INTERNATIONAL JOURNAL OF LEPROSY^

Volume 59, Number 4Primed in the U.S.A.

Effects of Cyclosporin A on Bacterial Growth andImmunological Responsiveness in BALB/c Mice

Infected with Mycobacterium LepraelMiyoko Tanaka, Yoshinori Tanaka, and

Kenji Kohsaka2

Cyclosporin A (CsA) is a well known im-munosuppressive agent, and is clinicallyused in the transplantation of various or-gans, such as the kidney, liver, heart and soon. It has suppressive effects on T-cell-de-pendent antibody formation and the cellu-lar immune response (2, 3, 8, 9, 27, 28).) Severalinvestigators have reported that CsA inhib-its the production oflymphokines by T lym-phocytes (4, 6, 27,28

) and that it inhibits T-cellgrowth factor gene expression (' 2) or impairsthe release of interleukin 1 (IL-1) and IL-2(4). Alberti, et al. (2) have presented the hy-pothesis that T-helper lymphocytes are thepreferential target of CsA immunodepres-sion. Mycobacterium leprae can multiplyonly in a limited number of experimentalanimals, one of which is the nude mouse(") which is defective in T-cell function.Therefore, we gave CsA to a normal mousein order to depress T-Iymphocyte function,and injected it with M. leprae to investigatethe growth of M. leprae and the immuno-logical responsiveness of CsA-treated Mice.

MATERIALS AND METHODSMicroorganism and bacterial suspension.

M. leprae were originally obtained from abiopsy of a subcutaneous leproma from apreviously untreated lepromatous leprosypatient and serially passed in the foot pads

' Received for publication on 12 December 1990;accepted for publication in revised form on 21 June1991.

2 M. Tanaka, B.Sc., Research Fellow; Y. Tanaka,M.D., D.Med.Sc., Professor, Department of Bacteri-ology, Faculty of Medicine, Tottori University, 86Nishi-machi, Yonago-shi, Tottori 683, Japan. K. Koh-saka, D.V.Sc., D.Med.Sc., Director, Department ofMicrobiology, National Institute for Leprosy Research,4-2-1 Aoba-cho, Higashimurayama-shi, Tokyo 189,Japan.

Reprint requests to Prof. Y. Tanaka.

of BALB/c nude mice. The foot pads frominfected mice were minced with scissors,ground, and added to phosphate bufferedsaline (PBS). The suspension was centri-fuged at 300 x g to remove debris. Thebacterial number was assessed in the su-pernatant by Shepard's method ( 23).

Reagents. Cyclosporin A (CsA), a prod-uct of Sandoz Pharmaceuticals, Ltd., wasdissolved in olive oil for oral administra-tion. Concanavalin A (ConA) was pur-chased from Sigma Chemical Company, St.Louis, Missouri, U.S.A., and 3 H-thymidinewas a product of New England Nuclear Re-search Products, Boston, Massachusetts,U.S.A. Rabbit anti-mouse immunoglobu-lin, fluorescein conjugated, and rabbit anti-mouse T-cell serum were products of Ce-darlane Laboratories, Ltd., Ontario, Cana-da, and fluorescein-conjugated goat anti-rabbit immunoglobulin was a product ofCooper Biomedical Inc., Malvern, Penn-sylvania, U.S.A. Dulbecco's modified Eaglemedium (DMEM) was from Flow Labora-tories, Inc., Tokyo, Japan.

Administration schedule. Six-week-old,female BALB/c mice (Sizuoka AnimalFarm) were separated into several groups.In the first experiment, one group receivedCsA 12 mg/kg body weight at day —2 and10 mg at day —1, then 1.5 x 10 6 M. lepraewere injected into the right hind foot pad atday 0. A dose of 8 mg/kg CsA was contin-ously administered six times weekly duringthe course of the experiments (for 19months). Another group of mice was treatedwith CsA in the same manner during thefirst 6 months, and received 3 mg/kg sixtimes weekly for the next 2 months. At var-ious intervals, the mice were sacrificed andthe number of acid-fast bacilli (AFB) in theinjection site was assessed by the methodof Shepard (23).

598

59, 4^Tanaka, et al.: Cyclosporin A Effect on M. leprae Growth^599

In the second experiment, doses of CsA12 mg/kg and 10 mg/kg body weight wereadministered to the first group of mice atdays —2 and —1, respectively, followed bysubcutaneous injection with 2.0 x 10 8 M.leprae and doses of oral CsA 8 mg/kg everyday for 1 week. The second group of micewas treated with 100 mg/kg daily from day—2 to day 7. Other groups consisted of micewith or without M. leprae injections whichdid not receive CsA. M. leprae-infected micewere treated with or without 100 mg/kg ofCsA for the observation of delayed-type hy-persensitivity.

Stimulants. M. leprae were suspended ata concentration of 5 x 10 7 per ml of PBS(M. leprae suspension; Ml). After sonicationat 100 W for 30 min, the suspension wascentrifuged at 1500 x g for 30 min. Thesupernatant was used as sonicated M. leprae(SS). ConA was used at a concentration of500 pig/m1 of PBS.

Immunofluorescent antibody method(IFA). To assay T cells, 100 ill of rabbitanti-mouse T-cell antiserum was added to100 Al DMEM containing 5 x 10 5 spleencells from mice infected for 19 months. Themixture was then incubated for 30 min at37°C in a humidified atmosphere containing5% CO,. The cells were washed three timeswith 0.15 M PBS containing 2% fetal bovineserum (FBS) and 0.02% NaN 3 , and resus-pended in 0.1 ml of 0.15 M PBS. Fluores-cein-conjugated goat anti-rabbit immuno-globulin (100 Al) was added, and the mixturewas incubated for 20 min at 4°C, followedby three washes with PBS. Fluorescein-la-beled cells were applied dropwise to glassslides, covered with a glass coverslip, sealedwith nail polish, and counted using a fluo-rescence microscope. To assay B cells, 0.1ml of fluorescein-conjugated rabbit anti-mouse immunoglobulin was added to spleencells which were incubated for 20 min at4°C and then washed three times. B cellswere counted in the same manner as T cells.The number of fluorescein-stained cells per300 or more cells was assessed in triplicateexperiments, and the T/B ratio was calcu-lated.

Lymphocyte blast transformation (LBT).Spleen cells from mice after various infec-tion periods were suspended in DMEM con-taining 10% FBS. Cells, 1 x 10 6 in 0.2 mlDMEM per well of a 96-well tissue culture

plate, were incubated at 37°C for 1 hr under5% CO, and then stimulated with 20 Al ofvarious mitogens followed by a 48-hr in-cubation. After labeling with 19 kBq 3 H-thymidine for 16 hr, the cells were harvestedon glass-fiber filters in a multichannel cellharvester. The radioactivity (counts perminute; cpm) of the 3 H-thymidine incor-porated into the cells in triplicate experi-ments was measured by a liquid scintilla-tion counter. The results are shown as thestimulation index (SI) determined by thefollowing formula:

3 H-thymidine cpm(stimulated with mitogen)

RESULTSGrowth of M. leprae in mouse foot pads

(Fig. 1). The number of AFB increasedgradually from the 12th to the 19th monthin the group of mice to which CsA was ad-ministered until the end of the experiment.However, there was a significant decreasein the number of AFB during the same pe-riod when the CsA treatment was discon-tinued by the 8th month (p < 0.01 for the16th and 19th months).

LBT and T/B ratios at 19th month. Asshown in Table 1, lymphocytes from micecontinuously treated with CsA had a di-minished response to SS and ConA com-pared with those from CsA-discontinuedmice. This is due to the continuous admin-istration of CsA. However, the L13T reac-tion in CsA-treated and M. /cprae-infectedmice was almost the same as that of unin-fected normal mice without CsA. It may bethat the 8 mg/kg dose of CsA was too smallto suppress the T-cell function. The T/Bratio of mice continuously treated with CsAwas significantly decreased compared with

SI —3H-thymidine cpm(without mitogen)

Foot pad swelling reaction. Three weeksafter subcutaneous injection on the back with2 x 10 8 M. leprae, the mice were challengedwith 20 of PBS containing 1 x 10 6 AI.leprae into the right hind foot pad and with20 Al of PBS alone in the left. The differencein thickness between the right and left footpads was measured with a dial thicknessgauge (0.01 mm; Peacock) 2 and 4 days afterchallenge.

600^ International Journal of Leprosy^ 1991

0

0

00z

12^16^20Months

FIG. I. Growth of M. leprae in the BALB/c mousefoot pad: 8 mg/kg CsA was administered 6 times week-ly during the course of experiments (0). As a controlgroup, the same dose of CsA was administered duringthe first 6 months, 3 mg/kg 6 times weekly for 2 monthsthereafter, and then stopped (•). Each point = mean± S.D. of the number of bacilli in the foot pad of eachmouse; bar = mean ± S.D. of the total. There is asignificant difference between the two at•the 16th and19th months (p < 0.01).

other groups (Fig. 2). Therefore, it is rea-sonable to consider that decreased totalT-cell functions are due to the decreasedT-cell number. In order to determine howlong CsA administration can be discontin-ued while retaining immune function sup-pression, we investigated the LBT reactionin the early stages of M. Ieprae infection.

LBT reaction after 9 days of CsA admin-istration. The lymphocytes from CsA-un-treated mice inoculated with M. leprae re-sponded to SS, Ml, and ConA more thanthose from uninfected control mice (Table2). When a 100 mg/kg dose of CsA wasadministered to mice for 9 days, lympho-cytes stimulated with SS and Ml incorpo-rated as much 3 H-thymidine as did those ofmice without CsA. However, responses toConA in lymphocytes from mice treatedwith CsA decreased remarkably as com-pared with those from infected mice with-

TABLE 1. Effect of Csil administration onlymphocyte blast transformations 19 monthsafter injection with 1.5 x 10 6 M. Ieprae.

Stimulants"CsA"

SS ConA

None 1.2 ± 0.2* 7.1 ± 5.0Discontinued 1.2 ± 0.1 11.9 ± 6.7Continued 1.0 ± 0.04 7.8 ± 3.3

" Schedule of CsA administration is described in thelegend of Figure I.

SS = sonicated supernatant of M. leprae suspen-sion; ConA = concanavalin A.

Lymphocyte blast transformation is shown as thestimulation index (mean ± S.D. of two to four mice).3 H-Thymidine uptake by the cells without stimulantwas 2673 ± 291 cpm.

out CsA. Thus, CsA only slightly suppressedthe immune response to the M. leprae an-tigens, SS and Ml, but did so distinctly toConA after being given to M. leprae- in-fected mice for 9 days (Table 2).

LBT reaction at week 4. Incorporationof 3 H-thymidine into lymphocytes in re-sponse to all three stimulants was greater inmice inoculated with M. leprae than in un-infected mice, and decreased in proportionto the dose of CsA administered to the in-fected mice (Table 3). These results indicatethat T-cell functions were activated in M.Ieprae-infected mice but that CsA sup-pressed these activities. It is noteworthy thatthe LBT response to ConA in mice treatedwith 100 mg/kg of CsA was remarkably de-creased compared with control mice.

LBT reaction at week 15. LBT responsesto SS and Ml in the cells from M. leprae-

Administrationof CsA

Discontinued

Cont inued

0^ 50^100T/ B ratios (^1

FIG. 2. T/B cell ratios of lymphocytes at 19th month:BALB/c mice were treated as shown in Figure 1; con-trol mice were not infected and not treated with CsA.Total spleen cells were 1.1 x 10 8 in control mice, 0.8x 10 8 in discontinued group, and 1.2 x 10' in contin-ued group.

0

c6.0

Cu0

5.0 ^0

59, 4^Tanaka, et al.: Cyclosporin A Effect on M. leprae Growth^601

TABLE 2. Lymphocyte blast transforma-tions in CsA-administered mice 7 days afterinjection with 2 x 108 M. leprae.

Stimulantsh

SS^MI^ConA

0 1.8 ± 0.4c 2.4 ± 0.5 7.7 ± 5.00 3.0 ± 1.6 4.7 ± 1.3 14.7 ± 4.1

+ 100 2.8 ±^1.3 4.5 ± 1.3 6.8 ± 4.1

Mice were given CsA on days —2, —1 before, andon days 0 to 6 after subcutaneous injection of M. leprae.

"SS = sonicated supernatant of Al. leprae suspen-sion, MI = Al. leprae suspension.

The stimulation index is shown as mean ± S.D. (N= 4). 'H-Thymidine uptake by the cells without stim-ulant was 1291 ± 297 cpm.

infected mice exceeded that of control mice(Table 4). However, the response to ConAin M. leprae-infected mice was lower thanthat of normal mice. In contrast with theLBT at week 4 (Table 3), spleen cells fromCsA-treated mice incorporated slightly (butnot significantly) more 3H-thymidine thanthose from Al. leprae-infected mice withoutCsA when stimulated with all three stimu-lants. When ConA and Ml were used tostimulate simultaneously, the stimulationindex (SI) showed an intermediate value be-tween that of Ml and ConA (Table 4). Thisindicates that stimulation by ConA was par-tially suppressed with M. leprae in all threegroups of mice in vitro.

Delayed-type hypersensitivity reaction.Foot-pad swelling was measured as the dif-ference between the thickness of the rightfoot pad which was challenged with M. lep-rae and that of the left which received PBS.CsA-treated mice had slightly less swellingthan untreated mice (Fig. 3).

DISCUSSIONThere have been several reports that in-

fi)ctions are complicated by CsA treatmentin experimental animals inoculated withvarious microorganisms. Most are exacer-bations of infectious lesions due to the in-hibition of development of protective im-munity ( I, 5, 10, 13, 17, 18, 26\/ thereare contradictions. CsA inhibited both thein vitro and in vivo growth of Cryptococcusneoformans ( 16). Survival of CsA-treatedmice infected with Trypanosoma museullwas somewhat prolonged ( 20). Cutaneous le-sions of Leishmania tropica-infected mice

Inoculation^Doseof Mleproe of CsA

long)0 IA)

0

100 10 811

0

100 10^20^1,0^60^80

Foot pad swelling ( 0.01 rni61

FIG. 3. Foot-pad swelling of mice 3 weeks afterinfection with 2 x 10 8 Al. leprae: BALB/c mice werechallenged with I x 10 6 AL leprae; 2 (A) and 4 (B)days later, foot-pad swelling was assayed by a dialthickness gauge. Each column shows mean ± S.D. of5 mice.

were suppressed with CsA ( 25). Grau, et al.(7) showed that CsA displayed a strong pro-tective effect on neurological complicationswhen low doses were given to Plasmodiumberghei ANKA-infected CBA/Ca mice, andthat protection was not seen when parasi-tocidal doses were used. In this study, weinvestigated the growth capability ofAl. lep-rae in mice treated with CsA.

When CsA was administered throughoutthe experiments, a small enhancement ofthe growth of Al. leprae was recognized (Fig.1). The mice were orally treated with 12 mg/kg and 10 mg/kg CsA at days —2 and —1,respectively, and with 8 mg/kg daily sixtimes weekly thereafter. This is a standarddosage finally proposed for human trans-plantation. Other authors have reported thateffective dosages of immune suppression inbacterial infection experiments were 50, 75,80, or 100 nig/kg ( I, 3, 18, 26, 27

) If CsA wasadministered at 100 mg/kg, M. kprae wouldlikely flourish since the LBT reaction in micetreated with 100 mg/kg CsA was less evi-dent than that in mice treated with 8 mg/kg CsA (Table 3), and the number of Al.leprae in the CsA-treated mouse foot padsshowed an increase compared with untreat-ed mice after 8 months (3.7 [± 1.9] x 10 5

bacilli in mice treated with CsA for 1 weekper month compared with 1.8 [± 0.4] x 10 5

bacilli in CsA-untreated mice).We tried to inoculate CsA-treated mice

with 106 M. leprae, since if CsA suppressedT-cell-dependent immunity, the number ofM. leprae would increase to over 10 6 , as

Af"^CsA'leprue doseinocu- (mg/kg)lation

CsA"dose ^ % Suppression°

(mg/kg)^SS^Ml

AI. lepraeinoculation

Stimulants

ConA^MI + ConA

602^ International Journal of Leprosy^ 1991

TABLE 3. Lymphocyte blast transforma-tions at week 4.

4f .^CsA"leprae dose^Stimulantsinocu-lation “115'^SS^MI^ConA

0 1.1 ± 0.2° 1.6 ± 0.4 52 ± 270 1.5 ± 0.2 3.0 ± 0.5 81 ± 288 1.3 ± 0.3 2.5 ± 0.1 48 ± 26

100 1.2 ± 0.6 2.1 ±^1.1 33 ± 33

Mice were administered CsA as shown in footnote" of Table 2.

'' The stimulation index is shown as mean ± S.D. (N= 4). I-Thymidine uptake by the cells without stim-ulant was 2660 ± 472 cpm.

shown in nude mice ("). Shepard, et al. (22 )

reported that 5 x 10 3 M. leprae increasedto approximately 10 6 , which was the ceilingof multiplication in normal mice. Accord-ingly, we should have inoculated with 10 3

M. lcprae for confirmation of bacterial mul-tiplication. However, growth of Al. lcpraedid not exceed 10 6 .

When 100 mg/kg CsA was administeredto normal mice for a week after infectionwith M. leprae, their lymphocyte responsesto stimulation were lower than those of un-treated mice at weeks 1 and 4, but not atweek 15. This suggests that CsA can sup-press T-cell-dependent immunity for at least4 weeks, but that after 15 weeks a reboundphenomenon specific to CsA appears. Tak-ashima, et a!. (27) suggested this phenome-non in their delayed-type hypersensitivityexperiments. Therefore, in order to sup-press the T-cell functions of normal mice,CsA should be continuously given for atleast 1 of every 4 weeks.

In the delayed-type hypersensitivity ex-periment, foot-pad swelling in CsA-treatedand M. lcprae-infected mice was smaller

than that in untreated and infected mice.Nomaguchi, et a!. observed delayed-typehypersensitivity by challenging 4 monthsafter immunization (unpublished data), andMatsuoka did so on the 20th week (unpub-lished data). A weak delayed-type hyper-sensitivity reaction was detected in untreat-ed mice 3 weeks after infection with M.leprae. The reaction was distinctly de-creased in CsA-treated mice.

We conjecture that immunization withlarge numbers of A/. leprae without CsAtreatment causes a specific immune re-sponse or immune tolerance. In our exper-iments, lymphocytes from mice subcuta-neously infected with 2 x 10 8 M. lepraeshowed an increased LBT response to Mlwhich was higher than that of the uninfectedcontrol mice (Table 4). However, in the samemice, Ml partially suppressed the LBT re-sponse to ConA on week 15 in all threegroups of mice, and the highest suppressionwas in the normal control group. Shepard,et al. (24) reported that mice intravenouslyinfected with 10 7 or 108 heat-killed M. lep-rae developed immune tolerance. Lcpro-min and phenolic glycolipid-I (PGL-I) sup-pressed the ConA response of peripheralmononuclear cells of lepromatous leprosypatients (14, 15, 21 , .) Nomaguchi, et al. ( 19 ) re-ported that a similar suppression occurredin mouse lymphocytes, and that it could berestored by the addition of mouse macro-phages. The conditions which induce im-mune response or immune tolerance shouldnow become clear.

In this study, there was less growth of M.lcprae in CsA-treated mice than in nudemice. However, the enhanced capability of

leprae to proliferate was suggested in thefoot pads of mice which had received CsAat least 1 week in every 4 weeks. We hope

TABLE 4. Lymphocyte blast transformations at week 15 after stimulating with M. lepraeantigens and/or ConA.

0 1.3 ± 0.3" 1.5 ± 0.3 45 ± 18 13 ± 2 67 ± 12+ 0 1.2 ± 0.2 2.0 ± 0.4 32 ± 9 10 ± 2 63 ± 11+ 100 1.5 ± 0.4 2.3 ± 0.3 34 ± 5 14 ± 7 58 ± 20

Mice were administered CsA as described in footnote of Table 2.b % Suppression = 1 - (cpm stimulated with MI + ConA/cpm stimulated with ConA) x 100.

The stimulation index is shown as mean ± S.D. (N = 4). 'H-Thymidine uptake by the cells without stimulantwas 2379 ± 546 cpm.

59, 4^Tanaka, et al.: Cyclosporin A Effect on M. leprae Growth^603

in the future that larger amounts of live M.leprae can be obtained more easily in ani-mals by using immunosuppressive agents orby inducing immunological tolerance.

SUMMARYWhen BALB/c mice were infected with

Mycobacterium leprae and orally treated 6times weekly with a dose of 8 mg/kg cy-closporin A (CsA) for 19 months, the num-ber of organisms was slightly higher at 19months as compared with mice in whichthe dose of CsA was gradually decreasedafter 6 months and discontinued at the 8thmonth (p < 0.01 for the 15th and 19thmonths). Lymphocyte blast transformation(LBT) showed that spleen cells from CsA-treated mice 4 weeks after infection with Al.leprae and 3 weeks after CsA treatment wasstopped responded to the sonicated super-natant of M. leprae suspension (SS), Al. lep-rae (Ml), and concanavalin A (ConA) lessthan those cells from mice not treated withCsA. This response was dose-dependent. Atweek 15, 14 weeks after CsA administrationwas stopped, the LBT response to SS andM1 by cells from M. leprae-infected miceexceeded that of mice without CsA treat-ment, and the response to ConA in Al. lep-rae- infected mice was less than that in un-infected mice without CsA-treatment. Thus,if CsA was administered, the T-cell func-tions were suppressed. However, when CsAtreatment was discontinued for longer pe-riods, the T-cell function was activated.From these results, we speculate that Al.leprae would have the capability of growingmore abundantly in mice treated with CsA100 mg/kg for 1 week every month.

RESUMENSe infectaron ratones BALB/c con Mycobacterium

leprae y se trataran 6 veces a la semana con dosis oratesde 8 mg/kg de ciclosporina A (CsA), durante 19 meses.El nnmero de microorganismos fue ligeramente mayoren los ratones tratados durante 19 meses que en aque-llos en los cuales la dosis de CsA se disminuy6 gra-dualmente despuês de 6 meses y se descontinuO al mesoctavo (p < 0.01 para los meses 15 y 19). La trans-formaciOn blastoide de linfocitos (TBL) a las 3 semanasdo haber suspendido el tratamiento con CsA, mostrOque las celulas esplenicas de los ratones tratados conla droga cuatro meses despuês de la infecciOn con Al.leprae, respondieron al sobrenadante sonicado de unasuspension de Al. leprae (SS), al Al. leprae (ML), y ala concanavalina A (ConA), en menor grado que las

celulas de los ratones no tratados con CsA; esta res-puesta fue dependiente de la dosis. Ala semana 15, 14semanas despuês de haber suspendido la administra-ci6n de CsA, la respuesta TBL de las celulas de losratones infectados con M. leprae contra el ss y el ML,excediO aquella respuesta de los ratones sin tratamientocon CsA. La respuesta a la ConA de las caulas de losratones infectados con M. leprae fue menor que la res-puesta de los ratones no infectados y no tratados conCsA. Asi, aunque la administraciOn de CsA suprimela funciOn de las celulas T, esta se regenera cuando eltratamiento con CsA se descontimia por periodos lar-gos de tiempo. A partir de estos resultados, especula-mos que el Al. leprae podria multiplicarse inns abun-dantemente en ratones tratados con 100 mg/kg de CsAdurante una semana de cada mes.

RESUMEDes souris BALB/c ont etc infectees par Mycobac-

terium leprae et traitbes par voic orate par 6 doses de8 mg/kg de cyclosporine A (CsA) par semaine pendant19 mois. Le nombre d'organismes Ctait legerement plusCleve apres 19 mois, par comparaison avec des sourischez lesquellcs la dose de CsA fut progressivementreduite apres 6 mois, et stoppee au 8eme mois (p <0.01 pour les 15eme et 1 9ème mois). La transformationlymphoblastique des lymphocytes (TBL) a montró queles cellules spleniques de souris traitees a la CsA 4semaines arcs ('infection par Al. leprae et 3 semainesapres l'arret du traitement a la CsA, repondaient moinsau surnageant sonique (SS) d'une suspension de Al.leprae, au Al. leprae(M1) et a la concanavaline A (ConA)que les cellules de souris non traitees a la CsA. Cettereponse êtait dependante de la dose. A la semaine 15,c'est-a-dire 14 semaines apres l'arret de l'administra-tion de CsA, la reponse TBL vis-a-vis de SS et MI parles cellules de souris infectees par Al. leprae, depassaitcelle do souris non traitees a la CsA, et Ia reponse a IaConA des souris infectees par Al. lepraeetait plus faibleque celle des souris non infectees et non traitees a IaCsA. Done si la CsA êtait administree, les fonctionsdes cellules T &talent supprimees. Cependant, quandle traitement a la CsA &wit arrete pour des periodesplus longues, la fonction des cellules T etait activee. Apartir de ces resultats, nous formulons l'hypothese que

leprae aurait la capacitC de se reproduirc plus ra-pidement chez des souris traitées a Ia CsA a la dose de100 mg/kg pendant 1 semaine chaque mois.

Acknowledgment. The authors are grateful to San-doz Pharmaceuticals Ltd. for the supply of cyclosporinA. This work was supported by a grant from the U.S.-Japan Cooperative Medical Science Program.

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