IN FERNATIONAL JOURNAL OF LEPROSY^ Volume 47, Number 3

Printed in the U.S.A.

Partial Inhibition of the Growth of Mycobacteriumlepraemurium in C3H Mice Immunized with

Cell Wall Skeletons'Yoshinori Tanaka"

Leprosy is a chronic infectious diseasewhich is related to a complex cell-mediatedimmunity against Mycobacterium leprae.According to various information, includ-ing clinical findings, histology and espe-cially skin test reactions to M. lepraeantigens, leprosy is classified into polarsubgroups, tuberculoid leprosy and lep-romatous leprosy. Tuberculoid leprosy isa relatively benign form and usually re-sponds well to chemotherapy. A patientwith tuberculoid leprosy shows positivelepromin reactivity. On the other hand,lepromatous leprosy is a malignant formand is associated with ineffectual cell-mediated resistance.

When we consider experimental leprosy,we usually find difficulty in searching for anexperimental animal because of the quitelimited host range of .11. leprae. It is knownthat M. lepraenutrium infection in mice isone of the useful models in experimentalleprosy. Through the study of .11. leprae-murium infection in nude mice and othermouse strains, Kawaguchi (I' Ii) observedthat experimental murine leprosy can beclassified into three clinical types, benign,intermediate and malignant, and suggestedthat mouse strain differences are related totheir cell-mediated immunity. The C3Hmouse is representative of the malignanttype. Therefore, M. lepraemurium infec-tion in C3H mice was used in this experi-ment as a model of lepromatous leprosy.

Immunotherapy has been employed inthe treatment of cancer in this decade, andvarious kinds of agents such as living orkilled bacterial cells, bacterial componentsand drugs have been used. Cell wall frac-

' Original manuscript received for publication on 2February 1979; revised manuscript received for pub-lication on 7 May 1979.

2 Y. Tanaka, M.D., Ph.D., Associate Professor,Department of Microbiology, Kurume UniversitySchool of Medicine, Kurume, Fukuoka, 830 Japan.

tion or cell wall skeleton (CWS), which hadbeen purified from Mycobacterium boricBCG cells, has been reported to be an ef-fective T cell stimulator ("•"). Cell wallcompositions of in vivo and in vitro grownM. lepraenutrium cells have been found tobe similar to those of M. boris BCG ( 1 . 7).Therefore, CWS of M. lepraemurium cells(LM-CWS) was used to stimulate T cellfunction in C3H mice.

C3H mice were either challenged with M.lepraemurium immediately after stimula-tion with LM-CWS or seven weeks afternonspecific stimulation with LM-CWS.Later the status of nonspecific delayed-typehypersensitivity (DTH) was determined.The growth of lepromata and the distribu-tion of murine leprosy bacilli in the variousorgans were then observed.

MATERIALS AND METHODSPurification of cell wall skeleton (LM-

CWS). Mycobacterium lepraemurium Ha-waii, grown on one percent Ogawa's eggyolk medium ( 17) for five to six weeks at37°C, were collected and washed in 0.067M phosphate buffer (PB) pH 6.8. Cells weredelipidated with acetone, ether-ethanol(1:1, v/v), chloroform, and chloroform-methanol (2:1, v/v). Purified cell wall skel-etons were prepared from the delipidatedcells by using the method of Azuma, et al.( 3 . 5). Three and two-tenths g of delipidatedcells was suspended in 100 ml of sterilizeddistilled water and disrupted by sonicationat four amperes (Kaijo Denki Co. Ltd., To-kyo, Japan) for 30 minutes in an ice bath(0°C). The disrupted cells were centrifugedseveral times at 600 x g for 20 minutes toremove the unbroken cells and cell debris,and the cell walls collected by centrifuga-tion of the supernatant at 20,000 x g forone hour. The precipitates were resuspend-ed in 0.067 M PB (pH 7.8) and treated insequence with trypsin, chymotrypsin and

487

488^ International Journal of Leprosy^ 1979

TABLE 1. Design of experiments.

Challenge

simulta-neously^after

^LM-CWS

Exp.^Groups^with IM"^IM''^

010^

LM-CWS boosters

A^

100^

Each 2 wk to 17th wk,then each 4 wk to 35th wk.

10^

Each 2 wk to 17th wk,then each 4 wk to 35th wk.

II.

B

III. (co nt rols )

IV. 1(X)^Ix at 5 wk, then each 4 wk.'

V.

" All mice challenged by live M. lepraemurium simultaneously with immunization.'' All mice challenged by live M. lepmemurium after seven weeks, i.e., after a preliminary immunization.

At seven weeks the level of nonspecific DTII WIIS measured in both groups IV and V by means of picrylchloride. The mice were then challenged and group IV immunized with LM-CWS injections. Group V micereceived PH on each occasion when group V was boosted.

pronase at 20°C for 24 hours, followed bywashing in turn with saline, water, acetone,ether, and chloroform-methanol (2:1). TheLM-CWS (450 mg) consists of a mycolicacid-arabinogalactan-mucopeptide com-plex and is analogous to the cell wall of thesame strain grown in vivo and of other my-cobacteria ( 1 . 7 . "' I).

Bacterial suspension for challenge. AC3H mouse, which had been inoculatedsubcutaneously with M. lepraemurium Ha-waii six months previously, was killed withchloroform and washed in cresol and thenin running water. A leproma was excisedfrom the mouse, cut into small pieces, andground in a mortar. The emulsion was di-luted 1,000 times with saline (ml/g). Thebacterial suspension used for this experi-ment was prepared from the supernatantafter letting the diluted solution stand forseveral hours in a refrigerator. It containedabout 1-5 x 10 7 cells per ml of suspension.

Animals. Inbred mice of the C3H strainwere used at 6-7 weeks of age. Four to sixmice of either sex were housed in a stain-less steel cage with sterile litter under con-ditions of controlled humidity at 23°C.

Preparation of oil-attached LM-CWS.LM-CWS was suspended in sterile liquidparaffin, followed by the addition of 0.2%Tween 80 solution, and mixed well by son-ication (s). The final concentration of LM-CWS was either 1 mg or 100 tig per ml. Onetenth ml of one of these emulsions was in-jected subcutaneously into the C3H mice.

Injection of LM-CWS and challenge of the

mice. Experiments were designed asshown in Table 1. In the first experiment(Experiment A), each of 24 C3H mice werechallenged subcutaneously in the breastwith 0.2 ml of M. lepraemurium suspensionand divided into three groups, I, II, and III.At the same time, eight mice of groups Iand II were injected subcutaneously intotheir backs with 100 ,ug or 10 ps of oil-at-tached LM-CWS per mouse, respectively.Mice of group III were not injected withLM-CWS (control group). Booster injec-tions of the same dose of LM-CWS intogroups I and II were performed every twoweeks until the 17th week; after that LM-CWS was injected every four weeks to theend of this experiment (at 35 weeks).

In another experiment (Experiment B),C3H mice were subcutaneously injected intheir backs with 100 //g each of oil-attachedLM-CWS and five weeks later reinjectedwith the same dose of LM-CWS (groupIV). Seven weeks after the first injection ofLM-CWS, the mice were checked for non-specific DTH to picryl chloride (see below)and then challenged subcutaneously in thebreast with 0.2 ml of M. lepraemurium sus-pension. Booster injections into the groupIV mice were performed every four weeks.Non-stimulated control mice (group V)were injected in their backs with 0.1 ml ofPB seven and two weeks before challenge.

Measurement of the size of lepromata andhistological examination. The length andbreadth of the murine lepromata were mea-sured by a slide caliper, and the size of the

47, 3^Tanaka: Inhibition of M. lepraemurium by cell wall skeletons^489

lepromata were expressed as the product oflength and breadth.

In Experiment A, four mice from each ofthe three groups were sacrificed at 17 and35 weeks, and the lepromata and variousorgans were carefully removed andweighed. Microscopic examination wascarried out by impression preparations.Lymph nodes, spleen, liver and lung werefreshly cut apart with a pair of scissors, andthe cut surfaces were stamped on glassslides, which were stained by Ziehl-Neel-son's method. Results were expressed bysymbol and numerical expressions. In Ex-periment B, autopsies and microscopic ex-aminations were carried out at 21 and 40weeks.

DTH to picryl chloride. DTH in themice was measured by the method ofAsherson and Ptak ( 2 . 20). About 0.1 ml of7% picryl chloride in ethanol was appliedto the skin of the clipped abdomen. Sevendays after this sensitization, both ears werechallenged with one per cent picryl chloridein olive oil. The thickness of the ear wasmeasured with a dial thickness gauge (Pea-cock Dial Thickness Gauge, Type G, OzakiSeisakusho Co. Ltd., Tokyo) before and 24hours after challenge with the antigen. Theresults were expressed as follows:

Ear swelling (%)= 1 x ( R — R„ L Lo ) x 1002 R„ L„

where R„ and R are thicknesses of the rightear before and 24 hours after challenge, re-spectively, and L„ and L are those of theleft ear.

Footpad swelling and organ weight datawere expressed as the mean and standarddeviation within the groups, and statisticalsignificance was determined using the Stu-dent's t test.

RESULTSThe progression of murine leprosy and

DTH to picryl chloride in mice injected si-multaneously with LM-CWS and bacilli.Murine lepromata were clearly recognized10 weeks after challenge with M. leprae-ma•am (Fig. 1). At 10 weeks the sizes ofthe lepromata were similar in the threegroups. At 13 and 17 weeks, the develop-ment of lepromata was supressed in groupsI and II and unimpaired in group III.

At 17 weeks four mice from each group

80

150 I

120 .13

1.190

60 0

30

020^

5Week s

FIG. 1. Relationship between DTH to picryl chlo-ride and the size of lepromata in mice injected simul-taneously with M. lepraemurium and LM-CWS. Eachanimal was challenged with 2-5 x 10" bacilli and treat-ed as described in Methods. Ear swelling of mice ingroups I (M), II (13), and III (^) are indicated in bars.The average size of the lepromata in groups I (•), II(A.), and IH (0) is shown as the mean value of theproduct of the length and breadth.

were sacrificed for autopsy. From that timeon, four remaining mice from each groupwere successively treated by injecting oil-attached LM-CWS every four weeks. Nosignificant difference in reactivity amongthe three groups was attributed to an over-dose of LM-CWS. At 22 and 28 weeks, themice in group III had developed the usualsoft lepromata expected in unimmunizedmice (I. ' 2 .9. During this time period, thehard lepromata in groups I and II were sig-nificantly smaller than those in group III.On the other hand, by 35 weeks, develop-ment of lepromata in mice in groups I andII was not inhibited. Thus the developmentof lepromata in these groups was only de-layed.

DTH to picryl chloride is shown in Fig.1. At 12 weeks the mean reactivity of micein group I was slightly higher than those ofmice in groups II and III. However, no sta-tistically significant differences were foundamong the three groups at 12 or 17 weeks.

At 23, 30, and 35 weeks, the ear swellingresponse of group I was similar to that ofgroup III. The reactivity of group II wasslightly greater than those of the other twogroups (Fig. 1).

Autopsy of mice at 17 weeks. At 17 weeksfour mice from each group were killed withchloroform, and the lepromata were care-fully removed and weighed (Table 2). Themean weights of the lepromata from thefour mice in group I was 0.10% of body

:3. 6 0

I 60

0"320

Axillary^InguinalWeeks^Groups"^Lymph Node Lymph Node

1 (100 jig)^1.6± 1.1^0.017^II (10 tkg)^2.5 ± 1.(1^1.8±1.01.0

111 (0 ttg)^3.0 ± 0.0^1.0 ± 0.7

1 (100 lig)^2.5 ± 1.0^0.5 ± 0.435^II (10 jig)^2.8 ± 0.5^1.6 ± 1.1

III (0 lag)^3.0 ± 0.0^1.7±0.60.6

490^ International Journal of Leprosy^ 1979

TABLE 2. Weights o/• lepromata and organs in Experiment A.

Groups^Lepromata^

Spleen^

Liver

1(100µg)^ -"

^

0.96 ± 0.07^

6.27 ± 0.16At 17 wk^

11(10 µg)^

0.36 ± 0.11^

0.67 ± 0.07^

5.48 ± 0.69III (0 tig)^

0.40 ± 0.31^

0.52 ± 0.09^

5.33 ± 0.74

I (100 ttg)^

0.55 ± 0.34^

1.06 ± 0.11^

6.33 ± 0.25At 35 wk^

II (10 jig)^

1.29 ± 1.03^

1.52 ± 0.42^

6.84 ± 1.50III (0 jig)^

2.05 ± 1.13^

0.87 ± 0.43^

5.60 ± (1.73

" Each value expressed as % of body weight. Data expressed as.i' ± S.D.; N = 4 in each group except groupIII at 35 weeks where N = 3.

weight and less than that of the other twogroups. The weights of the lepromata ofgroups II and III were almost the same. Inappearance, the lepromata in groups I andII were of the same extent (Fig. 1), butthere was a great difference in the weightsof the excised lepromata between groups Iand II, showing that the lepromata in groupI were extended but flat and that their vol-ume was very small.

Because it is well known that cell wallsof mycobacteria stimulate lymphoid tis-sues, the spleen and liver were removedfrom each of the mice and weighed. Thespleen weights of group I mice were 0.96%of body weight and greater than those ofthe other two groups (p < 0.01). The liverweights were 6.27% of body weight ingroup I animals, and this was not signifi-cantly greater than in the other two groups.There were no differences in spleen and liv-er weights between the mice in groups IIand III.

The right or left axillary lymph node,

which is the regional lymph node for theprimary lesion, inguinal lymph node,spleen, liver, and lung were evaluated forbacilli by impression preparations (Table3). Acid-fast bacilli were found essentiallyonly in the axillary lymph nodes of groupI mice at 17 weeks. However, in mice ingroups II and III, acid-fast bacilli werefound in more or less all of the organs ex-amined.

These results show that in group I miceinjection of oil-attached LM-CWS stimulat-ed lymphoid tissues and that the growthand dissemination of murine leprosy bacilliwere partially inhibited, and the bacilli re-mained in local lesions on the breast up to17 weeks after inoculation.

Autopsy of mice at 35 weeks. The lepro-mata were quite enlarged, and their weightsin groups I, II, and III were 0.55, 1.29, and2.05% of body weights, respectively.Spleen weights in group II mice averaged1.52% of body weights and were slightlygreater than those of groups I and III. The

TABLE 3. Acid fast bacilli in impression preparations of the various organs (Experi-ment A). Data expressed as x ± S.D. of numerical gradins,,s."

Organs

Spleen Liver Lung

0.0 0.3 ± 0.3 0.00.8 ± 0.3 1.5 ±^1.0 1.0 ± 0.01.1 ± 0.6 1.6 ± 0.8 1.4 ± 0.8

0.5 ± 0.0 0.5 ± 0.0 0.6 ± 0.30.9 ± 0.8 1.0 ± 0.7 1.1 ± 0.62.0 ± 0.0 2.0 ± 0.0 2.0 ± 0.0

" N = 4 in each group except group III at 35 weeks where N = 3." Number of acid-fast bacilli:

- (0) no acid-fast bacilli found in the microscopic fields examined.± (0.5) a very few bacilli found in several microscopic fields.+ (1.0) a few bacilli found in each microscopic field.

++ (2.0) 10 to 100 bacilli found in each microscopic field.+++ (3.0) many bacilli and globi.

At 21 wk

At 40 wk

IV (100 pg)V (0 t(g)

IV (100 pg)V (0 dig)

6.13 ± 0.385.57 ± 0.17

7.01 ± 0.8710.34 ± 3.41

0.64 ± 0.21"2.49 ± 0.89

4.11 ± 1.816.70 ± 4.37

0.82 ± 0.090.71 ± 0.19

^

0.88^0.16

^

2.96^0.67

47, 3^Tanaka: Inhibition of M. lepraemurium by cell wall skeletons^491

TABLE 4. Weights of' 1epromata and organs in Experiment B.

Groups^Lepromata^

Spleen^ Liver

" Each value was expressed as^of body weight. Data expressed as ± S.D.: N = 4 at 21 weeks and N = 3at 40 weeks.

apparent increase of this spleen weightmight be related to the fact that the DTHof group II mice was greater than that ofthe other two groups after 22 weeks (p <0.05). However, since many acid-fast ba-cilli were observed in the spleens byimpression preparations, the apparent in-crease in spleen weight might be due tomicrolepromata. Actually, one of the micein group II was found to have innumerablemacroscopic lepromata in both the spleenand the liver with associated increases inthe weights of these organs. There were nosignificant differences in liver weights amongthe three groups.

The axillary lymph nodes from all threegroups of mice became enlarged and con-tained many acid-fast bacilli microscopical-ly. Even in the inguinal lymph nodes,spleens, livers, and lungs of mice in groupsI and II, acid-fast bacilli were found regu-larly (Table 3).

These results differ from the results at 17weeks. It is obvious that at 35 weeks theincreases in weights of lepromata and thegrowth and dissemination of acid-fast ba-cilli into the various organs are seen notonly in group III (control animals) but alsoin groups I and II (animals treated with LM-CWS).

Observations in mice challenged with M.lepraemurium seven weeks after injection ofLM-CWS (Experiment B). As shown inFig. 2, nonspecific DTH was activated ingroup IV mice injected with oil-attachedLM-CWS seven and two weeks beforechallenge with .14. lepraemurium (at 0time). At 8, 13, and 21 weeks, DTH to pic-ryl chloride was significantly greater inmice in group IV than that in group V mice.Thereafter, no difference in DTH to picrylchloride was observed between the twogroups of mice.

At 9 and 13 weeks there were no differ-ences in the sizes of the lepromata between

the two groups. After 17 weeks the devel-opment of lepromata was suppressed anddelayed in group IV animals receiving LM-CWS similar to the observations made inthe LM-CWS treated mice described in Ex-periment A.

Three to four mice from each of the twogroups were sacrificed at 21 and 40 weeks.Results are shown in Tables 4 and 5 and arealmost the same as those in Experiment A(Tables 1 and 2). At 21 weeks the weightsof the lepromata in group V were muchgreater than those in group IV (p < 0.05).However, there were no significant differ-ences in the weights of the spleens and liv-ers between the two groups at 21 weeks.Multiplication of acid-fast bacilli and theirdissemination into various organs were par-tially inhibited in group IV mice. At 40weeks, the lepromata were quite large.There was enlargement of the spleen and

100

80

E" 60

di., 40

20

5^10^15^20^25^30^35^40Weeks offer inoculation of M. leproernorium

FiG. 2. Relationslip between DTH to picryl chlo-

ride and the size of lepromata in mice challenged withti. lepraemurium seven weeks after injection of LM-CWS. Each animal was stimulated nonspecifically by

injecting LM-CWS before challenge with 2-5 x 10'bacilli as described in Methods. Ear swelling of mice

in groups IV (El) and V (0) is shown by column. Theaverage size of lepromata in groups IV (•) and V

(0) is shown as the mean value of the product of the

length and breadth.

492^ International Journal of Leprosy^ 1979

TABLE 5. Acid-fast bacilli observed in the impression preparation of the various organs .

(Experiment B). Data expressed as 5( ± S.D. of numerical gradings as in Table 3.

Weeks^Groups"

I V ( I 00 fig)V (0 gig)

IV (100 gig)V (0 gig)

Organs

Axillary^InguinalLymph Node Lymph Node^Spleen

0.9 ± 0.32.8 ± 0.5

3.0 ± 0.03.0±0.00.0

layer^Lung

0.3 ± 0.3^0.5±0.(10.01.0 ± 0.7^0.9 ± 0.8

1.8 ± 1.3^1.7 ± 0.63.0 ± 0.0^3.0 ± 0.0

21

40

0.4 ± 0.3^

0.1 ± 0.30.5 ± 0.4

^1.0^0.7

2.7^0.6^

2.0 ± 1.03.0 ± 0.0

^3.0 ± 0.0

" N^4 at 21 wit_ks and N^3 at 40 weeks.

liver in group V animals, and these organswere found to have innumerable small le-promata macroscopically. Acid-fast bacilliwere observed microscopically in all theorgans examined.

DISCUSSION

The relationship between progression ofM. /epraenutrium infection and DTH hasbeen investigated by several authors

They inoculated murine leprosybacilli into the mouse footpad and detectedDTH to lepraemurium antigens. In themice infected with M. lepraemurium, DTHto antigens of I. lepraettutrium is a spe-cific and desirable reaction. However, ap-parent loss of DTH occurs after 8 weeks ofinfection in association with high levels ofsystemic antigen ( 1 "). Therefore, in thisstudy picryl chloride, a different antigenfrom M. lepraemierium, was used as theantigen for DTH. DTH to picryl chloridewas detected after over 20 weeks of infec-tion (Fig. 2).

Oil-attached CWS from mycobacteriastimulates T cell functions nonspecifically('s), and it can be expected that DTHagainst Al. lepraenutrium and picryl chlo-ride are activated when LM-CWS is inject-ed with both these antigens. When the non-infected normal C3H mice were injectedwith 10 or 100 Ag of oil-attached LM-CWS,DTH to picryl chloride was significantlydeveloped at 4 weeks, and an increasedlevel of DTH persisted over 20 weeks ascompared with the non-stimulated group(data not shown). In Experiment B, DTHto picryl chloride clearly developed afterover 20 weeks of infection. On the otherhand, in Experiment A, there were no sig-nificant differences in DTH at 12 and 17weeks among the three groups. These find-

ings indicate that DTH was more easily de-tected when the antigen challenge was ad-ministered after the animals were stimulatedwith LM-CWS.

In Experiment A, LM-CWS presumablyexerted a general immunostimulation, andvery few acid-fast bacilli were observed inthe inguinal lymph node, spleen, liver, andlung (Table 3). However, DTH to picrylchloride was not detected clearly at 12 and17 weeks. Because only a small amount ofpicryl chloride was in the circulation, it isnot sufficient to explain the facts on the the-ory of Schlossman, et al. (II), who reportedthat the presence of high concentrations ofspecific antigen in the circulation can resultin suppression of DTH. Two possible rea-sons may be considered: 1) the occurenceof suppressor T cells as recognized in tu-mor-bearing hosts (" 1 ' 22 ), and 2) toxic sub-stances which are produced by M. leprae-murium and which inhibit the expression ofDTH. These problems should be investi-gated in the future.

After 21 weeks of infection in both Ex-periments A and B, no differences in DTHwere observed between the groups, andfrom that time on lepromata grew largerand larger (Figs. 1 and 2). Poulter, et al.( 1 ") reported that a decay of DTH reactivitywas associated with a progressive increasein the number of M. lepraemurium. Itseems that the loss of detectable DTH topicryl chloride after 21 weeks was also as-sociated with an increase in the number ofbacilli.

C3H mice injected with oil-attached LM-CWS developed nonspecific DTH up toabout 20 weeks of infection. However, thegrowth of AI. lepraemurium was inhibitedonly in part, and both the increase in sizeof the lepromata and the multiplication of

47, 3^Thnaka: Inhibition of M. lepraemurium by cell wall skeletons^493

lepraemurium were observed aftersome delay. Since it is well known that theC3H mouse is a low responder to A/. hp-/wont/them antigen, any other mouse strainof higher responder status should also beused for this kind of experiment. After eval-uation of LM-CWS in such animals, the ap-propriateness of LM-CWS for the immu-notherapy of leprosy could be considered.

SUMMARYC31-I mice stimulated with Al. leprae-

murium cell wall skeletons (LM-CWS)were challenged with viable M. lepraemu-rbon, and delayed-type hypersensitivity(DTH) to picryl chloride was measured. Inone type of experiment the mice were chal-lenged at the time when stimulation of cell-mediated resistance by means of LM-CWSwas undertaken. The major purpose was toinvestigate principles pertaining to immu-notherapy. In contrast to the loss of a de-tectable DTH response to picryl chloride,development of murine leprosy was par-tially suppressed. In the second type of ex-periment the mice were stimulated with oil-attached LM-CWS seven weeks beforechallenge with ,11. lepraemurium. Findingswere: a) that nonspecific DTH as measuredby sensitization and challenge with picrylchloride was activiated before the infectionwith .11. lepraemurhon and b) that the DTHwhich developed was associated with par-tial protection against the growth of lepro-mata. The murine leprosy which developedin C3H mice stimulated with LM-CWS wasprogressive after some delay.

RESUMENSe estimularon ratones C3H con paredes de IL lep-

raemurium y posteriormente se inocularon (desafiaron)con A/. lepracmurium vivo. En estos ratones se indujo

un estado de hipersensibilidad retardada al cloruropicrilo que posteriormente se midiLi. En un tipo de

experimentos, los ratones se desafiaron al mismo tiem-Po que se hizo Ia estimulaciOn de Ia resistencia celular

con las paredes micobacterianas. El propOsito funda-mental de este tipo de experimentos fue el de inves-tigar aspectos relacionados con inmunoterapia. Encontraste con Ia perdida de la respuesta tardia hacia

el cloruro de picrilo, el desarrollo de la lepra murinasolo se suprimiO parcialmente. En el segundo tipo deexperimentos, los ratones se estimularon con paredesmicobacterianas emulsificadas con aceite, siete se-manas antes del desafio con lepraellutrium. Los

hallazgos fueron: a) que no se observti depresiOn ines-

pecifica de Ia reactividad tipo tardio (ya que esta se

pudo inducir con el cloruro de picrilo) antes de la in-fecciLin con el .11. lepraemurium y que Ia respuestatardia desarrollada estuvo asociada con unit protec-ciOn parcial contra el crecimiento de lepromas. La le-pra murina desarrollada por los ratones C311 estimu-

lados con paredes micobacterianas fue progresivadespues de cierto retardo.

RESUMEChez des souris de souche C311, stimulees par des

squclettes de paroi cellulaire de .11. hpraemurium

(LNI-CWS), on a etudie la reponse it ('administrationdune preparation de .1/. lepraemuritim viable. On aegalement mesure l'hypersensibilite de type retarde au

chlorure picrique. Dans une experience, on a etudielit reponse des souris au moment de la stimulation deIa resistance cellulaire an moyen Lie LN1-CWS.

L'objectif principal etait d'investiguer les principes qui

regissent l'immunotherapie. Par ailleurs, alors qu'il

etait devenu impossible de detester une reponsed'hypersensibilite retardee an chlorure picrique, le de-

veloppement de la lepre marine etait en partie sup-prime. Dans le second type d'experience, ICS SOUDS

etaient stimulees avec l'extrait LM-CWS en prepara-tion huileuse, sept semaines avant I' administration de1. /eproemurium. Les observations ont Cie les sui-

vantes: a) l'hypersensibilite de type retarde non

specitique, telle qu'elle est mesuree par In sensibi-lisation et par I' administration de chlorure picrique,

etait activee avant l'infection par M. lepraemurium:

b) l'hypersensibilite de type retarde qui s'est ainsi de-

veloppee, etait associee avec tine protection partiellecontre lit croissance de leprOmes. La lepre murine qui

s'est developpee dans les souris de souche C3H sti-mulee par la preparation LM-CWS, montrait apres uncertain delai, tine progression.

Acknowledgments. Grateful acknowledgment is ex-

pressed to Prof. M. Nakamura for his constant interest

during the course of this investigation and is also ex-pressed to Prof. I. AZUMa for his useful advice anddiscussion. I thank Dr. K. Chiba for his technical ad-

vice.

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