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Levamisole: A new treatment for recurrent aphthous stomatitis
oral medicine Editor: JAMES
W. LITTLE,
D.M.D.,
M.S.D.
Chairman and Professor Department of Oral Diagnosis and Oral Medicine University of Kentucky Lexington, Kentucky 40506
Levamisole: A new treatment for recurrent aphthous stomatitis James A. Olson, D.D.S., M.S., D. Cade Nelms, D.D.S., Ph.D., Sol Silverman, M.A., D.D.S., and Lynn E. Spitler, M.D., San Francisco,
Calif.
UNIVERSITY
OF CALIFORNIA
AND
OF MEDICINE
SCHOOL
AT
SAN
FRANCISCO
SCHOOL
OF DENTISTRY
Fifty patients with recurrent aphthous stomatitis were treated with levamisole, an anthelmintic which has been found to increase immune responses. The subjects had previously been refractory t,o all modalities of treatment other than prednisone, and 92 per cent had experienced monthly or continuous attacks of aphthae. The responses were grouped into three categories: 6 per cent appeared to go into permanent remission, 56 per cent were improved, and 38 per cent showed no response. Levamisole appeared to have a definite enhancing effect on the immune system, as measured by skin test reactivity, but did not affect in vitro parameters of cellular immunity.
R
ecurrent aphthous stomatitis is a chronic diseasecharacterized by painful, recurring ulcerations of the oral mucosa. Although the frequency of recurrent aphthous stomatitis in the general population is unknown, prevalence studies in special groups indica.te that the incidence ranges from 20 per cent in a hospital populationZ1* 24to 60 per cent among professional school studentsz2 There is considerable variation in the number, frequency, duration, and size of aphthous lesions. Aphthae usually occur as single or multiple shallow ulcerations, 2 to 4 mm. in diameter. Occasionally, they occur in large clusters. Ulcers exceeding 6 mm. in diameter produce more tissue destruction and are more persistent than those of small size. In the most severe variant, recurrent This research received support from the University of California California Dental Association, and the National Institutes of Health a gift from The Janssen Pharmaceutical Company. Dr. Spitler is supported by a Research Career Development Award
Board (NIDR (AI
of Regents, AI 10686)
43012).
the and
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Fig. 1. This patient has had similar aphthous attacks eight to ten times per year for the past 15 years. These ulcers were present for 3 days and healed in 2 weeks. All of these lesions were less than 6 mm. in diameter and were categorized as minor aphthae.
oral ulcerations are accompanied by ocular and genital involvement and occasionally by arthritis and other systemic manifestations. The triad of oral, ocular, and genital symptoms comprises Behcet’s syndrome.3l 4 The cause of recurrent aphthous stomatitis is unknown. Studies of the cellular immune systems, 15,l6 the humoral immune system,ll 6+I17I5 and lymphocytotoxicity9y *O have implicated immunologic variances in the pathogenesis of the disease. There is no standard treatment for aphthous ulcers, and clinical management is usually directed toward symptomatic relief. In small uncontrolled studies, levamisole (the levo isomer of racemic 2,3;5,6, tetrahydro 6 phenylimidaze (2,1-b) thiazole hydrochloride) has been reported to be effective in controlling attacks of recurrent aphthous stomatitis and in reducing subsequent episodes 26y3o Levamisole is an anthelmintic agent which has few toxic side effects. Animal studies have demonstrated that levamisole may increase both cellular and humoral immune responses,13s I’, I8 stimulate phagocytosis,14 and inhibit tumor growth.lg In man, levamisole has been reported to restore cutaneous activity to purified protein derivative (PPD) and/or dinitrochlorobenzene (DNCB) in some anergic patients.27-29 The purpose of this study was to determine the efficacy of levamisole in alleviating recurrent aphthous stomatitis by evaluating clinical responses to a specific drug schedule and by measuring alterations in selected immunologic parameters. MATERIALS Patients
AND
METHODS
Thirty-one female and nineteen male patients, ranging in age from 9 to 66 years, made up the study group. All patients had actively recurring aphthous ulcerations, established by history and clinical examination. One hundred normal subjects are included in this study as controls for the selected immunologic tests.
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et nl.
Fig. 8. This patient had a lo-year history 4 weeks, was more than 1 cm. in diameter
Laboratory
of similar aphthae. This lesion, which and was classified as a major aphthous
persisted ulcer.
study
Laboratory examinations included complete blood counts, exfoliative cytologic smears for evidence of cytopathogenic effects,23 direct culture for herpes virus, serologic examination for herpes antibody titers, and biopsies of early aphthous ulcers (1 to 3 days). Biopsy specimens were divided for both electron and light microscopic examinations. A battery of immunologic studies was performed prior to and 1 to 3 months after initiation of levamisole treatment. These studies were selected to determine the immunopotentiating effects of levamisole and included skin tests, lymphocytestimulation studies,l* human leukocyte migration inhibition factor test (LIF) ,’ and tests for chemotaxis.5s7 Skin tests
Each patient was tested with a battery of six skin-test antigens-candida (Dermatophytin 0, Hollister-Stier Labs, Spokane, Washington), 1 :lOO and 1 :lO; coccidioidin (Cutter Laboratories, Berkeley, California), 1 :lOO and 1 :lO; mumps (Eli Lilly, Indianapolis, Indiana) ; purified protein derivative from tubercle bacillus (PPD, Connaught Laboratories, Toronto, Canada), intermediate and second strength; streptokinase-streptodornase (Varidase, Lederle Labs, American Cyanamid, Pearl River, New York), 4/l U. and 40/10 U. ; and trichophyton (Dermatophytin, Hollister-Stier Labs, Spokane, Washington), 1:30. lymphocyte
stimulation
This test was performed by the method of Spitler and associates.25Lymphocytes were obtained from peripheral huffy coat cells by passing over a nylon fiber column. The cells were cultured in triplicate tubes with 2 x 10” cells in 2 ml. culture medium (minimal essential medium) containing antibiotics, Lglutamine, and 10 per cent homologous serum from male donors of AB blood type. Antigen (candida, coccidioidin, PPD, or SK-SD) or PHA was added to replicate tubes at the initiation of the cultures. PHA was added in doses of
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Fig. J. This 3%year-old female patient in numerous small clusters. She also had was diagnosed as Behqet’s syndrome.
for
recurrent
aphthous
stomatitis
had continuous attacks of aphthae which ocular and genital involvement, and her
591
occurred problem
500, 250, 12’5, and 62.5 mg. per tube. C?” thymidine was added to the cultures containing antigens after 5 days of incubation, and the cultures were harvested on the sixth day. Control and PHA-stimulated cultures were harvested after 3and 6-day incubation periods. Results were assessed by counting in a liquid scintillation counter. leukocyte
migration
inhibition
The technique of Astor and colleagues* was used to measure leukocyte migration inhibition. Buffy coat leukocytes were obtained by dextran sedimentation and adjusted to a concentration of 220 x lo6 per milliliter. Fifty microliter aliquots were dispersed into Falcon plastic tubes, and the following was added sequentially in 5 microliter amounts: saline, PPD 30 mg./ml., SK-SD 6000 U/ml., candida 1:2 dilution or coccidioidin. The cell preparations were incubated at 37O C. for 30 minutes. Agarose was freshly prepared by dispensing 60 mg. agarose in 6 ml. distilled water with 0.6 ml. filtered horse serum, 0.6 ml. Medium 199 (10X), and sodium bicarbonate, The agarose was poured into Falcon Petri dishes, and wells 2.3 mm. in diameter were cut. Seven microliters of each cell preparation was placed in triplicate wells, and the plates were incubated at 37O C. for 18 to 24 hours. The plates were placed in water at 80° to 90’ C. to fix the cells to the plates and to cause the agarose to partially melt and flow off. The areas of migration were determined by reading two diameters with an ocular micrometer. A positive response in the LIF test was considered to be at least a 20 per cent decrease from the control area of migration. Test for chemotaxis
This test was performed by the De Meo and Anderson? modification of the Boyden technique. Ten milliliters of venous blood containing 0.3 ml. sodium heparin (1,000 units per milliliter) was drawn and allowed to sediment with dextran in an upright position for 45 minutes at 37O C. An additional 10 ml. of blood was drawn and allowed to clot, and the serum was used for the chemotactic tests. The polymorphonuclear leukocytes (polys) were collected
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Fig. 4. A, Two major aphthae had been present for 2% months on the lower lip of this El-year-old boy. B, Two weeks after administration of levamisole there was complete healing of the ulcers. This patient has had similar healing with subsequent courses of levamisole therapy.
and counted, and the concentration was adjusted to 5,000 to 10,000 cells/mm.3 The miniaturized chemotaxis chamber (Mark-It Engineering Corporation) was assembled with a 3~ pore diameter filter placed between the upper and lower members. The bottom chamber was filled with 0.2 ml. dilute 502A staphylococcus filtrate and serum or saline solution, and the top chamber with 0.4 ml. of poly suspension. The chamber was incubated for 2 hours at 37O C. Following incubation, the top chamber was emptied with a gauze sponge down to the filter. The filter was removed, fixed in paraformaldehyde, and mounted on a glass slide. The number of polymorphonuclear leukocytes migrating through the filter per high-power field was counted. Three chemotactic tests were compared before and after levamisole therapy: (1) the patient’s white blood cells (WBC) with their own serum and antigen, (2) the patient’s WBC with control serum and antigen, and (3) the patient’s WBC with saline solution as a control. The control for each test was obtained from frozen samples originally drawn from a single male donor with type AB blood. Therapy
regimen
Following clinical and laboratory documentation of recurrent aphthous stomatitis and determination of immunologic status, each patient entered an open clinical therapeutic trial with levamisole. The drug was administered by mouth at a level of 2.5 mg. per kilogram of body weight per day, with the dosage rounded to the nearest 50 mg. The usual adult dose was 150 mg. per day. An abortive therapy schedule was used for all patients in this study. If ulcers were present, patients were given levamisole 2 successive days per week. If ulcers were not present, the drug was not given until new lesions developed, at which time the drug was given for 2 sequential days per week. If patients had continuous aphthae, levamisole was administered 2 days per week for approximately 4 months. If there was no improvement after 4 months, the therapy was discontinued. If improvement was noted, treatment was continued for the duration of the study. Patients who had remissions of more than 1
Volume 41 Number 5 Table
New treatment for recurrent aphthous stomatitis
I. Clinical findings in fifty patients with recurrent aphthous stomatitis Male (19patients)
Size of lesions Minor aphthae* Major aphthae$ Duration
‘2
131
2
(4)
I
ANpatients (JOpatients)
34 (68) 16 (32)
0
(0)
‘;i
[:08{
2
$5 ‘q
‘Z
(4) g;
ofrecurrences
Less than 2 per month 2 to 4 per month More than 4 per month or continuous Behcet’s
1’0 ‘(:!I)+
Female (31 patients)
of each lesion
Less than 10 days 11 to21 days More than 3 weeks Frequency
593
disease
3 (6) 10 (20)
6 (121
0
(01
1 (2) 22 (44
4 (8) 32 (64)
8 (16)
1; ‘:;I’
4
18)
*Minor aphthae: lesions 6 mm. or less in diameter. tNumbers in parentheses indicate the percentage of all patients category. $Major aphthae: lesions more than 6 mm. in diameter.
falling
in the indicated
week’s duration did not receive another dose of levamisole until new lesions again developed. Analysis
of results
The response to levamisole therapy was determined by: (1) clinical immunologic tests, (2) objective assessmentof the clinical course of the disease, and (3) the patient’s subjective assessmentof therapy. Ulcer assessmentwas recorded by photographs, mucosal distribution, average diameter of lesions, number of lesions per attack, and frequency of attacks. Patients mailed in special postcards recording the frequency and duration of aphthae between visits. Because of the extreme variations in aphthous manifestations, a classification was devised to measure the severity and treatment response. Since size and duration of aphthous ulcers appeared to be the most critical factors, three categories were included: (1) minor aphthae-6 mm. or less in diameter with individual ulcer duration of less than 3 weeks, (2) major aphthae-greater than 6 mm. in diameter with duration greater than 3 weeks, and (3) Behcet’s disease-ulceration of the oral mucosa with either ocular and/or genital involvement, arthritis, or other systemic manifestations. Statistical analyses were performed by the paired or unpaired Student t test when appropriate. RESULTS Patient population
Table I describes the clinical status of the aphthous patients. Approximately two thirds of the patients had minor aphthae (6 mm. or less), and one third had major aphthae (over 6 mm.). The majority of patients (64 per cent) had two to four aphthae per month. In most casesthe ulcers persisted for 2 to 3 weeks. Eight per cent of the patients had Behcet’s disease.
594
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Olson et 01.
IMay,
II. Clinical results of levamisole aphthous stomatitis
Table
Major
uphthae
Female
“Cured”
0
therapy
in fifty patients Minor
Mule
to)*
Female
1976
with recurrent
uphthae 1
3 (6)
Mule
Allpatients
0 (0) 14 (28)
3
(6)
Improved No change I:; i [:; :: f:i{ 37 (14) (6) 2Y (6) 11 (22) Total lo 6 Y 25 50 *Numbers in parentheses indicate percentage of all patients in the indicated category. Table
Ill. Side effects in fifty patients with recurrent
undergoing
Drowsiness Taste and smell alteration Skin rash Headache *Numbers in parentheses indicate category. laboratory
aphthous stomatitis
levamisole therapy
percentage
of all patients
falling
3 3 2 2 into
(6)* (6) (4) (4) the indicated
studies
Exfoliative cytologic examination and direct culture for herpes virus were negative for the presence of virus. Herpes antibody titers were low, indicating that the lesions were not due to active herpes infections. Evaluation of biopsy specimens with the light microscope confirmed the early lymphocytic infiltration in the disease with no distinctive diagnostic features. Ultrastructure observations did not reveal any specific features relative to cause or categorization. Clinical
results
Table II shows the clinical results of therapy. Three patients have been free of aphthous ulcerations for more than 6 months since receiving levamisole. Twenty-eight patients demonstrated improvement, marked by a decrease in the number, frequency, size, and duration of aphthous ulcers. Neither the severity of lesions nor the sex of the patient appeared to be significantly correlated with the response to levamisole. Table III illustrates the side effects of levamisole therapy observed in these patients. Twenty per cent of the patient,s experienced side effects of therapy. Drowsiness and mild fatigue were noted by three patients. This was ameliorated by administering the drug at bedtime. Three patients noted alteration in taste and smell, and two noted headaches. Two patients developed skin rashes; both have subsequently taken the drug without incident. In none of the patients were symptoms severe enough to warrant discontinuat.ion of therapy. Skin test results
Before therapy, skin test reactivity to the panel of akin test antigens was not significantly different in the patients with recurrent aphthous stomatitis as compared to normal control subjeets. More than 95 per cent of the patients tested showed a significant increase (more than 5 mm. induration) in skin test
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New treatment for recurred
IV. Changes in skin test reactivity twenty-six patients Table
Decrease Per cent
No.*
595
before and after levamisole therapy in
Increase Antigen
aphthous stomatitis
No.
No change Per cent
No.
Per cent
19 13 SK/SD 1; ii : Candida 11 4: 6 11 Mumps 14 ; ;: PPD i tt Coccidioidin 5 :; 1 ; 0 0 Trichophyton Gi ii i3 ii 31 Total *Numbers of subjects showing a 5 mm. or greater change in induration at the 48 hours after injection of the test antigen.
ii 23 54 ii 54 test site
reactivity to one or more antigens following levamisole therapy. Forty-five per cent of the patients showed a significant increase in reactivity to two or more antigens. Table IV demonstrates the changes for the individual test antigens. Significant increases in reactivity were recorded with all antigens in varying percentages of patients. The most frequent increase was seen in reactivity to SK-SD, to which 50 per cent of the patients showed increased reactivity after levamisole therapy. On the other hand, decreased reactivity to individual antigens was also observed in some patients. There was no correlation between conversion of immunologic reactivity and the patients’ clinical course. Over-all, the mean skin reactivity for all patients increased with candida, PPD, and SK-SD and decreased with mumps (Fig. 5). The changes in reactivity observed with candida and PPD were statistically significant (p < 0.02)) but those for SK-SD (p = 0.10) and mumps (p = 0.20) were not. Too few patients responded to trichophytin and coccidioidin to permit us to draw meaningful conclusions. lymphocyte
stimulation
results
There was no significant change in the background counts per minute (CPM) when results obtained before and after levamisole therapy were compared. A significant change in lymphocyte stimulation was considered to be a fivefold or greater change in the stimulation index, and an index of less than I was considered to be 1 for calculations. Only four tests with specific antigens showed a change in the thirty-five tests performed. One showed an increase and one a decrease to candida, and two showed a decrease to SK/SD. Over-all, the mean stimulation indices for three antigens tested (PPD, SK-SD, and candida) were not significantly changed after therapy (p = 0.2). The stimulation index was not significantly influenced by the addition to the culture& of levamisole, alone or with specific antigens (Fig. 6). leukocyte
migration
inhibition
test results
Comparison of the areas of migration in control wells without antigen before and after levamisole therapy can be used to describe an index of random migration of polymorphonuclear leukocytes (polys) .zo In twenty patients tested,
596
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et
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al.
50
4o
/
30
SK'SD
CANDIDA PPD MUMPS
SIZE (UUIII
20
10
BBPORE LBVAMIsoLB
AFTER LBVAHISOLE
Fig. 5. Results of skin test reactivity in patients with recurrent aphthous stomatitis before and after levamisole therapy. Numbers indicate millimeters of induration at the test site 48 hours after injection of the test antigen. SK/1sD, Streptokinase-streptodornase. PPD, Purified protein derivative.
SK/SD PPD CANDIDA STIMULATION INDEX
1:; 1
fl
3.0 I 2.0 LEVAMISOLE
1.0
I
BEFORE LBVAMISOLS
AFTER LEVAMISOLE
Fig. 6. Results of lymphocyte DNA synthesis (radioactive thymidine incorporation) in response to specific antigens in patients with recurrent aphthous stomatitis before and after levamisole therapy. Stimulation index equals the counts per minute in experimental tubes with antigen divided by the counts per minute in control tubes without antigen.
the findings were as follows: Six showed an increase, ten showed a decrease, and four showed no change in random migration of polys. When cells were pre-incubated with levamisole alone and compared to control cells incubated with saline solution, there was no inhibition or stimulation of migration. Of the fifty-eight tests performed with specific antigen, forty-seven showed no change, eight converted from positive to negative, and three converted from negative to positive. No change indicates that those tests that were positive before therapy remained positive and those that were negative reamined
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New treatment for recurrent aphthous stomatitis
597
100 I 90
-
I
Migration
LEVAMISOLE
40 I 30 20 10 I 1
BEFORE
LEVAI4ISOLS
AFTER L!NAMISOLE
Fig. 7. Results of leukocyte migration inhibition tests before and after levamisole therapy in patients with recurrent aphthous stomatitis. Per cent migration indicates the ratio of the area of leukocyte migration tested with specific antigen to the control area of migration without antigen.
12 HOMOLOGOUS SERUM #l AUTOLOGOUS SERUM
I
BBPORE t5vANISOm
AFTER LEVAH1s0LE
Fig. 8. Results of chemotactic tests in patienta with recurrent aphthous stomatitis before and after levamisole therapy. The polymorphonuclear leukocytes in five high-power fields are counted and averaged to give the number of white blood cells per high-power field.
negative. Statistical analysis of the over-all migration inhibition produced by each of the three test antigens indicated no significant difference in the results obtained after therapy as compared with results before therapy (Fig. 7). In vivo and
in vitro
correlations
Of thirty-eight significant changes in skin test reactivity to candida, PPD, and SK-SD in twenty patients, there are a total of fifty-three corresponding in vitro test results to specific antigens. Changes in only ten of the fifty-three in vitro tests paralleled the changes in skin test reactivity. Eight of the thirty-
598
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four LIF tests and two of nineteen lymphocyte to respective skin test changes. Chemotaxis
stimulation
Surg. 1976
tests corresponded
results
Fourteen patients were studied. After levamisole therapy, there was an increase in chemotaxis in both autologous and homologous serum, but the increase was statistically significant for studies performed in homologous serum only (p = 0.013) (Fig. 8). DISCUSSION Clinical effects
Levamisole produced a beneficial effect in reducing the number, frequency, and severity of recurrent aphthous stomatitis attacks in 62 per cent of the patients tested. Other studies 26s3o have similarly reported improvement in approximately two thirds of the subjects treated. The drug appeared safe, but long-range effects, if any, are unknown. A major advantage of levamisole over systemic corticosteroids in control of aphthae would be the possibility of permanent control without serious side effects. Although long-term control was observed in only 6 per cent of the patients treated, this percentage might be improved by altering the dosage and/or the frequency of administration. The mechanism by which levamisole produces clinical benefit in patients with recurrent aphthous stomatitis is not clear. It is well recognized that corticosteroids produce symptomatic relief and diminished signs in patients with this disease. Corticosteroids have a variety of biologic effects, immunosuppression being one of them, In this regard, the action of levamisole is exactly opposite; it increases rather than decreases immune reactivity. Immunologic
effects
Immunologic studies revealed that all patients were immunocompetent, but their reactivity was slightly decreased before treatment with levamisole when compared with the reactivity of .the controls. The criterion for immunologic competency is a response to at least two skin test antigens. Previous studies with levamisole have shown increased skin test reactivity to PPD in 22 per cent to 41 per cent of the subjects tested. 28,29 Another studyzT showed a 45 per cent conversion of skin test reactivity with dinitrochlorobenzene (DNCB) . Ninetyfive per cent of the patients in our study showed an increase in skin test reactivity to at least one antigen. Increased responses were seen primarily with candida, PPD, and SK-SD following levamisole therapy. On the other hand, a decrease in skin test reactivity to the various antigens, especially mumps, was also noted in some patients. This observation deserves further evaluation in a larger series of subjects. The findings of increased skin test reactivity after levamisole therapy without a concomitant increase in the lymphocyte stimulation index and without a decrease in the per cent migration suggests several possible modes of action. Levamisole or some metabolic product may act on cells involved in the non-
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specific inflammatory process and not directly on lymphocytes. The site of action may be the macrophage or polymorphonuclear leukocyte, thereby affecting chemotaxis and/or phagocytosis. The observation that levamisole increased the chemotactic response in these patients suggests that this may be the case. The drug may influence macrophage processing of antigen. Alternately, levamisole may affect the local vascularity at the test site (inflammation) by acting on the vascular endothelium to produce hyperemia. It is possible that other in vitro studies with different time sequences might show that levamisole does affect the lymphocyte function. However, our in vitro tests were performed at the same time that the changes in skin test reactivity occurred. We conclude that levamisole therapy produced definite immunologic changes and was of benefit in patients with recurrent aphthous stomatitis. Randomized double-blind studies are necessary to resolve definitively the question of clinical efficacy, and such studies appear to be warranted. REFERENCES
The Antinuclear Factor, J. Dent. Res. 1. Addy, M., ar$i Dolby, A. D.: Aphthous Ulceration: 51: 1594-1595, 1972. 5. H., Spitler, L. E., Frick, 0. L., and Fudenberg, H. H.: Human Leukocyte 2. Astor, Migration in Agarose Using Four Antigens: Correlation With Skin Reactivity, J. Immunol. 110: 1174.1179, 1973. 3. Behset, H.: tiber regitlvierende, aphthoese, durch ein Virus verursachte Geachwure am Mund, am Ange und an den Genitalient, Dermatol. Wochenschr. 10s: 1152,1937. 4. Berlin, C.: BehCet’s Disease as a Multiple Symptom Complex, Arch. Dermatol. 82: 127, 1960. 5. Boyden, Chemotactic Effect of Mixtures of Antibody and Antigen on S. : The Polymorphonuelear Leukocytes, J. Exp. Med. 115: 453-466, 1962. to Genital Mucosa in Patients With 6. Cummings, N. A., and Graykowski, E. A.: Antibodies Aphthous Stomatitis (AS), IADR Abstr. 915, 1974. 7. De Meo, A. N., and Anderson, B. R.: Defective Chemotaxis Associated With a Serum Inhibitor in Cirrhotic Patients, N. Engl. J. Med. 286: 735-740, 1972. 8. Dolby! A. E., and Allison, R. T.: Quantitative Changes in the Mast Cell Population in Mikulmz’s Recurrent Oral Aphthae, J. Dent. Res. 48: 901-903,1969. 9. Dolby, A. E.: Recurrent Aphthous Ulceration, Effect of Sera and Peripheral Blood Lymphocytes Upon Oral Epithelial Tissue Culture Cells, Immunology 17: 709,1969. 10. Dolby, A. E.: Recurrent Mikulicz’s Oral Aphthae, Br. Dent. J. 124: 359-360, 1968. 11. Donatsky, O., and Dablesteen, E.: An Immunofluorescent Study on the Humoral Immunity to Strep 2A in Recurrent Aphthous Stomatitis, Acta Pathol. Microbial. 8% 107-112, 1974. 12. Dutton! R. W., and Eady, J. D.: An In Vitro System for the Study of the Mechanism of Antlgenic Stimulation in the Secondary Response, Immunology 7: 40-53, 1964. 13. Fischer, G. W., Oi, V. T., Ampaya, E. P., Helley, J. L., and Bass, J. W.: Enhanced Host Defense Mechanism With Phenylimidothiozole, Pediatr. Res. 8: 4, 1974. 14. Hoebeke, J., and Franchi, G.: Influence of Tetramisole and Its Optical Isomers on the Mononuclear Phagocytic System. 1. Effect on Carbon Clearance in Mice, J. Reticuloendothel. Sot. 14: 317, 1973. 15. Lehner, T.: Immunologic Aspects of Recurrent Oral Ulcers, ORAL SURG. 33: 80-85, 1972. 16. Marquardt, J. L., Snyderman, R., and Oppenheim, J. J.: Depression of Lymphocyte Transformation and Exacerbation of BehGet’s Syndrome by Ingestion of English Walnuts, Cell Immunol. 9: 263-272, 1973. 17. Renoux! M. G., and Renoux, M. : Immunostimulant Effect of an Imidothiazole in the Immunization of Mice Infected With Brucella abortus, C. R. Acad. Sci. (Paris) 278: 349, 1971. 18. Renoux, G., and Renoux, M.: Immunology-Effect of Phenylimidothiazole (Tetramisole) on the Graft-Versus-Host Reactions, C. R. Acad. Sci. (Paris) 274: 3320-3323? 1972. 19. Renoux, G., and Renoux, M.: Levamisole Inhibits and Cures a Solid Malignant Tumor and Its Pulmonary Metastases in Mice, Nature [New Bio1.1240: 217-218, 1972. 20. Rogers, R. S., Sams, W. M., and Shorter, R. G.: Lymphoeytotoxicity in Recurrent Aphthous Stomatitis, Arch Dermatol. 100: 361-363, 1974.
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21. Shauiro. S.. Olson. D. L.. and Chellemi. S. J.: The Association Between SmokinLrD and Aphthous Ulcers, URAL S&G. 30: 624-636, 1970. 22. Ship, I. I., Morris, A. L., Durocher, R. T., and Burket, L. W.: Recurrent Aphthous Ulcerations and Recurrent Herpes Labialis in a Professional School Student Population. I-III. Experience, Medical History, Oral Examinations, ORAL SURF. 13: 1191-1202, 1317-1329, 14381444, 1960; 14: 30-39, 1961. 23. Silverman, S., and Beumer, J.: Primary Herpetic Gingivastomatitis of Adult Onset, ORAL SURG. 36: 496-505, 1974. 24. Sircus, W., Church, R., and Kelleher, J.: Recurrent Aphthous Ulcerations of the Mouth, Quart. J. Med. 26: 235-249, 1957. 25. Spitler, L. E., Levin, A. S., and Fudenberg, H. H.: Human Lymphocyte Transfer Factor. In Busch, H. (editor) : Methods in Cancer Research, New York, 1973, Academic Press, Inc., vol. 8, pp. 59-106. 26. Symoens, J., and Brugmans, J.: Treatment of Recurrent Aphthous Stomatitis and Herpes With Levamisole. Br. Med. J. 4: 592. 1974. 27. Tripodi, D., Parks, L. C., and B&mans, J.: Drug-Induced Restoration of Cutaneous Delayed Hypersensitivitv in Anereic Patients With Cancer. N. Enal. J. Med. 289: 354-357, 197% 28. Verhagen, H., Decree, J., DeCock, W., and Verbruggen, F.: Levamisole and the Immune Response, N. Engl. J. Med. 289: 11481149, 1973. 29. Verhagen, H., Decree, J., Verbruggen, F., Hoebeke, J., De Brabander, M., and Brugmans, J.: Immune Responses in Elderly Cuti-Negative Subjects and Effect of Levamisole, Verh. Dtsch. Ges. Inn. Med. 79: 623-628, 1973. 30. Verhagen, H., Decree, J., and Brugmans, J.: Treatment of Aphthous Stomatitis, Lancet 2: 842, 1973. Reprint requests to: Dr. James A. Olson School of Dentistry University of California San Francisco, Calif. 94143
W. LITTLE,
D.M.D.,
M.S.D.
Chairman and Professor Department of Oral Diagnosis and Oral Medicine University of Kentucky Lexington, Kentucky 40506
Levamisole: A new treatment for recurrent aphthous stomatitis James A. Olson, D.D.S., M.S., D. Cade Nelms, D.D.S., Ph.D., Sol Silverman, M.A., D.D.S., and Lynn E. Spitler, M.D., San Francisco,
Calif.
UNIVERSITY
OF CALIFORNIA
AND
OF MEDICINE
SCHOOL
AT
SAN
FRANCISCO
SCHOOL
OF DENTISTRY
Fifty patients with recurrent aphthous stomatitis were treated with levamisole, an anthelmintic which has been found to increase immune responses. The subjects had previously been refractory t,o all modalities of treatment other than prednisone, and 92 per cent had experienced monthly or continuous attacks of aphthae. The responses were grouped into three categories: 6 per cent appeared to go into permanent remission, 56 per cent were improved, and 38 per cent showed no response. Levamisole appeared to have a definite enhancing effect on the immune system, as measured by skin test reactivity, but did not affect in vitro parameters of cellular immunity.
R
ecurrent aphthous stomatitis is a chronic diseasecharacterized by painful, recurring ulcerations of the oral mucosa. Although the frequency of recurrent aphthous stomatitis in the general population is unknown, prevalence studies in special groups indica.te that the incidence ranges from 20 per cent in a hospital populationZ1* 24to 60 per cent among professional school studentsz2 There is considerable variation in the number, frequency, duration, and size of aphthous lesions. Aphthae usually occur as single or multiple shallow ulcerations, 2 to 4 mm. in diameter. Occasionally, they occur in large clusters. Ulcers exceeding 6 mm. in diameter produce more tissue destruction and are more persistent than those of small size. In the most severe variant, recurrent This research received support from the University of California California Dental Association, and the National Institutes of Health a gift from The Janssen Pharmaceutical Company. Dr. Spitler is supported by a Research Career Development Award
Board (NIDR (AI
of Regents, AI 10686)
43012).
the and
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for recurrent
aphthous
stomatitis
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Fig. 1. This patient has had similar aphthous attacks eight to ten times per year for the past 15 years. These ulcers were present for 3 days and healed in 2 weeks. All of these lesions were less than 6 mm. in diameter and were categorized as minor aphthae.
oral ulcerations are accompanied by ocular and genital involvement and occasionally by arthritis and other systemic manifestations. The triad of oral, ocular, and genital symptoms comprises Behcet’s syndrome.3l 4 The cause of recurrent aphthous stomatitis is unknown. Studies of the cellular immune systems, 15,l6 the humoral immune system,ll 6+I17I5 and lymphocytotoxicity9y *O have implicated immunologic variances in the pathogenesis of the disease. There is no standard treatment for aphthous ulcers, and clinical management is usually directed toward symptomatic relief. In small uncontrolled studies, levamisole (the levo isomer of racemic 2,3;5,6, tetrahydro 6 phenylimidaze (2,1-b) thiazole hydrochloride) has been reported to be effective in controlling attacks of recurrent aphthous stomatitis and in reducing subsequent episodes 26y3o Levamisole is an anthelmintic agent which has few toxic side effects. Animal studies have demonstrated that levamisole may increase both cellular and humoral immune responses,13s I’, I8 stimulate phagocytosis,14 and inhibit tumor growth.lg In man, levamisole has been reported to restore cutaneous activity to purified protein derivative (PPD) and/or dinitrochlorobenzene (DNCB) in some anergic patients.27-29 The purpose of this study was to determine the efficacy of levamisole in alleviating recurrent aphthous stomatitis by evaluating clinical responses to a specific drug schedule and by measuring alterations in selected immunologic parameters. MATERIALS Patients
AND
METHODS
Thirty-one female and nineteen male patients, ranging in age from 9 to 66 years, made up the study group. All patients had actively recurring aphthous ulcerations, established by history and clinical examination. One hundred normal subjects are included in this study as controls for the selected immunologic tests.
590
for
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et nl.
Fig. 8. This patient had a lo-year history 4 weeks, was more than 1 cm. in diameter
Laboratory
of similar aphthae. This lesion, which and was classified as a major aphthous
persisted ulcer.
study
Laboratory examinations included complete blood counts, exfoliative cytologic smears for evidence of cytopathogenic effects,23 direct culture for herpes virus, serologic examination for herpes antibody titers, and biopsies of early aphthous ulcers (1 to 3 days). Biopsy specimens were divided for both electron and light microscopic examinations. A battery of immunologic studies was performed prior to and 1 to 3 months after initiation of levamisole treatment. These studies were selected to determine the immunopotentiating effects of levamisole and included skin tests, lymphocytestimulation studies,l* human leukocyte migration inhibition factor test (LIF) ,’ and tests for chemotaxis.5s7 Skin tests
Each patient was tested with a battery of six skin-test antigens-candida (Dermatophytin 0, Hollister-Stier Labs, Spokane, Washington), 1 :lOO and 1 :lO; coccidioidin (Cutter Laboratories, Berkeley, California), 1 :lOO and 1 :lO; mumps (Eli Lilly, Indianapolis, Indiana) ; purified protein derivative from tubercle bacillus (PPD, Connaught Laboratories, Toronto, Canada), intermediate and second strength; streptokinase-streptodornase (Varidase, Lederle Labs, American Cyanamid, Pearl River, New York), 4/l U. and 40/10 U. ; and trichophyton (Dermatophytin, Hollister-Stier Labs, Spokane, Washington), 1:30. lymphocyte
stimulation
This test was performed by the method of Spitler and associates.25Lymphocytes were obtained from peripheral huffy coat cells by passing over a nylon fiber column. The cells were cultured in triplicate tubes with 2 x 10” cells in 2 ml. culture medium (minimal essential medium) containing antibiotics, Lglutamine, and 10 per cent homologous serum from male donors of AB blood type. Antigen (candida, coccidioidin, PPD, or SK-SD) or PHA was added to replicate tubes at the initiation of the cultures. PHA was added in doses of
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Fig. J. This 3%year-old female patient in numerous small clusters. She also had was diagnosed as Behqet’s syndrome.
for
recurrent
aphthous
stomatitis
had continuous attacks of aphthae which ocular and genital involvement, and her
591
occurred problem
500, 250, 12’5, and 62.5 mg. per tube. C?” thymidine was added to the cultures containing antigens after 5 days of incubation, and the cultures were harvested on the sixth day. Control and PHA-stimulated cultures were harvested after 3and 6-day incubation periods. Results were assessed by counting in a liquid scintillation counter. leukocyte
migration
inhibition
The technique of Astor and colleagues* was used to measure leukocyte migration inhibition. Buffy coat leukocytes were obtained by dextran sedimentation and adjusted to a concentration of 220 x lo6 per milliliter. Fifty microliter aliquots were dispersed into Falcon plastic tubes, and the following was added sequentially in 5 microliter amounts: saline, PPD 30 mg./ml., SK-SD 6000 U/ml., candida 1:2 dilution or coccidioidin. The cell preparations were incubated at 37O C. for 30 minutes. Agarose was freshly prepared by dispensing 60 mg. agarose in 6 ml. distilled water with 0.6 ml. filtered horse serum, 0.6 ml. Medium 199 (10X), and sodium bicarbonate, The agarose was poured into Falcon Petri dishes, and wells 2.3 mm. in diameter were cut. Seven microliters of each cell preparation was placed in triplicate wells, and the plates were incubated at 37O C. for 18 to 24 hours. The plates were placed in water at 80° to 90’ C. to fix the cells to the plates and to cause the agarose to partially melt and flow off. The areas of migration were determined by reading two diameters with an ocular micrometer. A positive response in the LIF test was considered to be at least a 20 per cent decrease from the control area of migration. Test for chemotaxis
This test was performed by the De Meo and Anderson? modification of the Boyden technique. Ten milliliters of venous blood containing 0.3 ml. sodium heparin (1,000 units per milliliter) was drawn and allowed to sediment with dextran in an upright position for 45 minutes at 37O C. An additional 10 ml. of blood was drawn and allowed to clot, and the serum was used for the chemotactic tests. The polymorphonuclear leukocytes (polys) were collected
592
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Fig. 4. A, Two major aphthae had been present for 2% months on the lower lip of this El-year-old boy. B, Two weeks after administration of levamisole there was complete healing of the ulcers. This patient has had similar healing with subsequent courses of levamisole therapy.
and counted, and the concentration was adjusted to 5,000 to 10,000 cells/mm.3 The miniaturized chemotaxis chamber (Mark-It Engineering Corporation) was assembled with a 3~ pore diameter filter placed between the upper and lower members. The bottom chamber was filled with 0.2 ml. dilute 502A staphylococcus filtrate and serum or saline solution, and the top chamber with 0.4 ml. of poly suspension. The chamber was incubated for 2 hours at 37O C. Following incubation, the top chamber was emptied with a gauze sponge down to the filter. The filter was removed, fixed in paraformaldehyde, and mounted on a glass slide. The number of polymorphonuclear leukocytes migrating through the filter per high-power field was counted. Three chemotactic tests were compared before and after levamisole therapy: (1) the patient’s white blood cells (WBC) with their own serum and antigen, (2) the patient’s WBC with control serum and antigen, and (3) the patient’s WBC with saline solution as a control. The control for each test was obtained from frozen samples originally drawn from a single male donor with type AB blood. Therapy
regimen
Following clinical and laboratory documentation of recurrent aphthous stomatitis and determination of immunologic status, each patient entered an open clinical therapeutic trial with levamisole. The drug was administered by mouth at a level of 2.5 mg. per kilogram of body weight per day, with the dosage rounded to the nearest 50 mg. The usual adult dose was 150 mg. per day. An abortive therapy schedule was used for all patients in this study. If ulcers were present, patients were given levamisole 2 successive days per week. If ulcers were not present, the drug was not given until new lesions developed, at which time the drug was given for 2 sequential days per week. If patients had continuous aphthae, levamisole was administered 2 days per week for approximately 4 months. If there was no improvement after 4 months, the therapy was discontinued. If improvement was noted, treatment was continued for the duration of the study. Patients who had remissions of more than 1
Volume 41 Number 5 Table
New treatment for recurrent aphthous stomatitis
I. Clinical findings in fifty patients with recurrent aphthous stomatitis Male (19patients)
Size of lesions Minor aphthae* Major aphthae$ Duration
‘2
131
2
(4)
I
ANpatients (JOpatients)
34 (68) 16 (32)
0
(0)
‘;i
[:08{
2
$5 ‘q
‘Z
(4) g;
ofrecurrences
Less than 2 per month 2 to 4 per month More than 4 per month or continuous Behcet’s
1’0 ‘(:!I)+
Female (31 patients)
of each lesion
Less than 10 days 11 to21 days More than 3 weeks Frequency
593
disease
3 (6) 10 (20)
6 (121
0
(01
1 (2) 22 (44
4 (8) 32 (64)
8 (16)
1; ‘:;I’
4
18)
*Minor aphthae: lesions 6 mm. or less in diameter. tNumbers in parentheses indicate the percentage of all patients category. $Major aphthae: lesions more than 6 mm. in diameter.
falling
in the indicated
week’s duration did not receive another dose of levamisole until new lesions again developed. Analysis
of results
The response to levamisole therapy was determined by: (1) clinical immunologic tests, (2) objective assessmentof the clinical course of the disease, and (3) the patient’s subjective assessmentof therapy. Ulcer assessmentwas recorded by photographs, mucosal distribution, average diameter of lesions, number of lesions per attack, and frequency of attacks. Patients mailed in special postcards recording the frequency and duration of aphthae between visits. Because of the extreme variations in aphthous manifestations, a classification was devised to measure the severity and treatment response. Since size and duration of aphthous ulcers appeared to be the most critical factors, three categories were included: (1) minor aphthae-6 mm. or less in diameter with individual ulcer duration of less than 3 weeks, (2) major aphthae-greater than 6 mm. in diameter with duration greater than 3 weeks, and (3) Behcet’s disease-ulceration of the oral mucosa with either ocular and/or genital involvement, arthritis, or other systemic manifestations. Statistical analyses were performed by the paired or unpaired Student t test when appropriate. RESULTS Patient population
Table I describes the clinical status of the aphthous patients. Approximately two thirds of the patients had minor aphthae (6 mm. or less), and one third had major aphthae (over 6 mm.). The majority of patients (64 per cent) had two to four aphthae per month. In most casesthe ulcers persisted for 2 to 3 weeks. Eight per cent of the patients had Behcet’s disease.
594
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Olson et 01.
IMay,
II. Clinical results of levamisole aphthous stomatitis
Table
Major
uphthae
Female
“Cured”
0
therapy
in fifty patients Minor
Mule
to)*
Female
1976
with recurrent
uphthae 1
3 (6)
Mule
Allpatients
0 (0) 14 (28)
3
(6)
Improved No change I:; i [:; :: f:i{ 37 (14) (6) 2Y (6) 11 (22) Total lo 6 Y 25 50 *Numbers in parentheses indicate percentage of all patients in the indicated category. Table
Ill. Side effects in fifty patients with recurrent
undergoing
Drowsiness Taste and smell alteration Skin rash Headache *Numbers in parentheses indicate category. laboratory
aphthous stomatitis
levamisole therapy
percentage
of all patients
falling
3 3 2 2 into
(6)* (6) (4) (4) the indicated
studies
Exfoliative cytologic examination and direct culture for herpes virus were negative for the presence of virus. Herpes antibody titers were low, indicating that the lesions were not due to active herpes infections. Evaluation of biopsy specimens with the light microscope confirmed the early lymphocytic infiltration in the disease with no distinctive diagnostic features. Ultrastructure observations did not reveal any specific features relative to cause or categorization. Clinical
results
Table II shows the clinical results of therapy. Three patients have been free of aphthous ulcerations for more than 6 months since receiving levamisole. Twenty-eight patients demonstrated improvement, marked by a decrease in the number, frequency, size, and duration of aphthous ulcers. Neither the severity of lesions nor the sex of the patient appeared to be significantly correlated with the response to levamisole. Table III illustrates the side effects of levamisole therapy observed in these patients. Twenty per cent of the patient,s experienced side effects of therapy. Drowsiness and mild fatigue were noted by three patients. This was ameliorated by administering the drug at bedtime. Three patients noted alteration in taste and smell, and two noted headaches. Two patients developed skin rashes; both have subsequently taken the drug without incident. In none of the patients were symptoms severe enough to warrant discontinuat.ion of therapy. Skin test results
Before therapy, skin test reactivity to the panel of akin test antigens was not significantly different in the patients with recurrent aphthous stomatitis as compared to normal control subjeets. More than 95 per cent of the patients tested showed a significant increase (more than 5 mm. induration) in skin test
Volume 41 Number 5
New treatment for recurred
IV. Changes in skin test reactivity twenty-six patients Table
Decrease Per cent
No.*
595
before and after levamisole therapy in
Increase Antigen
aphthous stomatitis
No.
No change Per cent
No.
Per cent
19 13 SK/SD 1; ii : Candida 11 4: 6 11 Mumps 14 ; ;: PPD i tt Coccidioidin 5 :; 1 ; 0 0 Trichophyton Gi ii i3 ii 31 Total *Numbers of subjects showing a 5 mm. or greater change in induration at the 48 hours after injection of the test antigen.
ii 23 54 ii 54 test site
reactivity to one or more antigens following levamisole therapy. Forty-five per cent of the patients showed a significant increase in reactivity to two or more antigens. Table IV demonstrates the changes for the individual test antigens. Significant increases in reactivity were recorded with all antigens in varying percentages of patients. The most frequent increase was seen in reactivity to SK-SD, to which 50 per cent of the patients showed increased reactivity after levamisole therapy. On the other hand, decreased reactivity to individual antigens was also observed in some patients. There was no correlation between conversion of immunologic reactivity and the patients’ clinical course. Over-all, the mean skin reactivity for all patients increased with candida, PPD, and SK-SD and decreased with mumps (Fig. 5). The changes in reactivity observed with candida and PPD were statistically significant (p < 0.02)) but those for SK-SD (p = 0.10) and mumps (p = 0.20) were not. Too few patients responded to trichophytin and coccidioidin to permit us to draw meaningful conclusions. lymphocyte
stimulation
results
There was no significant change in the background counts per minute (CPM) when results obtained before and after levamisole therapy were compared. A significant change in lymphocyte stimulation was considered to be a fivefold or greater change in the stimulation index, and an index of less than I was considered to be 1 for calculations. Only four tests with specific antigens showed a change in the thirty-five tests performed. One showed an increase and one a decrease to candida, and two showed a decrease to SK/SD. Over-all, the mean stimulation indices for three antigens tested (PPD, SK-SD, and candida) were not significantly changed after therapy (p = 0.2). The stimulation index was not significantly influenced by the addition to the culture& of levamisole, alone or with specific antigens (Fig. 6). leukocyte
migration
inhibition
test results
Comparison of the areas of migration in control wells without antigen before and after levamisole therapy can be used to describe an index of random migration of polymorphonuclear leukocytes (polys) .zo In twenty patients tested,
596
Olson
et
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al.
50
4o
/
30
SK'SD
CANDIDA PPD MUMPS
SIZE (UUIII
20
10
BBPORE LBVAMIsoLB
AFTER LBVAHISOLE
Fig. 5. Results of skin test reactivity in patients with recurrent aphthous stomatitis before and after levamisole therapy. Numbers indicate millimeters of induration at the test site 48 hours after injection of the test antigen. SK/1sD, Streptokinase-streptodornase. PPD, Purified protein derivative.
SK/SD PPD CANDIDA STIMULATION INDEX
1:; 1
fl
3.0 I 2.0 LEVAMISOLE
1.0
I
BEFORE LBVAMISOLS
AFTER LEVAMISOLE
Fig. 6. Results of lymphocyte DNA synthesis (radioactive thymidine incorporation) in response to specific antigens in patients with recurrent aphthous stomatitis before and after levamisole therapy. Stimulation index equals the counts per minute in experimental tubes with antigen divided by the counts per minute in control tubes without antigen.
the findings were as follows: Six showed an increase, ten showed a decrease, and four showed no change in random migration of polys. When cells were pre-incubated with levamisole alone and compared to control cells incubated with saline solution, there was no inhibition or stimulation of migration. Of the fifty-eight tests performed with specific antigen, forty-seven showed no change, eight converted from positive to negative, and three converted from negative to positive. No change indicates that those tests that were positive before therapy remained positive and those that were negative reamined
Volume 41 Number 5
New treatment for recurrent aphthous stomatitis
597
100 I 90
-
I
Migration
LEVAMISOLE
40 I 30 20 10 I 1
BEFORE
LEVAI4ISOLS
AFTER L!NAMISOLE
Fig. 7. Results of leukocyte migration inhibition tests before and after levamisole therapy in patients with recurrent aphthous stomatitis. Per cent migration indicates the ratio of the area of leukocyte migration tested with specific antigen to the control area of migration without antigen.
12 HOMOLOGOUS SERUM #l AUTOLOGOUS SERUM
I
BBPORE t5vANISOm
AFTER LEVAH1s0LE
Fig. 8. Results of chemotactic tests in patienta with recurrent aphthous stomatitis before and after levamisole therapy. The polymorphonuclear leukocytes in five high-power fields are counted and averaged to give the number of white blood cells per high-power field.
negative. Statistical analysis of the over-all migration inhibition produced by each of the three test antigens indicated no significant difference in the results obtained after therapy as compared with results before therapy (Fig. 7). In vivo and
in vitro
correlations
Of thirty-eight significant changes in skin test reactivity to candida, PPD, and SK-SD in twenty patients, there are a total of fifty-three corresponding in vitro test results to specific antigens. Changes in only ten of the fifty-three in vitro tests paralleled the changes in skin test reactivity. Eight of the thirty-
598
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four LIF tests and two of nineteen lymphocyte to respective skin test changes. Chemotaxis
stimulation
Surg. 1976
tests corresponded
results
Fourteen patients were studied. After levamisole therapy, there was an increase in chemotaxis in both autologous and homologous serum, but the increase was statistically significant for studies performed in homologous serum only (p = 0.013) (Fig. 8). DISCUSSION Clinical effects
Levamisole produced a beneficial effect in reducing the number, frequency, and severity of recurrent aphthous stomatitis attacks in 62 per cent of the patients tested. Other studies 26s3o have similarly reported improvement in approximately two thirds of the subjects treated. The drug appeared safe, but long-range effects, if any, are unknown. A major advantage of levamisole over systemic corticosteroids in control of aphthae would be the possibility of permanent control without serious side effects. Although long-term control was observed in only 6 per cent of the patients treated, this percentage might be improved by altering the dosage and/or the frequency of administration. The mechanism by which levamisole produces clinical benefit in patients with recurrent aphthous stomatitis is not clear. It is well recognized that corticosteroids produce symptomatic relief and diminished signs in patients with this disease. Corticosteroids have a variety of biologic effects, immunosuppression being one of them, In this regard, the action of levamisole is exactly opposite; it increases rather than decreases immune reactivity. Immunologic
effects
Immunologic studies revealed that all patients were immunocompetent, but their reactivity was slightly decreased before treatment with levamisole when compared with the reactivity of .the controls. The criterion for immunologic competency is a response to at least two skin test antigens. Previous studies with levamisole have shown increased skin test reactivity to PPD in 22 per cent to 41 per cent of the subjects tested. 28,29 Another studyzT showed a 45 per cent conversion of skin test reactivity with dinitrochlorobenzene (DNCB) . Ninetyfive per cent of the patients in our study showed an increase in skin test reactivity to at least one antigen. Increased responses were seen primarily with candida, PPD, and SK-SD following levamisole therapy. On the other hand, a decrease in skin test reactivity to the various antigens, especially mumps, was also noted in some patients. This observation deserves further evaluation in a larger series of subjects. The findings of increased skin test reactivity after levamisole therapy without a concomitant increase in the lymphocyte stimulation index and without a decrease in the per cent migration suggests several possible modes of action. Levamisole or some metabolic product may act on cells involved in the non-
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specific inflammatory process and not directly on lymphocytes. The site of action may be the macrophage or polymorphonuclear leukocyte, thereby affecting chemotaxis and/or phagocytosis. The observation that levamisole increased the chemotactic response in these patients suggests that this may be the case. The drug may influence macrophage processing of antigen. Alternately, levamisole may affect the local vascularity at the test site (inflammation) by acting on the vascular endothelium to produce hyperemia. It is possible that other in vitro studies with different time sequences might show that levamisole does affect the lymphocyte function. However, our in vitro tests were performed at the same time that the changes in skin test reactivity occurred. We conclude that levamisole therapy produced definite immunologic changes and was of benefit in patients with recurrent aphthous stomatitis. Randomized double-blind studies are necessary to resolve definitively the question of clinical efficacy, and such studies appear to be warranted. REFERENCES
The Antinuclear Factor, J. Dent. Res. 1. Addy, M., ar$i Dolby, A. D.: Aphthous Ulceration: 51: 1594-1595, 1972. 5. H., Spitler, L. E., Frick, 0. L., and Fudenberg, H. H.: Human Leukocyte 2. Astor, Migration in Agarose Using Four Antigens: Correlation With Skin Reactivity, J. Immunol. 110: 1174.1179, 1973. 3. Behset, H.: tiber regitlvierende, aphthoese, durch ein Virus verursachte Geachwure am Mund, am Ange und an den Genitalient, Dermatol. Wochenschr. 10s: 1152,1937. 4. Berlin, C.: BehCet’s Disease as a Multiple Symptom Complex, Arch. Dermatol. 82: 127, 1960. 5. Boyden, Chemotactic Effect of Mixtures of Antibody and Antigen on S. : The Polymorphonuelear Leukocytes, J. Exp. Med. 115: 453-466, 1962. to Genital Mucosa in Patients With 6. Cummings, N. A., and Graykowski, E. A.: Antibodies Aphthous Stomatitis (AS), IADR Abstr. 915, 1974. 7. De Meo, A. N., and Anderson, B. R.: Defective Chemotaxis Associated With a Serum Inhibitor in Cirrhotic Patients, N. Engl. J. Med. 286: 735-740, 1972. 8. Dolby! A. E., and Allison, R. T.: Quantitative Changes in the Mast Cell Population in Mikulmz’s Recurrent Oral Aphthae, J. Dent. Res. 48: 901-903,1969. 9. Dolby, A. E.: Recurrent Aphthous Ulceration, Effect of Sera and Peripheral Blood Lymphocytes Upon Oral Epithelial Tissue Culture Cells, Immunology 17: 709,1969. 10. Dolby, A. E.: Recurrent Mikulicz’s Oral Aphthae, Br. Dent. J. 124: 359-360, 1968. 11. Donatsky, O., and Dablesteen, E.: An Immunofluorescent Study on the Humoral Immunity to Strep 2A in Recurrent Aphthous Stomatitis, Acta Pathol. Microbial. 8% 107-112, 1974. 12. Dutton! R. W., and Eady, J. D.: An In Vitro System for the Study of the Mechanism of Antlgenic Stimulation in the Secondary Response, Immunology 7: 40-53, 1964. 13. Fischer, G. W., Oi, V. T., Ampaya, E. P., Helley, J. L., and Bass, J. W.: Enhanced Host Defense Mechanism With Phenylimidothiozole, Pediatr. Res. 8: 4, 1974. 14. Hoebeke, J., and Franchi, G.: Influence of Tetramisole and Its Optical Isomers on the Mononuclear Phagocytic System. 1. Effect on Carbon Clearance in Mice, J. Reticuloendothel. Sot. 14: 317, 1973. 15. Lehner, T.: Immunologic Aspects of Recurrent Oral Ulcers, ORAL SURG. 33: 80-85, 1972. 16. Marquardt, J. L., Snyderman, R., and Oppenheim, J. J.: Depression of Lymphocyte Transformation and Exacerbation of BehGet’s Syndrome by Ingestion of English Walnuts, Cell Immunol. 9: 263-272, 1973. 17. Renoux! M. G., and Renoux, M. : Immunostimulant Effect of an Imidothiazole in the Immunization of Mice Infected With Brucella abortus, C. R. Acad. Sci. (Paris) 278: 349, 1971. 18. Renoux, G., and Renoux, M.: Immunology-Effect of Phenylimidothiazole (Tetramisole) on the Graft-Versus-Host Reactions, C. R. Acad. Sci. (Paris) 274: 3320-3323? 1972. 19. Renoux, G., and Renoux, M.: Levamisole Inhibits and Cures a Solid Malignant Tumor and Its Pulmonary Metastases in Mice, Nature [New Bio1.1240: 217-218, 1972. 20. Rogers, R. S., Sams, W. M., and Shorter, R. G.: Lymphoeytotoxicity in Recurrent Aphthous Stomatitis, Arch Dermatol. 100: 361-363, 1974.
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21. Shauiro. S.. Olson. D. L.. and Chellemi. S. J.: The Association Between SmokinLrD and Aphthous Ulcers, URAL S&G. 30: 624-636, 1970. 22. Ship, I. I., Morris, A. L., Durocher, R. T., and Burket, L. W.: Recurrent Aphthous Ulcerations and Recurrent Herpes Labialis in a Professional School Student Population. I-III. Experience, Medical History, Oral Examinations, ORAL SURF. 13: 1191-1202, 1317-1329, 14381444, 1960; 14: 30-39, 1961. 23. Silverman, S., and Beumer, J.: Primary Herpetic Gingivastomatitis of Adult Onset, ORAL SURG. 36: 496-505, 1974. 24. Sircus, W., Church, R., and Kelleher, J.: Recurrent Aphthous Ulcerations of the Mouth, Quart. J. Med. 26: 235-249, 1957. 25. Spitler, L. E., Levin, A. S., and Fudenberg, H. H.: Human Lymphocyte Transfer Factor. In Busch, H. (editor) : Methods in Cancer Research, New York, 1973, Academic Press, Inc., vol. 8, pp. 59-106. 26. Symoens, J., and Brugmans, J.: Treatment of Recurrent Aphthous Stomatitis and Herpes With Levamisole. Br. Med. J. 4: 592. 1974. 27. Tripodi, D., Parks, L. C., and B&mans, J.: Drug-Induced Restoration of Cutaneous Delayed Hypersensitivitv in Anereic Patients With Cancer. N. Enal. J. Med. 289: 354-357, 197% 28. Verhagen, H., Decree, J., DeCock, W., and Verbruggen, F.: Levamisole and the Immune Response, N. Engl. J. Med. 289: 11481149, 1973. 29. Verhagen, H., Decree, J., Verbruggen, F., Hoebeke, J., De Brabander, M., and Brugmans, J.: Immune Responses in Elderly Cuti-Negative Subjects and Effect of Levamisole, Verh. Dtsch. Ges. Inn. Med. 79: 623-628, 1973. 30. Verhagen, H., Decree, J., and Brugmans, J.: Treatment of Aphthous Stomatitis, Lancet 2: 842, 1973. Reprint requests to: Dr. James A. Olson School of Dentistry University of California San Francisco, Calif. 94143