INTERNATIONAL. JOURNAL. OF LEPROSY Volume 69, Number 4

Printed in the U.S.A.(ISSN 0148-916X)

THIRTY-SIXTH U.S.-JAPANTUBERCULOSIS-LEPROSYRESEARCH CONFERENCEOmni Royal Orleans Hotel

New Orleans, Louisiana, U.S.A.15-17 July 2001

sponsored by theU.S.-Japan Cooperative Medical Science ProgramNational Institute of Allergy and Infectious Diseases

National Institutes of Health

U.S. Tuberculosis-Leprosy Panel

CHAIR

Dr. Philip C. HopewellUCSF School of MedicineSan Francisco General Hospital1001 Potrero Avenue, Room 2A21San Francisco, CA 94110TEL: (415) 206-8509FAX: 415/285-2037EMAIL: phopewel@sfghdean.ucsf.edu

MEMBERS

Dr. Clifton E. Barry, IIITuberculosis Research SectionLaboratory of Host DefensesNational Institutes of HealthTwinbrook II, Room 239, MSC 818012441 Parklawn DriveRockville, MD 20852-1742TEL: 301-435-7509FAX: 301-402-0993EMAIL: clifton_barry@nih.gov

Dr. Thomas P. GillisMolecular Biology Research DepartmentLaboratory Research BranchNational Hansen's Disease Center at

Louisiana State UniversityP.O. Box 25072Baton Rouge, LA 70894TEL: (225) 578-9836FAX: (225) 578-9856EMAIL: tgillis@Isu.edu

Dr. Gilla KaplanLaboratory of Cellular Physiology and

ImmunologyThe Rockefeller University1230 York AvenueNew York, NY 10021TEL: 212-327-8375FAX: 212-327-8376EMAIL: kaplang@rockvax.rockefeller.edu

Dr. David N. McMurrayMedical Microbiology and Immunology

DepartmentTexas A&M University SystemHealth Science CenterReynolds Medical Building, Mail Stop I 114College Station, TX 77843-1 1 14TEL: 409/845-1367FAX: 409/845-3479EMAIL: mcmurray@medicine.tamu.edu

367

368 International Journal of Leprosy 2001

,Japanese Tuberculosis-Leprosy Panel

CHAIR

Dr. Masao MitsuyamaDepartment of MicrobiologyGraduate School of MedicineKyoto UniversityYoshidakonoe-cho, Sakyo-kuKyoto 606-8501TEL: +81-75-753-4441FAX: +81-75-7.53-4446e-mail: mituyama@mb.med.kyoto-u.ac. j jp

MEMBER S

Dr. Kazuo KobayashiDepartment of Host DefenseGraduate School of MedicineOsaka City University1-4-3 Asahi -machi, Abeno-kuOsaka 545-8585TEL: +81-6-6645-3745FAX: +81 -6 -6646 -3662 ,e-mail: kobayak@med.osaka-cu.ac.jp

Dr. Masamichi GotoDepartment of PathologyFaculty of MedicineKagoshima University8-35-1 SakuragaokaKagoshima 890-852()TEL: +81-99-275-5270FAX: +81-99-265-7235e-mail:masagoto@ m2.kufm.kagoshi ma- u.ac.jp

Dr. Kiyoshi TakatsuDepartment of ImmunologyInstitute of Medical ScienceUniversity of Tokyo4-6-1 S h i rokanedai, M i nato-k uTokyo 108-8639TEL: +81-3-5449-5260FAX: +81-3-5449-5407Email: takatsuk@ims.u-tokyo.acm

Dr. Hatstimi TaniguchiDepartment of MicrobiologyUniversity of Occupational and EnvironmentalHealthIseigaoka, Yahatanishi-ku,Kitakyushu 807-8555TEL: +81-93-691-7242FAX: +81-93-602-4799e-mail: hatsumi@med.uoeh-u.ac.jp

THIRTY-SIXTH U.S.-JAPAN TUBERCULOSIS - LEPROSYRESEARCH CONFERENCE

The 36th Research Conference on Tuber-culosis and Leprosy of the U.S.-Japan Co-operative Medical Sciences Program washeld at the Omni Royal Orleans Hotel inNew Orleans, Louisiana, from 15-17 July2001. Dr. James L. Krahenbuhl and his stafffrom the National Hansen's Disease Labo-ratory Research Branch organized the meet-ing. The U.S. Tuberculosis-Leprosy Panelwas comprised of Drs. Clifton E. Barry, III,Thomas P. Gillis, Gilla Kaplan and DavidN. McMurray, with Dr. Philip C. Hopewell

serving as the U.S. Panel Chairman. Drs.Kasuo Kobayashi, Masamichi Goto,Kiyoshi Takatsu and Hatsumi Taniguchicomprised the Japanese Panel with Dr.Masao Mitsuyama serving as the JapanesePanel Chairman.

The abstracts of oral presentations in thearea of leprosy research are presented al-phabetically by first author below, followedby poster presentations. Abstracts of pre-sentations on tuberculosis research will ap-pear in Tubercle and Lung Disease.

69, 4 36th U.S.-.Johan Tuberculosis-Leprosy Research Conference 369

ABSTRACTS

Adams, L. B., Ray, N. A. and Krahen-buhl, J. L. Lack of superoxide produc-tion does not impair on effective host re-sponse to experimental Mycobacteriumleprae infection.

Activated macrophages produce reactiveoxygen intermediates (ROl) and reactivenitrogen intermediates (RNI), which areimportant in host resistance to pathogens.X-CGD mice, a model for X-Iinked chronicgranulomatous disease, have a disruptionin the gp91P'"" subunit of NADPH oxidaseand cannot produce superoxide. Wheninfected with Mycobacterium leprae, nor-mal macrophages from X-CGD mice sup-ported bacterial viability, whereas activatedmacrophages inhibited M. leprae metabo-lism and produced elevated levels of nitrite.Macrophage-mediated killing of M. lepraewas abrogated, however, by incubationwith inhibitors of RNI production. To deter-mine if X-CGD mice would show an im-paired immune response to M. leprae invivo, we examined growth of the bacilli inmouse foot pads. Multiplication of M. lep-rae in X-CGD mice showed a similar pat-tern of growth throughout the infection asthat observed in B6 mice. Thus, a defi-ciency in the production of powerful ROIby phagocytic leukocytes in X-CGD micedid not render them more susceptible to ex-perimental leprosy.

Fukutomi, Y., Kimura, H., Toratini, S.and Matsuoka, NI. Involvement ofcAMP in IL-10 production by macro-phages stimulated with M. leprae.

In response to M. leprae, mouse macro-phages released cytokines, such as TNF-a,IL-10 and IL-1(3, and Prostaglandin E2(PGE2), a cyclooxygenase product. PGE2was required for the induction of IL-10.Forskolin, which activated adenylate cy-clase, enhanced IL- I0 production and con-current suppression of TNF production.Rolipram, which inhibits the activity ofphosphodiesterase, similarly enhanced IL-I() and suppressed TNF. Neither forskolinnor rolipram enhanced the PGE2 produc-

lion by M. leprae-stimulated macrophages.It is well known that PGE2 elevates cAMPin cells by stimulating adenylate cyclase.Phosphodiesterase represents the cAMP-hydrolysing activity. Therefore, it is likelythat the elevation of cAMP triggers M.leprae-induced IL-10 production and the el-evated production of cAMP, either directlyor indirectly through the enhancement ofIL- I0 production, results in the suppressionof TNF production.

Gillis, T. P., Elzer, P. H., Shurig, G. G.,Rambukkana, A. and Krahenbuhl, J.L. Development of new vaccines for lep-rosy and tuberculosis.

We report here the results of two vaccinetrials for leprosy and one for tuberculosis inBALB/c mice. The first vaccine was basedon the M. leprae laminin binding protein-21(ML-LBP 21) thought to be involved in M./eprae—Schwann cell interactions. The vac-cine was given as a purified recombinantprotein made in E. coil mixed with Freund'sincomplete adjuvant. Lymphocyte transfor-mation tests from immunized mice indi-cated sensitization to ML-LBP 21, how-ever, immunized mice were unable to con-trol the infection as judged by growth of Al.leprae in the foot pad. The second vaccinetrial compared protection afforded by Brit-cello abortos vaccine strain RB5 I engi-neered to express either antigen 85A(Ag85A) or ESAT-6 cloned originally fromAl. bovis BCG and Al. bovis, respectively.While each vaccine construct demonstratedprotection over nonvaccinated controlschallenged by the aerosol route with M. tu-berculosis, the best protection was affordedthrough the co-administration of the recom-binant vaccines yielding approximately 1log y „ protection. A separate group of micevaccinated with the recombinant RB5 I con-struct expressing Ag85A showed no protec-tion when challenged with M. leprae.

Goto, M., Nomoto, M., Kitajima, S. andYonezawa, S. Pathogenesis of silentneuropathy of leprosy a morphometric

370 International Journal of Leprosy 2001

analysis of Pgp9.5 positive dermalnerves in autopsy.

Nerve damage should increase after ef-fective leprosy chemotherapy. In a consid-erable number of cured cases, however,neuropathy continues to advance (silentneuropathy). To better understand its patho-genesis, a basic study of dermal nerves wasperformed. Paraffin sections of lower ab-dominal skin taken at autopsy from 9 con-trol cases, 8 cured tuberculoid leprosy casesand 12 lepromatous leprosy cases werestained by Fite's acid-fast staining, anti-BCG and nerve specific anti-PGP9.5 im-munohistochemistry. One of the leproma-tous leprosy cases showed leprous neuritis.The remaining 19 cases showed no neuritisand all cases were negative for Fite andanti-BCG. Correlation of age and PGP9.5positive area was studied. All the tubercu-loid cases and 7/12 of lepromatous caseswere within the normal range, however.5/12 of lepromatous leprosy revealed a re-markable decrease in PGP9.5 positive areabeginning at approximately age 60. Usu-ally, the lower abdomen is spared from lep-rous neuropathy, but this study disclosedloss of abdominal dermal nerve in almosthalf of clinically cured lepromatous leprosycases.

Hagge, D. A., McCormick, G., Scollard,D. and Williams, D. L. An improvedmodel for studying M. leprae/Schwanncell interactions.

The viability of Mycobacterium leprae(ML) may have a major effect on the out-come of infection, therefore, an improvedmodel for studying the effects of ML onSchwann cells (SC), its neural target,should include infection at 33°C. This tem-perature is a permissive temperature forshort-term maintenance of ML viability,however, the effects of 33°C on ML viabil-ity within SC or on SC activity are un-known. The purpose of this study was todetermine the effects of 33°C on neonatalrat SC and embryonic neurons and on MLviability within SC. Results showed that SCmaintained at 33°C appeared to survive, ex-pressed specific gene transcripts, and inter-

acted with axons of embryonic neuronscomparable to those maintained at 37°C.ML retained 58% of their original viabilityup to 21 days post infection within SC at33°C versus 3.6% at 37°C. Preliminary ex-periments suggested that infection at 33°Cleads to disruption of SC monolayers, butdoes not appear to grossly affect SC/axoninteractions when infected cells wereseeded onto axons of embryonic neurons.Therefore, we present an improved modelfor the study of MLISC interactions.

Hagge, D. A., McCormick, G., Scollard,D. and Williams, 1). L. The effect ofMycobacterium leprae infection on cellsurface adhesion molecules of culturedrat Schwann cells.

Previously we have shown that Mycobac-terium leprae (ML) infection of Schwanncells (SC) at an MOI = 100:1 at 33°C re-sulted in the loss of the typical SC `swirled'monolayer morphology. SC appeared to re-tract from the monolayer into large cell ag-gregates and the expression of genes encod-ing the neural cell adhesion molecule(NCAM) and glial fibrillary acidic protein(GFAP) was altered in these cells. Sincethese molecules are important mediators ofSC/SC and SC/axon interactions, these dataindicated that adhesion molecules wereaffected by infection. However, when in-fected SC were seeded onto embryonic neu-rons, they were able to adhere, align, prolif-erate and myelinate axons. This indicatedthat infected SC could participate in nerveregeneration processes. To further investi-gate the effects of ML infection on SCadhesion molecules and SC/axon interac-tions, the expression of several other SCgenes encoding surface adherence mole-cules was studied in SC cultures using RTPCR and the effect of infection on intact,myel i nated SC/axon units was studied us-ing electron microscopy. Results showedthat in addition to the alteration of NCAMand GFAP, the expression of N-cadheringene was down-regulated and 1CAM wasup-regulated in cultured SC while expres-sion of neural adhesion molecule LI wasnot altered. ML were found in the SC ofthese cultures but were not found in axons

69, 4 36th U.S.-.Japan Tuberculosis-Leprosy Research Conference 37I

or in neuronal cell bodies. In addition, in-fection did not appear to affect the ability ofSC to maintain the architecture of myelinsheaths. These cells were able to maintainaxonal contact in a comparable manner tothat of uninfected cells. Therefore, ML in-fection did not appear to impair the func-tional capability of mature, myelinating SC,suggesting that other factors, such as an ag-gressive immune response, are responsiblefor nerve fiber destruction.

Haslett, A. J., Manandhar, R., Shrestha,N., Roche, P., Albert, M., Macdonald,M., Butlin, C. R. and Kaplan, G. Com-plex Mycobacterium leprae antigensstimulate interferon-gamma productionby T cells bearing both gamma-delta andalpha-beta T cell receptors.

Erythema nodosum leprosum (ENL), adebilitating inflammatory complication ofmultibacillary leprosy, is associated withtransient increases in Mycobacteriumleprae-specific T cell immunity in patientsotherwise anergic to M. leprae antigens. Wequantified the M. lep rae-specific T cellresponse in ENL patients utilizing theenzyme-linked immunospot (ELISPOT)technique. Following stimulation withMLSA-LAM, a delipidated cytosolic anti-gen obtained from M. leprae, a surprisinglyhigh frequency of interferon-y (IFN-y)-producing peripheral blood mononuclearcells was observed. This response seemeddisproportional to the modest lymphoprolif-erative responses that have been reportedpreviously in ENL. Further flow-cytometry-based analysis of intracellular cytokineproduction showed that MLSA-LAM-stimulated IFN-y production was predomi-nantly by T cells expressing the yb receptor(TCR-yy), with a minimal contribution byTCR-u..(3 T cells. Similar patterns of IFN-yproduction were noted in normal controlsfrom leprosy endemic areas. Our findingssuggest a possible role for TCR-y6 T cellsin the immunopathogenesis of ENL.

Matsuoka, M., Kashiwabara, Y., Nakata,Bormate, A. B., Wiens, C. and

Legua, P. Genomic diversity of Myco-

bacterium leprae and geographic distri-butions.

Geographic distribution of different rpoTgenotypes of Mycobacterium leprae iso-lated in Paraguay and Peru was investigatedin connection with human prehistoric mi-gration. All M. leprae genotypes of theupoT gene isolated in the two countriesshowed three tandem repeats of 6bp. Noisolates were detected of the rpoT genegenotype with four tandem repeats of 6bp,which is dominant in Japanese and Koreanisolates. Although it was assumed that lep-rosy was carried with ancient Mongoloidsto Latin America, the history of the expan-sion of leprosy in this continent was ob-scure. Polymorphism caused by the 78 baseinsertion sequence was detected in one iso-late based on the results of an arbitrarilyprimed polymerase chain reaction. The dif-ference in nucleotide sequence at this posi-tion seemed not to be applicable for thegenotyping since this unique insertion se-quence was not generally found amongstrains of M. leprae except for one isolate.M. leprae isolates showed various geno-types of TTC repeat but the strain seriallypassed 7 or 11 generations in nude micefootpads showed the same genotype withbacterial material obtained from third gen-eration strain. Genomic diversity of this re-gion seemed to be useful for typing M. lep-rue isolates.

Ohyama, H., Ohira, T., Takeuchi, K.,Nishimura, F., Hirosue, M., Kitanaka,M., Takashiba, S., Murayama, Y. andMatsushita, S. IL-12-induced IFN-yproductivity in T lymphocytes from hu-mans with leprosy.

The objective of this study is to compareT cell responses to interleukin-12 (IL-12)among LL, TT leprosy patients and healthysubjects. The amounts of IFN-y producedfrom T cells stimulated with PHA in thepresence of IL- 12 were measured. Thepolymorphism of the IL-12R131 gene wasanalyzed by using direct sequencing tech-nique to determine the effect of the SNPson IFN-y production by T cells. The resultsof the study are as follows. 1) All T cells

372 International Journal o f Leprosy 2001

isolated from LL and TT patients producedsmall amounts of IFN-y even ha the pres-ence of IL-12, whereas IFN-y productionvaried among T cells isolated from healthydonors. 2) Most subjects with leprosy car-ried the coding SNPs on the IL-12RB1gene. 3) Most activated T cells isolatedfrom donors with the coding SNPs on the1L-12RB 1 gene produced small amounts ofIFN-y in the presence of IL-12. These re-sults suggest that the difference of suscepti-bility to leprosy may be determined by theIFN-y production from activated T cells inthe presence of IL-12, and that the differ-ence of IFN-y productivity might he par-tially explained by the coding SNPs on the1L-12RB 1 gene.

Spencer, J. S., Marques, A. M., Lima, M.C. B. S., Junqueira-Kipnis, A. P., Tru-man, R. W. and Brennan, P. J. Charac-terization of B and T cell epitopes of theM. leprae homologue to ESAT-6.

ESAT-6 from M. tuberculosis is a wellcharacterized immunodominant 6 kDa pro-tein antigen found in the culture filtrate

which was shown to elicit a very potentearly iFN-y response in T cells froni M.tuberculosis-infected mice and humans. Ithas shown promise as a replacement for thePPD skin test since the genetic region thatcodes for this gene is deleted in all strainsof BCG, and thus is not recognized by indi-viduals who have been BCG vaccinated.The sequence of the M. leprae homologueto ESAT-6 shows only 36% homology atthe protein level compared with that of M.tuberculosis. We decided to examine thelevel of immunologic cross-reactivity of theM. leprae ESAT-6 with its M. tuberculosiscounterpart. Polyclonal antisera were ob-tained from slice immunized with the re-combinant forms of both ESAT-6 proteins.The antisera raised against each ESAT-6 re-acted only with the homologous protein.Synthesis of overlapping peptides for theM. leprae ESAT-6 allowed the identifica-tion of both B and T cell epitopes recog-nized by mice. in addition, using polyclonaland monoclonal antibodies specific for M.leprae ESAT-6, we were able to positivelyidentify reactivity by Western blot in sub-cellular preparations of M. leprae.

POSTER PRESENTATIONS

Costa, M. B., Martelli, C. M. T., Stefani,M. M. A., Neto, F. F. C., Maceira, J. P.,Schettini, A. P. M., Nery, J. A. C. andScollard, D. M. Distinct histopathologi-cal patterns in single lesion leprosy: theBrazilian Multicenter Study.

Single lesion leprosy has been identifiedas a clinical entity by the WHO, proposingthat this represents very early, paucibacil-lary (SSL-PB) disease, for which a singletreatment with rifampin, ofloxacin, andmi nocycl i ne (ROM) has been recom-mended. To determine whether clinicalSSL-PB lesions are histopathologically ho-mogeneous, biopsies of lesions from amulti-center ROM trial in Brazil wereevaluated and correlations with Mitsudaskin tests and with circulating antibody toM. leprae PGL-1 were examined. Partici-pants were recruited from clinics in 4

Brazilian States--Amazonas, Rondonia,Rio de Janeiro, and Goiás from October1997—December 1998. Punch biopsies wereobtained from SSL-PB lesions, and H/Eand Fite-Faraco-stained sections were clas-sified by one pathologist (MBC), withoutany clinical information. A second patholo-gist (DMS) reviewed H/E sections of 15%of these, without knowledge of any priorfindings. Biopsies were obtained from278/299 clinically SSL-PB patients; 259were consistent with leprosy, 7 of multi-bacillary disease; 12 had other diseases, and6 were unsuitable for assessment. Lesionswere classified into 5 groups: (1) wellcircumscribed epithelioid cell granuloma;(2) less circumscribed epithelioid cellgranuloma; (3) mononuclear inflammatoryinfiltrate permeated with epithelioid cells;(4) perivascular/periadnexal mononucleal-inflamniatory infiltrate; and (5) minimal or

69, 4 36111 U.S.-Japan Tuberculosis-Leprosy Research Conference 373

no morphological alteration detected. Thedistribution of biopsies within these groupswas as follows:

Group 1 2 3 4 5Number 87 56 31 77 8(%) (33.6) (21.6) (12) (29.7) (3.1)

Nerve involvement was observed in 127biopsies (49.8%), and rare acid-fast organ-isms were found in 17 (6.7%). Close agree-ment (81.6%; 1C95% = 65.7-92.3) was ob-served between pathologists regardingSSL-PB leprosy. An association was ob-served between a positive Mitsuda skin testand patients in Group I (p

374 International Journal of Lepro.sy 2001I

lively transferred cells in vivo without inter-fering with their function, for example cy-totoxicity. The tracking dyes do not inter-fere with doubling times of labeled cellsand the dye appears to be equally parti-tioned between daughter cells when a la-beled cell divides. More recently, a numberof reports have employed tracker dyes to la-bel prokaryote cells such as yeasts and bac-teria. Studies were carried out to determineif fresh, viable nu/nu mouse derived M. lep-rae could be labeled with fluorescenttracker dyes and employed in vitro and invivo. Two tracker dyes, PHK26 (red) andPKH65 (green) were employed to label M.leprae at concentrations ranging from 1:250to 1:1000 and yielded bacilli clearly fluo-rescent extracellularly and intracellularly incultured mouse macrophages. The viabilityof labeled M. leprae appeared to be com-pletely retained as determined by ra-diorespirometry, a quantitative measure ofmetabolism that we have shown to be di-rectly correlated with viability. The viabil-ity of labeled leprosy bacilli was subse-quently confirmed by titration of M. lepraein the mouse foot pad model of infection.Growth of M. leprae labeled at various con-centrations of PKH26 was indistinguishablefrom the growth of control unlabeledbacilli. These findings complement our lab-oratory's goal of characterizing fresh, abun-dant nu/nu mouse derived M. leprae as a re-search resource and will offer an importanttool to investigators interested in the intra-cellular interaction of the live (or dead) lep-rosy bacillus with subcellular componentsand organelles of its various host cells.

Stefani, M. M. A., Robbins, N., Martelli,C. M. T., Pereira, G. A. S., Gillis, T. P.,Krahenbul J. L. and the BrazilianLeprosy Study Group. Cytokine profileand Mycobacterium leprae DNA detec-tion in biopsies of single skin lesion pau-cibacillary (SSL-PB) patients.

Measurements of local cell-mediated im-munity and the presence of M. leprae insingle skill lesion paucibacillary (SSL-PB)leprosy patients were studied to establishthe immunological and bacteriologicalcharacteristics of patients designated to re-

ceive single dose ROM therapy. mRNA forcytokines (IFN-gamma, IL 12, IL 10, IL4and TNF-alpha) was quantitated from skinbiopsies (n = 39) by real time PCR and thepresence of M. leprae DNA was assessedusing an M. leprae-specific PCR (ML-PCR) test. Cytokine results were expressedas a raw data ratio relative to 18S rRNAquantities found in each biopsy and valueswere l og ,

0 transformed to correct for

skewed distributions. High levels of TNF-alpha, low levels of 1L12 and an absence ofIL4 were found in the majority of biopsies.Stratification of the SSL-PB patients intotuberculoid (TT), borderline tuberculoid(BT) and Indeterminate (I) showed that thehighest values for IFN-gamma were amongthe TT group (median = 1.77) followed byBT (median = 1.08) and the I group (me-dian = 0.02). ML-PCR results stratified bythe same criteria showed TT lesions yieldeda lower percentage of positivity (38.5%) forM. leprae as compared to BT lesions(83.3%). Taken together these results are inconcordance with the highly organized na-ture of granuloma seen in TT and strongCMI in these patients. The lowest valuesfor all cytokines was in the indeterminategroup of patients correlating with lower eel-lularity and more undifferentiated infiltratecharacteristic of indeterminate leprosy. Aweak but significant correlation was ob-served (r = 0.4, p = 0.01) between IFN-gamma and IL 12 in the 39 patients suggest-ing that IL12 may drive IFN-gamma pro-duction in these early, single lesions. Astrong correlation was observed (r = 0.7, p

69, 4 36tlr U.S.-Japan Tuberculosis-Leprosy Research Conference 375

ripheral nerves in experimentally infectedarmadillos have revealed conspicuous in-fection of epineurial and endoneurial EC,suggesting that M. leprae enter the en-doneurial compartment through its vascula-ture. Binding and ingestion of M. leprae byhuman umbilical vein endothelial cells(HUVEC) has also been demonstrated. Up-take was slower than reported by EC forother organisms but was saturable in bothtime and dose-response studies, suggestingthat binding is specific. We therefore soughtto determine whether or not activation ofEC would affect the binding and uptake ofM. leprae in vitro. Primary HUVEC wereisolated from umbilical cords and culturedat 37°C in gelatin-coated 96 well plates, or13 mm cover slips, and used after they hadbecome confluent (4-7 days). HUVECwere activated by addition of recombinanthuman TNFa, 50 ng/ml, or endotoxin(LPS), 1000 ng/ml. Activation was con-firmed by a cellular ELISA assay for ex-pression of ICAM- I (CD54), VCAM- l ,and E-selectin (CD62E). Fresh M. lepraewere obtained from nude mouse footpads,' 4C-labeled by incubation in commercialmedium (BACTEC) for 10-12 days,washed x 4 by centrifugation, and manuallycounted. ' 4C-M. leprae were added toHUVEC at a ratio of 100:1. Unboundbacilli were collected on a filter-pad aftervigorous washing using an automated cellharvester; bound and ingested bacilli weresimilarly collected after lysing the cellswith 0.01% SDS. The total radioactivity ofeach fraction was determined by liquidscintillation counting. Uptake of ' 4C-M.leprae was low at 3-6 hr. and became max-imal at 18-24 hr. No difference was ob-served in cultures performed at 33°C vs37°C, and no increase in uptake over base-line was observed with cells cultured at4°C. Activation of cells by TNFa or LPSproduced maximal increases in expressionof adhesion molecules at different times, aspreviously described. Neither agent, how-ever, produced any increase in uptake of'C M. leprae in any combination of pre-

and post-incubation regimens, from 3-18hr. Similarly, no increase in uptake of la-beled organisms could he demonstrated af-ter pre-incubation with unlabeled M. lepraefor 1-18 hr.

Shannon, E. J. and Sandoval, F. Thalido-mide's ability to enhance correlates ofT-cell activation in vitro is dependent onthe stimulant.

Thalidomide's mechanism of action inarresting ENL is unknown. Reports onthalidomide's effect on lymphocytes arecontradictory. The purpose of this studywas to determine if thalidomide influencedactivation of lymphocytes and to partiallycharacterize the phenotype of the respond-ing cell. Peripheral blood mononuclearcells (PBMC) were incubated in thepresence or absence of thalidomide andthen stimulated with SEA, anti-CD3, Con-A, or PHA. The culture supernatant wasassayed for IL-2 and IFN-y. The cells wereharvested to assess proliferation. Thethalidomide-treated-mitogen-stimulatedgroup produced significantly more IL-2than the control group. The PBMC treatedwith thalidomide and stimulated with anti-CD3 or Con-A produced more IFN-y thanthe control group. The PBMC treated withthalidomide and stimulated with SEA orPHA were suppressed in their ability to in-corporate [H3]-thymidine; whereas, thalido-mide enhanced the incorporation of [H 31-thymidine when the PBMC were stimulatedwith anti-CD3. The PBMC were sorted bynegative selection using micro beads conju-gated with anti-human CD8 or anti-humanCD4. When SEA or anti-CD3 were used tostimulate the thalidomide-treated PBMC orCD4+ or CD8+ cells, the PBMC respondedbest in the synthesis of IL-2 and incorpo-rated more [H 3]-thymidine than the CD4+which responded far better than the CD8+cells. Among mitogen stimulated PBMC,thalidomide acts synergistically with themitogen to stimulate the production of IL-2.The particular lymphocyte targeted bythalidomide is CD4+. The type of responsedepended on the nature of the stimulatingagent.

Truman, R. Trends in viability and thehi stopathological response to live M.leprae after intradermal inoculation inleprosy susceptible and resistant ar-madillos.

376 International Journal of Leprosy 2001

We examined the granulomas formed inthe skin of armadillos in response to intra-dermal inoculation of highly viable M. lep-rae and to killed leprosy bacilli. We foundthat the granulomas formed against live M.leprae were significantly larger than thoseproduced to M. leprae killed by heat,gamma irradiation or by freeze/thaw.Among Mitsuda(—) animals (n = 20) granu-lomas involving viable bacilli ranged 2-12times larger in size those made to killed M.leprae, but their cellular composition waslittle changed. Mitsuda(+) animals showedsimilar enhancement with little qualitativedifference in cellular composition. We usedRadiorespirometry (RR) and conventionalmouse foot pad technique (MFP) to exam-ine the viability of M. leprae recoveredfrom these intradermal inoculations. M. lep-rae viabilities fell markedly after initial in-oculation and then stabilized. Bacilli recov-ered from living-Mitsuda reactions showeda broad range of viabilities with variationaccording to the Ridley-Jopling classifica-tion. Highest viabilities were found amongbacilli from multibacillary hosts and the

lowest from among BT's. Over a six weekperiod, the viability of intradermal M. lep-rae among most multibacillary animalstended to increase. They tended to decreaseor remain very low among BT and otherpaucibacillary hosts. The pattern for intra-dermal M. leprae viability among leprosyresistant Mitsuda(—) animals (N = 4) resem-bled that seen for BT hosts, with initial via-bilities waning over time. The trends for in-tradermal viability also correlated with theoutcome of systemic infection. After intra-venous challenge with 10 x l0`' M. leprae,the LL-spectrum animals that had showedhigh intradermal viabilities developed signsof fully disseminated disease, while theMitsuda(—) resistant and paucibacillary ani-mals remained free of leprosy. Actively me-tabolizing bacilli may produce antigens thatare not present among killed bacillarypreparations, and better understanding ofthe M. leprae antigens involved in resis-tance to leprosy by armadillos could signif-icantly benefit our ability to identify andtreat potentially susceptible human con-tacts.

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