422^ International Journal of Leprosy^ 1984

It would be of importance of' performsimilar studies in other populations to fur-ther elucidate the mechanism(s) by whichsuppression is exerted to evaluate the po-tential use of these findings with regard totherapy.

- Tom H. M. Ottenhoff, M.D.- Dienne G. Elferink- René R. P. de Vries, Ph.D.

Department of Iminunohematologyattd Bloodbank

ACadClltisch /iekenhuis LeidenRiPisburgenveg 102333 AA LeidenThe Netherlands

REFERENCESI. BJUNE, 0., BARNETSON, R. ST. C., RIDI FY, D. S.

and KRONVALL, G. Lymphocyte transformationtest in leprosy: Correlation of the response withinflammation of lesions. Clin. Exp. Immunol. 25(1976) 85-94.

2. DRAPER, P. Studies on Mycobacterium leprae frominfected armadillos. In: Leprosy. Proc. XI Inter-national Leprosy Congress. Latapi, F., et al., eds.Amsterdam: Excerpta Medica, 1980, p. 97.

3. GoDAL, T. Immunological aspects of leprosypresent status. Prog. Allergy 25 (1978) 211-242.

4. GODAL, T., MYKLESEAD, B., SAMUEL, D. R. andMYRVANG, B. Characterization of the cellular im-mune defect in lepromatous leprosy: A specific

lack of circulating Mycobacterium /eprae-reactivelymphocytes. Clin. Exp. Immunol. 9 (1971) 821-831.

5. GULLBERG, M. and LARRSON, E. L. Studies oninduction and effector functions of concanavalinA-induced suppressor cells that limit TCGF pro-duction. J. Immunol. 128 (1982) 746-750.

6. HAREGEwoiN, A., GoDAE, T., MUSTAFA, A..S., BE-Emu, A., and YLNIANEBERILAN, T. T cell condi-tioned media reverse T cell unresponsiveness inlepromatous leprosy. Nature 303 (1983) 342-344.

7. HUN I ER, S. W. and BRENNAN, 1'. J. A novel phe-nolic glycolipid from Mycobacterium leprae pos-sibly involved in immunogenicity and pathogen-icity. J. 13acteriol. 147 (1981) 728-735.

8. KRANIER, M. and KOSZINOWSKI, U. T cell-specificsuppressor factor(s) with regulatory influence oninterleukin 2 production and function. J. Immu-nol. 128 (1982) 784-790.

9. MALKOVSKY, M., AsliERsoN, G. L., STOCKINGER,B. and WATKINS, M. C. Nonspecific inhibitor re-leased by T acceptor cells reduces the productionof interleukin-2. Nature 300 (1982) 652-655.

10. NATIE I. Immunology of human leprosy-currentstatus. Lepr. Rev. Special Issue (1983) 31S-45S.

11. REES, R. J. W. The significance of the leprominreaction in man. Prog. Allergy 8 (1964) 224-258.

12. TURK, J. L. and BRYCESON, A. D. M. Immuno-logical phenomena in leprosy and related diseases.Adv. Immunol. 13 (1971) 209-266.

13. VAN EDEN, W. 'ILA and leprosy. .1 mode/for thestudy of genetic control of Minnow response in aman, thesis, Leiden, 1983.

Response to Letter of Dr. Ottenhoff, et al.

To THE EDITOR:We are grateful for your invitation to

comment on the Letter to the Editor fromOttenhoff, et al. As discussed by Ottenhoff,et al., the discrepancies between our pub-lished data (Haregewoin, et al., Nature 303[1983] 342-344) and theirs may be due toeither methodological differences or differ-ences in the patient groups.

With the kind assistance of Dr. Leiker,we have had the opportunity of studyingseveral patients from Holland. Our resultsare shown in Table 1. As can be seen fromTable 1, we have also found a poor responseof such patients to T-cell conditioned me-dium (TCM) (Lymphocult T, Biotest). Only2 out of 8 patients gave a significant re-sponse (>5000 A cpm). However, both of

the responders also responded to Al. lepraealone. Thus, our data with TCM are quiteanalogous to those of Ottenhoff, et al. Theonly notable difference between their dataand ours is that, while in their assay theoverall response to PPD was quite low, wehad good responses to BCG in the threepatients tested. This difference may indicatethat significant methodological differencesexist between our two laboratories, but theirrelevance to the interleukin 2 (IL2) effect inlepromatous leprosy remains unclear. Incontrast to TCM, recombinant IL2 appearsto have significant effects in 3 out of 4 testedpatients. We hope that Ottenhoff, et al. willhave the opportunity also to study recom-binant IL2. We agree with Ottenhoff, et al.that the most likely explanation for the dis-

W. >95% F1.2 53.3

D.H. >95% 0.9^----73

N.G. 80% 1.7 3.8

T.O. >95% 13.9 11.8

L.I. >95% 11.2 8.7

H.E. >95% 1.3 4.0

M.A. _g 0.04 0.3

S.Y. - 0.06 0.7

1 - 74.517.7^29.027.9^43.8

25.1^115.1 1 15.1^68.4 - 131.3

^

9.3^7.4^10.2^11.7

^

23.6^26.8

^

5.2^8.7

^

13.7^14.4

^

4.1^2.9

^

13.7^12.8

1 0.08^41.2

0.1^37.0j

44.414.9^24.6

6.1^8.7^5.7^6.5

2.2^3.8^113.6^22.2

52, 3^ Correspondence^ 423

TABLE 1. Responsea of patients from Holland' to M. leprae, M. leprae + TCM,e M.leprae + IIPLC + purified IL2," and M. leprae + recombinant IL2 (REC IL2).c

3H-thymidine incorporation (cpm x 10-3)

CellPatient^via-

bilityCells^Cells +alone^Al. leprae

Cells +1/200TCM

Cells +Cells + Cells + HPLC1/200 HPLC^IL2

TCM + 1L2^80/m1Al. leprae 80/m1 + M.

leprae

Cells +Cells + REC IL2

REC IL2 6 U/ml6 U/ml + Al.

leprae

Cells +BCG

Peripheral blood leukocytes (PBLs) were separated within 12 hr after blood collection; 2 x 105 PBL incomplete medium (RPM! 1640 + 15% AB serum + 1% penicillin and streptomycin) were added to each wellof round-bottom microtiter plates. Armadillo-derived whole Al. leprae were added at a final concentration of5 x 10' bacilli/ml. IL2 preparations were used at indicated concentrations. Total culture volume was kept at200 pl. Cultures were pulsed with 1 pCi 31-1-thymidine on day 5 and harvested 20 hr later. Incorporatedradioactivity was determined by liquid scintillation counting. Median cpm from triplicate cultures were tabulated.

The patient material was obtained from Royal Tropical Institute, Amsterdam, Holland. Patients were di-agnosed and the blood was sent under the kind supervision of Dr. Derk L. Leiker.

TCM was Lymphocult T purchased from Biotest.d HPLC IL2 was high-performance liquid chromatography purified IL2, kindly gifted by Dr. B. R. Bloom,

Albert Einstein College of Medicine, New York, U.S.A.° Recombinant IL2 was a kind gift from Cetus Corporation, California, U.S.A.

cpm 5000 are boxed.- = not done.

TABLE 2. Number of lepromatous patients from different countries showing positiveresponse to M. leprae in the presence of TCM, HPLC-purified IL2, and recombinant IL2.a

Country Al. leprae +TCM"

Al. leprae +HPLC IL2

Al. leprae +REC IL2d

England 1/4° - f 4/4Ethiopia 3/4 - -Holland 0/6 1/4 3/4Norway 2/4 2/3 3/4Total 6/18 3/7 10/12

The patient material was obtained from ALERT/AHRI, Addis Ababa, Ethiopia; Ulleväl Hospital, Oslo,Norway; Royal Tropical Institute, Amsterdam, Holland, and Hospital for Tropical Diseases, London, England.The patients were diagnosed and the blood was sent under the kind supervisions of Dr. Abebe Haregewoin, Dr.Ivar Helle, Dr. Derk L. Leiker, and Dr. Michael F. R. Waters. PBLs were separated within 12-24 hr after bloodcollection. The experiments were done as described in Table I.

TCM was used at a final dilution of 1/200.HPLC IL2 was added at a concentration of 8 U/ml.

" Recombinant 1L2 was a kind gift from Cetus Corporation, California, U.S.A., and was used at 6 U/ml.' Positive/tested. Positive responders were sorted on the basis of cpm of >5000, i.e., difference in cpm

between cultures with M. leprae and IL2 preparations - Al. leprae alone.- = not done.

424^ International Journal of Leprosy^ 1984

crepancies observed may be related to thepatient population. Indeed, when we ana-lyze our results based on patients from dif-ferent locations, we find differences partic-ularly with TCM (Table 2). While thematerial is too limited to draw definite con-clusions, the data suggest that the lepro-matous group is heterogeneous with regardto their resonse to TCM. This heterogeneity

appears to become reduced when using re-combinant IL2.

—Abu Salim Mustafa, Ph.D.—Abebe Haregewoin, M.D.—Tore Godal, M.D., Ph.D.

Laboratory for ImmunologyThe Norwegian Radium HospitalOslo, Norway

Venous Involvement in Nonlepromatous Leprosy

To THE EDITOR:Our earlier report (') of venous involve-

ment in lepromatous leprosy (LL) patientsshowed a very high (94%) incidence of lep-rous phlebitis in this group. The study hassince been extended to cover the non-LLforms of leprosy, and 24 BT patients wereselected for vein biopsy. Cases were selectedonly when a subcutaneous vein was seenbeneath or lying just adjacent to a lesion. In18 cases the lesions selected were on theextremities, while in six cases they were onthe back. Resection of a 0.5-1 cm segmentof vein was done along with a skin biopsy.Both the skin and vein biopsies were fixedin formalin, processed, and 5 i thick par-affin sections were stained with hematoxylinand eosin (H&E), Fite's, and V.V.G. stains.On histological study, only 1 out of the 24cases showed evidence of vein wall involve-ment. This case was graded BT-BB clini-cally and histologically. Sections from theresected vein showed three focal epithelioidcell granulomata located in the intima justbeneath the endothelial layer (The Figure).Each granuloma caused a hump-like pro-tuberance into the vein lumen. The endo-thelium was continuous over each hump.The endothelial cells were flat and did notshow any signs of activation. The media andadventitia showed infiltration by epithe-lioid cells only at the base of the largestgranuloma. Most of the cells forming thegranuloma were epithelioid in nature. Lym-phocytes were scant and no giant cells wereseen. Acid-fast bacilli (AFB) could not befound, even after repeated examinationswith Fite's stain.

The present study shows that venous le-

THE FIGURE. Photomicrograph of one of the gran-ulomata showing epithelioid cells in the intimal layer(H&E x 300).

sions may be seen in nonlepromatous formsof leprosy. However, their frequency is muchless than in LL cases. It is also possible thatthe biopsy may not have picked up the well-localized venous lesion in some of the cases.Although AFB were not demonstrated inthese lesions, BT-BB leprosy is adequateproof of the presence of AFB in the veinwall at some time or other. The location ofthe lesions indicates that in this case alsothe mode of entry was from the vessel lu-men. Episodes of bacteremia are known tooccur in BT patients (2), and may have ledto the deposition of organisms and granu-loma formation in the vessel wall. The sig-nificance of these lesions vis-à-vis diseasedissemination, granuloma formation, andas sites for persister organisms needs furtherstudy.

—A. Mukherjee, M.B.B.S., M.D.—B. K. Girdhar, M.B.B.S., M.D.

Institute of PathologyIndian Council of Medical Research