses when cells from patients with active MS were stimulated with measles antigen. A number of investigators have recently reported cellular immune responses towards a variety of paramyxoviruses, including parainfluenza (Ciongoli et al., 1973; Ciongoli et al., 1975), measles (Ciongoli et al., 1973; Utermohlen & Zabriskie, 1973; Fuccillo et al., 1975), mumps (Ciongoli et al., 1973; Cunningham-Rundles et al., 1975), and rubella (Ciongoli et al., 1973), to be depressed in some MS patients. With the exception of the report by Cunningham-Rundles et al. (1973), these later studies have employed leucocyte-migration-inhibition tests, instead of the lymphocyte-stimulation test. These two aspects of cellular immune function might not be concomitantly depressed. In the present study we undertake the evaluation of cellular immune responses towards antigens associated with human embryonic fibroblasts infected by the LEC (ter Meulen, Katz & Kdckell, 1973) and Norrby (Norrby, 1967) strains of measles virus, mumps virus, and vaccinia virus and show that they are increased by treatment of MS patients with the immunopotentiating drug levamisole. Likewise, delayed skin test responses to a battery of five other bacterial and fungal antigens are increased. MATERIALS AND METHODS Lymphocyte stimulation test. Peripheral blood leucocytes were processed and cultured as previously described (Dau & Peterson, 1970), except that nylon fibre instead of cotton fibre columns were used to purify the lymphocytes. Each triplicate culture contained 1 x 106 lymphocytes and 2-5-5 x 10' polymorphonuclear leucocytes in 1 ml of Eagle's MEM-S containing 50 u penicillin, 50 pg streptomycin, 2 mm 1-glutamine, and 10% heat-inactivated autologous serum. Following a 16-h terminal-labelling period with 0 05 ,Ci of [l4C]thymidine (specific activity 59.2 mCi/M) (New England Nuclear, Boston, Correspondence: Dr Peter C. Dau, Laboratory of Cellular Immunology, Children's Hospital of San Francisco, 3700 California Street, San Francisco, California 94118, U.S.A.

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Massachusetts) per culture, the cells were harvested on the sixth day by filtering them onto glass fibre filters (Microbiological Associates, Bethesda, Maryland) with a MASH automatic harvester (Microbiological Associates). The filters were dried, placed into glass counting vials together with 10 ml of a scintillation cocktail (Econofluor, New England Nuclear), and counted for 10 min in a liquid scintillation counter. Results are expressed as the ratio of ct/min in lymphocyte cultures stimulated with virus-infected cells to ct/min in lymphocyte cultures stimulated with uninfected cells of the same type (stimulation index). Patients entered into this study had not taken any immunosuppressive agents during the preceding month. Virus-infected cells. Confluent cultures of human embryonic lung fibroblasts (Flow 2000 line, Flow Laboratories, Rockville Maryland), growing in 150-mm2 T flasks in L-15 medium containing 2% foetal calf serum and antibiotics, were infected with either 104 TCID50 of a newly isolated wild mumps virus, 8-2x 104 PFU of vaccinia virus, or 104 TCID5o of the LEC strain of measles virus and were harvested when a significant cytopathic effect including at least 10-20% cell loss was observed. Surface virus antigen expression was monitored in parallel virus-infected cultures which were haemadsorbed with Simian (measles-infected cultures) or guinea-pig (mumps-infected cultures) erythrocytes at the time of collection. Cultures were only processed for use in the lymphocyte stimulation test if the parallel cultures showed haemadsorption over at least 75% of their surface area. The LU 106 strain of human embryonic lung cells chronically infected with the Edmonston strain of measles virus, as described by Norrby (1967), was also studied; uninfected cultures of both types were processed for use in control cultures. Cultures of LU 106 cells are chronically infected with mycoplasma; Flow 2000 cells are mycoplasma free. To prepare the cells for use in the lymphocyte-stimulation test, the cultures were first incubated with 25 pg/ml of mitomycin C for 30 min at 37'C. The culture medium was then removed; the flasks were rinsed twice with 0 05% trypsin-0-02% EDTA in modified Puck's saline A and placed at 370C for 1 min, and the cells were detached from the bottom of the flasks by gentle shaking in Hanks's balanced salt solution (HBSS) containing 5% pooled, inactivated AB serum. The cells were pelleted by centrifugation of 150 g for 10 min, resuspended in 1 ml of HBSS-5% AB serum, and layered onto the bottom of a 60 x 15 mm plastic petri dish. Any remaining infectious virus associated with the cells was inactivated with u.v. light (253.7 nm) from a General Electric G8T5 germicidal lamp of a distance of 4 cm for 5 min with frequent manual agitation. No infectious virus remained after this treatment. The cells were washed twice with HBSS-5% AB serum, adjusted to the proper concentration in the same medium, dispensed in a volume of 100,1l into sterile, disposable, glass tissue-culture tubes (Bellco, Vineland, New Jersey), and stored at - 70'C until use. Measles antibody titres. Haemagglutinin inhibition (HI) titres were measured on fasting, heat-inactivated sera, according to the method of Norrby (1962). Rosette-forming cells. The percentage of long- and short-incubation E rosette-forming cells was measured according to Wybran et al. (1973). Skin tests. Testing was carried out with a battery of five antigens injected in a volume of 0 1 ml intracutaneously: candidin 1:100 (Hollister-Stier Laboratories, Spokane, Washington), coccidioidin 1:100 (Cutter Laboratories, Berkeley, California), PPD intermediate strength (Connaught Laboratories, Toronto, Canada), streptokinase-streptodornase 40/10 u (Lederle Laboratories, Pearl River, New York), and trichophytin 1:30 (Hollister-Stier Laboratories). A positive response was considered to be 5 mm or more of induration at either 24 or 48 hr. Levamisole therapy. Levamisole (a gift from Janssen Pharmaceutica, Brussels Belgium) was administered in a dose of 50 mg t.i.d. for 3 consecutive days every other week for 12 weeks. Clinically definite MS patients who received the drug were unselected volunteers with long-standing disease and, for the most part, major disability who were referred from the neurology section of the Palo Alto Medical Clinic, Palo Alto, California. Informed consent explaining the theoretical possibility of their condition becoming worse under the influence of an immunopotentiating drug was obtained in each case. Their clinical status was evaluated before and after completion of levamisole therapy by members of the neurological staff of the Palo Alto Medical Clinic or by one of us (P.C.D.). Disability was rated according to Kurtzke (1955). Patients who developed new or rapidly progressive neurological defects while taking levamisole were dropped from the study. Lymphocyte stimulation tests and skin tests were carried out prior to initiating levamisole therapy and 5 days after completing the first week's course of therapy; the total interval between testing was 14 days. Measles HI titres were measured before therapy and after completion of the entire course of therapy 3 months later. Statistical analysis. Differences between independent means were tested for significance by Student's t-test, and differences between observations on the same individual (paired data) by the t-test, difference method.

RESULTS Lymphocyte stimulation tests Dose-response curves with VCAA were carried out with the leucocytes from four male and five female volunteers, mean age 32 years (Fig. 1). A uniform dose of 1 x 105 stimulating cells/culture was selected for the further study of all four VCAA. Prior to levamisole therapy the mean responses of leucocytes from MS patients to VCAA (Table 1) were all lower than the responses of the control group; but because of the small number of patients studied, the difference was only significant (P< 005) in the case of the vaccinia-infected cells. All of the

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25 100 200 SO Number Of stknuk ;ng cells Per cufture (X 10-3) FIG. 1. Stimulation of I x 106 normal human lymphocytes by VCAA. The stimulating cells were human embryonic fibroblasts infected with the Norrby strain of measles virus (open columns), the LEC strain of measles virus (solid columns), mumps virus (hatched columns), and vaccinia virus (stippled columns). 2-5

responses towards NVCAA rose when tested after I week of levamisole therapy, and the increase was significant (P