67, 4^ Correspondence^ 473

Reprint requests to Dr. S. Agrawal at theabove address or FAX 977-25-20251;e-mail: sudha92@hotmail.com

REFERENCES• A1.1, P M. The age at onset of leprosy. Lepr. Rev.

35 (1964) 193-197.2. JOPLING, W. 11. and McDou0ALL, A. C. Delinition,

epidemiology and world distribution. In: Hand-book of Leprosy. 4th edn. Oxford: HeinemannMedical Books, 1988, pp. 1-9.

3. MONTEIRO, L. G., CAmPos, W. R., ()Rum:, F. andGRossi, M. A. F. Study of ocular changes in lep-rosy patients. Indian J. Lepr. 70 (1998) 197-202.

4. NoomaN, S. K. The epideiniology of leprosy. In:Leprosv. 2nd edn. Hastings, R. C., ed. London:Churchill Livingstone, 1992, pp. 29-45.

Apoptosis in Leprosy PatientsTo THE EDrfoR:

The protection-associated host responseto Mrcobacterium leproe infection isstrongly dependent upon subsequent cellu-lar immunity ('). Apoptosis appears as aphysiological mechanism that leads to cellelimination without inducing inflammationor damage to contiguous cells (3.5). In thisstudy, we have tested the hypothesis thatamong the various factors potentially re-sponsible for the lymphocyte alterations inleprosy, apoptosis could be implicated.Along this line, we evaluated the impact ofleprosy infection on the levei of sponta-neous apoptosis and the type of cells(CD4', CD8+, CD19+) that were likely con-cerned.

After informed consent, 19 newly de-tected leprosy patients (11 males, 8 fe-males) were included in the study before re-ceiving any treatment. Nine of them hadmultibacillary (MB) leprosy and 10 hadpaucibacillary (PB) leprosy. Seven (5males, 2 females) Senegalese healthy adultdonors were enrolled as controls.

Peripheral blood mononuclear cells wereisolated from heparinized whole blood byHistopaque®-1077 density gradient (SigmaDiagnosis, St. Louis, Missouri, U.S.A.) andcultured at 10" cells/ml in 24-well platesunder unstimulated (medium afoite) condi-tions. The plates were thereafter incubatedfor 2 clays (determined after a preliminarykinetic study) at 37°C in a water-saturatedatmosphere containing 5% CO,. Quantifica-tion of apoptosis was performed by 11ov cy-tometry as already described by stainingthe lymphocytes with 7 amino-actino-mycinD (7AAD; Sigma) which discrimi-nates live from early apoptotic cells. CD4',

CD8' and CDI9 cells were identitied usingmonoclonal antibodies (Becton DickinsonImmunocytometry Systems, San Jose, Cali-fornia, U.S.A.). The nonparametricKruskal-Wallis test was used to comparethe data between the different groups; a pvalue of <0.05 was considered as signifi-cant.

The proportions of apoptotic lympho-cytes were compared in leprosy patientsand controls. A highly sig,niticant increase(p = 0.01) in the levei of spontaneous apop-tosis in leprosy patients was found as com-pared to controls, suggesting a notable im-pact of the Al. leprae infection. Hence,apoptosis seems to be an active phenome-non in leprosy as previously found for sev-eral other intracellular infections (7.'"), in-cluding inalaria ()). However, the percent-age of 7AAD-positive cells was notsigniticantly different between the twogroups of PB and MB patients. Such obser-vation has to be contirmed with a greaternumber of patients.

The relative distribution of the lympho-cyte subset within apoptotic cells was stud-ied (ratio of the percentage of the apoptoticsubpopulation studied on the percentage oftotal apoptotic cells of the culture). Wefound that the distribution of CD4', CD8'and CD19+ cells within apoptotic cells didnot differ between patients and controls,suggesting that the infection simply in-duced an exaggeration of a naturally occur-ring mechanism.

Another type of analysis was performedin calculating the ratio of the percentage ofthe apoptotic subpopulation on the percent-age of the population concerned. This al-lowed us to determine the number of apop-totic cells within each subset separately.

474^ hnernational Journal of. Leprosy^ 1999

THE TABLE. Percentage oJ apoptoticcellsl,vithin cat.]] Irniphocyte subset.

CD4^CDS^CDI 9% -±-^r/f. ± S.D.^% ± S.D.

Controls 5.9± 1.9 6 ± 2.2 4.8 ± 3.7MB 9.4 ± 4.4 12.4 ± 7.5 13.5 ± 13PB 9.8 ± 4 14.6 ± 7 13± 10.3

This analysis showed that CD8+ andCD 19+ cells presented a higher proportion(p <0.004) of apoptotic cells than did theCD4+ cells in leprosy patients in compari-son with controls (The Table), suggesting apossible preferential lymphocyte subsetget in 114. /eprae-induced apoptosis.

Taken together, our results show thatapoptosis is an existing mechanism of celldestruction in leprosy. It might represent astrategy of the immune system to eliminateinfected cells. However, implications of sev-eral different fiictors in the fine mechanisminducing lymphocyte apoptosis, such aslymphocyte activation, cytokines, effect offree oxygen radicais or the effects of super-antigens already described for other intra-cellular infections (=.41, have to be studiedto further delineate the possible 'role ofapoptosis in the physiopathology of leprosy.

-Mbayame Ndiaye Niang, Pharm.D.Aissatou Toure Balde, Pharm.D.

Ronald Perraut, Pharm.D.Laboratoire cl 'ImmunologieInstitut Pasteur de Dakar36 Avenue Pastem-BP 220Dakar, Senegal

-Ibrahima Mane, M.D.Jean Louis Cartel, M.D.

Institut de LeprologieAppliquee de Dakar

Dakar, Senegal

Reprint requests to Dr. Niang at theabove address or FAX 221-39-92-10.

Acknowledgment. The authors are very gratefulto Ababacar Diouf and Babacar Diouf for their experttechnical contributions and to ali the workers of the In-stitut de Leprologie Appliquée de Dakar.

REFERENCESBLoom, B. R. and GODAI., T. Selective primaryhealth care: stratel.,lies for control of diseases in thedeveloping world. V. Leprosy. Rev. Infect. Dis. 5(1983) 765-771.

2. Bu-r-rKE, T. M. and SANDSTRONI, P. A. Oxidativestress as a mediator of apoptosis. Immunol. Today15 (1994) 7-10.

3. CoilkN, J. J. and DURE, R. C. Apoptosis and pro-grammed eell death in immunity. Ann. Re v. Int-munol. 10 (1992) 267-293.

4. DuRRBAum-LANimANN, I., GERCKEN, J., RAD, H.D. and ERNST, M. Effeet of^varo infection ofhuman monocytes with low numbers of ycobac-

tuberculosis bacteria on monocyte apopto-sis. Infect. Immun. 64 (1996) 5384-5389.

5. DuvALL, E. and WYLLIE, A. H. Death and the cell.Immonol. Today 7 (1986) 115-119.

6. KEANE, J., BALCEWICZ-SAIII.INSKA, M. K., RE-moLD, H. G., Cum', G. L., MEEK, B., FENTON, M.J. and KoRNFELD, 1-1. Infection by Mycobacteriumtuberculosis promotes human alveolar macro-phage apoptosis. Infect. Immun. 65 (1997)298-304.

7. KtiEur, N., ZYCHLINSKY, A. and Guiso, N. BOr-d e ella pertussis induces apoptosis in macro-phages: role of adenylate cyclase hemolysin. In-fect. Immun. 61 (1995) 4064-4070.

8. Sciimip, I., KRAI.I., W J., UITTENBOGAART, C. H.,BRAUN, J. and Gomil, J. V. Dead cell discrimina-tion with 7-amino actinomycin D in combinationwith dual color immunofluorescence in singlelaser flow cytometry. Cytometry 13 (1992)204-208.

9. TouRUBALDÉ, A., SAl21.11011, J. L. and Roussin-110N, C. Acute "P/a.vmodium fa/ciparum" infee-tion is associated with inereased percentages ofapoptotic cells. Immunol. Lett. 46 (1995) 59-62.

10. ZYCIII,INSKY, A., PRI:vosT, M. C. and SANSONETTI,P. J. Shigella fle.vneri induces apoptosis in in-feeted macrophages. Nature 358 (1992) 167-169.