INTERNATIONAL JOURNAL OF LEPROSY^ Volume 65, Number 4

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

THIRTY-SECOND U.S.-JAPANTUBERCULOSIS AND LEPROSY

RESEARCH CONFERENCECase Western Reserve University

Cleveland, Ohio, U.S.A.21-23 July 1997sponsored by the

U.S.-Japan Cooperative Medical Science ProgramNational Institute of Allergy and Infectious Diseases

National Institutes of Health

U.S. PanelCo-CI (AIRS

Dr. Jerrold J. EllnerDept. of MedicineCase Western Reserve University2109 Adelbert Road1st Flr., West Wing, Room W113Cleveland, OH 44109

Dr. Patrick J. BennanDept. of MicrobioloyCollege of Veterinary Medicine

and Biomedical SciencesColorado State UniversityFort Collins, CO 80523

MEMBERS

Dr. Thomas P. GillisImmunology Research Dept.Laboratory Research BranchGWL Hansen's Disease Center at LSUP.O. Box 25072Baton Rouge, LA 70894

Dr. Philip C. HopewellChest ServiceSan Francisco General HospitalUniversity of California1001 Potrero Ave., Room 5K1San Francisco, CA 94110

Dr. Gilla KaplanLaboratory of Cellular

Physiology and ImmunologyThe Rockefeller University1230 York AvenueNew York, NY 10021

Dr. David N. McMurrayMedical Microbiology and

Immunology Dept.Texas A&M UniversityMail Stop 1114College Station, TX 77843-1114

546

65,4^U.S.-Japan Cooperative Medical Science Program^547

Japanese Panel

Co-CHAIRS

Dr. Takeshi YamadaDept. of Oral MicrobiologyNagasaki University School

of Dentistry1-7-1 SakamotoNagasaki 852

Dr. Izuo TsuyuguchiDept. of Internal MedicineOsaka Prefectural Habikino

Hospital3-7-1 Habikino, Habikino-shiOsaka 583

MEMI3ERS

Dr. Chiyoji Abe^ Dr. Matsuo MitsuyamaResearch Institute of Tuberculosis^Dept. of BacteriologyJapan Anti-Tuberculosis Assn.^Niigata University School3-1-24 Matsuyama^ of MedicineKiyose-shi^

1-757 Asahimachi-doriTokyo 204^

Niigata 951

Dr. Yasuo Fukutomi^

Dr. Hajime SaitoLeprosy Research Center^ Hiroshima Environment andNational Institute of^

Health Assn.Infectious Diseases^ 9-1 Hirosekita-machi

4-2-1 Aoba-cho^ Naka-kuHigashimurayama-shi^

Tokyo 189Hiroshima 730

THIRTY-SECOND U.S.-JAPAN TUBERCULOSIS-LEPROSY RESEARCH CONFERENCE

The 32nd meeting of the U.S.-Japan Co-operative Medical Sciences Program (Lep-rosy Panel) was held in Cleveland, Ohio,U.S.A., under the auspices of the NationalInstitute of Allergy and Infectious Diseases,the National Institutes of Health, and CaseWestern Reserve University, on 21-23 July1997. Since the Leprosy and TuberculosisPanels of the U.S.-Japan Cooperative Med-ical Sciences Program amalgamated lastyear, the format has changed. The Panelsthemselves now have Co-Chairs and twoother members, equally representing lep-rosy and tuberculosis research. The Panelmembers are: Dr. J. J. Ellner (Co-Chair),Dr. D. N. McMurray and Dr. P. C. Hope-well, representing tuberculosis research onthe U.S. side; Dr. P. J. Brennan (Co-Chair),Dr. T. P. Gillis and Dr. G. Kaplan represent-ing leprosy research on the U.S. side; Dr. I.Tsuyuguchi (Co-Chair), Dr. C. Abe and Dr.

M. Mitsuyama, representing tuberculosisresearch on the Japanese side; and Dr. T.Yamada (Co-Chair), Dr. H. Saito and Dr. Y.Fukotomi, representing leprosy research onthe Japanese side.

Abstracts of presentations on leprosy arepresented below. The abstracts of presenta-tions on tuberculosis research will be pub-lished in Tubercle and Lung Disease.

Dr. Brennan, in the course of his scien-tific presentation, said that, in his experi-ence, there was no apparent role for basicbiological research in the context of the im-mediate goal of leprosy elimination. Hesaid that he was speaking in the context ofhis active participation in the recent meet-ing of the WHO Leprosy Elimination Advi-sory Group and the Joint WHO-ILEPWorkshop on Reaching Undetected Patientsin Endemic Countries. However, he saw animportant role in the post-elimination era,

548^ International Journal of Leprosy^ 1997

when the goal of statistical elimination isachieved and as we strive for disease eradi-cation or deal with the consequences of lep-rosy again becoming a neglected disease.He exhorted basic researchers to be fully

and enthusiastically supportive of the elim-ination goal but to be visionary and to beprepared for subsequent events and to availthemselves of all forums to try to define aglobal research strategy.

ABSTRACTS

Steel, W. F., Williams, I). L., Dallas, W. S.and Gillis, T. P. The foIP of M. lepraeand dapsone resistance.Biomedical evidence suggests that dap-

sone (DDS) resistance in Mycobacteriumleprae is related to the enzyme dihy-dropteroate synthase (DHPS). We have in-vestigated the relationship between DHPSand DDS resistance by characterizing thefolP homolog from resistant and susceptibleM. leprae. The folP from a DDS-suscepti-ble strain of M. leprae was amplified bypolymerase chain reaction (PCR), clonedinto the TA cloning vector pCR-2.1 and theDNA sequence of the folP was confirmedby direct DNA cycle sequencing. The resul-tant recombinant plasmid was transformedinto a DHPS temperature-sensitive mutantof Escherichia coli (MC4100ts3). Cloneswere obtained which complemented the fo-late pathway at 42"C, providing evidencethat the M. leprae folP encodes the DHPS.Two possible mechanisms of DDS resis-tance in M. leprae were then investigated:1) mutations in the folP homolog, and 2)mutations in the promoter region or openreading frame (ORF1) immediately up-stream of the folP homolog. The folP ofthree DDS-resistant and two DDS-suscepti-ble M. leprae strains were amplified byPCR, and the DNA sequence determined.The folP sequences from all strains wereidentical. To determine if mutations withinpromoter-like regions upstream of theORF I contribute to DDS resistance, a puta-tive promoter region was identified basedon sequence comparison with known my-cobacterial promoters. This region was am-plified by PCR, and the DNA sequence wasobtained from a DDS-susceptible strain andcompared to a known DDS-resistant strain.No sequence differences in this region were

observed. In summary, these studies indi-cate that resistance to DDS does not appearto be associated with mutations in either thestructural gene or the promoter regions ofthe gene encoding DHPS in M. leprae.-[Department of Microbiology, LouisianaState University, Baton Rouge; MolecularBiology Research Department, LaboratoryResearch Branch, GWL Hansen's DiseaseCenter at Louisiana State University, BatonRouge, LA; Molecular Science, GlaxoWellcome Inc., Research Triangle, NC,U.S.A.]

Silbay, F. S., TerLouw, S. M., Cho, S.-N.and Brennan, P. J. M. leprae rpsL andrp/R genes.We describe the sequences of two My-

cobacterium leprae ribosomal genes: rpsLthat encodes the S 12 ribosomal protein (stroperon) which binds to the 16S RNA andrplR that encodes the L18 ribosomal pro-teins (spc operon) which is one of three pro-teins that mediate the attachment of the 5SRNA into the large ribosomal subunit. Theresults show that the S12 and L18 ribo-somal proteins are conserved between my-cobacterial species and between mycobac-teria and other prokaryotes. However, theM. leprae rpsL sequence reveals two ATGcodons separated by an 87-bp fragment atthe 5' end. This fragment was absent fromthe corresponding genes of M. bovis, M. in-tracellulare, Micrococcus luteus, Esch-erichia coli and other prokaryotes. In spiteof this 87-bp difference, the sequence of theM. leprae rpsL gene had a high homologyto the M. bovis rpsL gene (87%). A similarhomology was also found between M. lep-rae and M. tuberculosis rplR (83%). Ex-pression of the M. leprae rpsL gene from

65, 4^U.S.-Japan Cooperative Medical Science Program^549

the two start codons produces two differentproteins, 17 kDa (153 aa) and 14 kDa (124aa), sizes that correlate with the deducedamino acid sequences from the two startcodons. An oligopeptide was synthesizedfrom the first 29 aa of the S 12 protein, andserology tests with leprosy patient antibod-ies showed positive signals to the oligopep-tide, i.e., the first 29 aa of the S 12 ribosomalprotein. The major implication of this workis that the M. leprae S12 ribosomal proteinis bigger than that expected from the de-duced amino acids of the M. leprae rpsLgene, published previously, and the corre-sponding genes from other prokaryotes.This difference may have a major effect onthe slow growth rate and the low optimumtemperature of M. leprae.—[Department ofMicrobiology, Colorado State University,Fort Collins, CO, U.S.A.]

Marques, M. A. M., Pessolani, M. C. V.,Cho, S.-N. and Brennan, P. J. Themapping and identification of the ex-pressed proteins of M. leprae.

Mycobacterium leprae purified from in-fected armadillos was disrupted by sonica-tion and the subcellular fractions—mem-brane, cell wall and cytosol—were obtainedby repeated differential centrifugation andsucrose gradient centrifugation. The sub-fractions were then submitted to the follow-ing: a) protein profiling by polyacrylamidegel electrophoresis in the presence of SDS(SDS-PAGE), and by bidimensional elec-trophoresis; b) glycolipid mapping by amodified silver staining procedure; c)monosaccharide profiling by gas-liquidchromatography; d) quantification of di-aminopimelic acid (DAP) as a marker ofcell wall by gas-liquid chromatography; e)reactivity to a set of monoclonal antibodiesby immunoblotting; and f) determination ofphenolic glycolipid-I (PGL-I) content as acapsule/"integument" marker by an ELISA.A total of 374 different major proteins wereidentified in the three fractions, with eachfraction showing a unique and consistentprotein profile. The previously describedGroES (10 kDa), MMPI (35 kDa) andGroEL (65 kDa) proteins were detectedpreferentially in the cytosol, membrane andcell wall, respectively, and proved to be re-

liable protein markers of these compart-ments. The glycolipids, lipoarabinoman-nan, lipomannan and phosphatidylinositolmannosides were observed in all fractions.Analysis for monosaccharides and DAPprovided excellent quality control in thatthere was no galactose (from the cell wall)in the cytosol fraction and little mannose inthe cell wall (hence, LAM/LM is mostlymembrane/cytosolic). DAP and PGL-I pre-dominated in the cell wall fractions. Severalof the new proteins from the cell wall wereidentified by N-terminal sequence analysis.This exercise provides us with added in-sight into the physiological functions ofsub-cellular proteins.—[Colorado StateUniversity, Fort Collins, CO, U.S.A.; Os-waldo Cruz Foundation, Rio de Janeiro,Brazil]

Adams, L. B., Scollard, D. M. and Kra-henbuhl, J. L. Reactive nitrogen inter-mediates regulate granuloma formationin M. leprae-infected mice.Production of reactive nitrogen interme-

diates (RNI) by inducible nitric oxide syn-thase (iNOS) is a major antimicrobialmechanism of activated macrophages.C57BL/6 mice (B6), B6 mice givenaminoguanidine (AG), an inhibitor ofiNOS, and iNOS gene knockout mice(iNOS KO) were infected i.v. with My-cobacterium leprae. Liver tissue from B6showed slow development of granulomas,with moderate-size, nonorganized, inflam-matory infiltrates surrounding the bacilli.AG mice and iNOS KO mice exhibitedlarge, focally organized granulomas con-taining multinucleated giant cells. CD4 andCD8 immunostainings were similar to hu-man tuberculoid granulomas, with CD4+cells dispersed throughout the granulomasand CD8+ cells located peripherally. iNOSmRNA was detected in granulomatous livertissue from B6, but none was found in theiNOS KO mice. Interestingly, high levels ofiNOS mRNA were detected in AG mice. Invitro, iNOS KO activated macrophages andB6 activated macrophages treated with AGproduced negligible nitrite and could notkill M. leprae. Like activated B6 macro-phages, though, activated iNOS KO macro-phages generated tumor necrosis factor-

550^ International Journal of Leprosy^ 1997

alpha (TNF- a). Intracellular mycobacteri-cidal events may be suppressed in vivo inthe absence of RNI and, in compensation,granuloma formation may be greatly en-hanced. This suggests that production ofRNI is an important regulator of cell-medi-ated immunity to M. leprae.—[LaboratoryResearch Branch, GWL Hansen's DiseaseCenter at Louisiana State University, BatonRouge, LA, U.S.A.]

Ohara, N., Matsuoka, M., Nomaguchi,H., Yukitake, H., Matsumoto, S.,Naito, M., Mise, K. and Yamada, 'F.Protection of the infection of M. lepraeby ribosomal particle, culture filtrate,and a antigen from BCG.Immunization of mice with the ribosomal

fraction from ruptured Mycobacterium bo-vis BCG (BCG), the culture filtrate, andcomponents of the antigen 85 complex(85A, 85B = a antigen, and 85C) remark-ably reduced the multiplication of M. lep-rae in the foot pads of mice. This is the firstreported case of the protective activityagainst M. leprae multiplication in mice ofBCG ribosomal fraction and the culture fil-trate. The inhibition was most evident withthe 85A components of the antigen 85 com-plex. When the ribosomal proteins sepa-rated from ribosomal RNA were injectedinto mice, only a slight inhibition was ob-served. Ribosomal RNA alone did not in-hibit at all, in contrast to the conclusion re-ported by Youmans and Youmans.—[Na-gasaki University School of Dentistry,Nagasaki; Leprosy Research Center, Na-tional Institute of Infectious Diseases,Tokyo; National Institute of Health Sci-ences, Tokyo, Japan]

Ohyama, H., Matsushita, S., Hatano, K.,Makino, M., Kato, N., Oyaizu, K.,Takashiba, S. and Murayama, Y. Hu-man T-cell responses to major membraneprotein II (MMP II) derived from M.leprae.

M. leprae is reported to have two immuno-dominant outer membrane proteins, MMP Iand MMP II, which were reported to behighly antigenic in leprosy patients. MMPII, a 22-kDa protein, was identified initially

as a bacterioferritin of M. leprae by Pes-solani, et al. Recently, it was reported thatmost leprosy patients showed a higher IgGtiter in their sera than did healthy controls,and it was hypothesized that the immune re-sponses to MMP II might reflect the im-mune system responses to M. leprae in lep-rosy patients.

In this study, we have evaluated T-cell re-sponses to MMP II in nine subjects, includ-ing leprosy patients. Among them, periph-eral blood mononuclear cells (PBMC) de-rived from one tuberculoid-type patient(DRBI*1501-DQAI*0102-DQB1*0602/DRB I* I 10I-DQA I *0501-DQB1*0302)showed remarkable proliferation whenchallenged with a mixture of each overlap-ping peptide covering the entire MMP IIamino acid sequence. We established aMMP II-specific T-cell line (KT line) fromPBMC in this tuberculoid patient and estab-lished the major T-cell epitopes of MMP II.This KT line reacted to two peptides, MMPII p67-87, including the DRB1*1501 bind-ing motif, and MMP II p89-109, includingthe DRB1*1101 binding motif. MMP IIp89-109 induced a much higher prolifera-tive response in the KT line than did MMPII p67-87. Moreover, anti-DRB I mono-clonal antibody markedly inhibited MMPII-induced proliferation of the KT line. It isconcluded that HLA-DRB1*1101 is one ofthe immune response (Ir) genes which in-duces a high T-cell response to MMP II.—[National Leprosarium Oku Komyo-En,Okayama; Department of Periodontologyand Endodontology, Okayama UniversityDental School, Okayama; Division of Im-munogenetics, Department of Neuroscienceand Immunology, Kumamoto UniversityGraduate School of Medical Sciences, Ku-mamoto-shi, Japan]

Goto, M., Matsuoka, M. and Kitajima,5.4. Animal model of leprous neuritis bythe transfer of CD4+ lymphocytes intoM. leprae-inoculated nude mice.In order to establish animal models of

leprous neuritis and to analyze the patho-genesis, Mycobacterium leprae-inoculatedBALB/C-nu/nu mice were challenged byM. leprae-immunized BALB/C mousespleen-derived CD4+ cells, and the results

65, 4^U.S.-Japan Cooperative Medical Science Program^551

were compared with a naive BALB/Cmouse spleen-derived CD4+ cell changemodel and a M. leprae-immunized wholespleen cell challenge model. In the celltransfer experiments, nude mouse foodpads showed increased swelling. Histologi-cal examination of foot pad nerve fasciclesshowed infiltration of inflammatory cellsand a decrease of the myelinated axons inthe immunized CD4+ cell transfer mice andin the immunized whole spleen cell transfermice. In the nerves, acid-fast bacilli showedfragmentation. In the naive CD4+ celltransfer mice, the nerve lesion was similarbut mild. These results indicate that CD4+lymphocytes play an important role in ex-perimental leprous neuritis formation.—[National Leprosarium Hoshizuka-Keiai-En, Kaya-shi; Leprosy Research Center,National Institute of Infectious Diseases,Tokyo, Japan]

Fukutomi, Y., Toratani, S., Matsuki, G.and Matsuoka, M. Involvement of nitricoxide in induction of anti-M. leprae activ-ity of mouse macrophages and cytokinesthat induce or suppress the activity.Mycobacterium leprae, the causative

agent of leprosy, is an obligate intracellularpathogen that prefers the mononuclearphagocyte as its host cell. A major topic isthe study of the mechanisms of macro-phages to cope with M. leprae in order tounderstand host resistance to leprosy. In thepresent study, M. leprae-infected mousemacrophages were incubated in vitro in thepresence of various cytokines followed bymeasurement of metabolic activity of thebacilli for evaluation of viability. Gamma-interferon (IFN-y) addition to the culture in-duced activation of the macrophages ac-companied by enhanced anti-M. leprae ac-tivity and accumulation in the culturemedium of nitrite/nitrate which were stableproducts of nitric oxide generated by the ac-tivated macrophages. Released tumornecrosis factor (TNF) by macrophages inresponse to M. leprae was also indispens-able in the induction of anti-M. leprae ac-tivity as a second signal with IFN-y prim-ing. T-cell growth factor-beta (TGF-P) in-hibited both anti-M. leprae activity andnitric oxide production. Interleukin-10 (IL-

10), on the other hand, could not suppressthe activity. Rather, it enhanced the activitywith the enhanced nitric oxide production.Concerning TNF production induced inmacrophages by M. leprae phagocytosis,IL-10 suppressed both TNF protein produc-tion and TNF mRNA expression in contrastto the enhanced TNF production by TGF-P.From these results, we conclude that sup-pressed nitric oxide production induced byTGF-P could result in suppression of theanti-M. leprae response of macrophages.The amount of TNF-/3 released in the cul-ture supernatant did not directly affect theextent of the activation.—[Leprosy Re-search Center, National Institute of Infec-tious Diseases, Tokyo, Japan]

Matsuoka, M., Gidoh, M., Kashiwabara,Y., Nagao, E. and Kinjoh, K. Rifampin-resistant isolates from Japanese leprosypatients and mutation in the rpoB gene.Rifampin-resistant Mycobacterium lep-

rae from Japanese patients suffering from arelapse were isolated in nude mouse footpads. Susceptibility against antileprosydrugs was examined either by the mousefoot pad method or the Buddemeyer sys-tem. An isolate exhibited no difference inbacillary growth in the foot pad of the micebetween nontreated mice, log 8.71 ± 0.197,and mice fed diets mixed with rifampin in aconcentration of 0.01% at 50 weeks of in-fection. The isolate was regarded as ri-fampin resistant. The isolate was also resis-tant against KRM 1648, since no reductionof radioactivity was shown at any concen-tration examined in the Buddemeyer sys-tem. A susceptibility test for dapsone(DDS) analyzed by the foot pad methodalso indicated that the isolate was DDS re-sistant at a low level. Direct sequencing ofthe variant revealed a single base changefrom C to T in codon 531 of the rpoB gene,as shown in many other rifampin-resistantisolates. Another biopsy specimen was ob-tained from a patient suspected to be ri-fampin resistant and inoculated, 1.0 x104/0.05 ml, into the nude mouse foot pads.Bacillary numbers in the foot pads of thecontrol mice were log 8.74 ± 0.26 and log5.78 ± 0.10 in the foot pads of the micetreated with rifampin. This isolate is re-

552^ International Journal of Leprosy^ 1997

garded as rifampin resistant at a low level.Silent mutation, a single base change fromC to T, was observed in codon 523 of therpoB gene. No mutation was revealed atany rifampin-binding sites of the rpoB geneof this isolate. The clinical history sug-gested that this isolate is multidrug resis-tant, and another reason that confers mul-tidrug resistance must be considered, suchas discharge of the drugs by the effluxpump.

The banding pattern of PCR-SSCP wasdifferent between the variant and the wildtype. A simple mutation detection methoddirected to the previously revealed hot spotin the rpoB gene is anticipated to be appli-cable for the detection of most rifampin-re-sistant cases in Japan. The result of thesilent mutation indicated that the sampleswhich harbor the mutation in screeningtests, such as PCR-SSCP, must be deter-mined by sequencing for rifampin resis-tance.—[Leprosy Research Center, Na-tional Institute of Infectious Diseases,Tokyo; National Leprosarium OkinawaAiraku-En, Nago-shi, Japan]

Okamura, H., Nomaguchi, H., Kawatsu,K. and Izumi, S. Relationship of leprosytypes and the serum levels of IL-18.Leprosy is classified into tuberculoid (T),

borderline (B), and lepromatous (L) typesaccording to pathological features. Recentinvestigations have clarified that there arebiases in the cytokine types appearing in thelesions of the patients. Gamma-interferon(IFN-y) appears dominantly in the lesionsof T patients, and interleukin-4 (IL-4), -5and -10 in L patients, although these cy-tokines are usually not detectable in the cir-culation. IL-18 (IFN-rinducing factor), arecently cloned cytokine, is produced byactivated macrophages and induces IFN-y,augments natural killer (NK) activity ofsplenocytes, and induces Fas ligand in T orNK cells. A marked synergism between IL-18 and IL-12 is observable for these ac-tions. It has also been clarified that the IL-18 precursor has an unusual leader se-quence, like IL-113, and is processed by anIL-1 P3-converting enzyme (caspase 1) to theactive form. Moreover, IL-18 has beenproved to be produced by various types ofcells other than macrophages, such as ker-

atinocytes, osteoblasts, intestinal epithelialcells, and pituitary or adrenal cortex glandcells. Since IL- 18 strongly induces the Tillcytokine IFN-y, it was expected that IL-18is expressed dominantly in the T type ofleprosy. Thus, in the present study, the lev-els of IL-18 in the circulation of varioustypes of patients were examined.

Human IL-18 was assayed using anELISA kit (Hayashibara, Okayama, Japan).IL-10 and IL-12 were measured by thecommercial ELISA kit, and the titration ofanti-phenolic glycolipid-I (PGL-I) IgM an-tibody was carried out by an ELISA or anagglutination method. Unexpectedly, theserum levels of IL-18 were much higher inthe blood of L patients than in that of T andB patients, which had the same levels as thehealthy controls. Moreover, IL-18 levels inthe circulation of L patients who were curedby successful treatment were significantlylower than those patients with active lepro-matous leprosy. It was of interest that serumIL-18 levels were apparently in parallelwith the levels of anti-PGL-I IgM levels.IL- I 0 and IL-12 in the blood were almostundetectable for any patient, and failed tobe elucidated for their relationship to theleprosy types. It could be considered fromthese results that IL-18 is produced irre-spectively of the dominancy of the Th sub-types. The patho-physiological roles of IL-18 have to be clarified. However, the long-existing IL-18 in the circulation of Lpatients may be related to the pathologicalaspects of leprosy. IL-18 has strong activityto induce the molecules concerned with tis-sue injury in T or NK cells. It also affectsthe secretion of some hormones. Thus, it isstill probable that IL-18 is involved in thepathogenesis of leprosy. This cytokine maybecome one of the tools for the elucidationof the pathogenic mechanism of this diffi-cult disease.—[Laboratory of Host De-fenses, Hyogo College of Medicine, Nishi-nomiya; Institute for Hansen Disease,Tokyo, Japan]

Kobayashi, K., Gidoh, M., Kai, M. andSaito, J. Role of type 1 helper T-cellgamma-interferon-(IFN-y)-inducing cy-tokines in experimental M. leprae infec-tion in mice.Cell-mediated immunity (CMI) partici-

pates in the host defense against mycobac-

65,4^U.S.-Japan Cooperative Medical Science Program^553

terial infection. Both interleukin-12 (IL-12)and interferon-gamma-inducing factor (IGIF/IL-18), produced mainly by macrophages,play critical roles in the expression of CIVILTo investigate the role of IL-12 and IGIF/IL-l8 in vivo, we have analyzed the cy-tokine profile and bacterial growth in genet-ically susceptible and resistant mice in-fected with Mycobacterium leprae. Theearly expression of IL-12 p40 and IGIF/IL-18 at the site of inoculation was found in re-sistant mice 3-72 hr after infection, but notin susceptible mice. Both strains of micedid not show expression of IL-4. Replace-ment IL-12 therapy resulted in the re-duction of bacterial counts in mice with es-tablished M. leprae infection. The resultsimply that susceptible mice exhibit a de-creased type 1 helper T (Th 1) lymphocyteresponse without a reciprocal increased Th2response, and that the intrinsic defect ofmacrophages is responsible for the suscep-tibility. In addition, IL-12 therapy may be apossible rationale for treatment of M. lep-rae infection.—[Departments of Bioregula-tion and Microbiology, Leprosy ResearchCenter, National Institute of Infectious Dis-eases, Tokyo, Japan]

Scollard, D. M., Gillis, T. P. and Williams,D. L. PCR assay for the detection andidentification of M. leprae in the U.S.The differentiation of leprosy from other

cutaneous granulomatous diseases is rou-tinely based on characteristic histopatho-logic features and the demonstration of My-cobacterium leprae by acid-fast staining.Increased ascertainment of other mycobac-terial infections in the skin has made thistask more difficult, but the distinction re-mains fundamental in selecting appropriatetreatment. Experience with formalin-fixedand paraffin-embedded tissues, frozen tis-sues, and tissue lysates referred for M. lep-rae-polymerase chain reaction (PCR) dur-ing the past 4 years has been reviewed.PCR was performed using primers andprobes previously developed to amplify a360-bp fragment of the gene and for an 18-kDa protein of M. leprae. Among 32 biop-sies, PCR was positive in 8/16 diagnosed asleprosy, and in 0/13 diagnosed as non-leprosy by histopathologic criteria. PCRalso identified M. leprae in 1/3 biopsies in

which acid-fast organisms were seen butleprosy was not diagnosed histologically. Ina nonendemic population, the sensitivityand specificity of this technique recom-mend its use primarily to identify M. lepraewhen acid-fast organisms are discerniblebut atypical clinical or histopathologic fea-tures obscure the diagnosis.—[LaboratoryResearch Branch, GWL Hansen's DiseaseCenter at Louisiana State University, BatonRouge, LA, U.S.A.]

Scollard, D. M., McCormick, G. T. andAllen, J. L. Early localization of M. lep-rae to epineural blood vessels.Infection of the peripheral nerve by My-

cobacterium leprae is the histopathologichallmark of leprosy, and is a major factor inthe deformity and social opprobrium of thisdisease, but the mechanisms by which thebacillus localizes to the peripheral nerve arenot known. Nerve involvement in experi-mentally infected armadillos is very similarto that in man; notably, the early accumula-tion of bacilli in this model is to theepineurium. To identify the cellular locationof epineural M. leprae, 1-cm segments ofproximal portions of seven peripheralnerves from five infected armadillos wereprocessed in Spurr resin for both high reso-lution light microscopy and ultrastructuralexamination. M. leprae were found in 18/47specimens (38%), of which 16 (89%) werein the epineurium. The bacilli were usuallylocated in the endothelium or in the adven-titia of small blood vessels. M. leprae werenot found in the epineural lymphatics.These results confirm earlier observationsthat M. leprae localize to the epineuriumearly in the pathogenesis of nerve involve-ment in leprosy, and support the hypothesisthat this is due to interaction with vascularendothelium.—[Department of ResearchPathology, Laboratory Research Branch,GWL Hansen's Disease Center at LouisianaState University, Baton Rouge, LA, U.S.A.]

Scollard, D. M., Gillis, T. P., Gormus, B.J., Lathrop, G. and Murphey-Corb,M. Simian immunodeficiency virus(SIV)-associated delay of CD4 cell accu-mulation at M. leprae sites in vivo is ac-companied by a delay in lymphocyte re-sponses in vitro.

554^ International Journal of Leprosy^ 1997

The mechanism by which immunity tomycobacteria is deranged in HIV-infectedindividuals (and SIV-infected rhesus mon-keys) is not known. Biopsies were taken atcutaneous Mycobacterium leprae inocula-tion sites in four Sly-negative control rhesusmonkeys and eight SIV-positive monkeyswith advanced SAIDS. The percentages ofCD4 and CD8 cells were determined by im-munostaining of frozen sections, and inblood by FACS analysis. Specific lympho-cyte responsiveness to M. leprae by 3 H-thymidine after 6 days of culture with M.leprae sonicate antigen (MLS) was alsomeasured. The percentage of CD4 lympho-cytes peaked at 5 days in healthy, SIV-neg-ative rhesus monkeys, but was delayed un-til 27 days in the eight SIV-positive animalswith advanced SAIDS.

In four Sly-negative animals, lympho-cyte responsiveness increased steadily from3 weeks onward; all four were responsive at15 weeks. In eight SIV-positive animals, re-sponsiveness was not observed until 5weeks (1/6 animals), and only 4/5 were re-sponsive at 15 weeks. A delay has been ob-served in the early CD4 participation in situin SIV-positive animals. Robust mecha-nisms of recruitment appear to remain inSIV-positive animals since the percentageof CD4 cells at inoculation sites ultimatelyreaches levels similar to the maximum inSIV-negative animals. The delay in CD4 re-cruitment appears to be associated with adelay in the initiation of specific, systemicimmunity to M. leprae in SIV-positive ani-mals. This suggests that early events in theresponse to mycobacteria, which may becritical in developing effective immunity,are disrupted by prior infection with SIV.M. leprae infection in rhesus monkeys maybe a useful model with which to study theimmunologic aberrations induced by retro-viruses.—[Laboratory Research Branch,GWL Hansen's Disease Center at LouisianaState University, Baton Rouge, LA; Tulane

Regional Primate Center, Covington, LA,U.S.A.]

Shannon, E. J., Aseffa, A., Sandoval, F.,Shitaye, A. and Webster, K. Thalido-mide enhances the synthesis of IL-2 inENL patients.Background: There is a renewed interest

in thalidomide due to its successful use inthe treatment of dermatological conditionsother than erythema nodosum leprosum(ENL). Although the exact mechanism(s)by which thalidomide attenuates inflamma-tion is unknown, modulation of the immuneresponse plays a role.

Design: Peripheral blood mononuclearcells (PBMC) obtained from patients duringENL (prior to treatment with thalidomide)were exposed for 2 hr to 4.0 pg/ml ofthalidomide and stimulated with 50 ng/mlof staphylococcal enterotoxin A (SEA). Af-ter 16 hr the culture supernatant was as-sessed for interleukin-12 (IL-2) by anELISA.

Results: Compared to SEA-stimulatedcells there was an increase of 127 ± 17pg/ml in IL-2 secreted by the thalidomide-SEA-treated cells. When compared toplasma from untreated patients, and afteringesting 300 mg of thalidomide, there wasa mean increase of 5.4 pg/ml of IL-2.

Conclusion: Besides enhancement ofIL-2 by thalidomide in ENL patients, wehave observed thalidomide to enhance IL-2in SEA-stimulated PBMC from HIV-seropositive patients and PBMC fromhealthy individuals. Ostraat, et al. (Trans-plant Immunol. 4:117, 1996) report similarIL-2 enhancement by thalidomide.—[Labo-ratory Research Branch, GWL Hansen'sDisease Center at Louisiana State Univer-sity, Baton Rouge, LA; Gondar College ofMedical Sciences, Gondar, Ethiopia; Clini-cal Branch, GWL Hansen's Disease Center,Carville, LA, U.S.A.]